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2022-11-16T15:17:22.508Z
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2022-11-15T00:00:00.000Z
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253526400
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s2orc/train
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Case report: Rechallenge with EGFR–TKIs after immunotherapy in EGFR–mutated non–small cell lung cancer with leptomeningeal metastasis
Rechallenge of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) after PD-1 blockade failure was an effective therapy for non-small cell lung cancer (NSCLC) patients with resistance to EGFR-TKIs. The third-generation TKIs, like osimertinib and furmonertinib, can reach higher concentration in the cerebrospinal fluid (CSF) than other TKIs, and exhibit a beneficial effect in NSCLC patients with leptomeningeal metastases (LM) harboring sensitive EGFR mutation. Here, we report that two-stage IV pulmonary adenocarcinoma patients with LM harboring an EGFR L858R mutation benefit from the third-generation EGFR-TKIs rechallenge after immune checkpoint inhibitor (ICI) and anti-angiogenic agent combination therapy. Complete response (CR) to partial response (PR) of central nervous system (CNS) response was achieved immediately after the administration of furmonertinib and osimertinib. We conducted next-generation sequencing (NGS) and IHC to elucidate the evolution of driver mutations and the immune microenvironment. In conclusion, these two cases might provide a therapeutic strategy for further clinical practice. More research was needed to elucidate the resistance mechanisms and improve current treatment strategies in EGFR-mutated patients with LM.
Introduction
Although patients with advanced NSCLC harboring drugsensitive EGFR mutations benefit from the use of EGFR-TKIs, most of them progress within 12 months from the start of treatment due to acquired resistance (1). In addition, approximately 40% of EGFR-mutated NSCLC patients present with disease progression in the central nervous system (CNS), either as brain metastases (BM) or leptomeningeal metastases (LM) after initial EGFR-TKI treatment failure (2). LMs are associated with poor p r o g n o s i s i n N S C L C p a t i e n t s ( 3 ) . A l t h o u g h underdiagnosed, about 10% of EGFR-mutated NSCLC patients experience LM during systemic TKI therapy, leading to dismal outcomes with a median survival of fewer than 6 months (4,5). In clinical practice, many physicians frequently provide a chemotherapy or immunotherapy break followed by EGFR-TKI retreatment (3,6). However, the optimal treatment for patients with EGFR-mutated NSCLC and LM that develops after EGFR-TKI therapy failure remains unclear. It is essential to explore the effectiveness of subsequent EGFR-TKI rechallenges after initial TKI failure in patients with NSCLC and LM.
In this study, we report two cases to evaluate the effectiveness of EGFR-TKIs rechallenge in EGFR-mutated NSCLC patients with LM after TKI failure and interspersed immunotherapy.
Case report 1
A 62-year-old Chinese female without a smoking history was admitted for a cough in September 2018. A computed tomography (CT) of the patient's chest showed a mass in the right upper lung and diffuse micronodules in both lungs ( Figure 1A). A needle biopsy of the mass revealed lung adenocarcinoma. Pemetrexed plus cisplatin were started as first line chemotherapy for two cycles until the disease progressed. CT examination showed that the right upper lung lesion was enlarged and the carcinoembryonic antigen (CEA) level was increased. Then docetaxel and carboplatin were chosen as the second-line treatments, and stable disease (SD) was achieved. Meanwhile, subsequent NGS of lung tissue identified the EGFR L858R mutation and the TP53 exon 8 mutation. PR was achieved after gefitinib (250 mg once daily) was added to the treatment for two cycles ( Figure 1B). After 9 months, anlotinib (12 mg once daily) plus gefitinib (250 mg once daily) were initiated for pulmonary lesion progression, and no mutation was detected in plasma. PR was observed according to the CT scan ( Figure 1C). Seven months later, re-examined CT suggested multiple metastases in both lungs, and the blood CEA was higher than before. Considering the progression of the disease, a pulmonary puncture was performed to search for drug-resistant genes, but the size of the puncture specimens was too small to conduct gene testing. For 1 month, empiric osimertinib was administered, but the disease still progressed. A lung biopsy was performed again on the right upper lung lesion and NGS was performed. The NGS results showed that EGFR L858R mutation, TP53 exon 8 missense mutation, and PD-L1 high expression (TPS = 60%), without EGFR T790M mutation. Toripalimab combined with albumin-bound paclitaxel was administered for two cycles. SD was achieved. But due to the intolerance of chemotherapy toxicity, the treatment regimen was changed to toripalimab plus bevacizumab. PR was achieved after two cycles of treatment ( Figure 1D). Approximately 12 months later, she developed dizziness, headache, and vomiting and was diagnosed with LM based on cranial enhanced magnetic resonance imaging (MRI) and CSF cytological analysis. DNA sequencing of CSF and plasma specimens revealed an EGFR L858R mutation. The EGFR T790M mutation was undetectable. After 3 days of administration of furmonertinib (160 mg once daily), the neurological symptoms disappeared completely. After 1 month of EGFR-TKI rechallenge, CR of intracranial lesions was further confirmed ( Figure 1E). The patient had received furmonertinib for more than 6 months, and no evidence of malignancy recurrence was found by brain MRI ( Figure 1F). The treatment history and gene test results of this patient are presented in Figure 1G.
Case report 2
A 49-year-old Chinese male with a smoking history was admitted to the hospital due to left lung adenocarcinoma with multiple bone metastases in January 2019. Then two cycles of pemetrexed plus carboplatin were initiated. SD was achieved in the pulmonary lesion. However, brain metastasis at the right frontoparietal junction was found by cranial enhanced MRI, and subsequent NGS of lung tissue identified an EGFR L858R mutation, without a T790M mutation. Then, the patient received second-line osimertinib treatment and cranial SBRT local radiotherapy (DT: 50 Gy/10 F) at the same time. Progression of lung lesions was observed by chest CT after 6 months. So docetaxel plus bevacizumab was started as the thirdline treatment for 6 months. Subsequently, bevacizumab therapy was maintained for another 3 months. As pulmonary metastases and bone metastases progressed, another lung biopsy NGS testing revealed an EGFR L858R mutation and high PD-L1 expression (TPS = 80%). The EGFR T790M mutation was undetectable. Therefore, the patient commenced on four-line treatment with anlotinib and durvalumab. PR was observed after two cycles (Figure 2A). But the patient developed a headache, with vomiting and a static tremor 8 months later. Metastatic adenocarcinoma cells were observed by IHC and EGFR L858R, TP53, and KRAS amplifications were obtained by NGS in CSF samples. Based on contrast-enhanced MRI, LM was indicated. The osimertinib rechallenge significantly relieved the headache after one week. Moreover, PR of LM occurred after two cycles of treatment ( Figure 2B). Finally, the osimertinib rechallenge was maintained for more than 11 months and the patient was still in close follow-up ( Figure 2C). The treatment history and gene test results are presented in Figure 2D.
Discussion
To the best of our knowledge, this is the first report to evaluate the efficacy of third-generation EGFR-TKI rechallenge for EGFR L858R mutated NSCLC patients with LM after immunotherapy. This report shows that EGFR-TKI rechallenge after ICI failure provides a prolonged PR in intracranial lesions for NSCLC patients with LM. Case 2 showed that rechallenge with a previously administered EGFR-TKI after the onset of LM may be an effective treatment strategy, not just switching to an unadministered EGFR-TKI like in Case 1. Besides, the use of CSF as a liquid biopsy specimen may facilitate precise diagnosis and personalized treatment for NSCLC with LM. In our cases, the PD-L1 expression level increased a lot after EGFR-TKI treatment resistance ( Figures 3A, B), and a favorable response to subsequent immunotherapy was achieved, as reported by others (7). Because NSCLC patients with EGFR mutation naïve for EGFR-TKI cannot benefit from immunotherapy, the efficacy of immunotherapy could be influenced by tumor microenvironment (TME) changes during the EGFR-TKI treatment (8). Therefore, EGFR-TKIs and immunotherapy may have potential synergistic effects.
The mechanisms of efficacy for furmonertinib or osimertinib rechallenge may be attributed to higher penetration into the CSF (9), recovery of TKI sensitive tumor clones (10), and the histological heterogeneity of intracranial lesions.
However, the precise mechanism of EGFR-TKI rechallenge after immunotherapy failure is unclear. We speculate that two kinds of cell clones may coexist in our cases: one is the EGFR mutant clone (the abundance of clones decreased significantly after TKI treatment, the inferior clone), and the other is the PD-L1 high expression clone (the adaptive production or increase after TKI treatment, the dominant clone). After the immunotherapy, the PD-L1 high-expression clone was at a disadvantage due to its sensitivity to immunotherapy, while the EGFR mutant clone was insensitive to or resistant to immune checkpoint inhibitors. Therefore, the EGFR mutant clone became the dominant clone and then transferred to the leptomeninges (cerebrospinal fluid/blood NGS after immunotherapy resistance also confirmed a significant increase in EGFR abundance), at which time the sensitivity to EGFR-TKI may be restored. So, the EGFR-TKI rechallenge could be effective after immunotherapy failure.
Though irAE did not occur in our cases, the potential toxicity of sequential ICI followed by osimertinib should be monitored closely (11). More clinical studies are needed to explore effective therapy for advanced NSCLC with LM after multi-line treatments.
Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.
Ethics statement
The studies involving human participants were reviewed and approved by the Nanjing Chest Hospital, The Affiliated Brain Hospital of Nanjing Medical University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Author contributions
CQ and SF designed the study. YZ collected the clinical data. QZ and ML participated in collecting the NGS data. YZ and WC analyzed the data. SF reviewed and analyzed data. CQ, YZ, and SF drafted the manuscript. SF supervised the entire study. All authors contributed to the article and approved the submitted version.
Funding
This work was supported by the "Six One Projects" in Jiangsu Province (LGY2019006). We are thankful to all referring surgeons, pathologists, and specialists for their contributions to this study.
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v2
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2022-11-16T16:22:25.321Z
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2022-11-15T00:00:00.000Z
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253542817
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s2ag/train
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Successful diagnosis and treatment of pheochromocytoma during severe coronavirus disease 2019 (COVID-19): a case report.
Pheochromocytoma is a rare but life-threatening condition due to catecholamine release induced by drug treatments such as β-blockers or glucocorticoids. We present a case of hypertensive crisis due to pheochromocytoma, induced after the initiation of dexamethasone and landiolol during intensive care for severe coronavirus disease 2019 (COVID-19). Based on a detailed medical history review, the patient was previously diagnosed with primary aldosteronism by confirmatory tests, moreover, an abdominal computed tomography scan identified an adrenal tumor 2 years before current admission. We tentatively diagnosed the patient with pheochromocytoma and initiated α-blockers without conducting a catecholamine report, leading to stable hemodynamics. We present a successfully managed case of pheochromocytoma concomitant with COVID-19, which has become a global crisis.
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v2
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2022-11-16T16:33:07.236Z
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2022-11-15T00:00:00.000Z
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253531087
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s2orc/train
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Oxidative imbalance increases the risk for colonic polyp and colorectal cancer development
BACKGROUND The role of oxidative stress in the pathogenesis of colorectal carcinoma (CRC) has garnered considerable interest recently. Specific oxidative factors have been implicated in the pathogenesis of adenomatous polyps and ultimately adenocarcinoma. AIM To evaluate the effect of oxidative imbalance as quantified by specific serological markers in the development of sporadic colon adenocarcinoma. METHODS A total of 170 patients that underwent endoscopy of the lower gastrointestinal tract in a tertiary center within 3 years were included in the study. They were allocated in three groups; those with sporadic colon adenocarcinoma (n = 56, 32.9%), those with colonic polyps (n = 33, 19.4%) and healthy controls (n = 81, 47.7%). All patients were evaluated for oxidant activity and antioxidant capacity with serum measurements of specific markers such as vitamins A, 25(OH) D3, E, C, B12, folic acid, glutathione, selenium (Se), zinc (Zn), free iron (Fe2+), and malondialdehyde and results were compared between groups. RESULTS Serum levels of vitamins C, E, D, Se, Zn, vitamin B12 and total antioxidant capacity were significantly lower in the combined neoplasia/polyp group than in the control group (P = 0.002, P = 0.009, P < 0.001, P < 0.001, P < 0.001, P = 0.020 and P < 0.001, correspondingly). Increased levels of vitamin E (P = 0.004), vitamin D (P < 0.001), Se (P < 0.001) and Zn (P < 0.001) seem to bestow a protective effect on the development of CRC. For vitamin D (P < 0.001) and Zn (P = 0.036), this effect seems to extend to the development of colon polyps as well. On the other hand, elevated serum levels of malondialdehyde are associated with a higher risk of CRC (OR = 2.09 compared to controls, P = 0.004). Regarding colonic polyp development, increased concentrations of vitamin Α and Fe2+ are associated with a higher risk, whereas lower levels of malondialdehyde with a lower risk. CONCLUSION Increased oxidative stress may play an important role in the pathogenesis and progression of CRC. Antioxidants’ presence may exert a protective effect in the very early stages of colon carcinogenesis.
INTRODUCTION
The term oxidative stress or oxidative imbalance refers to a series of intracellular complex metabolic processes that lead to overproduction and accumulation of oxidative products, otherwise called free radicals or reactive oxygen species (ROS), including hydrogen peroxide, hydroxyl radical, superoxide anion and peroxynitrite [1]. These, in turn, collaborate to overcome the protective action of existing intracellular antioxidant mechanisms. The overproduction of ROS has been shown to ultimately result in toxicity inimical practically to every cellular macromolecule including all intracellular organelles with a special emphasis on membranes and mitochondria. The net effect of this process is local and eventually generalized impairment of human organ system function, either in the form of inflammation or carcinogenesis [1].
The pathogenesis of sporadic adenocarcinoma (adenoCa) of the large intestine is a complex process involving both genetic and epigenetic factors [2][3][4]. The genetic component refers to the gradual accumulation of multiple genetic mutations in key growth regulatory genes [5], leading to two main types of gene instability (chromosome and microsatellite) which characterize sporadic adenoCa and define its biological behavior [4]. Furthermore, multiple other conditions have been identified as implicated in the pathogenesis of large bowel adenoCa, possibly through chemical modification of DNA bases during the replication process, especially in the stage of aberrant crypt foci (ACF) formation [5,6]. ACF represents the earliest histological alterations in the formation of colorectal neoplasia [6]. Risk factors associated with colorectal cancer include inflammatory bowel disease, nutrition habits (western type of diet), type II diabetes mellitus, sedentary (e.g., low exercise) lifestyle, professional exposure to specific irritants (e.g., mutagenic chemicals such as asbestos), previous medical procedures (e.g., cholecystectomy, radiotherapy of the pelvis, ureterocolic anastomosis), as well as smoking and obesity.
Among the aforementioned risk factors, nutritional habits and their potential influence in particular on the equilibrium between oxidative and antioxidative substances have garnered a significant amount of interest recently. Epidemiological studies indicate that colon adenocarcinoma is observed more frequently among individuals with a diet characterized by a low intake of fiber and calcium and a higher consumption of saturated fatty acids and proteins, especially of bovine origin (red meat)[7-10]. This dietary profile has been associated with increased production of potentially carcinogenic substances such as toxic bile acids and free iron [11]. Furthermore, the resulting positive energy balance (due to the high accumulation of calories when following the western type of diet) and subsequent obesity have been suggested to lead to metabolic stress including overproduction of ROS or other organic compounds, such as malondialdehyde (MDA) [12]. Contrariwise, antioxidant substances including polyphenols, tocopherols, carotenoids, curcumin, vitamin A, vitamin C and vitamin D seem to obtain a protective role against colorectal cancer [1]. It is intriguing to ascertain the exact role of oxidative imbalance in the pathogenesis of initially precancerous lesions such as colonic polyps and ultimately colon cancer. To our knowledge, there are only a few studies that have tried to detect a pattern of total oxidant activity and antioxidant capacity among patients with established sporadic adenocarcinoma of the large intestine, as this may be assessed through the measurement of specific serum compounds and these studies have produced conflicting results [13,14]. Thus, we designed a prospective, case-control, single-center study aiming to evaluate the role of oxidative imbalance and its effect as a possible primary agent in the development of sporadic adenocarcinoma of the large intestine. We tried to achieve this by determining serum levels of specific markers that reflect oxidant capacity in patients with established colorectal carcinoma in comparison to patients with colonic polyps and healthy controls.
Study population
A total of 6500 patients that were over 50-years-old and successfully underwent colonoscopy within 3 years in a major tertiary Greek hospital were screened for participation in the study. A set of specific exclusion criteria were used to curtail the influence of confounding risk factors in data analysis (Table 1). Ultimately 170 patients were included in our study. Among them, three specific groups were defined, those with a histologically confirmed sporadic adenoCa of the colon, those with a diagnosis of colonic polyps and a healthy control group consisting of patients with no significant findings or findings irrelevant to the development of CRC (e.g., diverticula).
Study protocol
All patients gave informed consent for their participation in the study and were interviewed by a gastroenterologist not involved in their endoscopic management. Relevant demographic, epidemiological and clinical characteristics were recorded. Full colonoscopy was performed on all patients following the established sedation, preparation and safety protocols in our Centre following international guidelines. All procedures were performed by the same experienced gastroenterologist (with more than 2000 colonoscopies/year and a rate of over 98% for successful completion of the procedure). When endoscopic findings, suggestive of adenocarcinoma or polyps of the large intestine were identified, single or multiple biopsy samples were obtained and sent to the Pathology department of our hospital. Biopsy specimens were assessed by two independent and experienced pathologists, unaware of the endoscopic findings, with a high inter-observer agreement (> 90%). Sporadic adenocarcinoma was evaluated for the following parameters: (1) Staging according to the Astler-Coller system of classification[15]; (2) Grading according to a three-degree system of differentiation based on the architectural model of development of sporadic adenoCa defined by the presence of adenoCa blasts, as follows: poorly differentiated (0%-49% adenoCa blasts), moderately differentiated (50%-95% adenoCa blasts), well-differentiated (> 95% adenoCa blasts); (3) size of adenoCa (< 1 cm, 1 cm, > 1 cm); (4) the number of adenoCa (1, > 1-synchronous adenoCa); (5) pathologic classification as ulcerative, fungating and polypshaped (either with a stalk or not); and (6) location in the colon. Colonic polyps were evaluated for the following parameters: (1) Histological classification of adenoma according to World Health Organization (WHO) classification as tubular, villous, tubulovillous[16]; (2) grading of adenoma's dysplasia (mild, moderate and severe according to WHO criteria); (3) size of polyp (< 0.5 cm, > 0.5 cm); (4) number of polyps (1, > 1); (5) presence of stalk or not; and (6) location in the colon. Smoking duration in yr (mean ± SD) 37.7 ± 10.0 33.5 ± 9.1 40.3 ± 6.2 Values are presented as n (%) or mean ± SD.
The definite diagnosis of either sporadic adenocarcinoma or colonic polyp was made within a maximum of 2 d from endoscopy. Until definite pathological diagnosis, patients underwent fasting (nil per os), after which blood samples were obtained.
The study protocol was approved by the Ethics Board of our Hospital.
Measurement of serum markers
Calculation of oxidative agents' levels took place as follows: Oxidant activity was measured by using a colorimetric test system for the quantitative determination of total lipid peroxides in serum (PerOx Assay, Immundiagnostik AG, Bensheim, Germany). Malondialdehyde was quantitatively measured using the reverse-phase high-performance liquid chromatography method (HPLC-Analytik, Immundiagnostik AG, Bensheim, Germany) and the Vp Series HPLC System (Series LC-10 ADVp Gradient pump, Series RF-10AXL Fluorescence detector, Series SPD-10ADVp UV detector, Shimadzu, Germany). Ferrum (Fe 2+ ) levels were calculated using a colorimetric assay [FerroZine, Roche Diagnostics GmbH (COBAS), Mannheim, Germany]. Triglycerides were quantitatively calculated by using a colorimetric enzymatic test [Trinder endpoint reaction, Roche Diagnostics GmbH (COBAS), Mannheim, Germany]. Calculation of antioxidative agents' concentrations took place as follows: Total antioxidant capacity was measured by using a colorimetric test system (Imanox Assay) for the quantitative determination of the residual exogenous provided hydrogen peroxide (H 2 O 2 ) that could not be eliminated from the total antioxidants in serum, after having completed an eliminating reaction with a certain amount of exogenously provided hydrogen peroxide (Immundiagnostik AG, Bensheim, Germany). Fat-soluble vitamins A (retinol) and E (α-tocopherol) levels were determined simultaneously in human serum using the reverse-phase HPLC method with the 22000 ClinRep Kit (Recipe Chemicals + Instruments GmbH & Co KG Labortechnik, Munich, Germany). Retinol was monitored at 325 nm and α-tocopherol at 295 nm with the Up series HPLC System (Series LC-10ADVp Gradient pump, Series RF-10AXL Fluorescence detector, Series SPD-10ADVp UV detector, Shimadzu, Germany). Fat-soluble vitamin D (25-OH vitamin D) was measured using a chemiluminescent immunoassay method (DiaSorin LIAISON 25 OH Vitamin D Total Assay, DiaSorin S.p.A, Italy). Water soluble vitamin C was quantitatively measured using the reverse-phase HPLC method (Immundiagnostik AG, Bensheim, Germany). Glutathione (GSH) levels were calculated as the ratio between GSH reduced/GSH total using the reverse-phase HPLC method (Immundiagnostik AG, Bensheim, Germany). Cobalamin/vitamin B12 and folate acid concentrations were calculated using a radioimmunoassay method from MP Biomedicals Inc., New York, United States, with the Packard cobra autogamma g counter from Packard, United States. Selenium (Se) and zinc (Zn) serum levels were determined using a graphite furnace atomic absorption spectrophotometry method with the AA spectrophotometer model 2100 (Perkin Elmer, Norwalk CT, United States). November 15, 2022 Volume 14 Issue 11
Statistical analysis
Statistical analyses were conducted using the SPSS statistical package (IBM Statistical Package for Social Sciences v. 19.0, Chicago, Illinois, United States). At first, cases were distributed according to demographic characteristics and smoking habits as well as levels of the studied compounds (median, 25 th and 75 th percentile for each compound and each patient category). As most variables measured followed a skewed distribution (with the notable exception of triglyceride levels), non-parametric tests (Mann-Whitney and Kruskal-Wallis) were used to compare means between different groups. In order to minimize the effect of possible confounding factors in our results, we then used multiple regression to compare log-transformed serum compound levels between patients with diagnosed pathology (either adenocarcinoma or polyp) vs controls (using two dummy variables for pathology diagnoses correspondingly), controlling for age (as a continuous variable), sex (male vs female), blood triglyceride (as a continuous variable) and smoking habits (as smokers vs non-smokers). Finally, we applied multiple logistic regression models to investigate the association between levels of the measured compounds and either risk of adenocarcinoma or risk of polyp development, controlling for the same variables as in the log-linear models. A two-tailed P value of < 0.05 was considered statistically significant for all comparisons.
RESULTS
The patients' main characteristics are summarized in Table 1. Briefly, our study included 56 patients with adenocarcinoma, 33 patients with colonic polyps and 81 patients in the control group (colonoscopy negative for cancer or precancerous lesions). Patients with adenocarcinoma tended to be relatively older (71.5 ± 6.6 years, mean ± SD), compared to those with colon polyps (66.1 ± 9.4 years) or with the control group (68.4 ± 8.5 years). Among patients with adenocarcinoma or polyps, men presented a clear majority (62.5% and 63.6% respectively) compared to women. In the adenocarcinoma subgroup, 89.3% of patients were smokers (from which 72% with a high rate of use, e.g., more than 20 cigarettes per day) compared to 51.5% of patients who were smokers in the polyp group, whereas smoking habits in the control subgroup bore a close resemblance to those of the adenocarcinoma subgroup (84% smokers from which 70.6% smoked more than 20 cigarettes/day). When comparing patients with adenocarcinoma or polyp(s) with the control group there were notable differences in practically all antioxidant markers (Table 2). Thus, serum levels of vitamins C, E, D, as well as Se, Zn and B12 and total antioxidant capacity were significantly lower in the combined neoplasia/polyp group than in the control group (P = 0.002, P = 0.009, P < 0.001, P < 0.001, P < 0.001, P = 0.02 and P < 0.001, correspondingly). For the antioxidant capacity, in particular, there is a clear picture of higher measurements in the control group when compared to a patient with neoplastic lesions (Figure 1). On the other hand, serum levels of oxidant activity presented the opposite pattern (P < 0.001 for the difference among the three groups) ( Figure 2). In summary, all antioxidant substances were statistically significantly lower among patients with adenocarcinoma compared to controls, except vitamin Α which did not present any differentiation (Table 3). Vitamin D presented the greatest difference since it was lower by 56.8% (95%CI: 50.2% to 62.6%). Although no statistically significant differences regarding the levels of each measured oxidant substance in isolation were observed, total oxidant activity was statistically significantly increased among adenocarcinoma patients.
Next, multivariate analyses were conducted to ascertain which variables presented a true correlation with the neoplastic process. Results were similar between univariate and multivariate analyses regarding the risk of adenoCa (Table 4, which presents the risk of adenocarcinoma in comparison to controls for change in the levels of the measured compounds equal to one standard deviation of its distribution). A significant protective effect was shown especially for vitamin D (OR = 0.04, 95%CI: 0.02 to 0.12) and Zn (OR = 0.16, 95%CI: 0.09 to 0.31) ( Figure 5) but also for vitamin E (OR = 0.57, 95%CI: 0.39 to 0.84) and Se (OR = 0.35, 95%CI: 0.22 to 0.55). An increase of the levels of the abovementioned substances equal to one standard deviation reduced the risk of colon adenocarcinoma to about 50%. The relation between low levels of the aforementioned antioxidants and increased risk of adenocarcinoma remained significant after mutual adjustment, e.g., OR for Se becomes 0.34 (95%CI: 0.13 to 0.88 P = 0.027). As far as the oxidant substances are concerned, the solitary finding was that a doubling of malondialdehyde serum concentration is associated with an approximately twofold increase in the risk for development of colon adenocarcinoma (OR = 2.09 95%CI: 1.27 to 3.45 P = 0.004). Due to the small number of patients in the colon polyp subgroup, fewer associations retained their significance for the development of colon polyps (Table 5). Nevertheless, similarly to results from the CRC group increased levels of vitamin D (OR = 0.27, 95%CI: 0.15 to 0.48, P < 0.001) and Zn (OR = 0.39, 95%CI: 0.16 to 0.94, P = 0.036) exhibited an association with a reduced risk for colon polyp development, whereas it is worthy of note that increased levels of vitamin A were associated with almost 9 times higher risk of colon polyps compared to controls (OR = 8.84, 95%CI: 3.76 to 20.74, P < 0.001). Moreover, November 15, 2022 Volume 14 Issue 11 confers a significant financial burden on health budgets) remains an important challenge. Comparative international epidemiological data indicate that the difference between the highest and lowest sporadic colon cancer incidence is approximately 10-fold, suggesting that environmental factors in the pathogenesis of colon cancer occupy a more prominent role than their genetic counterparts. The dominant environmental factor identified so far is the low-fiber, high-fat diet of Western industrialized countries [17,18].
Although numerous studies dedicated to elucidating the exact role of dietary factors in CRC pathogenesis, and research conducted in a variety of in-vitro and in-vivo animal models strongly hint in favor of a protective effect of antioxidants regarding CRC development, even in the stage of ACF formation, results derived from human populations are not as clear-cut in their results and are in fact at times conflicting [11,12,19,20].
In our study, a basic assumption was made that serum levels of oxidant/antioxidant compounds may accurately reflect the dietary intake habits of individuals and thus could be used to evaluate oxidative imbalance [12]. Over fifty natural and synthetic compounds have been shown to exert a relevant chemotherapeutic effect, but since for the majority of these agents, the literature concerning their role is comparatively scarce, we opted to focus on a variety of compounds with a reasonably established place in the management of the oxidative/anti-oxidative equilibrium [ Regarding the oxidant markers, MDA is an endogenous genotoxic end product of lipid peroxidation by ROS and has been utilized in vivo as a bio-marker of oxidative stress (peroxidability index) [21]. It is thought to participate in harmful processes that lead to DNA damage and mutation mainly through the formation of DNA adducts [22]. MDA-induced DNA lesions, called DNA interstrand cross-link seems to be implicated in the gene-toxic effects associated with lipid peroxidation and oxidative stress [21]. Therefore, MDA has been suggested to be strongly associated with CRC pathogenesis, a suggestion that the results of our study strongly endorse. However, the results from the polyp subgroup present a different picture as MDA levels were significantly lower than those in the control group. This latter finding seems odd, considering the fact that colonic adenomas are established precursors of sporadic CRC, but firstly the small number of cases in this subgroup may skew the results in an unexpected direction, and secondly, this finding may account for a more prominent role of MDA in the second part of the neoplastic process that leads to the evolution of precancerous lesions such as polyps to adenoCa.
On the other hand, elevated levels of free iron (labile iron), another recognized strong oxidant, actually doubled the risk of colon polyp development in our cohort whereas no similar correlation was adducts or by increasing the formation of lipid peroxyl radicals (ferroptosis), such as malondialdehyde and 4-hydroxynonenal which are potent carcinogens [24][25][26]. These findings provide a strong association between excessive intestinal heme iron and colorectal cancer. However, no sufficient evidence is available to our knowledge that links a mechanism of nonheme iron and colorectal cancer [25]. However, emerging evidence suggests that reduced iron intake and low systemic iron levels are also associated with the pathogenesis of colorectal cancer[25]. This is important because patients with colorectal cancer often present with iron deficiency. The mechanism supporting iron deficiency and colorectal cancer development is not fully understood; it may involve cellular functions' requirement for iron, which, when deficient, may hinder immune cells' ability to protect against cancer, providing the potential for a suppressed immunosurveillance response, affecting growth and differentiation of immune cells, as well as influencing cell-mediated immune response and cytokines activities which may contribute to tumor immune-cell evasion and inadequate tumor cell destruction [27]. On the contrary, the association between high iron concentration and the risk of formation of adenomatous (colonic) polyps is ambiguous [28]. Several studies suggest that the presence of high levels of Fe 2+ may induce the formation of colonic polyps, suggesting a potent involvement in the early rather than later steps of colorectal carcinogenesis [1,28,29]. Regarding the antioxidant markers, Vitamin A (retinol) did not exhibit any protective role in our study population. Elevated vitamin A concentrations were associated with almost 9 times higher risk of colon polyps compared to controls with no significant effect observed in the adenoCa subgroup. This is not as confusing as it may seem as results regarding the role of vitamin A in the prevention or recurrence of adenomas have been conflicting so far. Several studies [30][31][32] suggested a protective effect of vitamin A and its derivatives (retinoids) against CRC, whereas Andersen et al [33] failed to establish a beneficial role for vitamin A in CRC. On the contrary, recent studies on the metabolism of vitamin A in CRC imply that despite the presence of high concentrations of retinol or all-trans-retinoic acid (ATRA), CRC has been promoted instead of obtaining decreasing cancer cell proliferation [34][35][36]. The growth and differentiation of the colonic epithelial cells are strongly controlled by retinoid-activated genes which contain retinoic acid receptors (RARs) in their promoter regions. RARs bind to ATRA to induce the transcription of these genes. In many epithelial-derived adenomas and carcinomas, the expression of one or more RAR is lost and the cell loses its ability to regulate normal growth, a phenomenon called "ATRA-resistance". In addition, as CRC progresses, colorectal tumor cells lose the ability to produce ATRA [34]. Kropotova et al [37] claimed that these dysregulated pathways were more observed in adenomas rather than in more advanced carcinomas [37]. Consequently, the high levels of vitamin A in the polyp group in our study might reveal the inadequate protective mechanism of retinol, possibly due to decreased ATRA production and the loss of RAR in the colonic epithelial cells.
On the other hand, Vitamin D was by far the compound with the most significant decrease in concentration among patients with adenocarcinoma or colorectal polyps when compared to controls in our analysis. It should be noted that we assessed vitamin D levels by measuring circulating 25(OH)D3 (calcidiol) levels, thus providing an overall estimate of vitamin D status, as described elsewhere [38]. Since 1980, a large number of epidemiological and experimental studies support the association of vitamin D deficiency with a large variety of human diseases, including an increased incidence of colorectal cancer [39]. The most active metabolite of vitamin D which is 1a,25-dihydroxy vitamin D3 [1,25(OH)2D3, (calcitriol)], is synthesized in a highly regulated multi-step process by mitochondrial 25(OH)D3-1a-hydroxylase [38]. Several cell types, including colon cells have been described to contain vitamin D receptors (VDRs) [38]. When these receptors are activated by calcitriol, they are thought to induce differentiation, regulate detoxification metabolism, sensitize cells to apoptosis and inhibit proliferation, invasiveness, angiogenesis and metastatic potential [39]. In general, according to epidemiological studies, vitamin D deficiency may be linked to a higher risk for neoplasia. A recent meta-analysis of case-control and cohort demonstrated a consistent inverse relationship between serum 25(OH)D3 levels and CRC risk [40]. Another systematic review of studies evaluating the association of vitamin D intake or serum levels of 25(OH)D3 and the risk of CRC suggests as well an inverse correlation between CRC risk and both serum 25(OH)D3 and vitamin D intake [41]. This mostly positive observational data have failed to be confirmed by human intervention studies in which supplemental vitamin D administration was found to be ineffective in reducing colon cancer risk in contrast with dietary sources of vitamin D. These disappointing results may be explained by the timing of administration indicating that colon lesions may progress to a stage where they become unresponsive to vitamin D, bearing, therefore, the hallmarks of an epigenetic change [42]. Moreover, gene expression and activity controlled by VDRs have been described as up-regulated at the early stages of colorectal tumorigenesis with a subsequent sharp decline in advanced CRC [43]. Further investigations of VDR expression at different stages of colon cancer development have come to a consensus that VDR expression is frequently increased at the preneoplastic ACF and the early stages before being lost in more advanced lesions, suggesting a possible role for vitamin D supplementation in early stages with no benefit conferred in advanced cases of this neoplasia [44,45].
Vitamin E is a generic term that describes a group of lipid-soluble chain-breaking antioxidants that exist in nature as eight structurally related forms with α-tocopherol as the isomer found in the highest concentrations in serum and dietary supplements[1]. The results of our study show a potent protective effect for vitamin E in the adenocarcinoma group of patients compared to controls, although studies focusing on vitamin E have produced conflicting results so far [46,47]. Non-significant trends toward reduced blood concentrations of α-tocopherol have been observed in subjects subsequently developing colorectal cancer when compared with controls [47]. Conversely, intakes of other forms of vitamin E ( γtocopherol, δ-tocopherol, γ-tocotrienol and δ-tocotrienol) suggest a highly significant inverse trend between serum concentration of vitamin E and cancer risk (P < 0.001) [30,47]. In a recent interventional study, the administration of a combination of resveratrol and vitamin E to prevent the development of colonic adenomas exhibited clear benefits [48]. Therefore, our findings, though interesting, must be further evaluated within larger sample size studies.
Se, an essential trace element, is one of the most extensively studied anti-oxidant compounds [8]. A protective effect of Se for the prevention of colorectal adenomas development has been convincingly described [49][50][51][52]. Data from the European Prospective Investigation into Cancer and Nutrition cohort that evaluated the effect of Se supplementation according to the dose supplied, demonstrated a statistically significant decrease in the incidence of CRC, although only for a subgroup of subjects with baseline Se concentration < 100 μg/L [52]. These reports are in agreement with our results of a protective effect of higher Se levels regarding CRC risk. In summary, it can be concluded that an inverse doseresponse correlation between the level of Se in serum and the risk of colorectal cancer may exist, albeit this association may be stronger in particular subgroups of patients.
Zn is another potent compound that has been found to play a crucial role mainly in antioxidant defense systems, as a specific activator of many enzymatic reactions (e.g., CuZn Superoxide dismutases), in DNA synthesis as well as in immune functions [53]. Zn has also been shown to inhibit chemically induced neoplastic progression in the colon and to promote the cell cycle arrest of colon cancer cells in animal models [53,54]. Reports regarding Zn levels in biological fluids from CRC patients have been limited but encouraging [54]. In a large Mendelian randomization study, the analysis suggested that increased dietary Zn intake may be associated with a decreased risk of both proximal and distal colon cancer [55]. Similar findings were reported by a recent meta-analysis of nineteen studies that suggested a statistically significant inverse dose-response association of Zn intake with CRC risk [54]. The aforementioned findings are in agreement with our results that hint at a protective role for elevated serum levels of Zn in regards to CRC pathogenesis both in the early and later steps of this process.
There are several limitations to this study. From the antioxidant compounds analyzed in our study, we observed that vitamin D provided the strongest argument in favor of a protective role in the prevention of CRC. An important issue though, that should be taken into account concerning vitamin D assessment is the age of the participants as a potential confounder since it is known that vitamin D insufficiency is strongly associated with increasing age [56][57][58][59]. Of particular interest is also the finding that elevated serum levels of Fe 2+ were associated with a twofold increase in the risk of colon polyp development suggesting a possible role in the formation of colonic polyps and more specifically involvement in early rather than late stages of colorectal carcinogenesis. To our knowledge, there are not many studies in the literature in favor of this association, probably because in most of them, boundand not free-iron was under scrutiny [29]. In our analysis, we also noticed a trend for certain antioxidant substances to be associated with a lower risk of colonic polyp rather than CRC subgroups of patients. This possibly could be explained by a more prominent role in the protective effect of these antioxidants in the early stages of CRC pathogenesis, i.e. before the formation of precancerous cells. This effect, when overcome by the sum of tumorigenic factors will then be attenuated when the adenoma stage is reached rendering interventions such as nutritional antioxidant supplementation incapable of stabilizing or reversing the neoplastic phenotype. This is an attractive theory, especially considering the often inconsistent and even negative results from intervention trials with antioxidant supplementation[1, 60,61]. It is known that selecting the exact timing and duration of the intervention (e.g., the age of the patient at enrollment and the supplementation period) is challenging [62,63]. It remains unclear if interventions given for a relatively short period, as in most of the trials due to practical reasons, have the potential to interrupt the tumorigenic sequence. Furthermore, it is difficult to ascertain the optimal follow-up duration for such a trial to detect an effect on the incidence of a disease such as CRC with a time-extensive pathogenetic process. Apart from that, clinical trials cannot provide evidence concerning the exact point at which chemoprevention begins to take effect concerning the start of treatment or concerning the precise nature of this effect (whether this is gradual or constant) [61,[63][64][65]. In most studies, the relative risk predicted for the incidence of colonic polyp formation or CRC is assumed to be constant because of a lack of data to the contrary, thus suggesting that chemoprevention does not offer any cumulative protection[64,65]. Our study followed the "top-down" approach to studying the exosomal risk factors for CRC onset[2,66]. Thus, it suffers from the known limitations of this approach which we mentioned earlier, mainly that the time-points for specific marker measurements were limited and that the crucial pathophysiological effects regarding CRC pathogenesis may have already taken place. On the other hand, it presents a clear and unbiased approach to the biochemical serum profile of several factors important to the oxidative balance in a sizeable CRC cohort. Thus, while a causal effect can by no means be proven for these compounds, intriguing correlations emerge from our analysis that may be the trigger for further research and new insights.
CONCLUSION
In summary, we describe a possible protective effect for Se, Zn, vitamin E and vitamin D regarding CRC pathogenesis, while elevated levels of MDA were associated with a two-fold increase in the risk for CRC. Regarding the development of colonic polyps, higher serum levels of vitamin D and Zn correlated with a decreased risk of adenoma, whereas elevated levels of vitamin Α and Fe 2+ bestowed a higher risk. Interestingly, lower levels of MDA were found in patients with polyps when compared to controls. Our findings indicate that increased oxidative stress and a reduced antioxidant defense mechanism as assessed by a variety of serum compounds may participate in CRC pathogenesis and progression. Moreover, the possible protective effect of antioxidants may be more important in the very early stages of colon carcinogenesis, probably through an interactive mechanism in the early stages of ACF formation[1,6]. Total antioxidant intake may represent a better predictor of colorectal cancer risk as opposed to specific foods and nutrients [12]. Further trials are needed that should focus on the effect of total antioxidant intake in high-risk for CRC populations but prevention of CRC through manipulation of the oxidative balance in the human body via nutritional supplementation may represent a worthwhile future research target.
Research background
The role of oxidative stress in the pathogenesis of colorectal cancer (CRC) has recently attracted considerable interest. Specific oxidative factors have been implicated in the pathogenesis of adenomatous polyps and ultimately adenocarcinoma.
Research motivation
Several studies have evaluated the association between oxidative imbalance and the development of colorectal adenocarcinoma although the results are conflicting. Thus, the study was designed to assess the correlation between the dietary intake habits of individuals with either colonic polyps or CRC through measurements of oxidant/antioxidant serological markers aiming to introduce novel serum indicators of colonic cancer even in the stage of aberrant crypt foci.
Research objectives
The main objective of the study was to evaluate the effect of total oxidant activity and antioxidant capacity in the development of sporadic colon adenocarcinoma.
Research methods
A total of 170 patients that underwent endoscopy of the lower gastrointestinal tract in a tertiary center within 3 years were included in the study. They were allocated in three groups; those with sporadic colon adenocarcinoma (n = 56, 32.9%), those with colonic polyps (n = 33, 19.4%) and healthy controls (n = 81, 47.7%). All patients were evaluated for oxidant activity and antioxidant capacity with serum measurements of specific markers such as vitamins A, 25(OH) D3, E, C, B12, folic acid, glutathione, selenium (Se), zinc (Zn), free iron (Fe 2+ ) and malondialdehyde and results were compared between groups.
Research results
Serum levels of vitamins C, E, D, Se, Zn, vitamin B12 and total antioxidant capacity were significantly lower in the combined neoplasia/polyp group than in the control group (P = 0.002, P = 0.009, P < 0.001, P < 0.001, P < 0.001, P = 0.020 and P < 0.001, correspondingly). Increased levels of vitamin E (P = 0.004), vitamin D (P < 0.001), Se (P < 0.001) and Zn (P < 0.001) seem to bestow a protective effect on the development of CRC. For vitamin D (P < 0.001) and Zn (P = 0.036), this effect seems to extend to the development of colon polyps as well. On the other hand, elevated serum levels of malondialdehyde are associated with a higher risk of CRC (OR = 2.09 compared to controls, P = 0.004). Regarding colonic polyp development, increased concentrations of vitamin Α and Fe 2+ are associated with a higher risk whereas lower levels of malondialdehyde with a lower risk.
Research conclusions
In conclusion, increased oxidative stress may play an essential role in the pathogenesis and progression of CRC. Antioxidants' presence may exert a protective effect in the early stages of colon carcinogenesis.
Research perspectives
Further research in high-risk CRC populations is needed in order to assess the role of oxidative imbalance in the development of CRC and the potential for colonic cancer by dietary modifications regarding specific oxidative serum markers.
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v2
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2022-11-17T06:18:03.678Z
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2022-11-15T00:00:00.000Z
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253553102
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s2ag/train
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An emergency department nurse led intervention to facilitate serious illness conversations among seriously ill older adults: A feasibility study.
BACKGROUND
Serious illness conversations may lead to care consistent with patients' goals near the end of life. The emergency department could serve as an important time and location for these conversations.
AIM
To determine the feasibility of an emergency department-based, brief motivational interview to stimulate serious illness conversations among seriously ill older adults by trained nurses.
DESIGN
A pre-/post-intervention study.
SETTINGS/PARTICIPANTS
In an urban, tertiary care, academic medical center and a community hospital from January 2021 to January 2022, we prospectively enrolled adults ⩾50 years of age with serious illness and an expected prognosis <1 year. We measured feasibility outcomes using the standardized framework for feasibility studies. In addition, we also collected the validated 4-item Advance Care Planning Engagement Survey (a 5-point Likert scale) at baseline and 4-week follow-up and reviewing the electronic medical record for documentation related to newly completed serious illness conversations.
RESULTS
Among 116 eligible patients who were willing and able to participate, 76 enrolled (65% recruitment rate), and 68 completed the follow-up (91% retention rate). Mean patient age was 64.4 years (SD 8.4), 49% were female, and 58% had metastatic cancer. In all, 16 nurses conducted the intervention, and all participants completed the intervention with a median duration of 27 min. Self-reported Advance Care Planning Engagement increased from 2.78 pre to 3.31 post intervention (readiness to "talk to doctors about end-of-life wishes," p < 0.008). Documentation of health care proxy forms increased (62-70%) as did Medical Order for Life Sustaining Treatment (1-11%) during the 6 months after the emergency department visit.
CONCLUSION
A novel, emergency department-based, nurse-led brief motivational interview to stimulate serious illness conversations is feasible and may improve advance care planning engagement and documentation in seriously ill older adults.
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v2
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2022-11-17T06:18:05.179Z
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2022-11-15T00:00:00.000Z
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253551625
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s2ag/train
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HPV-associated Vulvar Intraepithelial Carcinoma With Sebaceous Differentiation: Report of 2 Cases.
Sebaceous carcinoma (SC) is a malignant neoplasm demonstrating sebocytic differentiation, commonly in the periocular area. Sebocytic differentiation is recognized by multivesicular cytoplasmic clearing with frequent nuclear scalloping. The vesicles can be highlighted by immunohistochemical stains against the perilipin family proteins including adipophilin. Extraocular SC is uncommon but well reported, often in the setting of Muir-Torre syndrome; however, vulvar SC is exceptionally rare. The literature review yielded only 12 prior cases of vulvar SC, all of which showed invasion. Here we report 2 additional similar cases from 2 different institutions of an intraepithelial carcinoma with sebaceous differentiation. Histologic examination of multiple specimens from both patients showed similar features: a multifocal intraepithelial basaloid nodular neoplasm sparing the basal layer with occasional pagetoid spread. The tumor cells demonstrated a high nuclear to cytoplasmic ratio, mitoses, variably foamy vacuolated cytoplasm, and nuclear indentation. Multiple specimens from both patients showed evidence of sebaceous differentiation (substantiated by adipophilin positivity in a membranous vesicular pattern in case 1 and by androgen receptor and epithelial membrane antigen positivity in case 2), and squamous differentiation (substantiated by p63/p40 and weak CK 5/6 expression), as well as human papillomavirus (HPV) association (substantiated by p16 block positivity and detection of high-risk HPV by in situ hybridization). One case was a true in situ lesion without evidence of invasion, and the other case was predominantly an in situ carcinoma with prominent adnexal extension and focal superficial invasion of <1 mm seen in one of multiple specimens. To our knowledge, these 2 cases are the first to show a vulvar SC/carcinoma with sebaceous differentiation that is predominantly limited to the epidermis, and the first documentation of HPV infection in vulvar sebaceous neoplasms. Vulvar intraepithelial carcinoma with sebaceous differentiation is the umbrella term we chose for this entity. Whether this is a true SC in situ that is HPV positive/driven, or a vulvar intraepithelial neoplasia with sebaceous differentiation, is not entirely clear. We emphasize the importance of looking for this morphology to avoid misclassification. Due to the rarity of cases, optimal treatment at this site has not been established.
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v2
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2022-11-17T16:03:49.438Z
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2022-11-15T00:00:00.000Z
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253569678
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s2ag/train
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Abstract A048: Impact of COVID-19 on pancreatic cancer outcomes in Florida
Introduction: Pancreatic cancer (PC) is currently the third-leading cause of cancer deaths in the United States. African Americans with PC have an increased incidence and worse survival outcome when compared to other racial groups. During the COVID-19 pandemic, there is evidence that hospital resources were allocated to treating immediate life-threatening conditions. Some of the daily highest case numbers were reported in the state of Florida with several peaks throughout 2020 and 2021. Additionally, the state of Florida has the second-highest rate of new cases of PC within the United States with an incidence of 4860/100,000. Our specific aim is to define the impact of COVID-19 between race, age, income, and gender on the survival time of newly diagnosed patients with pancreatic cancer in Florida. Materials and Methods: Patients with pancreatic adenocarcinoma diagnosed from January 1st, 2017 to October 31st, 2020 were identified through the statewide clinical research and network database called OneFlorida Clinical Consortium by using the ICD10 diagnosis code for pancreatic cancer. Patients were then placed into 3 cohorts based on date of pancreatic cancer diagnosis: pre-pandemic (01/01/2017- 09/30/2019), transition (10/01/2019-02/28/2020), and pandemic (03/1/2020-10/31/2020). Patients with a diagnosis of neuroendocrine carcinoma were excluded. Patients were followed for at least one year unless a death occurred. Summary statistics were reported for demographic variables (age, sex, income, gender). Kaplan-Meier analysis with log-rank test was performed to compare the difference in overall survival time among groups. Results: This retrospective study had a total of 934 unique patients available for analysis. Of the 934 patients, 81.3% were in the pre-pandemic cohort (n= 759), 8.2% transition cohort (n=77), and 10.5% pandemic cohort (n=98). There was a decrease in the rate of diagnosis from the pre-pandemic (23 per month) to pandemic cohort (12.2 per month). The demographic distribution of the sample was 23.4% Black, 68.7% White and 7.9% Other. The median age was 67 years (27–89). There were 49.8% women and 50.2% men. The median income was $52,915 ($23,704–$124,821). The differences in overall survival time were not significant for age and gender across the 3 cohorts. Income <$53,000 had significantly lower survival time across the 3 cohorts. African Americans had significantly lower survival time for pre-pandemic and transition cohort (p< .005), but Caucasians had the lowest survival time for the pandemic cohort (p <.005). When stratified for stage, the mean survival (in months) for White vs. Black populations was 37.8 vs. 26.1 for stage I, 37.6 vs. 27.3 for stage II, 28.5 vs.18.77 for stage III, and 20.7 vs. 21.7 for stage IV. Discussion: This study demonstrated a decrease in diagnosis & survival rate during the COVID-19 pandemic in Florida. Dissemination of resources should target these disparities in income and race.
Citation Format: Guettchina Telisnor, Alexander S. Lim, Zhongyue Zhang, XiangYang Lou, Ibrahim Nassour, Bo Han, Edward Agyare, Sherise C. Rogers. Impact of COVID-19 on pancreatic cancer outcomes in Florida [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A048.
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v2
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2022-11-17T16:05:25.209Z
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2022-11-15T00:00:00.000Z
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253561381
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s2ag/train
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Abstract C064: Prognostic impact of an immunomorphological signature integrating immune, fibroblast and tumor markers in a series of 217 patients operated on for pancreatic adenocarcinoma
An integrative approach taking into consideration the tumor microenvironment, in particular cancer-associated fibroblasts (CAFs) and tumor-infiltrating lymphocytes (TILs) and its interaction with tumor cells offers a more precise reflect of pancreatic ductal adenocarcinomas (PDACs) biology. In this setting, an immunomorphological signature established from bioinformatics data could thus represent an attractive approach. Moreover to molecular signatures, on which there is still no consensus. This study aimed to evaluate the prognostic value of a panel of morphological and immunohistochemical (IHC) markers on the immune, fibroblastic and epithelial contingents of PDACs for overall (OS) and relapse-free (RFS) survival. Two hundred and seventeen patients operated for PDAC between 2000 and 2017 were included for a tissue microarray (TMA) analysis. A panel of antibodies and special stains was selected based on a previous bioinformatics study (under review) literature data. Slides were digitized and lymphocytes expressing CD3, CD8 and CD20 were quantified. A pixel classifier was used to assess the percentage of collagen and fibroblasts expressing anti-LRRC15, HSP47, α-SMA and FAP antibodies involved in the stroma reaction. Due to the co-marking of stromal and epithelial cells of PTK7 and β-catenin, respectively implicated in embryogenesis and the canonical Wnt pathway, these markers were interpreted semi-quantitatively in a double-blind fashion. After dichotomization using the maximized log-rank method, the association of these parameters with OS and RFS was assessed by univariate and multivariate Cox models. Five years after surgical resection, 26 patients (12.0%) were free of recurrence and 56 (25.8%) were alive. In multivariate analysis, the following parameters were associated with reduced OS and RFS: poor differentiation, lymph node ratio greater than 0.2, loss of tumor β-catenin on tumor cells, a low density of tumor-infiltrating CD3 T lymphocytes and a high percentage of HSP47 fibroblasts. Reduced OS was also associated with an age greater than 65 years at the time of surgery and the absence of adjuvant treatment. In order to evaluate the joint effects of the three tumor compartments markers independentely related to OS and RFS (β-catenin, HSP47, CD3), we grouped them into 3 classes (0/1+, 2+, 3+) according to the number of good prognosis criteria (class 3+ with the greatest number of good prognosis criteria). The multivariate analysis shows an additive effect of these parameters in OS and RFS compared to class 3+ patients. These data underline the interest of simultaneously characterizing the three epithelial, immune and fibroblastic compartments in PDAC. Furthermore, we highlight for the first time the prognostic impact of HSP47 by immunohistochemistry and confirm the β-catenin one. This tissue approach with an IHC panel studied on an operating specimen could be a first step towards applicability on biopsies and lead to a better patient stratification in a therapeutic setting.
Citation Format: Franck Monnien, Chloé Molimard, Marine Abad, Marie-Paule Algros, Sophie Félix, Nikolaus Zirganos, Bruno Heyd, Angélique Vienot, Christophe Borg, Frédéric Bibeau. Prognostic impact of an immunomorphological signature integrating immune, fibroblast and tumor markers in a series of 217 patients operated on for pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C064.
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v2
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2022-11-17T16:05:25.235Z
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2022-11-15T00:00:00.000Z
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253569791
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s2ag/train
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Abstract C044: MICAL2 expression in pancreatic cancer cells modulates the tumor microenvironment through the TGF beta pathway
Introduction: Pancreatic cancer is characterized by a desmoplastic, fibroinflammatory stroma. The crosstalk between cancer cells and their surrounding tumor microenvironment (TME) promotes disease progression, metastasis, and chemoresistance. We identified MICAL2 as a super-enhancer associated gene in pancreatic cancer. MICAL2 (molecule interacting with Cas-L) proteins are Flavin monooxygenases that promotes actin depolymerization and indirectly regulate SRF transcription. In other work, we have found that MICAL2 promotes pancreatic cancer growth and progression. In this study, we evaluated how MICAL2 pancreatic cancer cell expression modulates the PDAC tumor microenvironment. Methods: RNA-Seq analysis performed on human pancreas cancer cells (AsPC) before and after MICAL2 knockdown revealed differential regulation of TGF-β and other proinflammatory cytokines. We validated the expression of TGF-β and other potent cytokines in multiple human and mouse pancreas cancer cell lines by q-PCR. We next exposed human and mouse pancreatic stellate cells (PSCs) to conditioned media from cancer cells before and after MICAL2 knockdown and checked the expression of TGF-β responsive genes by qPCR. KrasG12D/+; Trp53R172H/+; Pdx1-cre (KPC) cells with and without MICAL2 were orthotopically injected to assess the in vivo tumor growth and metastasis. We sorted epithelial cells and CAFs from KPC orthotopic tumors with and without MICAL2 by using EPCAM, PDGFR, and PDPN markers. Results: MICAL2 knockdown (KD) resulted in downregulation of TGF-β gene in both human and mouse pancreas cancer cell lines (50% reduction, p<0.05). Human hPSCs and mouse mPSCs co-cultured with pancreatic cancer cells showed a significant downregulation of myofibroblastic and inflammatory CAFs genes including a-SMA, fibronectin, IL1α and IL6 upon MICAL2 KD as compared to hPSCs and mPSCs co-cultured with shcontrol cells. Orthotopic injections of KPC MICAL2 KD cells led to decreased tumor growth as compared to shcontrol (0.26 gm vs. 1 gm, p = 0.008). Our Immunofluorescence revealed less collagen deposition and α-SMA expression and reduced secretion of IL6 by stromal cells in MICAL2 KD tumors. Flow sorting showed less percentage of epithelial and CAF population in MICAL2 KD orthotopic tumors as compared to Shcontrol tumors. A qPCR analysis of the sorted populations showed less expression of TGF-β on epithelial cells and reduced expression of IL6 on PDGFRα/PDPN+ CAFs extracted from MICAL2 KD tumors as compared to control tumors. Conclusion: These data reveal that MICAL2 expression mediates tumor-stromal crosstalk through TGF- β. Ongoing work is focused on dissecting the role of MICAL2 in priming the metastatic niche through remodeling of the TME.
Citation Format: Bharti Garg, Shweta Sharma, Sohini Khan, Edgar Esparza, Sarah Sass, Dawn Jacquish, Evangeline Mose, Herve Tiriac, Andrew Lowy. MICAL2 expression in pancreatic cancer cells modulates the tumor microenvironment through the TGF beta pathway [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C044.
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v2
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2022-11-17T16:08:11.186Z
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2022-11-15T00:00:00.000Z
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253566656
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s2orc/train
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Targeted therapy with histamine antagonists - New challenges to fight with breast cancer
Breast cancer (BC) is currently the most commonly diagnosed cancer in women. BC is most often derived from the epithelial tissue of the mammary gland and is a global problem due to the steady increase in morbidity and mortality in most countries. A particular problem today is the triple negative subtype (TNBC), which accounts for approximately 10-15% of breast cancer cases. BC occurs most frequently in young women and is characterised by various biological characteristics, an unfavourable clinical course and a poor prognosis. Recent studies to detect the effects of histamine receptors on breast cancer have shown that all H1R-H4R receptors are also hyperactive in the cancer microenvironment. Chronically maintaining a high level of histamine in the tumour-affected tissue contributes to increased angiogenesis at this site, induction of cancer cells proliferation and T lymphocyte dysfunction. The rising incidence of breast cancer is contributing to an increasing amount of research into targeted therapies. Studies on the effect of histamine antagonists through H1R-H4R receptors have proven their effectiveness in the treatment of breast cancer. Among those in the study, there was a reduction in tumour growth, cell proliferation and an increase in apoptosis. The use of histamine antagonists also contributed to a reduced risk of death from breast cancer and increased overall survival (OS). Therefore, targeted therapy is needed to improve the prognosis of patients with breast cancer.
Introduction and purpose
Breast cancer (BC) originates from the epithelium of the mammary gland [1] and is now the most commonly diagnosed cancer in women [2]. Currently there is a continuous increase in its morbidity [3] and mortality [4]. According to a recent report by the World Health Organisation, in as many as 91 countries, cancer mortality in the under-70s population was the 1st or 2nd cause of death [5]. The high incidence of the disease has contributed to a growing body of research into the possibility of alternative forms of treatment [6]. The triple-negative subtype (TNBC) represents approximately 10-15% of all cases of breast cancer. It is most common in young women and is characterised by different biological features, an unfavourable clinical course and a poor prognosis. This subtype of breast cancer represents an important area of research for a new specific targeted therapy due to the insufficient effectiveness of existing therapies [7,8]. It has been shown that the level of histamine in the affected tissues is an important element, especially in the case of breast cancer, but also of the skin, colon or lung or pancreas. The cancer microenvironment contains a large number of histamine receptors H1R, H2R, H3R and H4R and high histamine concentrations. Therefore, targeted therapy is needed to improve the prognosis of patients with breast cancer. Recently, an increasing number of studies confirm the positive effect of histamine antagonists in the treatment of breast cancer [9].
Mechanism of action of histamine and its receptors
Histamine is a biogenic amine that is produced primarily by mast cells and basophils. Platelets, lymphocytes and macrophages are also involved in its production in small amounts. Although histamine is present in the body in relatively low amounts it is probably one of the most pleiotropic molecules currently known in the human [10]. It plays a significant role in inflammatory processes [11], neurotransmission [12], pain perception [13] but is also involved in cancer processes [14]. the development and progression of cancer. There are currently four histamine receptors: H1, H2, H3, H4. They are named respectively according to the order in which they were discovered [15]. The enzyme responsible for histamine production is histidine decarboxylase (HDC). Moreover due to the increased expression of the HDC enzyme at the site of cancerogenesis, it is possible that it will be used as a marker in breast cancer [16,17] Additionally histamine is involved in tumour cell proliferation, ageing, and apoptosis which opens up another perspective for the introduction of new cancer therapies using antihistamines [18]. Research confirms that the microenvironment of cancerous tissues contains a high concentration of histamine and the number of H1R-H4R receptors. In addition, their high level may persist up to several months after the excision of the neoplastic lesion [9]. The figure below shows the pleiotropic mechanisms of action of histamine in the tumour-affected tissue.
Role of Mast Cells:
Mast cells (MC) are derived from hematopoietic stem cells and are primarily characterised by their pleiotropic action [19]. They are a crucial part of the immune response [20]. MCs are involved in inflammatory processes and allergic reactions [21], but it also turns out that they also contribute to processes that protect against cancer and are involved in their development [22]. However, the exact processes involving MC cannot currently be determined. The interference of mast cells in the spread of neoplastic processes occurs through enhanced angiogenesis and increased migration of neoplastic cells as a result of the destruction of the extracellular matrix [23]. Mast cells accumulate in the tumour stroma, influencing the formation of its microenvironment [24]. This is due to their interaction with tumour-affected cells, immune response cells, and the extracellular matrix [25]. According to studies, their increased number within the tumour may be associated with a better or worse prognosis, which depends on the type of tumour and its stage [24]. Extensive research is needed to understand the exact mechanisms of accumulation of mast cells and histamine and its H1R-H4R receptors, which could contribute to alternative forms of treatment for triple-negative breast cancer [26]. It is currently known that increased MC density in axillary lymph nodes in breast cancer has been associated with a better prognosis in many studies [27].
Correlation between histamine receptors and breast cancer
The tumour microenvironment in patients with breast cancer is characterised by hyperactivity of the HDC [28] enzyme compared to healthy tissue in the control group. This results in an increase in the level of histamine in the tissue affected by the tumour [29]. Previous studies to detect the effects of histamine receptors in breast cancer have shown that all H1R-H4R receptors are also overactive in the cancer microenvironment [30]. In addition, in mice, the histamine H1R and H2R receptors had a significant role in the progression and induction of breast tumours in mice. A reduction in the incidence of breast tumours has been reported as a result of the administration of H2R antagonists [31,32]. Additionally, H1R [33] and H2R are over productive which has been associated with a worse prognosis for patients [34]. The mechanism of action of H1R antagonists is to reduce cell proliferation. Also by interfering with the cell cycle, by increasing the G0 / G1 phase and reducing G2 / M which suggests that drugs that affect the H1 receptor may contribute to cell death in breast cancer [35]. It is also worth mentioning the EAG1 protein which induces breast cancer cells to the G1 phase of the cell cycle, which leads to carcinogenesis [36]. Some of the H1R antagonists have the ability to bind to EAG1 channels and inhibit the expression of this gene, which reduces the chances of induction of breast cancer [37].
According to the available sources, the unequivocal effect of histamine on the tissues covered by neoplastic cells cannot be determined. The amount of histamine at the site of the neoplasm affects the cell proliferation in breast tumours in different ways. The low amount acting through the H3R receptor limited cell proliferation [38]. Currently, research is mainly being conducted on the effects of the H4R receptor, which was discovered last. It is overexpressed in breast cancer, but also in other cancers, such as lung cancer, gastrointestinal cancer and skin cancer. It plays an important role in the immune response [17,39].Furthermore, it turns out that the H4R receptor may prove to be a useful marker in monitoring triple negative breast cancer (TNBC). Its increased expression correlated with increased patient survival. It is important to emphasise that there are no specific markers in TNBC at this time [40]. During the study, mice lacking the H4R receptor had significantly less tumour growth and percentage of CD4+ tumour-infiltrating T cells. However, the number of Natural Killer cells increased. In this regard a key role for targeted therapy in triple-negative breast cancer is probably to be played by the H4R receptor [39].
Effect of histamine antagonist on breast cancer
Histamine has been shown to play an important role in breast tumour growth and tumour cell proliferation in several mouse studies. It was possible to establish that the main mechanism acted through the H2R and H4H. Moreover, by administering H2R antagonists to mice, it was possible to reduce tumour growth and cell proliferation [27,41]. However, when Cimetidine (H2R antagonist) was tried on breast cancer patients, it did not improve prognosis [42]. H2R and H4R receptors are involved in the regulation of both CD4+ and CD8+ T lymphocytes. According to a breast cancer study, increased CD4+ lymphocytes in patients led to reduced overall survival (OS). Increased OS in patients was correlated with a low CD4/CD8 ratio as a result of the administration of H4R antagonist [43].
It's also worth mentioning that another study confirming the beneficial effects of histamine on reducing tumour size is one conducted on mice. Mice in this study were exogenously administered histamine dihydrochloride (HDC) which is an immunomodulator that affects the activity of the immune system. This increases the effectiveness of interleukin-2, which stimulates the immune system to attack cancer cells. Mice were administered the exogenous route of histamine dihydrochloride three times a week, resulting in a remarkable reduction in tumour size as compared to the control trial [44]. In addition, it has been shown that antagonists of the histamine H1R receptor limit cell growth and contribute to increased apoptosis through ERK activation in vitro in various tumour cell lines [45,46]. Moreover, these mechanisms only take place in the primary and HER2 breast cancer cells [47]. Among the drugs affecting the H1 receptor, desloratadine and loratadine play a crucial role, increasing survival in women with ER (-) and ER (+). The desloratadine and ebastine are also noteworthy due to the results which indicated a reduced risk of death from breast cancer in women before and after menopause [48]. The study of Astemizole, which is an H1R antagonist, also proved to be noteworthy. Through the mechanisms presented in Fig.2, it contributes to the increased survival of patients with breast cancer [35].
Combination therapy & histamine
Currently, a growing number of studies are aiming to find combination therapies associated with histamine antagonists but the main difficulty with finding an appropriate treatment is that histamine antagonists do not always improve patient outcomes when administered alone. However, the synergistic action of some of the drugs such as cetirizine with thalidomide makes it possible to reduce angiogenesis and tumour size. Moreover, neither of these drugs, when administered alone, had an effect on angiogenesis or reduction of tumour size [49]. The combination of immunotherapy with antihistamine in the treatment of triple-negative breast cancer also brings positive results through an immunostimulating effect and leading to the normalisation of the micro-environment of neoplastic cells [50]. It is also worth mentioning that high levels of histamine contribute to disorders of T lymphocyte function, induction of immunosuppression and increased resistance to immunotherapy. Recently it has been proven that pharmacological maintenance of histamine at a low level using histamine antagonists acting through H1R brings better results of immunotherapy in cancer patients [51]. Phagocytes inhibit signal transmission to NK cells, which accelerates neoplastic processes. However, this process is prevented by the administration of histamine with interferon-alpha, contributing to the killing of tumour cells by NK [52]. Besides this, research is also being conducted on the effect of histamine of radiotherapy. The results indicate that histamine can block epithelial-to-mesenchymal transition events in infiltrated tumour cells. This is another alternative treatment pathway suggesting the usefulness of using histamine in the treatment of breast cancer by radiotherapy [53].
SUMMARY:
At present, we do not know the complicated mechanisms of action of drugs affecting H1R-H4R receptors on the proliferation and proliferation of neoplastic cells in breast cancer. At this point in time, many research results are controversial and inconclusive. Drugs that interact with H1R-H4R receptors have different effects depending on tumour type and stage. Therefore additional multiple studies are necessary. Histamine is known to play an important role in the tumour microenvironment, which affects the subsequent prognosis of patients. The search for new alternative methods may contribute to increased 5-year survival in triple-negative breast cancers. Continued research on the histamine H4R receptor seems promising. Its overexpression is associated with a better prognosis of patients. It may prove to be particularly useful as a prognostic marker in triple negative breast cancer, which so far does not have any specific markers of its own. Work on an effective drug in the treatment of breast cancer should be continued due to the fact that antihistamines have a high level of safety and have a low cost of production.
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2022-11-17T16:11:03.030Z
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2022-11-15T00:00:00.000Z
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253563998
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s2ag/train
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Abstract B028: Bacterial cytotoxin therapy limits tumor growth for pancreatic ductal adenocarcinoma
Treating pancreatic ductal adenocarcinoma (PDAC) with systemic chemotherapeutic drugs has remained a challenge, due in part to the hypovascularized and poorly perfused nature of PDAC tumors, impeding the accumulation of systemically delivered drugs. Several clinical trials aimed at improving drug delivery in PDAC, through targeting of ECM components (HALO-301) or stromal angiogenic signaling (IPI-926-03) have unfortunately not been effective. However, the features that have interfered with systemic therapy in PDAC are potential advantages for the use of bacterial therapies, as bacteria can actively migrate through tissues, thrive in hypoxic microenvironments, and benefit from local immune suppression. Recent developments in the field of synthetic biology have made it possible to engineer complex logic circuits into bacteria, enabling the production of anticancer therapies directly within the tumor parenchyma. Furthermore, live bacteria, once colonized within the tumor niche, are capable of providing a stable source of anticancer compounds directly, rather than relying on repeated systemic doses. We have therefore worked to develop novel bacterial strains and demonstrate preclinical efficacy of a novel strain of therapeutic bacteria for targeting PDAC. We began by testing a range of bacteria-produced toxins and identified the pore-forming protein theta toxin as having the greatest effect in both 2D cell culture and PDAC explant (tissue slice) models. We then engineered a non-toxic probiotic bacteria, E. coli Nissle 1917, to produce either theta toxin or GFP following induction with acyl-homoserine lactone (AHL). To assess preclinical efficacy, we performed intratumoral injections of live GFP- and theta-expressing bacteria into the “KPC” genetically engineered mouse model (Kras LSL.G12D/+; Tp53 LSL.R172H/+; PdxCre tg/+). While GFP-producing bacteria did not induce a change in tumor growth kinetics, treatment with theta toxin-producing bacteria demonstrated prolonged stabilization of tumor growth, increasing the doubling time from 13.7 days (GFP) to 32.5 days (theta) without additional therapy. Indeed, one theta-treated KPC animal lived 113 days following a single bacterial injection, compared to a median of ~12 days for vehicle- or gemcitabine-treated historical controls. Histological analyses demonstrated that diffuse populations of bacteria co-localized with regions of tumor necrosis and cell death, but that bacterial presence and evidence of increased cell death was not observed in healthy tissues, such as the lung, liver, intestine, and diaphragm. Strikingly, while there was minimal spread of bacteria to non-tumor tissues, we observed translocation of the bacteria to regions of liver metastases and distant papillomas following injection of the primary pancreatic tumor, suggesting a mechanism for targeting both known and unknown metastases following local administration. Together these studies demonstrate potent preclinical activity of cytotoxic bacterial therapy as a novel strategy to circumvent the challenges of systemic treatment of PDAC.
Citation Format: Amanda R. Decker, Tetsuhiro Harimoto, Steve A. Sastra, Tal Danino, Kenneth Olive. Bacterial cytotoxin therapy limits tumor growth for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B028.
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2022-11-17T16:11:03.043Z
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2022-11-15T00:00:00.000Z
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253558517
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s2ag/train
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Abstract C032: Combination CAF/myeloid targeting in PDAC
Immunotherapy has revolutionized clinical care for many cancers, yet these treatments fail to control disease in many patients and new strategies are needed to improve anti-tumor immunity and enhance response rates. There is great need for an increased understanding of the cellular crosstalk within tumors and the identification of stromal and immune populations involved in shaping the tumor microenvironment (TME). Local immunosuppression (LIS) is one of the striking hallmarks of Pancreatic Ductal Adenocarcinoma (PDAC), a disease that is highly resistant to existing immunotherapies. Oncogenic Kras activation in tumor cells promotes the invasion and proliferation of tumor-supporting stromal cells, while excluding cancer-targeted cytotoxic T cells. LIS is mediated by multiple subtypes of cancer-associated fibroblasts (CAFs) and myeloid cells resident within the tumor parenchyma. Multiple prior attempts to reverse LIS in PDAC by targeting individual stromal cell populations have been unsuccessful, alluding to the complexity of stromal crosstalk within the TME. The stromal diversity of PDAC complicates investigating paracrine cascades involving multiple cell types. To decipher diverse drug effects on altering the TME, we employ in vivo studies in mouse models recapitulating the human disease, as well as a novel tumor explant model that enables the short-term culture of intact human or murine PDAC. Importantly, PDAC explants maintain their histopathological architecture and cellular diversity over time. This medium-throughput platform allows for testing of multiple drugs and mechanistic hypotheses in the native PDAC TME. We show in preliminary data that Smoothened inhibition (SMOi) decreases proliferation and activity of myCAFs, but provokes the expansion of CD11b-positive myeloid cells in vivo. Thus, we hypothesize that LIS in PDAC is maintained by a delicate balance between myCAFs and myeloid cells, preventing effective T cell invasion. Single cell RNA-seq data comparing ctrl vs. SMOi-treated murine PDAC elucidates stromal subpopulations involved in the LIS phenotype and guides the identification of myeloid subtypes emerging after SMOi. Strikingly, we demonstrated that simultaneous SMOi and targeting myeloid cells via anti-Gr1 or CCR1 inhibition (CCR1i) significantly elevates cytotoxic T cell numbers within the TME. We are currently investigating whether the activity of these T cells may be further potentiated through combination with immunomodulatory agents. By testing various treatment combination in the same TME, we will identify the best synergistic effects for future immunotherapy approaches in human PDAC. In summary, we are elucidating the complex mechanism behind LIS in PDAC by employing our novel explant culture system alongside in vivo studies. We aim to develop a translatable regimen to neutralize LIS, reactivating the cytotoxic T cells in the tumor periphery to invade, proliferate, and attack cancer cells.
Citation Format: Marie C. Hasselluhn, Lukas J. Vlahos, Dafydd Thomas, Alvaro Curiel Garcia, Amanda R. Decker, Tanner C. Dalton, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Combination CAF/myeloid targeting in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C032.
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2022-11-17T16:12:36.737Z
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2022-11-15T00:00:00.000Z
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253562996
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s2ag/train
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Perioperative ventilator-induced lung injury- An unexpected complication of post COVID-19 sequelae
In today’s era of the COVID-19 pandemic, post-covid lung sequelae increases the incidence of ventilator-induced lung injury in patients undergoing cancer surgeries. A 68 years old female patient underwent surgery for squamous cell carcinoma lower lip under general anesthesia. 10 minutes after reversal and adequate respiratory efforts, sudden desaturation with high peak airway pressures of 35-40 cmH2O was noticed. Bilateral air entry was markedly reduced with crepitus all over the chest and abdomen with stable hemodynamics. Chest X-ray revealed a bilateral deep sulcus sign suggesting bilateral pneumothorax and subcutaneous emphysema. Bilateral thoracostomy tubes were inserted immediately. The saturation and airway pressure improved, and she was extubated the next day. Retrospectively, a possible history of previous undiagnosed COVID-19 infection was sought and this emphasizes the importance of this history, in the ongoing pandemic. Previous history of COVID-19 predisposes patients to a high risk of ventilator-induced lung injury perioperatively.
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2022-11-17T16:15:37.255Z
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2022-11-15T00:00:00.000Z
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Abstract A023: First in-human, safety and preliminary efficacy study of (neo)adjuvant, model-based, whole-body hyperthermia treatment in advanced solid cancer patients or stage IV (TxNxM1) metastatic pancreatic adenocarcinoma patients: Liquid biopsies
Hyperthermia, the procedure of raising the temperature of a part of the entire body above normal for a defined period of time, is applied alone or as an adjunctive treatment to various established cancer treatment modalities such as radiotherapy and chemotherapy. Whole-Body Hyperthermia (WBHT), in contrast to local or regional hyperthermia, represents the only hyperthermia modality available for patients with disseminated malignancies. The biological rationale for the treatment of malignant disease by heat is driven by a number of reasons; a) the survival of cells depends on the temperature and duration of heating in a predictable and repeatable way; b) the tumor cell environment (such as hypoxia, poor nutrition, and low pH) that negatively influences the tumor cell killing by ionizing radiation and some chemotherapy regimens, is beneficially influenced by heat therapy; c) the differential sensitivity of normal and tumor cells to heat is dependent on cell type and environmental conditions; d) heat treatment enhances the biological effect of both radiation and chemotherapy agents. The biological rationale is based on a direct cell-killing effect at temperatures in the range of 41– 42°C. A systematic review of van der Horst et al, 2018, addressed clinical trials that used local or whole-body hyperthermia treatment (at variable temperatures) in pancreatic cancer patients. In those described trials, the weighted estimate of the treated population median overall survival was 11.7 compared to 5.6 for the control cohorts. In addition, locoregional hyperthermia (42-44°C) clinical trials showed that the weighted estimate median overall survival of the treated population was 15 months compared to 9 months in control cohorts. The MATTERS trial is a first in-human clinical investigation in advanced solid cancer patients or pancreatic adenocarcinoma patients (TxNxM1). The justification of the design is based on evaluation of pre-clinical data and clinical evaluation of clinical data, safety and/or performance of similar devices/therapies. The study is a mono-centric, non-randomized trial in which the safety and preliminary efficacy of whole-body hyperthermia will be evidenced. Well designed and performed early-stage correlative studies have the potential to strongly influence further clinical development of oncology clinical trials, and correlative data obtained from early stage trials has the potential to provide important guidance on the design and ultimate success of later stage trials. Blood samples will be collected for analysis of immunological panels (e.g. cytokines, chemokines), exosome research, RNA expression profiles. Urine will be collected for analysis of exosome research. The samples will be collected during different timepoints (before, during and after treatment).
Citation Format: Ivana Gorbaslieva, Dana Mustafa, Robin Colenbier, Marc Peeters, Dirk Ysebaert, Vera Saldien, Luigi Brancato, Oleg Rudenko, Johan Van den Bossche, John Paul Bogers. First in-human, safety and preliminary efficacy study of (neo)adjuvant, model-based, whole-body hyperthermia treatment in advanced solid cancer patients or stage IV (TxNxM1) metastatic pancreatic adenocarcinoma patients: Liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A023.
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2022-11-17T16:16:59.223Z
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2022-11-15T00:00:00.000Z
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253559598
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s2ag/train
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Abstract B057: KRAS mutant-specific protein interactions reveal mechanisms in pancreatic cancer tumorigenesis and metabolic regulation
Pancreatic ductal adenocarcinoma (PDAC) is the third most deadly human cancer in the US with a five-year survival rate of 11%. KRAS is mutated in over 95% of PDAC patients and is a key driver of tumorigenesis. Despite the promise of targeted inhibition of the RAF-MEK-ERK MAPK signaling pathway, arguably the most critical KRAS-mediated signaling pathway, clinical trials targeting MEK/ERK signaling as a single-agent therapy have been unsuccessful, indicating the role of additional KRAS-specific signaling pathways. The most frequent KRAS mutations in PDAC are KRAS G12D (40%), KRAS G12V (33%) and KRAS G12R (17%). However, the KRAS G12R mutation is rare in lung and colorectal cancers (<1%), suggesting the presence of KRAS mutant-specific signaling, which remains poorly understood. While mutagenic processes may drive the observed mutation frequency data, many studies have demonstrated that mutant KRAS protein signaling drives the overall observed mutational frequencies. In agreement with this observation, the KRAS Q61L mutant is predicted to occur in PDAC but is rarely detected in the patient population. Therein, we hypothesize that mutation-specific signaling promotes tumorigenesis and that determination of the KRAS mutant-specific interactomes that promote pancreatic tumorigenesis in KRAS G12R yet hinder oncogenic fitness in KRAS Q61L will provide insight into the development of KRAS mutation-selective therapies in PDAC. Thus, we used doxycycline-inducible KRAS constructs combined with BioID proximity labeling to determine the mutant-selective interactomes of four KRAS mutant proteins in an isogenic immortalized pancreatic cell line. While we detected significant overlap in effector signaling, numerous mutant-selective differences were detected, including pathways regulating endocytosis and autophagy. Interestingly, the PDAC tumor microenvironment has been shown to have limited nutrient availability, which promotes macropinocytosis, the nonselective uptake of proteins and molecules from extracellular spaces, and autophagy, a mode of cellular recycling, to promote tumor proliferation. To replicate this environment in cell culture, we utilized a minimal glucose medium supplemented with albumin, a large protein that is absorbed via macropinocytosis. We show that this altered cell culture medium preferentially drives increased macropinocytosis and resistance to MEK MAPK and autophagy inhibition in KRAS G12R-mutant PDAC. Furthermore, while KRAS G12R PDAC cell lines continue to proliferate in the absence of glucose, many KRAS G12D mutant PDAC cell lines fail to sustain proliferation. To determine alternative potential therapeutic vulnerabilities, we have performed an RNA sequencing screen in high and low glucose medium, which has exposed an increase in receptor tyrosine kinase signaling and a reprogramming of metabolic processes in the tricarboxylic acid cycle. These studies provide a rationale for the limited success of MEK/ERK therapies in the clinic and we propose novel treatment strategies for KRAS G12R PDAC patients with elevated macropinocytosis.
Citation Format: Guy Aaron Hobbs, Rachel Burge, Amanda Linke, Kamala Sundararaj, John P. O'Bryan. KRAS mutant-specific protein interactions reveal mechanisms in pancreatic cancer tumorigenesis and metabolic regulation [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B057.
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2022-11-17T16:17:36.779Z
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2022-11-15T00:00:00.000Z
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253560927
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s2orc/train
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Recent advances of bioresponsive polymeric nanomedicine for cancer therapy
A bioresponsive polymeric nanocarrier for drug delivery is able to alter its physical and physicochemical properties in response to a variety of biological signals and pathological changes, and can exert its therapeutic efficacy within a confined space. These nanosystems can optimize the biodistribution and subcellular location of therapeutics by exploiting the differences in biochemical properties between tumors and normal tissues. Moreover, bioresponsive polymer-based nanosystems could be rationally designed as precision therapeutic platforms by optimizing the combination of responsive elements and therapeutic components according to the patient-specific disease type and stage. In this review, recent advances in smart bioresponsive polymeric nanosystems for cancer chemotherapy and immunotherapy will be summarized. We mainly discuss three categories, including acidity-sensitive, redox-responsive, and enzyme-triggered polymeric nanosystems. The important issues regarding clinical translation such as reproducibility, manufacture, and probable toxicity, are also commented.
Introduction
Tumor heterogeneity, unlimited proliferation capacity, and tumor metastasis pose great challenges to cancer treatment [1][2][3][4][5]. The therapeutic efficacy and safety profiles of current tumor treatments, such as immunotherapy and chemotherapy, are still unfavorable, which is largely ascribed to the poor biodistribution of therapeutic drugs [6,7]. Further, these therapeutic drugs may induce unwanted side effects like uncontrolled immune-related systemic cytokine storm and cardiotoxicity [8]. With regard to the therapeutic efficacy, multiple drug resistance and limited immune responsiveness severely hinder their further applications [9][10][11]. Due to this, smart drug delivery systems (DDSs) offer distinct strategies to address these issues.
Among them, nanomedicine has achieved tremendous progress in cancer management [12][13][14]. Nanoparticles can protect the cargo from degradation and facilitate their accumulation at tumor sites through passive diffusion or active targeting, optimizing the drug biodistribution [15][16][17][18][19]. Especially in the past few years, nanocarriers that can respond to biological cues in the tumor microenvironment (TME) attracted increasing attention [20][21][22]. As one representative, responsive polymeric nanoparticles can achieve the precise release of drugs at the tumor site by exploiting the differences in physicochemical properties between tumor tissues and normal tissues, including pH, redox state, as well as particular enzymes (Fig. 1) [23][24][25][26]. The low pH, high concentration of reactive oxygen species (ROS) and glutathione (GSH), as well as overexpression of matrix metalloproteinase (MMP) in tumor tissues or cancer cells can act as responsive factors to achieve precise drug delivery [27,28]. Along with monoresponsive polymers, dual-and multi-response polymeric nanomedicines have also been designed to counter more complicated intracellular and extracellular environmental cues, which enable more functional and controlled release of anticancer 2 Bioresponsive polymeric nanomedicine
pH-responsive polymeric nanomedicine
The extracellular pH (pH e ) of healthy tissues and blood is maintained at 7.4, while their intracellular pH (pH i ) is 7.2 [31,32]. Different from healthy tissues, tumor sites exhibit a lower pH e than healthy tissues, and the pH gradient in most malignancies is inverted (pH i > pH e ). pH i in human patients shows a mean pH value of 7.0, varying from 5.7 to 7.8. This variance is determined by the volume, histology, and detection location of tumors [33]. Nanoparticles can partly accumulate in tumor sites via blood circulation by the enhanced permeability and retention (EPR) effect [34]. And depending on their physical and physiochemical properties, they can either release the drug directly in the TME or be taken up by the tumor cells. The pH responsiveness of the polymeric nanomedicines can be achieved via either the protonation of ionizable groups or the degradation of their acid-cleavable bonds [35]. Accordingly, a range of pH-responsive polymeric nanomedicines have been created for realizing spatiotemporally-controlled drug release, which can be generally classified into extracellular pH-responsive and cytosolic pHresponsive polymers.
Extracellular pH-responsive polymeric nanomedicine
The poor biodistribution and restricted tumor penetration of nanoparticles are two major obstacles for cancer therapies [36]. Research showed that the stealth nanoparticles typically apply the EPR mechanism to target tumors with the help of their high tendency to extravasate across tumor vasculatures and accumulate around blood vessels [37,38]. However, the dense tumor matrix hinders the penetration and tumor accumulation of the large stealth nanoparticles [39,40]. In opposed to that, nanoparticles can penetrate tumors deeply with the help of their low diffusional restriction, but they exhibit a shorter circulating half-life and less accumulation around tumor [41][42][43]. Accordingly, Li et al. developed pH-sensitive size-switchable sensitive cluster nanobombs (SCNs)/Pt nanoparticles to enhance tumor penetration [44]. In this study, SCNs/Pt was fabricated via selfassembly from poly (ethylene glycol)-(2-azepane ethyl methacrylate)-modified poly(amidoamine) (PAMAM) dendrimers (PEG-b-PAEMA-PAMAM/Pt) ( Fig. 2(a)). At physiological pH, PAEMA was hydrophobic and promoted the assembly of PEG-b-PAEMA-PAMAM/Pt into SCNs/Pt. At the acidic TME, PAEMA swiftly protonated and became hydrophilic, resulting in the instant disintegration of SCNs/Pt into smaller particles for efficient tumor penetration (Figs. 2(b) and 2(c)). To verify this, BxPC-3 multicellular spheroids (MCSs) were used to test tumor penetration and tumor inhibition capability. As a consequence, even at a scanning depth of 85 μm, the red signals that represent nanoparticles within the MCSs were visible, which confirmed the bottomless penetration and consistent distribution capabilities of nanoparticles at acidic conditions. Additionally, SCNs/Pt achieved 82% tumor suppression with a 2 mg/kg dose in the mouse-bearing BxPC-3 xenograft tumor.
Adjusting the dimensions of nanoparticles to increase penetration depth into the tumor in responding to the pH variances is another strategy for improving delivery efficiency. The ability to regulate drug release precisely at the tumor site can inhibit drug leakage in normal tissues, thereby minimizing toxicity. However, when compared to the physiological pH (~ pH 7.4), the TME is slightly acidic (~ pH 7.0-6.5) [45]. Thus, more sensitive pH-responsive release systems are needed to meet this challenge.
Liu et al. recently developed a proton transistor nanodetergents (pTNTs) library. The pTNTs were capable of amplifying and transforming small pH disturbance into state transition in membranolytic activity, allowing selective plasma membrane rupture (PMR) for cancer treatment [46]. In this research, the pTNTs were built by a PEG block and a pH-responsive membranolytic block (MB). The MB contains ionizable tertiary amine segments (ethyl piperidine (C6)) and hydrophobic segments (C6-R x ). The mainstream of the tertiary amines was deprotonated and sheltered by a PEG shell, therefore retaining a low membranolytic state ("OFF" state). After a fast rise of C6 protonation in the low-pH environment, the pTNTs were converted into cationic nanoparticles, resulting in powerful membrane cleavage activity ("ON" state) ( Fig. 2(d)). It should be noted that the library of P(C6-R x ) copolymers was screened for an optimized pTNT against Panc02 tumor cells ( Fig. 2(e)). After 4 h of incubation, P(C6-Bn 20 ) displayed a more than 32-fold increase in cytotoxicity with a slight pH shift (0.1 pH). At pH 7.4, P(C6-Bn 20 ) exhibited a selective killing ability while causing minimal harm to cancer and normal cells. Following that, subsequent experiments showed that P(C6-Bn 20 ) exerted a cytotoxic outcome for tumor cells via a membranolytic manner under acidic conditions.
Cytosolic pH-responsive polymeric nanomedicine
Nanoparticles could be taken up by cells through the endocytic pathway [47]. These nanoparticles must overcome endosomal trapping to release cargo into the cell cytosol. In particular, nucleic acids like small interfering ribonucleic acid (siRNA) are insecure in acidic endosomal environments [48,49]. To break through this barrier, Ling et al. developed a pH-responsive, point-source burst nanoscale coordination polymer (NCP) particle named CbP/siRNA against PD (siPD)-L1@Dig, which contains carboplatin (Carb), digitoxin (Dig), and siPD-L1 [50]. In their research, when under acidic conditions, NCP generate phosphate ions and the produced osmotic stress could rupture endosomal Nano Res.
membranes, allowing siPD-L1 for efficient endosomal escape. As a result, western blot analysis revealed that at a 5 nM siRNA dose, both CbP/siPD-L1 and CbP/siPDL1@Dig effectively suppressed PD-L1 expression, achieving knockdown efficacy of > 95% in vitro. Furthermore, CbP/siPD-L1@Dig dramatically inhibited tumor development in CT26-bearing mice, with a tumor growth inhibition (TGI) of 80.8% ± 5.6%, and mice showed a median survival of 44 days and less peritoneal metastatic dissemination. Recently, increasing attention has been paid to the stimulator of interferon genes (STING), a cytosolic pattern recognition receptor that is crucial for eliciting spontaneous antitumor T-cell immunity [51,52]. 2′ ,5-3′ 5′ cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is an endogenous ligand for STING and is produced by the enzyme cyclic-GMP-AMP synthase in response to the existence of tumor-derived DNA in the cytoplasm [53][54][55]. T cells are primed by activating STING, initiating a complicated type I interferon (IFN-I)-driven inflammatory response [56]. However, cGAMP is restricted by its low bioavailability which hinders its access to the cytosol to engage with STING [57]. To solve this dilemma, Shae et al. developed polymersomes that contain an aqueous center and a vesicle membrane composed of pH-responsive, membrane-destabilizing amphiphilic diblock copolymer chains to realize its intracellular release and endosomal escape (Figs. 3(a) and 3(b)) [58]. Importantly, a molar ratio of cationic 2-(diethylamino) ethyl methacrylate (DEAEMA) groups to hydrophobic butyl methacrylate (BMA) moieties was previously revealed for the optimum of endosomal escape. As a result, they observed a 5-10 fold gene expression increase in mice treated with STINGnanoparticle (NP) in comparison with free cGAMP, along with a 35-fold increase of Cxcl1 and a 20-fold rise of Ifna2. Meanwhile, by utilizing B16F10 melanoma models, they revealed that STING-NPs achieved an 11-fold inhibition of tumor growth and a remarkable prolongation in the survival time compared to the cGAMP group. To go further, Shae et al. combined STING agonist with antigenic peptides as a personalized cancer vaccine, termed as NanoSTING-vax ( Fig. 3(c)) [59]. This NanoSTING-vax elicited nearly 8% tumor-specific CD8 + T cells in the blood, twice as much as in the free peptide group and the soluble mixture of cGAMP and peptide group.
Given the above, many pH-responsive polymeric nanomedicines were developed in the past few years. The upcoming direction of pH-responsive polymeric nanomedicine in cancer treatment may need to step towards the concept of "library", such as classification for different subtypes, causes, and stages of same cancer, and classification for different cancer types. Of course, this requires a large enough number of trials and samples.
Redox-responsive polymeric nanomedicine
Due to the fact that altered levels of redox molecules are closely related to numerous diseases, redox-responsive polymeric nanomaterials (PNMs) are appealing targets for DDSs [60]. In comparison with normal cells, tumor cells have a highly reducing environment due to the excessive GSH synthesis inside the cell [61]. In addition, tumor cells can produce excessive ROS, resulting in an increase in oxidative stress [62]. Based on the unique intracellular and extracellular redox environments of tumors, an increasing number of redox-responsive polymeric systems were created [60]. In this part, we discuss anticancer drug delivery polymeric systems including GSH-responsive and ROS-responsive in the past few years and highlight recent novel strategies.
GSH-responsive polymeric nanomedicine
Although neutral and negatively charged nanoparticles can travel further in the bloodstream in comparison with those positively charged nanoparticles, their cellular absorption and tumor accumulation capability are restricted [63]. Alternatively, positively charged nanoparticles can assist endosomal escape and give rise to an enhanced tumor accumulation of therapeutics [64]. To address this issue, Jia et al. developed a GSH-responsive nanocarrier, which was composed of disulfide-doped organosilica-micellar hybrid nanoparticles. Also, the nanoparticles were modified with PEG and polyethyleneimine (PEI) (Fig. 4(a)) [65]. In this study, PEG shielded the positive charges of PEI and therefore prolonged the circulation of the nanocarrier via inhibiting the nonspecific protein adsorption. When the nanoparticles reach the TME, the extracellular GSH (2−20 μM) could induce the first-stage redox responsive activity, along with PEG chain separation. Then, the short-chain PEI was exposed to the environment and the nanocarrier carried positive charges, resulting in increased uptake of tumor cells via electrostatic interactions. Furthermore, owing to the proton sponge effect, the exposed PEI further promoted the endosomal escape of the nanocarrier and their entry into the cytoplasm [66,67]. After that, high intracellular GSH concentrations could trigger second-stage redox responsiveness via disulfide bond breakage in the silsesquioxane matrix, resulting in the release of the inner drugs. Consequently, exploiting hydroxycamptothecin (HCPT) as a model drug, HCPT@DOSN-PEI-SS-PEG exhibited 2.5-fold improved antitumor efficacy (56.8%) in comparison with HCPT@DOSN-PEI-SC-PEG (23.0%). Besides, conjugating two drugs to generate a dimeric prodrug is a promising method for achieving the combinatory therapeutic efficacy of both drugs. Previously, researchers demonstrated that using a dimeric prodrug can boost the efficiency of drug encapsulation and improve the loading stability by increasing the hydrophobic contact between drug molecules [68]. Also, the linkage between two drugs can be responsive to the redox state, such as the disulfide bond, which allows the drugs to be released in a specific environment. For example, Liu et al. developed a GSHresponsive prodrug that not only inhibit glycolysis of tumor cells but also reverse the immunosuppressive microenvironment ( Fig. 4(b)) [69]. In this study, lonidamine (LND) and NLG-919 were connected by a disulfide bond and were loaded in F127 micelles (donated as LSN@F127). After tumor cells uptaked the nanoparticles, the overexpressed GSH could cleave disulfide bonds and promote the drug release. The released LNDs further engaged with mitochondria and reduced HK II expression, therefore interfering with tumor cell glycolysis [70]. Meanwhile, the levels of ROS were considerably increased as a result of LND activation and disulfide bond consumption of GSH, which efficiently killed tumor cells through immunogenic cell death (ICD), prompting a subsequent immunological response. Combinatory use of NLG919 could alleviate tumor immunosuppressive microenvironment and thus dramatically suppress tumor growth. As a consequence, compared to the single drug, the produced dimeric prodrug could significantly boost drug loading effectiveness in F127 micelles.
Recently, adjuvant therapy such as injecting supportive cytokines (such as interleukins) or TME modulating substances, are frequently applied to improve the therapeutic potency of adoptive transfer of tumor-specific T cells (ACT) [71,72].
Regarding that systemic administration of immunomodulators might bring adverse events, delivering adjuvant agents to the ideal place is of necessity [73]. In the previous work of Irvine's group, they presented an alternative chemistry-based strategy to deliver adjuvant drugs during adoptive treatment by attaching the drugloaded lipid nanoparticles (dubbed "backpacks") to the plasma membrane of T cells via their surface thiol groups [74,75]. To further increase the controllability and efficiency of the drug release, they developed a strategy for chemically coupling adjuvant delivery and T cell activation using TCR-responsive nanoparticle backpacks [76]. Further in their research, they noticed that the reinforced redox activity on the primed CD8 + T cell surface could be used to induce adjuvant release in response to antigen stimulation. Accordingly, they synthesized a bis-N-hydroxy succinimide (NHS) cross-linker containing a disulfide bond (NHS-SS-NHS) and determined the conditions under which the solution-phase interaction of the cross-linker and cargo proteins could induce the formation of nanogels (NGs) (Fig. 4(c)). As a result, the disulfide bond was engineered to be cleaved in response to the reducing conditions like high GSH concentrations around the T cell surface, inducing the protein cargo release. Adopting human IL-15 superagonist (IL15Sa) as a tested drug cargo, they discovered that T cells packed with T-cell receptor (TCR)responsive NGs exhibited a 16-fold increase of proliferation in tumors than T cells driven by systemic cytokine administration. Moreover, this controlled release enabled an 8-fold increase in dosage limit compared to the free cytokine.
ROS-responsive polymeric nanomedicine
Numerous responsive DDSs based on the variance of ROS have been created to provide precise drug delivery to the disease lesions [77][78][79]. However, the short lifetime, narrow diffusion, limited effect range, and restricted intracellular ROS level severely impede their treatment efficacy [80,81]. Meanwhile, mitochondria takes a critical part in supplying cellular energy and inducing apoptosis [82]. Also, it is possible to generate large concentrations of mitochondrial ROS (mtROS) in situ and damage mitochondria, triggering programmed cell death [83,84]. Therefore, drug delivery methods that target mitochondria with ROS responsiveness were developed to increase efficacy and minimize the possible side effects. For instance, Zhang et al. developed a ROS-responsive nanocarrier with dual-targeting properties toward tumor cells and intracellular mitochondria, exhibiting selfcirculation of mitochondrial drug release with a burst of mtROS for enhanced cancer therapeutic efficacy [85]. In their study, the dual-targeted polymeric nanoparticles were composed of ROSresponsive camptothecin (CPT) prodrug monomer with a thioketal bond (DT-PNs) (Fig. 5(a)). The increase of mtROS in tumor cells could induce the release of CPT in mitochondria. In situ produced CPT further stimulated the circulation of mtROS, resulting in the further high-dosage release of CPT and a final burst of mtROS, both of which were capable of eliciting enduring high-oxidative stress and achieving efficient cancer cell elimination. As a result, the apoptotic ratio of DT-PNs group (45.73%) was substantially greater than CPT group (34.27%). And the in vivo test showed that DT-PNs achieved an 81% tumor inhibition ratio.
In the article discussed above [69], the GSH-responsive prodrug could achieve great therapeutic efficacy for tumor treatments. However, compared with this redox-responsive strategy, a ROSresponsive method is more tumor-specific and holds great promise to expand the exposure of tumor cells toward prodrugs due to the fact that the GSH concentrations between tumor cells and normal cells present insignificant variation [86][87][88]. For example, Xu et al. chose the anticancer drug mitoxantrone (MTO) and created a ROS-responsive MTO-based polyprodrug (iRGD-NPs). Mechanistically, MTO was copolymerized with a ROScleavable thioketal-containing linker to form the polyprodrug, termed as polyMTO ( Fig. 5(b)) [89]. The presence of ROS in tumor cells could induce the thioketal link break in the polyMTO, leading to the programmed release of intact MTO for interrupting DNA synthesis. As a consequence, the group treated with iRGD-NPs achieved superior therapeutic efficacy compared to free MTO. Moreover, tumor cells have a significant level of heterogeneity of redox potential [90]. The overproduced ROS and GSH vary in different types of tumor or in distinct locations within the same type of tumor. In addition, the fluctuation levels of GSH and ROS have been observed at distinct stages of tumor growth [90]. However, the mainstream of stimuli-responsive nanoparticulate DDSs were supposed to respond to either ROS or GSH, leaving limited therapeutic efficacy. Luo et al. expected that a single thioether could be more efficient as a dual-sensitive linkage than a dithioether. To verify their hypothesis, they devised and produced two new redox dual-sensitive PTXfatty acid conjugates (PTX-S-OA and PTX-2S-OA) by conjugating oleic acid (OA) to paclitaxel (PTX) through a single thioether bond or a dithioether bond respectively (Fig. 5(c)) [91]. Furthermore, the PTX-S-OA was significantly more superior than the PTX-2S-OA in the matter of tumoricidal potency. This work may explore the future avenue of redox dual-sensitive DDSs for achieving enhanced therapeutic efficacy.
On the whole, it is discovered that combination therapy shows a potential development direction for redox-responsive polymeric platforms in cancer treatment. The combination includes different treatments and multiple stimulus. In order to achieve enhanced efficacy, how the redox response interacts with various stimulus such as redox-pH and redox-enzyme needs to be more deeply learned. For example, diselenide bonds have a special dual redox response that may respond to both oxidants and reductants simultaneously.
Enzyme-responsive polymeric nanomedicine
The enzyme-responsive nanoplatform is also often developed for achieving precise drug delivery [92]. TME is enriched with various enzymes. Most malignant tumor cells express a high level of gelatinase-A (MMP-2), gelatinase-B (MMP-9), and esterase which can be exploited as the trigger [93,94].
MMP-responsive polymeric nanomedicine
MMP is a family of extracellular proteinases that degrade various proteins [95]. Among them, MMP-9 and MMP-2 are frequently overexpressed in malignant cancers such as breast cancer, pancreatic cancer, and lung cancer. They are tumor cell growth regulators and take a vital part in angiogenesis at the tumor site. Besides, other members of the MMP family, like MMP-12 and MMP-13, are highly expressed in lung cancer, and MMP-3 is found to put forward the development of breast cancer. The close relationship with tumorigenesis in the TME renders MMP a suitable target for designing responsive polymeric nanomedicine.
For instance, Cassandra et al. invented a general MMPresponsive nanoparticle to achieve the precise drug release in tumor sites [96]. This polymeric nanomedicine was synthesized through diblock copolymerization. PTX and MMP-responsive peptides were covalently linked to norbornene analogs monomers and copolymerized through ring-opening metathesis polymerization. PTX acted as the hydrophobic moiety of the polymer via a biodegradable ester and MMP substrate peptide functioned as the hydrophilic block. The polymer was capable of self-assembly into nanoparticles upon dialysis from dimethyl sulfoxide (DMSO) against aqueous solution. The MMPresponsive nanoparticle underwent dramatic morphology change within the TME as a consequence of MMP degradation of its peptide shell, and further released paclitaxel into the tumor cell via hydrolysis ( Fig. 6(a)). The nanoparticle with a great drug loading capability (63% by weight per polymer) exhibited improved biosafety and tumor inhibition efficacy. The maximum tolerated dose of the MMP-responsive nanoparticle (240 mg/kg in the mouse model) was 16 times higher than clinical PTX without overt toxicity. In the HT1080 xenograft model, efficacy was examined among responsive nanoparticles, non-responsive nanoparticles, and clinical PTX. At the dose of 15 mg/kg, the responsive nanoparticle exhibited slightly stronger inhibition than the non-responsive type. Moreover, scientists have developed MMP-2 and MMP-9 specific responsive units to target TME. For example, Han et al. engineered MMP-2 sensitive polymeric nanomedicine that enhanced drug penetration into solid tumors ( Fig. 6(b)) [97]. The monomer consists of a hyaluronic acid (HA) protective shell, amidogen of PAMAM core, and an MMP-2 cleavable peptide. They were linked via click reaction to form an HA-peptide-PAMAM macromolecule. And doxorubicin was incorporated within the PAMAM core. Upon MMP-2 degradation, the nanoparticle could precisely release its payload within the TME. This strategy showed an improvement in tumor growth inhibition of nearly 50% more than merely doxorubicin solution while alleviating systematic toxicity. Similarly, Gordon et al. created an MMP-9 responsive nanogel to increase tumorspecific cellular uptake (Fig. 7(a)) [98]. The outer layer of the polymer nanogel was covered with PEG-conjugated MMP-9 responsive peptide. Upon exposure to MMP-9, the PEG shell could degrade and the inner core was exposed. The positive charge-bearing polyamine-type surface therefore rapidly entered into the tumor cells, and the nanogel further released a noncovalent payload within cells. The MMP-9 responsive peptide decorated nanoparticle exhibited nearly 10 times of the cellular uptake of naked nanoparticles.
Esterase-responsive polymeric nanomedicine
Esterase is an intracellular enzyme, usually overexpressed in malignant tumors, such as in colorectal tumor [94]. Their high expression is intimately connected to the promotion of tumor growth and migration. Esterase's ability to catalyze the hydrolysis of various ester bonds is promising for liposome dissociation and cleavage of drug-drug conjugate via an ester bond. For polymeric nanomedicine, utilizing esterase to reverse carrier charge or degrade protective shell are emerging applications of esteraseresponsive units.
Qiu et al. designed an esterase-responsive nanomedicine for gene therapy to target tumor while avoiding fibroblast hyperactivation ( Fig. 7(b)) [99]. The polymer core was protected from extracellular esterase degradation via a 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) lipid and cholesteryl 3β-N-(dimethylaminoethyl) carbamate shell. Since esterase is overexpressed in cancer cells, the peripheral quaternary amines with N-propionic 4-acetoxybenzyl ester substituents quickly disassembled and released DNA due to charge conversion. However, such polymeric nanoplatform remained as unchanged cationic because of the lack of esterase in fibroblasts. Consequently, selective apoptosis of tumor cells was induced as plasmid encoding apoptosis-inducing ligand was delivered into the nucleus. The efficiency of this therapy was examined in an intra-peritoneal cervical tumor model. Such polymeric nanomedicine dramatically decreased the number of tumor nodules than common chemotherapy drugs including PTX, cisplatin, and irinotecan. WNT16B, a fibroblast hyperactivation signal, was found downregulated in comparison with chemotherapy, indicating its selectivity. Similarly, Saw et al.
developed an esterase-responsive polymer-prodrug for siRNA delivery, which allowed the nanoplatform to release its payload in tumor cells [100].
Besides its application for gene therapy, esterase-responsive units were also applied to deliver small molecules. In a recent study by Sui et al., natural product triptolide (TPL) was linked to the PDA-polyethylene glycol (PDA-PEG) polymer via an ester bond to avoid its systematic toxicity and improve its solubility ( Fig. 8(a)) [101]. The polymer self-crosslink with PDA-PEGlactobionic acid to form nanoparticles. Lactobionic acid was exposed to the outer layer of the nanoparticle to interact with β-Dgalactose receptors, which encouraged endocytosis by cancer cells. Subsequently, the linkage of polymer and TPL was cleaved through esterase degradation to reach the tumoricidal effect.
Gamma-glutamyl transpeptidase (GGT)-responsive polymeric nanomedicine
GGT is a type of membrane enzyme responsible for the transfer of a γ-glutamyl group to acceptor peptides and amino acids [102]. They could mediate transcytosis between densely packed cells, such as epithelial and endothelial cells in the lung, liver, and vascular systems. With their highest intracellular concentrations in the liver, they are regarded as sensitive biomarkers for liver pathologies [103]. As for nano-based strategy design, hijacking GGT to attain augmented drug penetration into tumor cells is a rising interest for nanoscientists.
To trigger GGT-mediated transcytosis, Zhou et al. designed a GGT-responsive zwitterionic polymeric nanocarrier to achieve deep penetration into the tumor cells (Fig. 8(c)) [104]. Adsorptionmediated transcytosis (AMT) could be encouraged efficiently by the cationization of nanoparticles [105]. The drug-conjugate PBEAGA-CPT produces primary amine through GGT-catalysed γ-glutamylamide hydrolysis, thus leading to a quick transcytosis and endocytosis and consequently enhancing the tumor penetration and therapeutic efficacy. Similarly, Wang et al. developed a GGT-responsive dendrimer-drug conjugate to achieve deep penetration into the liver tumor ( Fig. 8(b)) [106]. GSH was selected as the GGT-responsive unit for its superior activity as a GGT substrate, and CPT was connected to the polyamidoamine dendrimer via a ROS-sensitive thioketal linker. The outer layer of GSH could be recognized by GGTs on the vascular endothelial cells and degraded into the positive-charged polymer, allowing caveolae-mediated transcytosis for the nanoparticles delivery into TME. GSH-modified dendrimer achieved substantial inhibition of xenograft BxPC-3 tumors, while PEG-modified analogs still resulted in seven times increase of the tumor volume, suggesting the significance of the GGT-responsive mechanism on therapeutic efficacy.
As has been noted, most of the available enzyme-responsive polymeric delivery platforms are still in the proof-of-concept (POC) stage [107]. These platforms need to undergo additional biosafety testing including assessments of their immunogenicity, toxicity, pharmacokinetics, and biocompatibility before they may be potentially translated into clinical use.
Other bioresponsive materials for various diseases
Apart from pH-, redox-, and enzyme-responsive nanomedicines, bioresponsive materials are available in a wide range of various types and are employed in many diseases. When developing bioresponsive materials for disease treatments, endogenous variations are appealing targets since they are frequently significant markers for several sorts of diseases [108][109][110]. Meanwhile, other typical endogenous stimuli include glucose, ions, adenosine triphosphate (ATP), hypoxia, mechanical cues, as well as nucleic acids [111]. For example, to enable on-demand cargo release, the ATP-controlled DDSs usually employ ATPtargeted aptamers as mediums [112]. To be specific, ATP either drives conformational changes that produce structure-disorder forces or competitively binds to loading sites of cargo to initiate release. Based on these unique functions of ATP, there are many studies about ATP-controlled formulations in the past few years, including tubular structure, poly-ion micelles, aptamer-crosslinked DNA microcapsules, nanogels composed of DNA complexes, and silica [113][114][115][116][117]. More functions and types of different ATPresponsive materials and DDSs can be referred to Deng's review [118].
Conclusions and prospects
In this review, recent advances of bioresponsive polymer for cancer treatment are highlighted. With the rapid advancement of nanotechnology, diverse multifunctional agents at the nanoscale can be effectively synthesized. Polymers, in particular, have shown great potential in cancer immunotherapy and chemotherapy, as well as in their application as drug solubilizers and stabilizers. Bioresponsive polymers could enable more sophisticated control and realization of precise and efficient release by exploiting endogenous physiological properties as triggers. Such design facilitates the precise release of chemotherapeutic and immunotherapeutic agents at the specified tumor sites, offering the feasibility of specific and precise treatments.
Despite the significant progress made in this field, bioresponsive polymers must overcome certain barriers before clinical translation. First, the pH and redox state of tumors are often variable rather than constant in tumor cells, which may result in the false activation of such bioresponsive nanosystems. To solve this problem, a more sensitive bioresponsive polymer needs to be developed. For example, Liu et al. synthesized a P(C6-Rx) library to obtain the most sensitive pH-responsive material by screening the whole library [46]. Likewise, Chen et al. reported a pH-activatable nanophotosensitizer library that could be utilized to spatiotemporally target different phases of endosomal maturation, thus inducing adjustable cellular pyroptosis [119]. This kind of bio-signal amplification response components and library screening opens the future avenue for improvements. Furthermore, further knowledge of tumor biochemical properties is required to assist the design of bioresponsive polymers with augmental efficacy. Second, the bioresponsive polymer must be stable within the body's circulation before reaching the tumor site. To accomplish this, Jia et al. created multi-stage responsive polymers. They synthesized a gradient redox-responsive and twostage rocket-mimetic drug delivery system allowing longer blood circulation and improved tumor accumulation [65]. Third, as new biological characteristics of cells and cytokines are being discovered, it is important to assess the targetability of cells within the TME in order to identify potential targets for polymeric nanomedicine. For instance, Tang et al. aimed tumor-associated neutrophils with enzyme-responsive polymers through its specific enzyme myeloperoxidase (MPO) [120], indicating an innovative therapeutic strategy for immune modulation in TME. As an extension, the bioresponsive DDSs are not limited to cancer immunotherapy and chemotherapy. Moreover, response targets can be generalized and applied to other materials. Overall, by the incorporation of interdisciplinary technologies and personalized analytical tools, bioresponsive polymers have a significant clinical impact for precision medicine with enhanced efficacy and limited side effects.
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Abstract A080: METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8
Background: Our work focuses on the role of METTL21A, a member of the little studied seven β-strand family of candidate lysine methyltransferases (KMTs), and its role in the regulation of pancreatic ductal adenocarcinoma (PDAC) development. Using a meta-analysis of publicly available gene expression datasets, we found that METTL21A is significantly downregulated in PDAC versus normal tissue and the reduced expression predicts poor patient survival. Thus, there are intriguing correlations connecting METTL21A to PDAC pathogenesis; however, to date, there are no data on the role of METTL21A in cancer and an enzymatic function for METTL21A is poorly recognized. Methods: To test the hypothesis that METTL21A plays a role in PDAC initiation and progression, we generated conditional Mettl21a knockout mice and crossed to PDAC models: p48Cre/+ KrasG12D/+ (Kras vs. Kras;Mettl21a) and p48Cre/+ KrasG12D/+ p53fl/fl (Kras;p53 vs. Kras;p53;Mettl21a). Furthermore, we studied the impact of METTL21A depletion on human PDAC cell lines and PDX growth in vivo and in vitro. To identify the substrate of METTL21A enzymatic activity we performed methylation assays followed by mass spectrometry analysis. Results: Histopathological analysis of pancreatic tissues of Kras and Kras;Mettl21a mice at 6 months of age revealed that loss of METTL21A significantly accelerates pancreatic cancer initiation. Additional studies using Kras;p53 and Kras;p53;Mettl21a mice demonstrated that METTL21A depletion accelerates PDAC progression and results in a significantly shorter overall survival. In congruence with those observations, knockdown of METTL21A in human PDAC cell lines leads to the robust increase in cell proliferation, enhances colony formation ability in vitro and xenograft growth in vivo. In contrast, cells rescued with ectopic expression of wildtype METTL21A but not with enzyme-dead mutant METTL21A showed significant growth impairment in vitro and in vivo. Next, our in vivo methylation assays identified HSPA1 and HSPA8 (members of the HSP70 protein family) as substrates of METTL21A in PDAC cells. Our subsequent analysis showed that METTL21a specifically tri-methylates HSPA1 and HSPA8 at lysine 561 (K561me3). Next, to directly evaluate the role of METTL21A mediated methylation of HSPA1/A8 at K561 in regulating cancer phenotype, we knocked out HSPA1/A8 and complemented with expression of wildtype or K561A mutant HSPA1/A8 in PDAC cell lines. We found that only methylation-resistant HSPA1/A8 increased cell proliferation and tumor growth in vivo. Together, these results argue that in PDAC, the principal physiologic activity of METTL21A is generation of HSPA1/A8 K561me3, which suppresses cancer growth. Conclusion: Our research revealed that METTL21A is a novel tumor suppressor that inhibits the initiation and progression of PDAC through methylation of the heat-shock proteins HSPA1/8 substrate-binding domain.
Citation Format: Xiaojie Yang, Mohamad Zoabi, Simone Hausmann, Natasha M. Flores, Xiaoyin Lu, Jibo Wu, Ana Morales-Benitez, Or Gozani, Pawel K. Mazur. METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A080.
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2022-11-17T16:19:53.044Z
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Temperature and OCT imaging monitoring in photothermal therapy of breast cancer
Malignant tumor is a serious threat to human health. With the development of medical technology, a variety of treatment methods appear in clinic. As a non-invasive treatment, laser photothermal therapy is a treatment that kills cancer cells by converting light energy into heat energy through laser irradiation. Its advantage is protecting normal tissue while destroying cancerous tissue. However, it’s still not clear that the effect of heat generated by laser on tissue and temperature changes during photothermal treatment process. Optical coherence tomography (OCT) is a non-contract, real-time optical imaging technology. OCT has been widely used in clinical treatment and scientific research based on fast imaging speed and high detection sensitivity. In our study, breast cancer of mice was chosen as the research object. Combined infrared thermography and OCT were applied to monitor the dynamic changes of tumor tissue. The effect of photothermal from OCT image and temperature were obtained and analyzed. Specifically, we investigated the structural change characteristics and temperature distribution of tumor tissue with increasing laser power. And then, the temperature change of tumors of different sizes at power of 3W were further analyzed. The results show that combined with OCT images and temperature can be well used to guide the photothermal treatment process. It can serve as a basis for the method with safely, consistently and effectively.
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2022-11-17T16:20:25.966Z
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Abstract PR003: Race plays a role on the rate of transdifferentiation in human pancreatic acinar ductal metaplasia and its' drug response
Pancreatic diseases (acute/chronic pancreatitis, type 2 diabetes, pancreatic cancer) disproportionately affect the Black/African American community in comparison to non-White Hispanics and Whites. Acinar to ductal metaplasia (ADM), the process by which pancreatic acinar cells transdifferentiate into ductal epithelial cells, is believed to be an initiating event of pancreatic ductal adenocarcinoma. Our lab has developed a 3D organoid assay to display ADM using primary, human pancreatic acinar cells to study the rate of transdifferentiation among these three different races. Preliminary data shows that the rate of ADM is occurring significantly faster (p < 0.05) in Blacks/African Americans (White=11, Hispanic=10, Black/African American=5), which may explain the disproportionately behind the incidence and mortality rates for this race in pancreatic diseases. We additionally use nanoparticles to study the biomechanical properties (I.e., viscoelasticity, storage modulus) of the ADM microenvironment which shows a stiffer microenvironment in Blacks/African Americans than for the other races (White=4, Hispanic=4, Black/African American=1). Furthermore, I study the use of histone deacetylase (HDAC) inhibitors in reversing the process of ADM, which has consequently shown race-related outcomes, with Blacks/African Americans displaying a significant chemoresistance (p < 0.05) to HDAC treatment (White=6, Hispanic=6, Black/AA=3) by utilizing an ADM reversal index (ADMRI). Through further analysis, the plan is to continue procuring human samples from these three races to isolate an ADM-specific biomarker in relation race and drug reversal by studying the expression/activity of pancreatic associated genes by bulk-RNA and single-cell sequencing.
Citation Format: Corey Perkins, Jinmai Jiang, Hesam Hakimjavadi, David Quashie Jr, Yating Mao, Jamel Ali, Thomas D. Schmittgen. Race plays a role on the rate of transdifferentiation in human pancreatic acinar ductal metaplasia and its' drug response [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR003.
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2022-11-18T16:12:33.695Z
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Validation of a Lateral Flow Assay for Rapid Diagnosis of Histoplasmosis in Advanced HIV Disease, Buenos Aires, Argentina
Histoplasmosis is a major cause of mortality in individuals with advanced human immunodeficiency virus (HIV) disease (AHD). We evaluated in patients with AHD a lateral flow assay (LFA) developed by MiraVista® Diagnostics (MVD LFA). Histoplasmosis was defined based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) case definitions. We also compared the results of this LFA with those obtained using a commercial enzyme immunoassay (EIA) developed by IMMY, Clarus Histoplasma GM EIA, IMMY (HGM EIA). A retrospective observational study was conducted at Hospital Juan A. Fernández, located in Buenos Aires, Argentina. The study included 48 urine specimens from patients aged >18 years with AHD. Urine specimens included 17 patients with disseminated histoplasmosis and 31 specimens from patients without evidence of histoplasmosis. Specimens were tested using the MVD LFA and the HGM EIA. The MVD LFA and the HGM EIA had similar analytical performance, with a sensitivity of 94%, specificity of 100%, positive predictive value of 100%, negative predictive value of 97%, and an accuracy of 98%. Comparison of the MVD LFA with the HGM EIA demonstrated a Kappa agreement index of 0.906. The LFA evaluated in this study had high analytical performance; it provided rapid diagnosis of histoplasmosis with minimal requirements for laboratory training, equipment, and laboratory infrastructure.
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2022-11-18T16:19:51.575Z
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Lung Cancer Risk Prediction with Machine Learning Models
The lungs are the center of breath control and ensure that every cell in the body receives oxygen. At the same time, they filter the air to prevent the entry of useless substances and germs into the body. The human body has specially designed defence mechanisms that protect the lungs. However, they are not enough to completely eliminate the risk of various diseases that affect the lungs. Infections, inflammation or even more serious complications, such as the growth of a cancerous tumor, can affect the lungs. In this work, we used machine learning (ML) methods to build efficient models for identifying high-risk individuals for incurring lung cancer and, thus, making earlier interventions to avoid long-term complications. The suggestion of this article is the Rotation Forest that achieves high performance and is evaluated by well-known metrics, such as precision, recall, F-Measure, accuracy and area under the curve (AUC). More specifically, the evaluation of the experiments showed that the proposed model prevailed with an AUC of 99.3%, F-Measure, precision, recall and accuracy of 97.1%.
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2022-11-20T05:26:19.998Z
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2022-11-15T00:00:00.000Z
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253670470
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s2orc/train
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Unique Presentation of Bing–Neel Syndrome With Co-existing Chronic Lymphocytic Leukemia
ing–Neel syndrome (BNS) is a rare presentation of Waldenström’s macroglobulinemia (WM). 1 It results from an infiltrationof malignant lymphoplasmacytic cells into the central nervous system (CNS). CNS manifestation is more commonly seen as a feature of disease relapse, however, can also be present at initial diagnosis. 2 Incidence of BNS is unknown; however, a recent retrospective analysis of over 1500 patients with WM noted only 13 cases of BNS both at presentation and disease relapse. This equated to 0.8% of all cases. 2 We present a case of a 59-year-old man with co-ex-isting chronic lymphocytic leukemia/lymphoma (CLL) and lymphoplasmacytic lymphoma (LPL/BNS). To our knowledge, this has not been previously reported in the literature; however, 3 cases of CLL and LPL have been reported. This patient had been under investigation with neurology for a 19-year history of essential tremor with dystonic features in his upper limbs. the patient’s symptoms neces-sitating His condition he help Furthermore, patient bilateral paraesthesia affecting all fingers palms bilaterally weakness, global are- flexia, poor balance frequent and bulbar signs neces-sitating permanent us of a wheelchair. Examination revealed no cranial nerve deficits, bilateral but predominantly right sided postural tremor without any latency, upper limb dystonia, holding hands partially flexed at the wrists. There was posi-tional variability of tremor, being most prominent when arms abducted clonal populations as detected in the BM, BMT, and PB sam- ples. Molecular analyses to ascertain the mutational status of MYD88 L265P and the tumor suppressor gene TP53 were per- formed on both BM and CSF samples, with the MYD88 muta-tion present in both samples. However, both the BM and CSF were negative for TP53 mutation. Fluorescence in situ hybrid-ization (FISH) analysis of 200 nuclei from CD19+ purified cells from PB showed no evidence of deletions of ATM or TP53 . The patient was regional MDM CLL stem cell Examination
B ing-Neel syndrome (BNS) is a rare presentation of Waldenström's macroglobulinemia (WM). 1 It results from an infiltrationof malignant lymphoplasmacytic cells into the central nervous system (CNS). CNS manifestation is more commonly seen as a feature of disease relapse, however, can also be present at initial diagnosis. 2 Incidence of BNS is unknown; however, a recent retrospective analysis of over 1500 patients with WM noted only 13 cases of BNS both at presentation and disease relapse. This equated to 0.8% of all cases. 2 We present a case of a 59-year-old man with co-existing chronic lymphocytic leukemia/lymphoma (CLL) and lymphoplasmacytic lymphoma (LPL/BNS). To our knowledge, this has not been previously reported in the literature; however, 3 cases of CLL and LPL have been reported. This patient had been under investigation with neurology for a 19-year history of essential tremor with dystonic features in his upper limbs. Over the course of 2 years, the patient's symptoms continued to progress resulting in significant deterioration in mobility necessitating in-patient rehabilitation and use of a wheelchair. His condition continued to decline and he required significant help with personal care and living arrangements. Furthermore, this patient also developed bilateral limb paraesthesia affecting all fingers and his palms bilaterally as well as weakness, global areflexia, poor balance with frequent falls and bulbar signs necessitating permanent us of a wheelchair. Examination revealed no cranial nerve deficits, bilateral but predominantly right sided postural tremor without any latency, upper limb dystonia, holding hands partially flexed at the wrists. There was positional variability of tremor, being most prominent when arms abducted at the shoulders and partially flexed at the elbows. The tremor also involved directionally stereotyped repetitive movements, mainly involving wrist extension and pronation. He had an action tremor but no terminal worsening. Consequently, the patient underwent a number of biochemical investigations that identified an IgM kappa paraprotein of 21 g/L and free kappa light chains of 199 mg/L with a kappa:lambda ratio of 10.94. At this point, the patient was referred to hematology for review. There was no significant family history and Eastern Cooperative Performance Group performance status (ECOG) status was 3. He was a nonsmoker with minimal alcohol intake and his routine medications included primidone and clonazepam. Differential diagnoses included lymphoma (most likely lymphoplasmacytic lymphoma/WM) with associated CNS involvement in the form of BNS, paraneoplastic manifestation of possible malignancy or an incidental finding unrelated to his neurological symptoms. Initial investigations included computed tomography (CT) of the neck, chest, abdomen, and pelvis, magnetic resonance imaging (MRI) of head, whole spine and pelvis, bone marrow (BM) aspirate and trephine (BMT), cerebrospinal fluid (CSF) analysis, and lymph node (LN) biopsy.
The patient was mildly anemic with a hemoglobin of 127 g/L with no other cytopenias; lactate dehydrogenase (LDH) and calcium were normal. CT of the neck, chest, abdomen, and pelvis had shown widespread lymphadenopathy, both above and below the diaphragm without splenomegaly, with largest nodal size of 3.1 cm in pelvic nodes. Initial MRI showed bilateral symmetrical thickening of multiple cranial nerves and postganglionic, lumbar and brachial nerve roots as well as cauda equina nerve roots. The most common radiological manifestations of BNS are thought to be leptomeningeal/dural infiltration or parenchymal involvement of the brain or spinal cord. 3 BM had shown normal trilineage hematopoiesis with an increased population of small, mature lymphocytes, some of which had a plasmacytoid appearance. Flow cytometry of the BM identified an infiltrate of CD19+ B cells that accounted for 90% of CD45+ events, as well as 2 distinct clonal populations. First, a monoclonal CD5+, CD10−, kappa (weak)+, CD23+, FMC7−, CD79b−, CD200+ B-cell population with CLL score of 5/5. The second was a monoclonal CD5−, CD10−, kappa (strong)+ CD23−, FMC7−, CD38−, CD43−, CD200−, CD22+ B-cell population ( Figure 1A-H). Peripheral blood (PB) morphology was unremarkable and flow cytometry confirmed the 2 distinct clonal populations in keeping with those identified in the BM. Flow cytometry on the CSF sample identified the CD5−/CD10− kappa + population only ( Figure 1I-L). BMT histology showed normal trilineage hematopoiesis, several nodules co-expressing CD23 and the lymphoid enhancer binding factor 1 (LEF1) in B cells, which appeared to also stain positively with CD5 by immunohistochemistry (IHC). In keeping with BM and PB samples, the BMT also identified a co-existing second nodule, which was negative for CD5, CD23, and LEF1. Examination of the LN showed a cellular sample with numerous small mature lymphocytes. A population of these cells appeared to be plasmacytoid in appearance, and flow cytometry again identified 2 distinct clonal populations as detected in the BM, BMT, and PB samples. Molecular analyses to ascertain the mutational status of MYD88 L265P and the tumor suppressor gene TP53 were performed on both BM and CSF samples, with the MYD88 mutation present in both samples. However, both the BM and CSF were negative for TP53 mutation. Fluorescence in situ hybridization (FISH) analysis of 200 nuclei from CD19+ purified cells from PB showed no evidence of deletions of ATM or TP53.
The patient was discussed at regional lymphoma MDM and with a national expert at University College London. CLL was diagnosed concurrently with BNS at Binet stage B; however, did not meet criteria for treatment. An initial 2 cycles of modified MATRIX (methotrexate, cytarabine, rituximab but no thiopeta) chemotherapy regime were administered. More intensive treatment was chosen due to aggressive nature of initial presentation with plans for consolidation with autologous stem cell transplant in first remission. This was thought to be preferable with ibrutinib reserved for relapsed disease, especially in view of its CNS penetrance, 4,5 . Following completion of 2 cycles of chemotherapy, BM recovery, and intensive in-patient physiotherapy, the patient underwent repeat biochemical and radiological evaluations. He had good neurological improvement and is now mobilizing independently with a walking aid. Examination revealed bilateral but predominantly right upper limb tremor, with dystonic features and action tremor with mild ataxia. There was full power proximally in the upper limbs, but mild weakness at wrist extension and finger abduction. There was full power in lower limbs. There was global areflexia, but no objective sensory deficit. From review of previous power grading, there felt to be significant improvement. Biochemically, the paraprotein in PB had fallen by 33%. CSF protein had decreased by more than 50% with flow cytometry demonstrating a persistent, but reduced, clonal population of abnormal cells. MRI images were reviewed and had shown partial improvement in intracranial disease. The patient was rediscussed with the national expert and 2 further cycles of modified MATRIX were administered (a total of 4 cycles). To date he has achieved a very good partial response (VGPR) and continues to improve neurologically 6 ( Table 1).
Cases of BNS present clinical, diagnostic, and management challenges. Our patient's long history of neurological symptoms may have obscured initial diagnostic investigations. Extensive neurological follow-up, as well as immunoglobulin and light chain profiles raised a possibility of a lymphoproliferative disorder. The literature on the topic is limited but 2 relatively recent publications 2,4 guided both the diagnostic and management approach of this very rare condition. Review of flow cytometry and IHC data performed on multiple sample types noted a dual population of cells: a distinct CLL clone and a co-existing WM clone. Analysis of CSF using flow cytometry identified an isolated population of CD5− CD10− B-cells cells. There was no evidence that the CLL clone had crossed the blood-brain-barrier. Although a very common adult leukemia, CLL very rarely affects the central nervous system. Only about 1%-2% 7 of patients have been shown to have neurological symptoms related to CLL. The morphological presence of lymphoplasmacytoid lymphocytes in the CSF is currently gold standard for the diagnosis of BNS, 4 with positivity for MYD88 by molecular testing providing diagnostic support. CLL and WM are closely related conditions, they both arise from B cells at the late differentiation stage of their life cycle. 8,9 The co-existence of CLL in this patient's case added to the complexity and rarity of his presentation. As cases of BNS are relatively rare, there is no consensus or national/international guidance for treatment options. Treatment of choice focused on presenting symptoms and disease area to maximize chances of entering long-term remission as well as minimizing both short-and Table 1 Chronological summary of relevant disease parameters to compare disease response to treatment and then autologous stem cell transplant long-term side effects. Ibrutinib was initially considered as a treatment option, as it crosses the blood-brain-barrier; however, following discussion with the national expert a more intensive treatment regime was advised due to symptoms related to disease burden. Following completion of 4 cycles of MATRIX chemotherapy, he has recently attended for an in-patient assessment by hematology and neurology teams at University College London where he underwent a BM biopsy and lumbar puncture. Results of these 2 tests have demonstrated a deeper response, with undetectable MYD88 L265P but persistence of CLL clone in BM by flow cytometry. CSF analysis demonstrated no clonal B-cell population but persistence of MYD88 L265P positivity. Imaging showed no active disease. His ECOG has now been upgraded to 0. This patient is currently undergoing investigations and evaluations for carmustine and thiotepa (CARTH) chemotherapy with autologous stem cell transplant with the aim of obtaining long-term remission. This patient has continued to have significant improvement with mobility, requires less help with daily activities, and now mobilizes with only a rollator.
We have presented an unusual case of co-existing BNS/LPL and CLL or biclonal composite lymphoma, 10,11 in a patient with a long history of an underlying neurological disorder. Although three cases of composite LPL and CLL have been published, this is the first where LPL presentation includes BNS. As such, it has presented a diagnostic, investigative, and management challenge requiring additional investigations and collaboration with national experts to formulate an appropriate and individualized treatment plan.
AUTHOR CONTRIBUTIONS
KC and AA are co-lead authors. KC, MC, MM, and DD contributed to manuscript review.
DISCLOSURES
The authors have no conflicts of interest to disclose.
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v2
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2022-11-24T16:08:06.630Z
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2022-11-15T00:00:00.000Z
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253824301
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s2orc/train
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Clinicopathological Features of Breast Cancer in Relation to Exposure of Cycling Reproductive Hormones: A Multicenter Retrospective Study of 14 731 Patients Diagnosed with Invasive Breast Cancer
Background The reproductive period for women begins at menarche and ends at menopause, representing the total time period of exposure to cycling reproductive hormones. The potential associations between clinicopathological features and exposure of cycling reproductive hormones has not been extensively studied. This retrospective study enrolled 14 731 patients diagnosed with invasive breast cancer and was designed to evaluate factors associated with the reproductive period on breast cancer type and patient outcomes. Material/Methods A total of 14 731 female breast cancer patients from the Western China Clinical Cooperation Group from January 1, 2008, to December 31, 2017, were enrolled. Unconditional logistic regression was performed to assess the associations between clinicopathological features and menarche age, menopause age, and reproductive years. The differences in risk factors between lower and higher number of reproductive years (<35 and ≥35 years) were examined with the chi-square test. Results First, patients with late menarche age were more likely to present with tumors of higher histological grade and larger size. Second, the findings suggested a higher likelihood of smaller tumor size in postmenopausal patients with a greater length of reproductive years. Conversely, higher histological grade was associated with this group of patients, compared with their counterparts with shorter reproductive years. Third, patients with luminal breast cancer with a greater length of reproductive years were more likely to present larger tumors. Conclusions Our findings indicated that several clinicopathologic factors, including tumor size and histological grade, were associated with the length of reproductive years in patients diagnosed with breast cancer.
Background
Breast cancer is the most common cancer type and the leading cause of cancer-related death in women. In 2020, a total of 2 261 419 new cases of breast cancer were diagnosed worldwide, accounting for 24.5% of all cancer types and 684 996 resulting deaths (representing 15.5% of all deaths) reported among female patients [1]. Breast cancer is widely acknowledged as a serious threat to female health in developing and developed countries worldwide [2]. In China, 268 625 new cases of breast cancer were diagnosed (accounting for 15.09% of all cancer types) in women and 69 495 resulting deaths (6.92% of all cancer-related deaths) were reported during 2015 [3]. Clarification of the epidemiological and clinicopathological characteristics of breast cancer is essential to improve treatment options.
Menarche and menopause are markers of onset and cessation, respectively, of ovarian and reproduction-correlated endocrine activity [4]. After menarche, the ovaries begin to produce steroid hormones that directly affect the development of the breasts. After menopause, when steroid hormone levels plummet, the breast begins to enter into recession [5]. Previous studies have proposed that the risk of breast cancer in women increases with time of exposure to reproductive hormones [6]. Early age at menarche and late age at menopause may therefore be associated with an increased risk of breast cancer [7]. The reliability of these associations has been supported by a metaanalysis conducted by the Collaborative Group on Hormonal Factors in Breast Cancer [8]. In early epidemiological studies, the duration of reproductive years was defined as the period between menarche and menopause and was considered as a risk factor of breast cancer [9,10]. To some extent, the length of reproductive years represents the length of exposure to cycling reproductive hormones, calculated from age at menarche to age at menopause. Cycling reproductive hormones are recognized as a causal factor in the etiology of breast cancer and play an important role in the initiation and promotion of neoplastic growth [11,12]. However, the potential associations between clinicopathological features and the exposure of cycling reproductive hormones has not been extensively studied. In addition, previous studies have mainly focused on the associations between menarche/menopause age and breast cancer risk; whereas, the correlations between tumor clinicopathological features and menarche and menopause age have rarely been reported [4]. Therefore, this retrospective study examined data on 14 731 patients diagnosed with invasive breast cancer between January 2008 and December 2017 from 23 breast cancer centers in 9 provinces of China and aimed to evaluate factors associated with the reproductive period on the development of breast cancer type and patient outcomes.
Study Population
This multicenter retrospective study was supported by 23 breast cancer centers from 9 provinces of China (Chongqing, Yunnan, Sichuan, Guizhou, Gansu, Shanxi, Guangxi, Ningxia, and Xinjiang). On the basis of the exclusion criteria (Figure 1), a total of 14 731 female patients with invasive breast cancer diagnosed based on pathological examinations from January 1, 2008, to December 31, 2017, were enrolled. We restricted analyses comparing clinicopathological features of breast cancer in premenopausal and postmenopausal women with menarche from ages 11 to 18 years. Women who had natural menopause and had undergone bilateral oophorectomy were categorized as postmenopausal, otherwise they were categorized as premenopausal. In addition, women regularly using combined oral contraceptive pills and postmenopausal hormone replacement therapy were excluded. After screening, a total of 14 438 female patients were included and divided into 2 groups: premenopausal (n=8631) and postmenopausal (n=5807). All the study protocols were endorsed by the Ethics Committee of Chongqing Medical University and the other breast cancer centers.
Data Collection
Patients were queried about their pregnancies and outcomes, the time that each pregnancy was completed, whether the baby was breastfed, and the duration of breastfeeding. Additionally, patients were asked the age at first menstruation and whether they were postmenopausal. If so, they were queried to indicate the reason for menopause (natural, surgical, or other) and the age at which menopause happened. Records of other risk factors, initial disease symptoms and signs, clinical characteristics, pathological characteristics, and imageological features were obtained for all patients with invasive breast cancer from 23 breast cancer centers. Pre-and post-surgery pathological characteristics, including estrogen receptor (ER) expression, progesterone receptor (PR) expression, and human epidermal growth factor receptor 2 (HER2) expression were examined by pathologists with the aid of commercial immunohistochemistry (IHC) tests at the immunohistochemistry core laboratory of each breast cancer center.
Determination of Risk Factors
Data on risk factors of patients were obtained from electronic medical records by professional clinicians, following the same protocol. The reference time was the time of first diagnosis.
All risk factors were classified based on China's national conditions. Menopause status was evaluated on the basis of the e938619-2 inquiry at the time of first diagnosis. Based on the median age of menarche and menopause, the patients were divided into different groups. Increased risk of breast cancer in women has been suggested with longer exposure to cycling reproductive hormones. Therefore, a combination of early menarche and late menopause could increase the risk of breast cancer [4]. To some extent, the length of reproductive years represents the period of exposure to the cycling reproductive hormones, calculated as age at menarche to age at menopause [10]. In the current study, postmenopausal patients were subdivided into 2 groups: reproductive years <35 years (n=2417) and reproductive years ³35 years (n=2974). The average ages at diagnoses of the 2 groups were 57 and 60 years, respectively. Depending on the criteria set by the Chinese National Health and Family Planning Commission, body mass index was categorized into 4 grades: underweight (<18.5 kg/m 2 ), normal weight (18.5-23.9 kg/m 2 ), overweight (24-27.9 kg/m 2 ), and obese (³28 kg/m 2 ) [13]. In addition, the demographic composition of the Chinese population was fully considered during categorization of other risk factors. The race/ethnicity of included patients was classified into 4 groups: Han, Uighur, Hui, and others (including Tujia, Manchu, Bouyi, and other groups). Concomitantly, the time of pregnancy and parity were categorized as 0, 1, 2, or ³3. Ovariectomization, hysterectomy, and family history of cancer were binary variables, all of which were also considered.
Determination of Biological Characteristics
IHC tests were used to define ER, PR, and HER2 status. While HER2 results were generally defined based on IHC analyses, fluorescence in situ hybridization (FISH) was used in some cases, especially for patients with CerbB-2 (IHC) scores of 2+. Patients with CerbB-2 (IHC) scores of 3+ or FISH positivity were determined as HER2-positive and those with negative or 1+ CerbB-2 (IHC) scores or FISH negativity as HER2-negative. In cases in which CerbB-2 (IHC) and FISH data were inconsistent, FISH results were preferentially used. ER status and PR status were obtained from IHC analyses. In our study, ER status and PR status were successfully evaluated in nearly 95% of patients. Specifically, 7143 (49.47%) patients were determined as ER+ and PR+, 4294 (29.74%) as ER-and PR-, 1653 (11.45%) as ER+ and PR-, and 628 (4.35%) as ER-and PR+. Based on immunohistochemical staining of breast cancer tissues, postmenopausal patients were categorized as luminal (ER+/PR+), HER2-overexpressing (ER-/PR-/HER2+), and triple-negative (ER-/PR-/HER2-) subtypes [14][15][16]. The nuclear protein Ki67 and the tumor protein p53 (P53) labeling analyses were additionally included. According to the St. Gallen International Expert Consensus of 2013, the cutoff value of Ki67 was defined as 20% [17]. However, due to the significant amount of missing data on Ki67, both luminal A and B were classified as the luminal group. Compared with luminal tumors, triple-negative and HER2-overexpressing tumors are more aggressive subtypes related to poorer 5-year survival [18][19][20]. Subgroup analysis was based on 5391 patients, including 3201 luminal (59.38%), 396 HER2-overexpressing (7.35%), and 1139 triple-negative (21.13%) cases.
Statistical Analysis
The differences in risk factors between groups with shorter length of reproductive years (<35 years) and greater length of reproductive years (³35 years) were examined with the chisquare test. Case-case associations of clinicopathological features among early menarche (<14 years) vs late menarche (³14 years) premenopausal and postmenopausal women were evaluated using a multivariate logistic regression model. Additionally, early menopause (<50 years) vs late menopause (³50 years) and shorter length of reproductive years (<35 years) vs greater length of reproductive years (³35 years) groups were evaluated among postmenopausal women via multivariate logistic regression. Subgroup analyses included triple-negative breast cancer, luminal, and HER2-overexpression breast cancer; multivariate logistic regression analyses were applied to each individual subgroup during subgroup analysis. The variables identified as significant with univariate analysis (P<0.05) were included in the multivariate model. All statistical analyses were performed using SAS 9.4.0. (SAS Institute Inc, Cary, NC, USA). The statistical significance of our findings was evaluated using 2-tailed tests and the significance level set at P<0.05.
Cohort Characteristics
A total of 14 438 female breast cancer patients (including 8631 premenopausal and 5807 postmenopausal patients) were enrolled. The distribution of menopause and menarche ages is shown in Figure 2. The median age at menarche was 14 years from all patients, and 52.4% had onset of menstruation at 13 or 14 years of age (Figure 2A). In postmenopausal patients, median age at menopause was 50 years, with 61.5% reporting menopause at 48 to 52 years of age ( Figure 2B).
Menarche Age and Tumor Clinicopathological Features
In multivariate logistic regression analyses of the association between menarche age and tumor characteristics, more aggressive tumors were associated with late menarche age of patients with breast cancer. In particular, patients with late menarche
e938619-4
age were more likely to present with tumors of higher histological grade and later local staging (P=0.0027 and P=0.0317, respectively, Table 1). Multivariate logistic regression analyses conducted among premenopausal and postmenopausal patients consistently revealed higher histological grades in premenopausal patients with late menarche age. In contrast, no association between menarche age and clinicopathological features among postmenopausal patients was discovered ( Table 1). The crude analyses suggested an extremely weak association between menarche age and clinicopathological features of breast cancer among postmenopausal patients.
Menopause Age, Reproductive Year, and Tumor Clinicopathological Features
Accordingly, we further assessed the relationships of menopause age and length of reproductive years with tumor characteristics. The results showed no association between menopause age and clinicopathological features of breast cancer, except the expression of P53, but showed a definite correlation between length of reproductive years and tumor features. Similar to previous analyses of associations between menopause age and tumor features, our findings suggested a higher likelihood of smaller tumor size in postmenopausal patients with a greater length of reproductive years (P=0.0105, Figure 3). Conversely, higher histological grade was associated with this group of patients, compared with their counterparts with shorter reproductive years P=0.0218, Figure 3). In view of these findings, we speculated that the duration of reproductive years was a more valuable predictor than menarche and menopause ages, especially in postmenopausal patients.
It is well known that breastfeeding history and number of fullterm pregnancies impact the length of exposure to cycling reproductive hormones. To reduce the impact of heterogeneity, we conducted a comparison of hormonal risk factors, including parity and breastfeeding history, between the groups with different reproductive year lengths via the chi-square test. Analysis of the results indicated no differences in times of parity and breastfeeding of babies between the groups. While ethnic composition analysis suggested statistical differences between the 2 groups, the heterogeneity may have had no effect on the association between length of reproductive years and tumor characteristics, as more than 95% of patients were Han Chinese (P=0.0003, Table 2). In addition, the proportion of patients with ovariectomization or hysterectomy was higher in the shorter reproductive years group relative than in the greater length of reproductive years group (1.20% vs 0.64%, P=0.0158; 10.34% vs 2.69%, P<0.0001, Table 2).
Subgroup Analysis
Based on the categorization of postmenopausal patients with breast cancer, subgroup analysis was conducted. Most associations between length of reproductive years and tumor clinicopathological characteristics were observed for luminal and triple-negative breast cancer types. In analyses stratified by tumor subtype, a negative association between length of reproductive years and tumor size was detected specifically in luminal breast cancer (P=0.0328, Table 3). In triple-negative breast cancer, a greater number of reproductive years was associated with lower detection rate of calcification and reduced expression of P53, compared with the group with shorter length of reproductive years (P=0.0052 and P=0.0034, respectively, Figure 4). Multivariate logistic regression analyses suggested an association between patients with more reproductive years and advanced axillary lymph node metastasis (P=0.0429, Figure 4).
Discussion
Menarche, a marker of the onset of puberty, signifies the beginning of the female reproductive years. After menarche, the ovaries begin to produce steroid hormones that directly affect development of the breast. Conversely, menopause is the symbol of perpetual cessation of menstrual cycles. After menopause, circulating hormone levels are altered and various changes occur in the reproductive organs, such as ovarian aging, vulvovaginal atrophy, and breast atrophy [21][22][23][24][25].
Our hospital-based study involved 14 438 female breast cancer patients, including 8631 premenopausal and 5807 postmenopausal patients. We analyzed the differences in clinicopathological features between patients with different menarche and menopause ages and length of reproductive years. In multivariate logistic regression analyses, female breast cancer patients with late menarche age were more likely to have a higher histological grade and larger tumor size. By multivariate analysis, we indicated that patients with higher menarche age were more likely to have higher histological grade and larger size tumor. Similar to our study, a study by Song et al showed that a number of reproductive factors, including older age at menarche, shorter time since the last birth, and greater number of offspring, were correlated with poorer survival, with significant associations among hormone receptor and human epidermal growth factor 2-positive breast cancer cases [26]. Meanwhile, the study by Ritte el al showed women with an early menarche age (<13 years) had a 2-fold increased risk of ER+/PR+ breast cancer relative to those with late menarche age (³13 years) [27]. Although our study lacked prognostic evaluation, higher histological grade and larger tumor size are clearly suggestive of poor prognosis in breast cancer [28][29][30][31]. However, menarche age is not universally beneficial for prognosis. Analyses of premenopausal and postmenopausal patients showed that premenopausal patients with a late menarche age also had tumors with a higher histological grade. However, no associations were evident between menarche age and clinicopathological features among postmenopausal patients. To ascertain why menarche age was not related to tumor features and to identify the associated hormonal risk factors in postmenopausal patients, we further examined the correlations among menopause age, length of reproductive years, and tumor characteristics. Interestingly, our data indicated no associations between menopause age, histological grade, and tumor size. Notably, greater length of reproductive years was correlated with lower tumor size but higher histological grade. In postmenopausal female patients with breast cancer, the length of reproductive years, defined as the period between menarche and menopause ages, may be a more valuable prognostic factor of clinical outcome than either menarche or menopause age. Several studies reported some reproductive factors on risk of breast cancer, such as early menarche, nulliparity and late menopause, which is consistent with the findings from our study [32]. Furthermore, Olsson et al reported that cyclic hormonal stimulation of breast tissue is likely the most significant hormonal factor contributing to breast cancer. Additionally, the first full-term pregnancy affects the long-term hormonal levels including increased sex hormone-binding globulin and decreased prolactin and estrogen, which probably provide further protection against breast cancer [33]. Hormonal risk factors of breast cancer include late menopause, low and/or late parity, early menarche, postmenopausal hormone replacement therapy, and use of combined oral contraceptive pills, all of which affect survival [8,[34][35][36].
In this study, all hormonal risk factors, except menopause and menarche age (times of parity and breastfeeding baby), were confounders. To assess heterogeneity, we analyzed the differences in population characteristics between postmenopausal e938619-8 patients of different reproductive years. Evaluation of the results suggested no differences in times of parity and breastfeeding of babies among the groups of postmenopausal patients with differences in reproductive years. Although ethnic composition analysis revealed statistical differences between the 2 groups, heterogeneity may not affect the associations between length of reproductive years and tumor characteristics, because more than 95% of patients were Han Chinese.
The length of reproductive years is equivalent to the duration of lifetime endogenous estrogen exposure, especially after elimination of the confounding factors. Previous studies indicated that the effects of hormonal risk factors were mainly restricted to hormone receptor-positive tumors (ER+/PR+). For instance, multiple large case control and meta-analysis studies reported that the protection provided by parity was restricted to hormone receptor-positive tumors (ER+/PR+) [37][38][39]. Genetic studies suggested that polymorphisms within the ER
e938619-13
gene and other members of the ER signaling pathway are predominantly associated with age at natural menopause [40,41]. Consequently, we speculated that associations between the length of reproductive years and tumor features differ among various molecular subtypes of breast cancer. To examine this hypothesis, we conducted subgroup analysis according to various breast cancer molecular subtypes defined by hormone receptors status. In the luminal breast cancer subtype, postmenopausal patients with greater length of reproductive years were more likely to have smaller-sized tumors. Consistent with our findings, an earlier study by Song et al [42] suggested that hormone receptor-positive tumors are more significantly associated with higher duration of estrogen exposure. Spitale et al reported better survival in patients with hormone receptorpositive (ER+ and/or PR+) breast cancer than those with hormone receptor-negative breast cancer [43]. In addition, a study by Song et al involving 3430 breast cancer patients indicated better survival in patients with longer duration of estrogen exposure [26]. Similar to our study, Khalis et al reported that early menarche and nulliparity were remarkably associated to an increased risk of breast cancer [44]. Furthermore, an analysis based on 1126 patients diagnosed with invasive breast cancer and 2106 controls suggested that parity and extended breastfeeding were associated with decreased risks [45]. In conjunction with previous studies, our data supported the conclusion that length of reproductive years might be an available prognostic variable in postmenopausal women with luminal breast cancer. Previous studies on associations between duration of hormone exposure and breast cancer have usually focused on hormone-positive populations, and limited information is available for triple-negative populations. Data from the current investigation showed a lower detection rate of calcification in patients with longer reproductive years relative to their counterparts with a shorter length of reproductive years. However, we did not classify calcification as malignant or benign on the radiograph, so the result of calcification had no clinical value. Coincidentally, we observed that patients with a greater length of reproductive years were more likely to show more progressive axillary lymph node metastasis.
Accordingly, we propose that length of reproductive years is not a potential protective factor for triple-negative breast cancer,
e938619-15
and the mechanism is possibly associated with hormone receptors. However, due to the limitations in sample size, the reliability of our results remains open to debate.
To the best of our knowledge, the present study was the first to analyze the associations between length of exposure to cycling reproductive hormones and clinicopathological characteristics among women with breast cancer. To reduce heterogeneity, subgroup analysis was conducted according to various molecular subtypes of breast cancer, defined by hormone receptor status. The larger sample size should have provided greater statistical power, compared with previous studies. Information on most variables was obtained from the electronic medical records system in breast cancer centers and was therefore reliable. All missing data were removed during the analysis and should not have significantly impacted the interpretation of the results.
Our study had a number of limitations that should be considered when interpreting the results. First, the prognostic evaluation could not be carried out as a result of a lack of survival data. Additionally, some important confounding factors such as BRCA gene mutation status, status of Ki67 and P53, nuclear grade, socioeconomic status, and performance status, may have affected our results and resulted in bias to some extent. During the 10-year duration of the review, diagnostic methods and patient management have improved and affected patient outcome, which led to inevitable bias. Furthermore, because patients were registered from multiple hospitals, we cannot entirely control the quality of pathological diagnosis and primary data, which led to inevitable bias. Finally, for the correlations between clinicopathologic features and menarche age, and menopause age and reproductive period, the crosssectional analysis could not indicate that late menarche and greater length of reproductive years were the cause or consequence of these clinicopathologic features.
Conclusions
In conclusion, our data suggested that the length of reproductive years might present a positive association with histological grade but negative association with tumor size. Further research is warranted to ascertain the significance of the length of reproductive hormone exposure in breast cancer prognosis in the future.
Ethics Approval and Consent to Participate
All study protocols were approved by the ethics committee of each participating breast center, and all participants gave their written informed consent to participate.
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v2
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2022-12-04T18:55:31.028Z
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2022-11-15T00:00:00.000Z
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254210172
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s2ag/train
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A Phase 1 Study with the Novel B-Cell Lymphoma 2 (Bcl-2) Inhibitor Bgb-11417 As Monotherapy or in Combination with Zanubrutinib (ZANU) in Patients (Pts) with CLL/SLL: Preliminary Data
Background/introduction: The effectiveness of Bcl-2 inhibitors as a treatment for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) was established by the approval of venetoclax in pts with CLL/SLL across all lines of therapy. However, the related adverse events (AEs) and emergence of BCL2 mutations, resulting in resistance, can limit the utility of venetoclax. BGB-11417 is a highly selective Bcl-2 inhibitor with potency >10 times that of venetoclax in biochemical assays. BGB-11417 monotherapy is tolerable, with no maximum tolerated dose (MTD) reached after dose escalation through all planned doses to 640 mg once daily (QD) in pts with non-Hodgkin lymphoma (EHA 2022. Abstract P687). The combination of Bcl-2 and Bruton tyrosine kinase (BTK) inhibitors is tolerable with synergistic activity in CLL and mantle cell lymphoma (MCL) ( J Clin Oncol 2019;37:2722-9; N Engl J Med 2019;380:2095-103; EHA 2020. Abstract S158; N Engl J Med 2018;378:1211-23). ZANU, a next-generation BTK inhibitor, has shown favorable activity and safety in pts with CLL/SLL (EHA 2021. Abstract LB1900) and Waldenström macroglobulinemia ( Blood . 2020;136(18):2038-2050). BGB-11417-101 is an ongoing first-in-human phase 1/1b dose-escalation/expansion study (NCT04277637). Pts with various B-cell malignancies were enrolled; data from CLL/SLL cohorts are presented here. Methods: In separate monotherapy and combination therapy
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v2
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2022-12-07T19:35:18.928Z
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2022-11-15T00:00:00.000Z
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254357758
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s2ag/train
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Right foot – osteochondroma - A case report
Osteochondromas are thought of as benign bone tumours, however they are actually developmental defects.A long tubular bone that may be sessile or pedunculated has an outgrowth with cartilage covering it that is visible on radiographs. Additionally, the cartilage cap can harden. Any changes in radiological appearance are strongly indicative of chondrosarcoma, particularly those with ill-defined border development and thickening of the cartilage cap >15 mm.: An insidious onset, slow progression, and lack of aggravating or alleviating variables were all complaints made by a 21-year-old male patient with swelling and pain across the right foot dorsal side for the past two years Upon examination, there was a firm, irregularly shaped, 5 to 6 cm swelling over the dorsal part of the right foot that was attached to the underlying bone.: The majority of benign bone tumours, or 36% to 41% of all benign bone tumours, are conventional osteochondromas. Osteochondromas are uncommon in the foot and ankle regions, but if a big osteochondroma develops in these areas and is interfering with function, it should be removed. The amount of the lesion, any soft tissue involvement, and the depth and placement of the cartilage cap can all be seen on an MRI, which is helpful in the workup of an osteochondroma that is symptomatic or worrisome.According to histology, the osteochondroma's cap is made of hyaline cartilage, with well-differentiated cells abundantly spaced out by cartilage matrix and oriented in columns that mimic the epiphyseal growth plate in the deepest levels of the cap.
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2022-12-12T16:08:34.330Z
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2022-11-15T00:00:00.000Z
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254561661
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s2ag/train
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EOSINOPHILIC CYSTITIS MASQUERADING AS CARCINOMA URINARY BLADDER : A CASE REPORT
Eosinophilic cystitis is a rare entity that usually presents with hematuria and suprapubic pain and can
have the gross appearance of a bladder malignancy. Here we describe a case report of a patient that
presented with sub acute intestinal obstruction with the gross appearance of a bladder malignancy invading the sigmoid and
was later found to have eosinophilic cystitis.
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v2
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2022-12-15T16:01:11.980Z
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2022-11-15T00:00:00.000Z
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254668677
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s2ag/train
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ANALYSIS OF GENE EXPRESSION DATASETS TO DISCOVER BLOOD-BASED BIOMARKERS IN BREAST CANCER
Breast cancer has an immensely hazardous impact on the world population. Despite advances in surgery, radiation and
chemotherapy have been prevalent in the recent past, there still exists a need to study new biomarker (driving) genes to
contribute to the development of personalized cancer treatment and drugs. In this study, we aim to analyze gene
expression datasets for common differentially expressed genes (cDEGs) in the blood of stage 0-1 Breast cancer patients.
Datasets were collected from the public Gene Expression Omnibus (GEO) repository. Upon analysis, 23 DEGs passed
the cut-off criteria (p-value of < 0.5 and log fold change value of > 1.25). Common genes were identified from at least two
out of the three datasets. In order to identify network, pathway characteristics and hub genes, computational tools of
STRING and Jvenn were applied to a protein interaction network. Upon careful analysis and literature review, DDX6
(DEAD-Box Helicase 6) was found as a potential novel biomarker and warrants further study. Literature review confirmed
this gene had been identified in relation to other forms of cancer (excluding breast cancer) in previous studies, thus
showing novelty in relation to Breast cancer. Studying these 23 genes could illuminate a new direction of the
development of effective breast cancer treatment. Overall, in this study we present findings of different insights on
molecular mechanisms of Breast cancer and provide greater confidence on which genes are differentially expressed in
Breast cancer.
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v2
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2022-11-16T06:16:51.131Z
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2022-11-16T00:00:00.000Z
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253520923
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s2ag/train
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Potential mechanism of pyrotinib-induced diarrhea was explored by gut microbiome and ileum metabolomics.
BACKGROUND
Pyrotinib is a novel epidermal growth factor receptor/human epidermal growth factor receptor-2 (HER2) tyrosine kinase inhibitor that exhibited clinical efficacy in patients with HER2-positive breast cancer and HER2-mutant/amplified lung cancer. However, severe diarrhea adverse responses preclude its practical use. At present, the mechanism of pyrotinib-induced diarrhea is unknown and needs further study.
METHODS
First, to develop a suitable and reproducible animal model, we compared the effects of different doses of pyrotinib (20, 40, 60 and 80 mg/kg) in Wistar rats. Second, we used this model to examine the intestinal toxicity of pyrotinib. Finally, the mechanism underlying pyrotinib-induced diarrhea was fully studied using gut microbiome and host intestinal tissue metabolomics profiling.
RESULTS
Reproducible diarrhea occurred in rats when they were given an 80 mg/kg daily dose of pyrotinib. Using the pyrotinib-induced model, we observed that Lachnospiraceae and Acidaminococcaceae decreased in the pyrotinib groups, whereas Enterobacteriaceae, Helicobacteraceae and Clostridiaceae increased at the family level by 16S rRNA gene sequence. Multiple bioinformatics methods revealed that glycocholic acid, ursodeoxycholic acid and cyclic AMP increased in the pyrotinib groups, whereas kynurenic acid decreased, which may be related to the pathogenesis of pyrotinib-induced diarrhea. Additionally, pyrotinib-induced diarrhea may be associated with a number of metabolic changes mediated by the gut microbiome, such as Primary bile acid biosynthesis.
CONCLUSION
We reported the establishment of a reproducible pyrotinib-induced animal model for the first time. Furthermore, we concluded from this experiment that gut microbiome imbalance and changes in related metabolites are significant contributors to pyrotinib-induced diarrhea.
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2022-11-16T14:24:49.694Z
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2022-11-16T00:00:00.000Z
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253524671
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s2ag/train
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Targeted RET inhibitor therapy effective in treating multiple cancer types
DECEMBER 1, 2022 CANCER R ecent interim results from the international phase I/II ARROW trial show that pralsetinib, a highly selective RET inhibitor, confers robust and durable responses regardless of tumor type in patients with RET fusion-positive cancers. Published in Nature Medicine,1 the results build on prior data from the ARROW trial that showed the safety and efficacy of pralsetinib in patients with RET fusion-positive non–small cell lung cancer (NSCLC) and advanced RET-altered thyroid cancers. Those results led to pralsetinib’s US Food and Drug Administration approval for these indications.2,3 The phase I/II ARROW study is an open-label, single-arm study that enrolled patients with advanced RET-altered solid tumors. The first published results of the study focused on the safety and efficacy of pralsetinib in the cancer types in which RET alterations are most common (medullary thyroid cancers, papillary thyroid cancers, and NSCLC). In the current study, researchers examined whether the safety and efficacy of pralsetinib could extend to RET fusion-positive solid tumors other than NSCLC and thyroid cancers. A total of 29 patients who had any of 12 cancer types were enrolled in the study. The most common cancer types among the patients in the study were pancreatic cancer, cholangiocarcinoma, neuroendocrine tumors, and sarcoma; other types included head and neck cancer, colorectal cancer, small cell lung cancer, malignant mesenchymal tumor, mixed sarcoma and adenocarcinoma, malignant isolated fibroma, sweat gland cancer, and salivary duct cancer. Most patients were female (61%), White (65%), had metastatic disease (87%), and had received prior therapies (87%). The median age was 53 years, and the primary endpoint of the study was overall response rate. Of the 29 cancers, 23 were able to be evaluated for the efficacy of pralsetinib treatment. The study showed an overall response rate of 57% among the 23 patients across the range of cancer types. Overall, three (13%) patients had a complete response and 10 (43%) had a partial response. Patients with a response included all four patients with pancreatic cancer (including one complete response), two of three patients with cholangiocarcinoma, two of three patients with sarcoma (including one complete response), two of three patients with neuroendocrine cancer, one patient with head and neck cancer, and one patient with an unknown primary tumor (complete response). One patient with cholangiocarcinoma had a response at a single time point before discontinuing therapy after experiencing an adverse event. Of these 13 patients with a response, nine (69%) had a median duration of response of ≥6 months, two (15%) had a response of ≥18 months, and one (8%) had a response of ≥24 months. The overall median time to response was 1.9 months. The safety analysis done in 29 patients showed results consistent with previously reported results, according to the new study’s authors, and included treatment-related side effects in 25 (86%) patients, with 20 (69%) experiencing grade 3 or higher adverse events. Increased levels of liver enzymes (aspartate transaminase and alanine transaminase) and decreased levels of white blood cells were the most common side effects. Shortterm and permanent dose interruptions due to side effects were seen in 17 patients (59%) and 13 patients (45%), respectively. “These findings demonstrate the potential for RET inhibitors to benefit patients across tumor types and show the power of precision medicine to match patients to the right targeted therapy based on the unique features of their cancer,” said lead author of the study Vivek Subbiah, MD, associate professor in the department of investigational cancer therapeutics at The University of Texas MD Anderson Cancer Center, Houston, in a press release.4 Commenting on the study, Taofeek K. Owonikoko, MD, PhD, chief of the division of hematology/oncology at the University of Pittsburgh School of Medicine Hillman Cancer Center in Pennsylvania, described it as small with a high impact, demonstrating the success of a precision medicine approach with the use of pralsetinib in a tumor-agnostic manner. “These data are consistent with the findings with the other RET-specific inhibitor, [which led] to FDA approval of selpercatinib in RET-mutant tumors,” he said.
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2022-11-16T14:54:43.563Z
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2022-11-16T00:00:00.000Z
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253525176
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s2orc/train
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CT radiomics-based long-term survival prediction for locally advanced non-small cell lung cancer patients treated with concurrent chemoradiotherapy using features from tumor and tumor organismal environment
Background Definitive concurrent chemoradiotherapy (CCRT) is the standard treatment for locally advanced non-small cell lung cancer (LANSCLC) patients, but the treatment response and survival outcomes varied among these patients. We aimed to identify pretreatment computed tomography-based radiomics features extracted from tumor and tumor organismal environment (TOE) for long-term survival prediction in these patients treated with CCRT. Methods A total of 298 eligible patients were randomly assigned into the training cohort and validation cohort with a ratio 2:1. An integrated feature selection and model training approach using support vector machine combined with genetic algorithm was performed to predict 3-year overall survival (OS). Patients were stratified into the high-risk and low-risk group based on the predicted survival status. Pulmonary function test and blood gas analysis indicators were associated with radiomic features. Dynamic changes of peripheral blood lymphocytes counts before and after CCRT had been documented. Results Nine features including 5 tumor-related features and 4 pulmonary features were selected in the predictive model. The areas under the receiver operating characteristic curve for the training and validation cohort were 0.965 and 0.869, and were reduced by 0.179 and 0.223 when all pulmonary features were excluded. Based on radiomics-derived stratification, the low-risk group yielded better 3-year OS (68.4% vs. 3.3%, p < 0.001) than the high-risk group. Patients in the low-risk group had better baseline FEV1/FVC% (96.3% vs. 85.9%, p = 0.046), less Grade ≥ 3 lymphopenia during CCRT (63.2% vs. 83.3%, p = 0.031), better recovery of lymphopenia from CCRT (71.4% vs. 27.8%, p < 0.001), lower incidence of Grade ≥ 2 radiation-induced pneumonitis (31.6% vs. 53.3%, p = 0.040), superior tumor remission (84.2% vs. 66.7%, p = 0.003). Conclusion Pretreatment radiomics features from tumor and TOE could boost the long-term survival forecast accuracy in LANSCLC patients, and the predictive results could be utilized as an effective indicator for survival risk stratification. Low-risk patients might benefit more from radical CCRT and further adjuvant immunotherapy. Trial registration: retrospectively registered. Supplementary Information The online version contains supplementary material available at 10.1186/s13014-022-02136-w.
Introduction
Definitive concurrent chemoradiotherapy (CCRT) is the standard treatment for patients with unresectable locally advanced non-small cell lung cancer (LANSCLC). In the past two decades, concomitant regimens achieved promising tumor local control and long-term survival. With improved outcome, the maintenance of an adequate pulmonary function is essential to ensure acceptable quality of life and adjuvant immunotherapy. However, many patients with LANSCLC are diagnosed with pre-existing lung comorbidities, which significantly increases the risk for radiation-induced lung toxicity (RILT) [1,2].
Most existing RILT prediction models largely focused on clinical prognostic factors (CPFs) and dose-volume histogram parameters [3][4][5], but remained insufficient. Recently, machine learning methods have been reported to improve the capacity of the predictive modelling [6][7][8][9], compared with logistic regression widely used in normal tissue complication probability model.
Moreover, radiomics analysis, attempting to identify computational biomarkers potentially hidden within high-throughput imaging data [10,11], has been demonstrated the added predictive value for overall survival (OS) [12][13][14] or RILT [8,9]. However, most of them rely on the radiomic information from tumor or its surrounding peritumoral region, few studies have been designed based on the radiomics analysis of tumor organismal environment (TOE).
Similar to other published reports [15,16], our previous study [17] indicated that pulmonary function test (PFT) was significantly related to patients' long-term survival. However, it failed to predict progression-free survival (PFS). Even though patients with worse FEV1/ FVC% or DLCO% showed a high objective response rate (ORR) to CCRT, their survival outcomes were still poor, hinting that TOE, the status of lungs in the case of LAN-SCLC might play an indispensable role in the prognostic prediction after CCRT. As some patients could not tolerate well with PFT, radiomics analysis using machine learning method might be an effective technique to investigate the relationship of tumor and TOE, due to its accessibility.
In this study, we utilized computed tomography (CT) images before CCRT to develop an image-based machine learning framework to analyze the relationship of primary lung tumor and bilateral lungs for long-term survival prediction in LANSCLC. To balance the training accuracy and predictive capability using relative small number of patient samples, an integrated feature selection and model training (IFSMT) approach was developed to extract the most critical quantitative radiomic features from both tumor and lungs. A radiomic-based risk stratification was built to distinguish high-risk and low-risk patients and provided evidence for clinical decision making.
Study population
Consecutive patients irradiated for lung cancer from September 2011 to April 2019 in our institution were retrospectively screened. Inclusion criteria included: (1) histologically confirmed NSCLC; (2) unresectable stage III disease (AJCC/UICC 8th staging criteria) proven by chest and upper abdominal CT, brain magnetic resonance imaging (MRI), bone scan and/or positron emission tomography-computed tomography (PET-CT); (3) definitive radiotherapy with concurrent chemotherapy was administered; (4) stay followed-up no less than 6 months since the start of radiotherapy (unless death or disease progression was documented); (5) complete clinical records. Patients that met the inclusion criteria were randomly assigned into the training and validation cohort, with the numbers at a ratio 2:1.
Conclusion Pretreatment radiomics features from tumor and TOE could boost the long-term survival forecast accuracy in LANSCLC patients, and the predictive results could be utilized as an effective indicator for survival risk stratification. Low-risk patients might benefit more from radical CCRT and further adjuvant immunotherapy.
Trial registration: retrospectively registered.
Keywords Locally advanced non-small cell lung cancer, Radiomics, Machine learning, Long-term survival prediction, Tumor organismal environment.
Radiotherapy and concurrent chemotherapy
Patients were positioned supine and immobilized in a vacuum pad. They were scanned from the Atlas to the second lumbar vertebra level with 0.3-0.5 cm thickness slices to obtain the stimulation CT images. The respiration motion was recorded by performing 4DCT scanning. The maximum intensity projection images were reconstructed using the images collected in 10 phases of respiratory cycle. Gross tumor volume (GTV) was delineated to cover the tumor and involved regional nodes visible on each phase of the 4DCT. The total volumes of GTVs across the 10 respiratory phases CT composed the internal target volume (ITV). Planning target volumes (PTVs) were created by expanding GTV and clinical target volume with 6 mm. Lungs were delineated according to the atlases for organs at risk (OARs) in thoracic radiation therapy [18], but GTV was excluded from the lung delineation. A dose of 60-76 Gy was prescribed to PTV-GTV in 22-33 fractions, with 2-3 Gy per fraction performed once daily, using intensity modulated radiation therapy technique. The dose constraints for OARs were: V20 < 35% for lungs; mean lung dose < 19 Gy; maximun dose (Dmax) of esophagus < 105% prescription dose; Dmax of spinal cord < 46 Gy; V30 < 40% for heart.
All patients received platinum-based double agents weekly or every three weeks. The regimens included docetaxel/paclitaxel/etopside/pemetrexed plus platinum.
Evaluation and follow-up
The baseline characteristics of each patient before entry were reviewed attentively and extracted from their medical records, including blood tests, PFT, blood gas analysis (BGA) and radiologic tests. All included patients received regular radiologic follow-up, including chest and upper abdominal CT and brain MRI performed every 3 ~ 6 months in the first 2 years, and every 6 ~ 12 months thereafter. PET-CT, bone scan, and biopsy were recommended if clinically required. The responses to CCRT were first assessed by an independent radiation oncologist and confirmed by a senior physician at 4 ~ 6 weeks post CCRT, based on Response Evaluation Criteria in Solid Tumors 1.1. Another senior radiologist was consulted for disagreement. Therapeutic toxicities were graded and recorded according to Common Terminology Criteria for Adverse Events 4.0.
OS modelling procedures
The whole procedures were illustrated in Fig. 1. For both cohorts of patients, the regions of interest (ROIs) corresponding to GTV and lungs were delineated by an autocontouring software tool CezanneDraw™ v1.0 (Homology Medical, Ningbo, China, 2020) using the CT slices and manually modified by radiation oncologists if necessary. One 3D bounding box was fitted for each ROI. And inside the bounding box, the CT values of the ROI voxels were retained while the values of other voxels were marked by zero. CT values of voxels in each bounding box were then interpolated to a resolution of 1 mm×1 mm×5 mm and resampled into 400 discrete values (called bins) with absolute discretization from − 1000 to 3000 Hounsfield units, leading to a fixed bin size of 10 Hounsfield units.
A total of 92 tumor-related and lung-related features were then computed for both ROIs and used as the input feature pool for the machine learning framework by the LIFEx software (version 3.44) [19]. The imaging-based features covered two categories of texture features and first order features. The texture features consisted of four sub-categories of matrix based texture features. These matrices included the grey-level co-occurrence matrix (GLCM), neighborhood grey-level different matrix (NGLDM), grey-level run length matrix (GLRLM) and grey-level zone length matrix (GLZLM). The first order features included indices from shape, indices from histogram and conventional indices.
The machine learning based classification method used to predict the two-class 3-year survival status for each individual patient was support vector machine (SVM) [20]. The SVM mapped the features of training data into a high-dimensional feature space through a kernel function and utilizes a hyper-plane to optimally separate the training data points into two categories. To reduce the possibility of overfitting, only a subset of features from the feature pool could be selected for the input of SVM. In this study, the IFSMT approach was developed to maximize the fitting accuracy and minimize the overfitting potential. This posteriori approach applied the genetic algorithm (GA) for the feature selection, which was illustrated in Fig. 2 and Additional File 2. A chromosome represents a feature template working with SVM of certain configuration for diagnosing purpose. The SVM is implemented in leave-one-out cross-validation (LOOCV) fashion to score a chromosome. In each generation, the chromosomes of higher scores may go through mutation, partially changing feature encoding, and crossover, partially exchanging feature encoding, to make new ones to replace those of lower scores. Collect the chromosome of best score from each generation into a group. And the best one in the group is the result of the model. Manual reconfiguration of SVM is not included in the model.
Once the optimal set of features was determined, the SVM models were trained again on the training cohort. In this study, after extensive experimental comparisons, the linear kernel was chosen for SVM and optimal hyper parameters of the SVM (C, ε and γ) were determined through exhaustive search in the parametric space. Receiver operating characteristics (ROC) curves were obtained by varying threshold of the decision variable, the signed distance to decision hyper-plane. Area under curve (AUC) for each ROC was calculated for training cohort. The trained models were then used to predict the survival status for each individual patient in the validation cohort, and ROCs and their corresponding AUCs were also calculated. All the above feature selection and machine learning approaches were implemented on the cloud-based clinical data service platform iRAAS® v2.0 (Homology Medical, Ningbo, China, 2020). To assess the importance of each selected feature to the accurate prediction of the clinical outcome, a one-by-one feature evaluation procedure was designed. This procedure tested the importance of each feature by deleting each feature from the selected feature set and calculating the reduction of the AUC for the model trained with the original selected features except this specific feature. This reduction of model performance was used as the importance weight (IW) of this feature. All the selected features were then sorted according to their IWs. To further assess the importance organismal features, the AUC for the model trained with the original selected features excluding all the lung-related features were also calculated.
Statistical methods
OS was defined as the time from radiotherapy start to the last follow up, which ended at November 30th, 2021, or death. A t-test was used to determine if there was significant difference between the means of continuous variables, while Fisher's exact test was performed to reveal the difference in distribution between two groups of categories variables. The association between radiomic features and PFT/BGA indicators was examined using Pearson's correlation coefficient. A p-value < 0.05 (twosided) were considered as statistically significant. Missing data were excluded from the statistical analysis. Statistics were performed using SPSS 22.0 (IBM, Chicago, IL, USA).
To report the model fitting accuracy and the prediction capability, the true positive rate (TPR), true negative rate (TNR), F1 score, overall prediction accuracy, average prediction accuracy for the training cohort and validation cohort were calculated based on the SVM model. Herein, death is marked as the positive. The overall prediction accuracy was expressed as the number correctly predicted patients / the number of all patients; and the average prediction accuracy = (TPR + TNR)/2.
To assess the prognostic value of the survival status model, the predicted 3-year survival status was adopted respectively as the clinical risk estimator to stratify the patients into the high-risk and low-risk groups. Patients with negative predicted survival status were classified into the low-risk group and the others with positive predicted survival status into the high-risk group. Kaplan-Meier curves for both groups were displayed to illustrate its effectiveness and log-rank test was performed.
Patient characteristics
A total of 298 LANSCLC patients were included for analysis, with 200 in the training cohort and 98 in the validation cohort. The baseline and treatment-related characteristics were comparable between these two cohorts (Additional File 3). There were 57 females and 241 males in the whole cohort, with the median age of 59 years (range, 28-81 years). Squamous cell carcinoma was the predominant histologic type both in the training (46.5%) and validation (62.2%) cohorts.
As shown in Table 1, the overall prediction accuracy for 3-year survival status was 92.50% and 85.71%, and the AUC of the ROC was 0.965 and 0.869, respectively, in the training and validation cohort.
Stratification of patients in the validation cohort with machine learning model
In the validation cohort, 60 (61.2%) of 98 patients were stratified into the high-risk group and 38 (44.1%) into the low-risk group. CCRT was more successful in patients in the low-risk group than those in the high-risk group. The ORR was 84.2% (32/38) and 66.7% (40/60) in the low-risk and high-risk group, respectively (p = 0.003) (Additional File 4). And the low-risk group yielded better 3-year OS (68.4% versus 3.3%, p < 0.001, log-rank) than the highrisk group (Fig. 3B). What's more, the rate of Grade ≥ 2 pneumonitis was 31.6% (12/38), versus 53.3% (32/60) (p = 0.040) in the low-risk and high-risk group. The typical presentation of two patients in the low-risk and highrisk group was illustrated in Fig. 4.
Correlation of selected radiomic features to the model performance
A total of 9 features were selected in the proposed model, including 5 tumor-related features and 4 lung-related features. In Table 2, the IW of each selected feature for both training and validation cohorts were listed in the order from high to low. The imaging features from lungs ranked at 2nd, 4th, 5th and 8th in the all 9 features in the training cohort, and 1st, 3rd, 6th, and 8th in the validation cohort. When all pulmonary features were excluded from the selected feature set, the AUCs for the training and validation cohorts were reduced by 0.179 and 0.223, respectively (Fig. 5). Figure 4 showed two patients in the low-risk and high-risk groups.
Dynamic changes of lymphocyte counts before and after CCRT
Although there was no significant difference in lymphocyte counts before CCRT (median, 1650 vs. 1650 cells/mm 3 , p > 0.99) between the low-risk and highrisk group (Additional File 6), patients in the low-risk group had less Grade ≥ 3 lymphopenia (63.2% vs. 83.3%, p = 0.031) during CCRT, and more patients in the low-risk group could recover to normal level (≥ 1000 cells/mm 3 )
Discussions
Application of radiomics to the long-term survival prediction for LANSCLC after CCRT is a reasonable extension under the background of the field-wide adoption of machine learning methods. Other than previous works focused on the features from tumor and peritumoral tissue, the relationship between tumor and TOE is increasingly attached importance. Significant association was found between pulmonary function and radiomic features extracted from the lungs of CT images [21][22][23]. In current study, the long-term survival forecast accuracy of LANCLC patients after CCRT was demonstrated to be boosted by integrating primary tumor characteristics and pulmonary features from pretreatment CT images. Based on the CT-based predictive model, patients could be precisely stratified into the low-risk and high-risk group before treatment, which should be considered in individualized treatment decision-making process. From the importance rank of the selected features, it could be confirmed that two features from tumor, GLRLM_SRE and GLZLM_GLNUz which represent the inhomogeneity of CT images [19], remained important factors determining OS, which were consistent with published literatures [24,25]. Meanwhile, the ranking of pulmonary features underlined their indispensable role in the OS forecast. Our results of the significant difference between fitting and prediction accuracies with and without pulmonary features in model performance further support this finding, implying that the TOE, herein the pulmonary environment, might have a significant impact in LANSCLC patients with large tumor burden and limited pulmonary function. Accordingly, the relatively longer OS for patients with healthier pulmonary status could possibly contribute to their more tolerance to radical CCRT and less incidence of severe lung toxicities.
PFT have been reported to predict the risk of RILT after CCRT [26][27][28][29]. Our previous work showed that FEV1/FVC% and DLCO% were prognostic factors for long-term survival but not for PFS [17], implying that long-term survival outcomes might not be achievable due to detriment of pulmonary function even though patients had good early response to CCRT. To further interpret the underlying role of these selected radiomic lung features, the correlation between radiomic features and PFT/BGA indicators were explored in depth and it was confirmed that FEV1/FVC% was well correlated with radiomic pulmonary features. This correlation between the pulmonary ventilation function and selected Abbreviation: PFT, pulmonary function test; BGA, blood gas analysis radiomic pulmonary features for OS prediction reaffirms the findings in Occhipinti et al. 's study that the changes in lung function, such as bronchial thickening and honeycombing, can be mechanistically explained based on morphological CT features [23]. And it might additionally imply that the tumor not only interacts with cells in its immediate vicinity, but also communicates with the entire host organ [30], just as suggested by a prior study [31] that the tumor and TOE could possibly interact in a bi-directional way.
In the aspect of methodology, the machine learning framework in this study used SVM combined with the proposed IFSMT approach to iteratively select features using GA and improve the accuracy of the prediction model. Our avoidance of topical deep-learning frameworks, such as deep convolutional neural network, is due to the intrinsic weaknesses of overfitting and blackbox for these frameworks. To ease the problem of overfitting, the deep-learning frameworks are more suitable for the learning tasks armed with big data as learning samples. However, the number of patients in current study for model training was relatively small, which intensively restricts the application of deep-learning frameworks which may have millions of parameters and thousands of decision making variables. The SVM is equivalent to an optimized three-layer neural network with only one hidden layer. This simplified neural network architecture substantially reduces the potential of overfitting. Additionally, in contrast with the problem of blackbox for deep learning framework, the features used in modeling are explicitly created and selected with the IFSMT approach. Therefore, each feature had an explicit clinical or physical meaning relevant to image of a specific ROI, which made it easy to apprehend the behind-the-scene mechanism of the survival status prediction and directly related the comprehensible clinical and image oriented indices to the clinical outcome. The effectiveness of IFSMT approach had been demonstrated by high AUC values achieved for the survival status prediction.
The most recent work on prognostic model for the survival outcome for NSCLC patients treated with CCRT demonstrated that pretreatment CT texture features provided prognostic information beyond CPFs [12]. However, it didn't provide the result in terms of AUC or employ the validation cohort. In another predictive model conducted by Dehing-Oberije C et al. [32], which used CPF indices only, the AUC was 0.74 for the training cohort, 0.75 and 0.76 for the two separate validation cohorts. The improvement of model performance by imaging features in current study is discernible with the AUCs of 0.965 and 0.869 for the training and validation cohort, which could be attributed to inclusion of the image-based pulmonary features.
What's more, the predictive OS results using imaging features in our study with machine learning could be utilized as an effective indicator for the survival risk stratification of these patients, which could potentially individualize CCRT regimen and adjuvant treatment from the perspective of personalized medicine. For example, immunotherapy has evolved into a standard adjuvant treatment option for LANSCLC patients treated with definitive CCRT. Based on the promising results of the phase III PACIFIC study [33,34], adjuvant immunotherapy resulted in a significant prolonged PFS and OS for those patients. To be noticed, the most common grade 3 or 4 adverse event in the durvalumab arm was pneumonia (4.4%), followed by pneumonitis or radiation pneumonitis (3.4%), and Asian patients seemed to have a higher rate of any grade pneumonitis (73.6%) and severe pneumonitis (5.6%) [35]. Thus, based on the survival risk stratification of LANSCLC patients in this study, low-risk patients might have several potential advantages for adjuvant immunotherapy: (1) supporting role of better pulmonary function and quality of life; (2) superior tumor remission with less pulmonary toxicities; (3) less severe lymphopenia during CCRT and better recovery of lymphopenia from CCRT. However, for high-risk LANSCLC patients who had worse baseline FEV1/FVC%, higher rate of Grade ≥ 3 lymphopenia during CCRT, worse recovery of lymphopenia from CCRT, and higher incidence of radiation-induced pneumonitis, radical CCRT or further adjuvant immunotherapy might not be feasible because of poor organ functions and high probability of severe complications. Therefore, pretreatment radiomics-based risk stratification of LANSCLC patients using features from tumor and TOE could provide direct evidences to effectively support the treatment decision making.
It should also be noted that there were a few limitations in this study. First, the absence of external validation was the major disadvantage. Nevertheless, multiple CT simulation machines were available in our institution (Additional File 1). The high AUC values were generated from these different scanners with varied parameter settings, demonstrating the great robustness of our model. Besides, Zhao et al. considered that radiomic features in lung cancer were reproducible over a wide range of imaging settings [36]. Multicenter validations with larger samples are warranted for the ultimate application of this model clinically. Second, there might be some variability in multiple observer delineations in our study. E et al. reported that although the ROIs delineation tended to be different between individual experts, an overall high AUC value could still be achieved [37]. Third, we focused only on the radiomic analysis of pretreatment planning CT in this study, and other imaging modalities, such as PET-CT [38] and MRI, still need to be investigated as to whether they could also yield complementary information which would facilitate more accurate predictive models.
Conclusion
Pretreatment CT-based radiomics features from tumor and TOE could improve the long-term survival forecast accuracy in LANSCLC patients treated with CCRT using machine learning. The predictive results could be utilized as an effective indicator for the stratification of these patients into the low-risk and high-risk groups. It was further confirmed that patients in the low-risk group had better baseline FEV1/FVC%, less severe lymphopenia during CCRT, better recovery of lymphopenia from CCRT, lower incidence of radiation-induced pneumonitis, superior tumor remission and long-term survival, which might suggest more benefit for these patients from radical CCRT or further adjuvant immunotherapy.
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Endoscopy-assisted resection of a sphenoid-wing meningioma using a 3D-printed patient-specific pointer in a dog: A case report
A 9-year-old female mixed-breed dog presented for treatment of a presumed sphenoid-wing meningioma. Clinical signs included tonic-clonic seizures lasting <1 min, which had started 3 months previously. The physical examination results were unremarkable. An eccentrically located neoplastic cystic structure in the right sphenoid bone region suggestive of a meningioma and peritumoural brain oedema was observed in pre-operative magnetic resonance imaging (MRI). Prior to surgery, a three-dimensional (3D) patient-specific pointer (PSP) was designed using computed tomography (CT) images and computer-aided 3D design software. After a targeted approach and exposure of the lateral part of the right temporal lobe by a craniectomy guided by the 3D-PSP, complete macroscopic piecemeal resection of the meningioma could be performed using endoscopy-assisted brain surgery. Post-operative MRI confirmed complete excision of the tumor. Anticonvulsive therapy was discontinued after 90 days, and the dosage of anticonvulsants was tapered 2 weeks after surgery. At a follow-up examination 225 days post-operatively, recurrence of seizures was not observed, and the absence of tumor recurrence was confirmed by a repeat MRI examination. To the best of our knowledge, this is the first report in veterinary medicine describing a successful resection of a sphenoid-wing meningioma using a 3D-PSP. 3D-PSP-assisted craniectomy may be a surgical option for some canine skull-based tumors, such as sphenoid wing meningiomas.
Introduction
Meningiomas are the commonest primary intracranial canine tumors, especially in large-breed dogs, representing 49-51.5% of all intracranial neoplasms (1)(2)(3). Meningiomas can occur at many sites, including the olfactory/frontal region, the skull-base cavity, and the suprasellar and parasellar regions. However, skull-base So et al. .
/fvets. . meningiomas are rare in dogs and cats (2, 4), and the prognosis of these tumors depends on several factors such as location and therapeutic strategy (2-6). Most meningiomas in humans are treated with complete surgical resection, and the recurring tumors are treated with chemotherapy and/or radiotherapy (2, 3,7). However, pre-operative brain biopsies and complete perioperative surgical removal of tumors are challenging, and few reports describe their use in veterinary medicine (6,8).
Pre-operative surgical planning and accurate perioperative surgical techniques are essential to achieve good functional results (7,9,10). However, surgery of the canine skull base is challenging because of the complexity of the vessels and nerves in this region (10)(11)(12). Determination of the safest and most accurate approach is essential to minimize permanent surgical trauma to the normal brain (9,12,13). Neuronavigation (NN) and the endoscopic approach have been developed to increase the accuracy and safety of surgeries (10,14,15). However, NN has several limitations when applied to canines. NN can interfere with the surgical approach due to the relatively small size of the canine skull. Cost also plays a key role in limiting its adoption in veterinary medicine (9,10,(15)(16)(17). Recently, threedimensional (3D) printing has been widely used to overcome these limitations in neurosurgery (9,12,18). 3D-printed patientspecific skull-contoured brain biopsy guides have reportedly aided needle placement into the target in dogs (8). 3D patientspecific drill guides have also been reported to be widely used for accurate spinal screw placement (18,19).
Very few reports of veterinary skull-base meningiomas seem to exist (8,10,15). Moreover, unlike human medicine, no specific surgical strategies are available for individual tumor sites in dogs. Here, we describe (1) the feasibility of targeting sphenoid-wing meningiomas using a fabricated 3D-printed patient-specific pointer (3D-PSP), and (2) the endoscopyassisted surgical procedure that overcomes the limitation of a complex and challenging surgical site.
Case description
Case A 9-year-old, female mixed-breed dog with a body weight of 15.1 kg and a body condition score of 5/9 was referred for generalized seizures and meningioma. The reported clinical signs included tonic-clonic seizures lasting <1 min, which had started 3 months prior to presentation, and decreased appetite. Hematological and blood investigation results were within normal limits except for the liver panel findings. The levels of alanine transaminase, alkaline phosphatase, and γ-glutamyl transferase levels were mildly elevated.
At presentation, the physical and neurological examinations were normal. The computed tomography (CT) and magnetic resonance imaging (MRI) examinations performed at the referring veterinary clinic had discovered a large eccentrically located cystic structure under the right sphenoid wing. The marked uniform contrast enhancement of the heterogeneous cystic remainder was confirmed on CT and MRI. In addition, contrast enhancement revealed well-defined, broad-based tumor margins conforming to the meningeal plane. This structure showed heterogeneous, intratumoural fluid accumulation characterized by an enlarged, sharply defined outer margin with a significant hyperintense signal and peritumoural oedema on T2-weighted images. Based on the imaging characteristics ( Figure 1), cystic meningioma was suspected. Neither thoracic radiography nor abdominal ultrasonography revealed any abnormalities.
Choice of surgical technique
The lesion resulted in mass effect, intracerebral oedema, and associated signs such as generalized seizures, which were indications for intervention in this case. CT was used to measure the extent of bony invasion or hyperostosis due to the possibility of the tumor infiltrating the skull bone. T2-weighted MRI imaging identified intraparenchymal oedema and important structures such as the cavernous sinus and optic nerve. Schematic images were prepared for pre-operative planning using CT and MRI, which demonstrated the approximate relationships among the tumor, cerebral arteries, optic nerve, and bony landmarks (Figures 2A,B). The tumor was identified .
/fvets. . as sphenoid-wing meningioma of the skull base. A craniectomy with dura mater resection was planned.
In human medicine, lateral and middle sphenoid-wing meningiomas can be resected using pterional craniotomy, but this is a challenging technique due to closely located neurovascular structures such as the optic nerve, the cavernous sinus, and the middle cerebral artery (7). The relationships between the vascular and bone morphology and the anatomic landmarks have been clarified in human medicine, but little information of this kind is available to assist sphenoidwing meningioma surgery in veterinary medicine (7). Dogs have relatively massive temporal muscles and different sizes, locations, and morphologies of neurovascular structures and structures such as the zygomatic arch and the coronoid process of the mandibular bone (20-23). Therefore, for accurate and safe delineation of the surgical site, we decided to employ a 3D-printing technique similar to the use of a 3D-printed biopsy guide, since we lacked NN capability and sufficient experience with traditional in-silico planning and in-vivo measuring techniques.
Planning and production of the D-PSP
Surgical planning, including confirmation of the location, size, and shape of the 3D-PSP and identification of the exact location of the tumor, was performed using computer-aided 3D design software (3-DS Max; Autodesk, San Francisco, CA, USA) applied to the MRI and CT images (20,21). This was necessary because a PSP used in brain surgery can differ in detail from patient-specific guides used on the spine. Detailed guide concepts and methods have been described previously (20,21).
Postcontrast CT images (Alexion TM ; Canon Medical Systems Corporation, Otawara, Japan; slice thickness, 1 mm; operating parameters, 120 kV and 12 mA) of the patient were obtained in the DICOM file format (Digital Imaging and Communication in Medicine) and converted to stereolithography files. Segmentation, 3D model reconstruction, and prosthesis design were then performed using computeraided design software (Mimics; Materialize NV, Leuven, Belgium). The design process required ∼1 h (20).
The tumor was confirmed in a sagittal cut of the 3D bone model using the computer software. A 3D tumor-targeting cylinder passing through the sphenoid wing bone was created, and the diameter and location of the cylinder were designed to match those of the tumor ( Figures 2C-H). The diameter of the resulting pointer was twice that of the cylinder. The footprint of the pointer represented the margins of the planned craniectomy, and allowed sufficient surgical space for tumor resection. An attachment arm was created for the PSP, which was designed to fit the most characteristic surface of the frontal bone and sagittal crest. The surface of the arm had an inverted skull structure to ensure stable bone attachment. Once the arm was fixed, the pointer automatically indicated the craniectomy site under which the tumor was located.
To ensure accurate surgical planning, rehearsal surgery was performed using a 3D-printed bone model and the PSP. Both were printed using the RS6000 3D printer (Uniotech, Shanghai, China) according to a previously described protocol (20). Printing and sterilization of the 3D bone models and the PSP prior to the real surgery required ∼1 day (20).
Surgical technique
Midazolam ( The surgical procedure was based on sphenoid-wing craniectomy (22)(23)(24). The fur was clipped from the lateral canthus of the eyes to the occipital protuberance, and laterally to the zygomatic arches. The skin was aseptically prepared with chlorhexidine and disinfected with alcohol and povidoneiodine. The dog was placed in sternal recumbency with its head elevated to ∼30 • . A 6-cm vertical scalp incision was made dorsal to the zygomatic bone. The platysma was incised vertically. The superficial temporal nerve and rostral auricular nerve were retracted caudally, and the zygomatic branch of the facial nerve was retracted cranially. The zygomatic attachment of the temporal muscle was incised and bluntly elevated from the temporal bone. The scalp and temporal muscles were turned over and fixed laterally using a Senn and Gelpi retractor, and a wide spatula was used to maintain the surgical field (22)(23)(24). After separating the muscle per requirement, the pre-made 3D-PSP was applied to the skull landmarks described in Section Planning and Production of the 3D-PSP without complete removal of the periosteum (20) (Figure 3A). After dissection of the soft tissue of the skull, the craniectomy site was determined based on the 3D-PSP ( Figure 3B) (20). The craniectomy line was made along the outer boundary of the pointer using a surgical pen and electrocautery. The bone was resected using a 2.0 mm bone burr (2.0 mm burr/drill; Stryker Corp., Kalamazoo, MI, USA) along a tool path indicated by the 3D PSP, following which the tumor could be immediately identified (20,23).
After exposing the tumor using the pointer, we maximized visualization of the surgical site using an endoscope. Under endoscopic visualization, durotomy was performed using a #11 scalpel and micro-scissors, and the ventrolateral aspect of the lateral temporal lobe was exposed under an enhanced surgical field (20,22,23). To magnify the surgical site, a 10-mm, 0 • rigid telescope was connected to a high-definition camera system (Stryker Endoscopy, Stryker Corp., Kalamazoo, MI, USA). A mechanical endoscope holder was used to maintain the position of the endoscope. The tumor margins were then carefully dissected away from the normal brain tissue until macroscopic, piecemeal resection was complete ( Figure 3C). Although the location of neurovascular structures was not easily identified, there was no significant bleeding. Following tumor removal, we applied Floseal R (Baxter Healthcare Corporation, Fremont, CA, USA) (25). After confirming complete haemostasis of minor bleeding, the craniectomy area was covered with temporal muscle. The incised temporal and masseter muscle, subcutaneous tissues, and skin were closed per set guidelines (22,25). A biopsy specimen was removed from the tumor and fixed in 10% neutral-buffered formalin for histopathological examination. Eighteen-day post-operative MRI confirmed that the tumor was totally removed (Figure 4).
Outcomes, complication, and follow-up
Post-operatively, temperature, pulse, respiratory rate, capillary refill time, blood pressure, auscultation, and neurological status were monitored. Post-operative analgesia was provided with remifentanil ( Hana Pharm) was tapered over 3 months post-operatively. The clinical features and the results of histopathological examination of the surgically resected tissue samples were suggestive of a grade 1 meningothelial meningioma ( Figure 3D). Thirty-two days after surgery, the patient was readmitted to our clinic because of fluid fluctuations at the surgical site. The owner reported that the patient repetitively scratched the surgical site at home. Ultrasound was used to discriminate the fluid, and the presumptive diagnosis was seroma. The fluid was red, and analysis showed a haematocrit level of 4.5% and total nucleated cell count of 24,910 cells/µL. The seroma responded well to surgical debridement and application of a Barovac drainage tube (Barovac R ; Sewoon Medical, Seoul, Republic of Korea), which provided continuous negative pressure.
The follow-up observations comprised repeated neurological and MRI examinations for 296 days postsurgery. Recurrence of seizures was not observed, and complete tumor resection with resolution of peritumoural brain oedema was confirmed by repeated MRI (Figure 4).
Discussion
This is the first case report describing a feasible, endoscopy and 3D-PSP-assisted, sphenoid-wing meningioma resection in a canine patient. The PSP allowed us to perform the craniectomy targeting the brain tumor. Complete macroscopic piecemeal resection of the sphenoid-wing meningioma was performed accurately without injuring the main blood vessels. The patient had a good clinical outcome without significant complications over 296 days of follow-up.
Sphenoid-wing meningiomas are a type of skull-base tumor (2, 4). There are extremely limited reports of these tumors in dogs and cats. In humans, complete surgical resection is the recommended treatment (2, 3, 7). However, surgical treatment is an arduous task for several reasons. First, skull-base tumors are located deep under the brain; thus, it is challenging to target the tumor accurately and perform surgical resection in a limited operative space (10,11,26,27). In addition, several arteries and veins are present in complex areas of the skull base, and the restricted field of approach makes it difficult for the surgeon to perform complete tumor resection without vessel injury (22). Neurosurgical planning is vital to determine a precise and safe surgical approach for minimizing brain damage in patients with skull-base tumors because of the closely located, critical neurovascular structures (9,12,13). Therefore, the surgeon must be familiar with vascular and bone morphology as well as with anatomy. Visual interpretation of conventional MRI scans is usually sufficient for diagnosis, but the planning and execution of neurosurgical procedures require transformation of these data into a 3D space (1-4, 9, 28). In humans, NN .
has improved the 3D localization of brain tumors (10,14). NN captures vital neurovascular structures as well as critical neighboring structural and functional regions, and converts this information into digitized neuroradiological data. An optimized approach is determined through this simulated presurgical step, which can significantly facilitate skull-base surgeries (14). Furthermore, recent frameless NN systems have improved the usefulness of NN in skull-base tumor surgeries (17). In veterinary medicine, NN has reportedly allowed surgeons to approach the pituitary fossa with clinically acceptable accuracy (29). NN has also been used to obtain diagnostic brain biopsy samples (8,30). However, the relatively small size and thickness of the canine skull compared to humans makes it difficult for neurosurgeons to apply NN accurately in dogs (22,23,31); attempting NN in the presence of a large volume of temporal muscle and zygomatic bone could result in longer access times and narrower surgical fields. These limitations make it difficult for veterinary neurosurgeons to apply NN to sphenoid-wing meningioma resection. However, 3D-printed biopsy guides have been recently used for veterinary medicine without NN and have proven feasible in terms of outcomes (8). 3D-printing technology offers significant advantages for neurosurgery. 3D-printed material can facilitate presurgical planning by converting visual images into tactile models (18). Surgeons can rehearse realistic surgery because 3D-printed materials can be milled, cut, and drilled with the usual surgical instruments as often as required (18). Customized prosthetics, implants, fixtures, and 3D-printed biocompatible materials have been widely used recently (9,12,18). This 3D-printing technique can have several advantages when applied to brain surgeries (8,12,20). 3D printed guide techniques such as 3D brain biopsy can designate the surgical location more accurate and less-invasive (8). When resecting brain tumors, precise surgical site exposure is crucial to avoid excessive manipulation of the brain parenchyma (6, 8-13, 32, 33). If the surgical window is inaccurately delineated at the first time, expansion of the surgical window size would be inevitable (20). Moreover, it can be applied to the hands of less experienced surgeons (8,20,34). Surgeons with 3D PSP can be pre-operatively accustomed to patient specific circumstances, such as neurovascular structures and anatomic bony landmark in the course of 3D planning (12,34). Also surgeons would reduce the surgical time because they can skip the setting of the neuronavigation system and special imaging instruments perioperatively (8,20,30). In the present case, the exact craniectomy site could be easily and safely determined, avoiding in-surgery modification of the surgical window. The 3D-printed implant fitted perfectly to the skull surface only at the predefined craniectomy site; any slight displacement of the print would have led to an incongruency between the under-surface of the print and the surface of the skull, thereby indicating inaccurate positioning. The positioning of the 3D print was also limited by the narrow and deep approach to the sphenoid bone (20-22, 25, 26). However, in close-up positions, an endoscopy-assisted approach can provide a clear view of the surgical view (23). This advantage is beneficial during surgical procedures for deep-seated lesions in narrow spaces (15,22,35). Therefore, a 3D-PSP combined with an endoscope can facilitate the complex procedure of sphenoidwing meningioma resection in small-breed dogs.
In human medicine, the surgical management of sphenoidwing meningioma is reportedly challenging because of the deep and narrow surgical site in the skull base and the proximity of critical neurovascular structures such as optic nerves and arteries (7,23,29). In this case, the surgical approach, surgical view, and space for instruments were insufficient due to the tension around the temporal muscle and the coronoid process of the mandibular bone. Gross total resection requires elevation of all the tumor-infiltrated tissues, including the dura mater, to avoid tumor recurrence (36). Here, after tumor resection, the craniectomy window was covered with the temporal muscle; the dura mater could not be repaired with artificial graft because the working space was insufficient (35, 36). MRI showed cerebrospinal fluid (CSF) leakage with evidence of a CSF fistula outside the right frontal lobe 18 days post-operatively, which significantly improved 225 days post-operatively ( Figure 4). In humans, CSF leakage is reportedly a complication of cranial and spinal surgery, especially when the final structural repair of the dural defect is incomplete (37-39). Accordingly, it is reasonable to presume that the CSF leakage in our patient was caused by the dural defect. CSF leakage has been associated with secondary complications such as meningitis, encephalitis, and wound infection, and thus various treatment options have been established for persistent CSF leakage in humans, including conservative therapy, epidural blood patches, and surgical patches (39)(40)(41). However, reports describing treatments for CSF leakage in animals are rare (40, 41). Our patient was treated conservatively, as several studies in humans have suggested conservative treatment for almost all CSF leaks (42,43). In our case, it remained unclear if the seroma observed after 32 days was associated with the initial CSF leak.
This study had some limitations. First, the duration of time required to design and print the 3D-PSP models was relatively long (8,18,19). This represents a significant limitation on the use of this model in emergent situations such as intracerebral hemorrhages. Second, the surgeon's technique has a major influence on accurate pointer positioning. To ensure a perfect fit, the surgeon must separate the soft tissue sufficiently for adequate exposure of the area where the pointer will be applied (19). Slight deviations of the pointer during surgery can have serious consequences such as introducing errors in the craniectomy site (20). Improvements in software and adequate surgeon experience would increase the effectiveness of 3D-PSP applications. Third, we have included the findings of only one dog, although the feasibility of 3D-PSP-aided complete resection of sphenoid-wing meningioma has been demonstrated. Fourth, we could not evaluate the location or size .
of the surgical window, as previous studies have done, because post-operative CT was not performed (20,21). Although the 3D-printed pointer can be modified to fit the patient, further research is necessary to compare the accuracy and utility of this method with current best practices. Further studies with larger samples of dogs are also warranted to simplify the pointer manufacturing process and ensure the accuracy, reproducibility, and reliability of this technique. The development of additional methods and techniques is required to avoid damage to critical vessels such as the middle cerebral artery during resection of skull-base tumors. In humans, brain angiography has been successfully applied prior to surgery to determine the meningioma's relationship to the surrounding vasculature and their degree of encasement. However, reports describing these tumors in veterinary medicine have been very few and application of angiography proved impossible in the present case due to the owner's financial constraints (36,44). Further studies on the use of MRI-and CT-based brain angiography in veterinary medicine are warranted.
Conclusions
We demonstrated that sphenoid-wing meningioma could be safely approached by endoscopy-assisted 3D-PSP without requiring NN. Clear visualization resulted in complete resection of the skull-base meningioma. The described method is a viable option for performing skull-base craniectomy in dogs.
Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.
Ethics statement
Ethical review and approval was not required for the animal study because was a case report. We did the surgery. Written informed consent was obtained from the owners for the participation of their animals in this study. Written informed consent has been obtained from the owner of the animal to publish this paper.
Author contributions
JS, YR, and HL managed the case and wrote and edited the manuscript. HL, YR, and JS performed the surgeries. YR and FF critically reviewed and revised the manuscript. JJ supervised the clinical management of the patient. All authors contributed to the preparation of the manuscript and have approved its publication.
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2022-11-17T06:18:04.923Z
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The multidisciplinary management of cholangiocarcinoma
Cholangiocarcinoma is a lethal malignancy of the biliary epithelium that can arise anywhere along the biliary tract. Surgical resection confers the greatest likelihood of long‐term survivability. However, its insidious onset, difficult diagnostics, and resultant advanced presentation render the majority of patients unresectable, highlighting the importance of early detection with novel biomarkers. Developing liver‐directed therapies and emerging targeted therapeutics may offer improved survivability for patients with unresectable or advanced disease. In this article, the authors review the current multidisciplinary standards of care in resectable and unresectable cholangiocarcinoma, with an emphasis on novel biomarkers for early detection and nonsurgical locoregional therapy options.
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2022-11-17T06:18:05.207Z
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s2orc/train
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KRAS inhibitors: going noncovalent
KRAS G12D is the most frequent KRAS mutation in human cancer with particularly high frequencies in pancreatic and colorectal cancer. Informed by the structure of the KRASG12C inhibitor adagrasib, Hallin et al. have now, through multiple rounds of structure‐based drug design, identified and validated a potent, selective, and noncovalent KRASG12D inhibitor, MRTX1133. This study demonstrated that MRTX1133 inhibited both the inactive and active state of KRASG12D and showed potent antitumor activity in several preclinical models of pancreatic and colorectal cancer, especially when combined with cetuximab, a monoclonal antibody against the EGFR, or BYL‐719, a potent PI3Kα inhibitor.
A historical perspective of KRAStargeting drugs
KRAS oncogenes have been identified in a quarter of all human tumors and appear with high prevalence in some of the most lethal types of cancer such as pancreatic ductal adenocarcinoma (PDAC, 95%), colorectal carcinoma (CRC, 50%), and lung adenocarcinoma (LUAD, 30%). Yet, KRAS oncoproteins were considered undruggable for decades and patients with KRAS-mutant tumors remained excluded from personalized medicine approaches [1]. This notion changed recently thanks to the identification of a previously unrecognized pocket in the switch-II region (switch-II pocket) of KRAS. Moreover, by taking advantage of the reactive cysteine residue in one of the mutant KRAS isoforms (KRAS G12C ), Shokat and colleagues developed the first compound to directly block a KRAS oncoprotein [2]. Since the switch-II pocket is only accessible when KRAS G12C is bound to GDP and therefore inactive, binding of a covalent inhibitor requires a substantial degree of nucleotide cycling to effectively block this oncoprotein. Indeed, KRAS G12C retains a significant level of nucleotide cycling despite its insensitivity to classical GTPase-activating protein (GAP)-stimulated GTP hydrolysis which in this case is mediated via the noncanonical GAP RGS3 [3].
Further efforts in drug development in the following years led to the development and subsequent approval of sotorasib (AMG 510), a drug developed by Amgen (Thousand Oaks, CA, USA). Sotorasib forms a covalent bond with the KRAS G12C oncoprotein blocking it in its inactive state and has demonstrated clinical efficacy for a subset of patients with KRAS G12C -mutant LUAD as well as CRC [4]. A second covalent KRAS G12C inhibitor developed by Mirati Therapeutics, adagrasib (MRTX849), recently received the Breakthrough Therapy designation by the FDA [5]. Although the KRAS G12C mutation is particularly frequent in LUAD (40% of KRAS mutations, Fig. 1A), its overall prevalence only encompasses 13% of all KRAS-mutant tumors, indicating that there is an urgent requirement to target other mutant isoforms.
The most prevalent KRAS mutation in human cancer is KRAS G12D , present in 33% of all cases and the most frequent mutant KRAS allele in PDAC (46%, Fig. 1A). Consequently, the development of inhibitors targeting KRAS G12D has always been of particular interest since the identification of the switch-II pocket. Yet, this mutant isoform lacks the reactive cysteine residue present in KRAS G12C , thus imposing a significant challenge to design selective compounds that bind to this mutant isoform in a stable manner. In addition, the GTP hydrolysis rate of KRAS G12D is two to three times lower than that of KRAS G12C [6]. Nevertheless, informed by the structure of the KRAS G12C inhibitor adagrasib, scientists at Mirati Therapeutics were recently able to synthesize, through multiple rounds of structure-based drug design, a selective, noncovalent KRAS G12D inhibitor (MRTX1133) that is active at concentrations in the low nM range [7]. This drug binds to the switch-II pocket with extraordinary high affinity, thereby obviating the requirement for covalent interactions (Fig. 1B).
Validation of the KRAS G12D inhibitor MRTX1133
A more recent study has now evaluated the mechanism of action and antitumor activity of MRTX1133 [8]. First, the authors performed a series of assays to validate the binding efficacy of the drug to KRAS G12D when compared with wild-type KRAS. Homogenous time resolved fluorescence (HTRF) as well as surface plasmon resonance (SPR) assays confirmed an IC50 of < 2 nM and a binding KD of 0.2 pM, which are approximately 700-fold more selective for KRAS G12D over wild-type KRAS. In addition, MRTX1133 was able to prevent binding of a RAF1 RBD peptide to KRAS G12D preloaded with the nonhydrolyzable GTP analog GMPPCP with an IC50 of 9 nM. Together with the resolved structure of MRTX1133 associated with KRAS G12D bound to GDP or to GMPPCP, the authors observed a conformational change in the switch I and II regions that was incompatible with effector binding. Thus, this indicated that MRTX1133 inhibited both inactive and active KRAS G12D states (Fig. 1B). Although this compound inhibited the inactive form with higher potency, its additional activity against the active form is likely to contribute to its higher overall potency. The authors also tested the cellular activity of MRTX1133 in cell lines carrying the G12D mutation in KRAS [8]. MRTX1133 inhibited downstream signaling pathways in a concentration-dependent manner with an IC50 of < 3 nM. Moreover, this inhibitory effect was maintained for up to 48-72 h, at least when applied at higher concentrations. The authors also found that MRTX1133 inhibited ERK phosphorylation and cell growth in 2D as well as 3D cultures in 24 out of 25 KRAS G12D -mutant cell lines tested. In contrast, most non-KRAS G12D -mutant cell lines were not inhibited at all and only a few of them responded at higher concentrations. More importantly, MRTX1133 was active in mouse xenograft tumors in the range of 10-30 mgÁkg À1 . Of note, when tested in a series of 25 human cell line-and patient-derived xenografts (PDX) at 30 mgÁkg À1 , 11 of them displayed tumor regression rates of > 30%. Interestingly, although 73% of PDAC models responded to the treatment, only 25% of CRC models were affected. Whether this holds true in the clinic remains to be determined, but it could be a consequence of the fact that mutant KRAS acts as a primary driver in PDAC but not in CRC [8]. Finally, MRTX1133 showed a poor bioavailability when applied orally. Nevertheless, it was effective when administered via IP or IV routes. Whether this limitation will affect the clinical utility of this compound remains to be determined. Yet, data recently presented at the NCI RAS Initiative Symposium held in Frederick, MD suggested that formulation strategies to enhance oral absorption and/or increase IV half-life may increase the probability of augmenting the efficacy in the clinic.
Based on the limited efficacy in some of the preclinical in vivo models, the authors set out to explore factors that constrain the response to MRTX1133 as well as to identify collateral dependencies that could maximize its efficacy. To this end, they conducted a CRISPR/Cas9 sgRNA library screen both in vitro and in vivo. These experiments revealed several tumor suppressor genes such as PTEN, KEAP1, NF1, or RB1 that conferred at least partial resistance to MRTX1133. Interestingly, KEAP1 also scored strongly in one of the in vivo models of PDAC, suggesting that KEAP1 could be a key modifier of antitumor response.
Since KRAS inhibition as a monotherapy does not result in prolonged tumor regression in lung cancer [4,5], the authors anticipated that combination therapies would likely increase the therapeutic benefit of MRTX1133. The results of the CRISPR/Cas9 sgRNA library screen suggested that, among others, inhibition of EGFR and PTPN11 (SHP2) could synergize with MRTX1133. Informed by these results, the authors selected several compounds that could target these and other proteins and tested whether they could observe a synergistic effect with MRTX1133. Interestingly, combinatorial treatment with MEK, ERK, SHP2, or SOS1 inhibitors did not substantially enhance the activity of MRTX1133 to the same extent previously observed with KRAS G12C inhibitors. However, the HER2 family inhibitors, afatinib and cetuximab, as well as the selective PI3Ka inhibitor, BYL-719, did show a synergistic effect in PDAC and CRC cell lines. Based on these results, the authors combined MRTX1133 with cetuximab, an EGFR inhibitor approved for KRAS WT CRC, and with the PI3Ka inhibitor BYL-719. These combinations were even efficient in models in which either drug as a monotherapy had no effect. Importantly, both drug combinations did not cause significant body weight loss in mice when used at effective doses, suggesting that there could be therapeutic windows when used in human patients.
The findings described by Hallin et al. may have a significant impact for patients with KRAS G12D -mutant tumors [8]. Until now, the therapeutic options for patients with KRAS G12D -positive PDAC were limited to old chemotherapy strategies, such as gemcitabine, 5-fluorouracil, or taxanes. PDAC is one of the types of cancer with the lowest survival rates and accumulating evidence indicates that mutations in KRAS are an early event that drives this disease. Hence, a drug that could block the initiating event in those PDAC patients with KRAS G12D mutations could lead to substantially improved patient survival. As a proof-of-principle, recent clinical data with sotorasib (21% ORR) and adagrasib (50% ORR) in KRAS G12Cmutant PDAC provide initial evidence, although KRAS G12C mutations comprise less than 2% of KRAS-mutated PDAC (Fig. 1A) [9,10].
Several mechanisms have been described that, at least in cell culture and mouse models of PDAC, allow survival of tumor cells in the absence of mutant KRAS [11,12]. This is not unexpected since lung cancers treated with KRAS G12C inhibitors are also capable of rapidly developing resistance. Therefore, combination therapies are likely to be more effective and the current study by Hallin et al. already proposes several potent combinations [8]. In contrast to PDAC, KRAS mutations are usually not considered an initial driving event in CRC, instead being responsible for the progression of adenomas to malignant carcinomas. This property might be one of the reasons for the limited effect of KRAS G12C inhibitors in CRC patients [13]. Whether the combinatorial therapies of MRTX1133 outlined in this study will increase the efficacy of this novel KRAS inhibitor remains to be determined.
Finally, it will be of great interest to see how MRTX1133 will perform in clinical trials. Based on prior experience with sotorasib or adagrasib, it is likely that combination therapies will be much more effective. Until then, additional preclinical studies will shed light on potential mechanisms of resistance and guide clinicians to select appropriate combination therapies. Interrogation of resistance mechanisms in patients treated with sotorasib or adagrasib revealed secondary mutations in KRAS that prevented these drugs from binding to the switch-II pocket, as well as mutations in effectors of related signaling pathways such as EGFR, BRAF or activating mutations in other RAS paralogs [14,15]. Given the high affinity binding mode of MRTX1133, similar mutations, either preventing high-affinity binding to the switch-II pocket or activating other signaling pathways, can be expected. Yet, most tumors acquire resistance without the presence of novel mutations [14,15]. Understanding the molecular mechanisms responsible for the appearance of resistance will provide new treatment avenues to increase the clinical efficacy of these compounds. Regardless of these potential limitations, the development of MRTX1133 is undoubtedly a major step forward toward the implementation of much-needed effective therapies for patients with KRAS G12D -mutant tumors.
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v2
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2022-11-17T06:18:05.230Z
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2022-11-16T00:00:00.000Z
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253552286
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s2ag/train
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Laparoscopic Excision of Large Wilms Tumor in Children: A Single-Center Experience from a Resource-Challenged Nation.
Background: In this study, we aim to review the outcomes of children with Wilms tumor (WT) operated through the minimally invasive surgery (MIS) approach at our center. We also intend to highlight essential surgical steps during laparoscopic excision of large WTs. Methods: This retrospective study included children with unilateral WT who had undergone resection for a period of 4 years, w.e.f. July 2013 to July 2017. Simple maneuvers such as tilting the table in different positions and use of blunt metallic cannula to lift the tumor to access the hilar vessels were used to dissect large WT. An extended lumbotomy incision was used for retrieval of tumor and lymph-node sampling. Results: Eleven patients (male:female = 7:4) of WT, all having stage III disease, had undergone laparoscopic tumor resection at our center during the study period. The median age at presentation was 36 months (range = 17 months-5 years) and the median preoperative tumor volume was 1140 (range = 936-1560) cm3. The average length of the lumbotomy incision was 6.3 (range = 5-8.2) cm. The median hospital stay was 6 (range = 5-10) days. Two children developed complications (port-site recurrence and grade III surgical site infection in one each) during the postoperative period. All cases are long-term survivors after a median follow-up of 86 (range = 56-104) months. Conclusion: This study highlights the feasibility and safety of the removal of large WT through the MIS approach. Problems due to large-sized tumors in children can be overcome by simple maneuvers. Also, adequate lymph node sampling is possible with a suitably placed extended lumbotomy incision for tumor removal.
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v2
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2022-11-17T15:10:33.581Z
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2022-11-16T00:00:00.000Z
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253555538
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s2orc/train
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Cell membrane-coated human hair nanoparticles for precise disease therapies
Precision medicine is the ultimate goal for current disease therapies, including tumor and infection. The lack of specific targeted drugs for liver cancer and the lack of specific anti-infective drugs in the treatment of diabetic foot ulcer with infection (DFI) are the representative obstacles in those 2 major diseases currently plaguing human beings. Inventing natural biocompatible polymers derived from natural materials is one of the main development directions of current bio-medical materials. Though previous studies have demonstrated the potential application values of human black hair-derived nanoparticles (HNP) in cancer, methicillin-resistant Staphylococcus aureus (MRSA) infection, and thrombosis scenarios treatments, it still has not solved the problem of low local therapeutic concentration and general targeting ability. Here, we firstly modified the HNP with membrane encapsulations, which endowed these dual-pure natural bio-fabricated materials with better targeting ability at the disease sites with no reduction in photothermal therapy (PTT) effect. HNP coated by red blood cell membrane loaded with DSPE-PEG-cRGD peptide for the therapeutic application of liver cancer greatly prolonged in vivo circulation time and enhanced local targeting efficacy as well as low toxicity; HNP coated by the murine macrophage cell membrane (RAWM) for the DFIs treatment greatly promoted the adhesive ability of HNP on the bacteria and thereby improved the killing effect. Briefly, the appropriate cell membranes camouflaged HNP nanomedicine has the characteristics of excellent photothermal effect, an all-natural source with excellent biocompatibility and easy access, which is expected to have huge potential in both benign and malignant diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12951-022-01673-6.
Introduction
The spectrum of diseases that cause human death in different historical periods varies. With the development of science and technology, the diseases that cause death have changed from infectious diseases to "modern diseases" [1]. The number one killer today is cardiovascular and cerebrovascular diseases, followed by various types of malignant tumors (some cities and regions have become the number one killer), followed by diabetes and Alzheimer's disease [2]. In the global cancer burden data released in 2020, China ranks first in the world in the number of new cancer cases. Among them, hepatocellular carcinoma (HCC) ranks 6th in new cancer cases worldwide and has the third highest mortality rate [3]. In recent years, significant progress has been made in surgical techniques, interventional treatment, therapeutic reagents, and radiotherapy for HCC [4]. However, the monotherapy for HCC has appeared "ceiling effect", and more precise targeted therapy is urgently needed to further improve the curative effect of HCC [5].
While about 451 million adults worldwide have diabetes, by 2045, this number will rise to an estimation of 693 million. Patients with diabetes for more than 5 years have a 61% chance of getting complications; those with diabetes for more than 10 years rise to 98%, and the incidence of diabetic foot is as high as about 20% [6]. The diabetic foot has a very poor prognosis and even higher mortality and disability than most cancers, and remains an intractable complication of diabetes. Diabetic foot ulcer with infection (DFI) is one of the most important reasons for disease progression, amputation, and death in patients with diabetic foot [7]. Considering that DFI is a systemic disease, the current treatment of DFI is based on glycemic control, using debridement, dressing coverage, negative pressure suction, surgical treatment, antibacterial treatment, and other methods for foot wound care. When the ulcers have a lot of exudation, hydrocolloid or hydrogel dressings are usually chosen; while the infections are severe, silver ion dressings are generally used [8]. However, silver ion dressing is suitable for the healing of ulcers with infection in mice for it accelerates healing with a significant reduction in bioburden; while for diabetic mice, it can only promote healing but the antibacterial effect is average, for it cannot keep up with the speed of high blood sugar to promote the growth of bacteria [9]. Whether silver ion dressings have a great antibacterial effect on DFIs remains to be determined. As oral antibacterial therapy alone may not achieve the desired effect, antibacterial dressings are also applied in local wounds, which can delay the progress of infection and help wound healing [10]. However, due to the poor microcirculation of patients with DFI, intravenous or oral administration of antibiotics for local ulcers with infection often fails to achieve effective therapeutic concentrations. Therefore, it is imminent to design biomaterials that have broad-spectrum antibacterial properties and long-time effects.
Photothermal therapy (PTT), as an emerging tumor therapeutic approach, has received extensive attention from researchers. Nano-PTT technology has the advantages of wide applications, non-invasiveness, strong selectivity, ease of operation, and little damage to normal tissue [11]. However, excellent biodegradability and photothermal performance are often difficult to balance in current nano-photothermal conversion materials, which makes it difficult for them to obtain the approval of the Food and Drug Administration, as well as to be applied in clinical practice [12]. Therefore, nanomaterials with superior properties and biodegradability are bottlenecks to overcome. Some researchers have found that natural melanin nanoparticles (NPs) can be extracted from cuttlefish juice, and by using biomimetic technology, red blood cell membrane camouflaged melanin NPs (Mela-nin@RBC) can be prepared for enhanced PTT of tumors with prolonged circulation time and great degradability [13]. Studies have shown that human hair as a raw biomaterial is affordable and readily available, which has a micron-sized hierarchical superstructure that can tightly wrap melanosomes in the cortex. Hair is mainly composed of keratin and melanin. Keratin is fundamental for some natural biomimetic materials, for it has wide applications in bone regeneration and hemostasis [14,15]. Melanin also has good photothermal effects and has been reported to treat tumors and infections with considerable curative effect [16,17].
However, the application of human hair nanoparticles (HNP) in disease models still has limitations, considering that HNP has no tumor targeting ability in vivo, and is easily captured by the reticuloendothelial system [16]. Therefore, encapsulating HNP with a certain cell membrane can address this dilemma well. At the same time, studies have shown that the use of genetic engineering to extract the cell membrane which overexpressed vascular cell adhesion molecule-1 (VCAM-1) can adhere to the inflammation site and recruit immune cells to fight the infection [18,19]. Considering that macrophages are the inflammatory initiating cells during infection, if the murine macrophage cell membrane (RAWM) can be used as a cell membrane coating to camouflage HNP, it may be possible to simultaneously target the inflammatory site and exert local anti-inflammatory effects, which offers a promising strategy for DFIs treatment.
Here, we designed a kind of natural nanomaterials-HNP coated by the red blood cell membrane (RBCM) or RAWM. The HNP@RBCM-cRGD can specifically eliminate HCC cells via PTT treatment; the HNP@RAWM performs anti-bacterial ability and promote the wound healing rate of DFIs in the mice model (Scheme 1). This combination strategy was the first try to verify a universal strategy for HNP in cell membrane camouflages, to preserve its excellent PTT ability and enhance its targeting ability in different disease models.
Synthesis of HNP and characterization
Collected human black hair clippings from adults were added to a heated and boiling NaOH solution. A glass rod was stirred quickly to fully dissolve the hair, and then the hair solution was cooled to room temperature. The resulting hair solution was added to a dialysis bag and dialyzed against a phosphate-buffered saline (PBS) solution for 24 h to remove the NaOH. The hair solution was poured out and stirred at room temperature in a mixer for 1.5 h. After centrifugation at 2000 rpm/ min for 6 min, the supernatant was taken, and the centrifugation at 12,000 rpm/min for 10 min was repeated again after washing with PBS 3 times. The hair solution was obtained, which was evaporated to dryness to obtain hair micro-particles. After weighing the hair micro-particles, pure water was added to dissolve, and the hair micro-particles were crushed with an ultrasonic probe in an ice-water bath to obtain a hair nanoparticle solution, namely HNP.
Preparation of RBCM, RAWM, and membrane-coated HNP
Blood was collected from 8-week-old C57 male mice from mouse orbit in the ethylenediaminetetraacetic acid (EDTA) tubes, shaken and placed on ice. After centrifuging the collected blood at 3000 rpm for 15 min, we carefully aspirated the red blood cell (RBC) at the bottom without suctioning the light-yellow and white upper layers, placing the RBC in PBS and washing it for 3 times. Scheme 1 Schematic illustration of RBCM-cRGD/RAWM coated HNP hybrid nanovesicles in applications of malignant and infectious diseases. The cell membranes encapsulated HNP system exhibits excellent efficient PTT ability, with enhanced tumor targeting and bacterial adhesion ability of HNP After collecting the pellet, we used 25% PBS (v/v) to lyse RBC for 2 h, and mixed by pipetting every half an hour. The lysate was centrifuged at 12,000 rpm/min for 10 min and the supernatant was discarded. After washing with PBS twice, the solution was continuously extruded 11 times through a polycarbonate membrane microextruder (Avanti Polar Lipids, USA, CAT#610000-1Ea) to obtain RBCM in the form of vesicles, and the protein is quantified to determine the solution concentration. The RBCM was stored in a 4 °C refrigerator for short-time storage and a − 80 °C refrigerator for long-time storage.
The mouse macrophage cell line-RAW 264.7 was cultured with RAW 264.7 cell-specific medium (Procell, China, CAT#CM-0190) at 37 °C in a cell incubator with 5% CO 2 . When the cells grew to 50% density, we used 0.25% trypsin to digest the macrophages and neutralized them with RAW 264.7 special medium after about 1 min, and centrifuged at 800 rpm/min for 5 min to collect the pellet. After washing the pellet 3 times with PBS, we added 3 mL of strong radioimmunoprecipitation assay (RIPA) lysis solution (Beyotime, China, CAT#P0013B) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) to the pellet by gentle pipetting until the solution is clear, and lysed it on ice for 15 min. The solution was then shattered in an ice-water bath with a 45 W ultrasonic probe for 30 min, with sonication conditions of 2-s on and 3-s off to rupture the cell structure. The nucleus was removed by gradient centrifugation, and finally, ultracentrifugation was used to gain the cell membrane at 100,000 rpm/min for 45 min, namely RAWM. A transparent precipitate was uniformly pipetted with 1 mL of PBS and the solution was also continuously extruded 11 times through an Avanti extruder to obtain RAWM in the form of vesicles.
The DSPE-PEG-cRGD powder was weighed and added to the RBCM solution in the form of vesicles. An icewater bath ultrasound was applied for 5 min to obtain RBCM-cRGD. The HNP and RBCM-cRGD or RAWM were mixed and subjected to ultrasonic treatment to obtain membrane-coated HNP, namely HNP@RBCM-cRGD or HNP@RAWM.
Transmission electron microscopy (TEM) images were acquired by a Tecnai G2 Spirit 120 kV cryo-EM (FEI, The Netherlands). Scanning electron microscope (SEM) images were acquired by a Nova Nano 450 field emission SEM (FEI). The dynamic light scattering (DLS) and zeta potential values were evaluated on a Malvern Zetasizer Nano instrument (Malvern, UK). The absorption spectra were measured by a UV-2000 photo spectrometer with UVProbe v2.42 (Shimadzu, Japan).
Membrane protein characterization
Western blotting and Coomassie staining were used to determine whether the protein components contained in our collected cell membrane were consistent with those of RBC and RAW 264.7. Briefly, RBCM, RBCM-cRGD, HNP@RBCM-cRGD, RAWM, and HNP@ RAWM were lysed with RIPA lysate and the protein quantification was performed by the bicinchoninic acid kit (Beyotime, China, CAT# P0012S). All samples were mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Bio-Rad, USA) and heated in the metal bath at 100 °C for 10 min. Afterward, all samples were loaded on 10% SDS-PAGE gel with protein amounts of 30 µg/well and were run at 80 V for 20 min and 120 V for 1 h. The SDS-PAGE gel was stained in Coomassie for 1 h at room temperature and washed with deionized water to remove the residual dye by microwave heating. Also, the SDS-PAGE gel was transferred to polyvinylidene difluoride membranes for western blot analysis. Membranes-associated protein markers contained anti-CD47-Rabbit-mAb (Abclonal, China, CAT#A11382), β-actin (Abclonal, CAT#AC028), anti-integrin αv-Rabbit-pAb (Cell signaling technology, China, CAT#4711T), and anti-integrin alpha 4 (Proteintech, USA, CAT#19676-1-AP). The polyvinylidene difluoride membranes were blocked with a quick block solution (Beyotime, CAT#P0256) and incubated with related antibodies diluted in 1:1000 at 4 °C overnight, followed by incubation with horseradish peroxidaselabeled goat anti-rabbit or mouse lgG (H + L) secondary antibody (Abcam, UK; CAT#ab205719, CAT#ab205718, 1:10,000) for 1 h at room temperature. The strips were exposed to the Chemiluminescence Imager (Bio-Rad, CAT#17001402) after adding the chemiluminescence solution (Invitrogen, USA, CAT#34580).
In vitro subcellular localization of HNP@RBCM-cRGD
To determine the cellular uptake and subcellular localization of HNP@RBCM-cRGD, Hepa 1-6, CT26, and PANC-2 cells were seeded on 24-plate dishes and were incubated with the DiI (Beyotime, CAT# C1995S)-labeled HNP@RBCM-cRGD at 37 °C for 20 min. After 3 times of gentle washing with PBS, the cell nuclei were counterstained with Hoechst 33258 (Beyotime, CAT# C1011) for 15 min at 37 °C. The subcellular localization of HNP@ RBCM-cRGD was examined via a fluorescence microscope (Zeiss, Germany) at an excitation wavelength of 560 ± 20 nm and an emission wavelength of 650 ± 5 nm for DiI. An excitation wavelength of 360 ± 20 nm and an emission wavelength of 460 ± 25 nm were used to observe Hoechst fluorescence.
Measurements of photothermal performance
The temperature trends of HNP with different concentrations (0.2 and 0.5 mg/mL) were measured under irradiation by an 808 nm laser at 1.0 W/cm 2 for 7.5 min. The temperature alterations were monitored and captured by Fluke Ti540 SF6 (USA). Also, the photothermal conversion efficiencies (η) of HNP and HNP@RBCM-cRGD were calculated by the following equation [20]: where h represents the heat transfer coefficient, S indicates the surface area of the container, T max represents the maximum temperature, T surr indicates the room temperature, Q dis represents heat absorption of the EP tube (Eppendorf tube), I represents the laser power, and A 808 is the absorbance of NPs at 808 nm. In the second equation, when the heat input is equal to the heat output in the measurement system, where m D is the weight of water, C D indicates the specific heat capacity of water, and τs represents the time constant of NPs. τs can be measured by the linear regression curve via the above equation.
Tumor-targeting and PTT ability of HNP@RBCM-cRGD in vivo
A Hepa 1-6 tumor-bearing mouse model was used to verify the tumor targeting and PTT ability of HNP@ RBCM-cRGD in vivo. The study was approved by the Institute Ethics Committee at Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University (ethical code: SRRSH20210131). Hepa 1-6 cells (3 × 10 6 cells per mouse, 100 μL PBS each) were injected subcutaneously into the right hind legs of 32 male BALB/c nude mice (5 weeks old) to establish a tumor-bearing mouse model. BALB/c nude mice were randomly divided into 8 groups as follows: PBS, RBCM-cRGD, HNP, and HNP@RBCM-cRGD with or without laser. When the tumor volume reached 30-50 mm 3 , aqueous dispersion of PBS, RBCM-cRGD, HNP, and HNP@RBCM-cRGD (same concentration: 1.5 mg/mL of HNP, 200 μL) were injected into the tumor-bearing mice via tail vein. After 24 h, the laser groups were irradiated with an 808 nm laser for 0 min, 2.5 min, 5 min, 7.5 min, and 10 min to observe the PTT ability of materials in vivo.
Mouse cutaneous wound model and S. aureus wound infection model
All the animal experiments and procedures in this study were conducted with consent from the Committee of the Use of Live Animals in Teaching and Research at Sir Run Run Shaw Hospital (SRRSH). A cutaneous wound model was used to verify the antibacterial ability of HNP@ RAWM in vivo. Thirty-three C57BL/6 mice, 8 weeks of age, were housed in sterile and filter-capped cages for 1 week before establishing the diabetic model [21]. Mice with blood glucose levels of > 300 mg/dL at 2 weeks after the final injection of streptozotocin (STZ, Sigma, USA) were considered diabetic status. C57BL/6 mice were randomly divided into 8 groups as follows: PBS, RAWM, HNP, and HNP@ RAWM with or without laser. Later, cutaneous wounds were established based on the previous study [22]. Briefly, the mice were anesthetized by the intraperitoneal injection of 0.2 mL 1% pentobarbital (1 mL/kg, 20 g pentobarbital for each mouse) before operation. Then, the dorsal side of the mice was shaved with hair removal cream, cleaned, and sterilized. Next, we used a biopsy punch to perform 2 symmetrical round wounds with 8 mm in diameter on the dorsum, and fixed with rubber rings to prevent scratching. A dose of 10 7 CFU/mL S. aureus per wound (20 μL bacterial fluid) was dropped on the wound for 24 h, and the mouse cutaneous wound model was established.
To treat the mouse cutaneous wound model, the reagents were incubated on the wound for 1 h for them to be fully covered. The laser groups were irradiated with an 808 nm laser and maintained at 50 °C for 5 min. We assessed the healing rate by photographing the instant wound area on day 1, 4, and 7 versus day 0. Contemporarily, the bacterial counts in each wound were collected by sterile cotton swab and were cultured in agar plates for 12 h to assess the bacterial load.
After day 7, all the mice were sacrificed and the woundrelated skin tissues were removed from the dorsal side. The tissues were fixed in 4% paraformaldehyde solution before paraffin embedding and sectioning. To evaluate the wound closure rate at a micro-level, we performed hematoxylin & eosin (H&E) staining to compare the bacterial infection difference in 8 groups. In the meantime, the major organs, such as the heart, spleen, liver, lung, and kidney were also stained with H&E on day 7 for the biosafety background investigation.
Bacterial culture of DFIs pus and Proteus vulgaris wound infection model
This study was approved by the Institutional Review Board of the SRRSH (ethical code: 20200210-126) and written informed consent was obtained from the diabetes patient (consent number: CON501). Nine C57BL/6 mice with diabetic status were randomly divided into 3 groups as follows: PBS, HNP, and HNP@ RAWM with laser irradiation. Later, diabetic ulcer model mice were established as mentioned before. Then we took the local pus from patients with DFIs for culture to detect the main bacterial species by clinical examination. To establish the Proteus vulgaris wound infection model, we dropped the pus on the wound for 24 h.
To investigate the therapeutic effects, the reagents were incubated on the wound for 1 h. The laser groups were irradiated with an 808 nm laser (1.0 W/cm 2 , 50 °C, 5 min). The healing rate was assessed by the skin lesion size on day 1, 4, and 7 versus day 0. Meanwhile, we collected the bacterial counts in each wound and cultured them for 12 h to assess the bacterial load.
In vitro and in vivo biosafety of HNP@RBCM-cRGD
Hepa 1-6 cells were counted as 4 × 10 3 cells per well with 100 μL of Dulbecco's Modified Eagle Medium (DMEM) medium suspension and were seeded into 96-well plates overnight. Then, PBS, RBCM, HNP, and HNP@RBCM-cRGD were added at the concentration of 20 μg/mL into each well and further cultured for another 0, 12, 24, and 48 h before biosafety evaluation through the MTS assay (Promega, USA, CAT#G3582). A fresh medium containing 10% MTS reagent was added into the well for cell incubation at 37 °C for 2 h, followed by detection of absorbance at 490 nm via Multi detection microplate reader (Thermo, USA) to evaluate the cell viability.
ICR mice were intravenously injected with 200 μL PBS, RBCM-cRGD (0.75 mg/mL), HNP (1.5 mg/mL) and HNP@RBCM-cRGD (1.5 mg/mL), or drop with 20 μL PBS, RAWM (0.2 mg/mL), HNP (0.4 mg/mL) and HNP@ RAWM (0.4 mg/mL) on the wounds with or without laser. After day 1 and day 30, all the mice were sacrificed. The extracted serum was collected by eyeball extraction of blood samples, and hematology studies were performed. Alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CR, n = 3) were detected as the liver and kidney function indexes in the blood biochemistry test. The vital organs (heart, liver, spleen, lung, and kidney) were harvested, prior to being fixed with 4% paraformaldehyde overnight, then embedded in paraffin and sliced, later stained with H&E, and finally observed under an inverted optical microscope (Zeiss).
Statistical analysis
A student's t test was used to evaluate the statistical significance of the variance. One-way ANOVA method was used to analyze quantitative data and expressed as mean values ± standard deviations (s.d.). A P value < 0.05 were considered statistically significant.
Characterization of HNP@RBCM-cRGD and HNP@RAWM
As shown in Fig. 1A, the TEM revealed that HNP was distributed separately with a black round-like shape. HNP@RBCM-cRGD possessed HNPs with a layer of RBCM coating on the outside. HNP@RAWM appeared to encase HNPs in double cell membranes of macrophage cells (Additional file 1: Fig. S1A). Near-infrared (NIR) light irradiation produces higher photon energies and lower scattering, resulting in deeper tissue penetration than light in the UV-visible region, and is therefore considered more suitable for PTT [23][24][25]. As melanin is fully absorbed in NIR, nearly 44% of HNP consisted of melanin, suggesting that HNP had a great potential in becoming biocompatible photothermal material under NIR irradiation [26]. The molecular structure of melanin is shown in Fig. 1B. To explore whether the HNP@ RBCM-cRGD and HNP@RAWM hybrid NPs were successfully prepared, dynamic light scattering (DLS) and zeta potential were applied for the physical property evaluation, and western blot analyses were performed for the protein component detection. Fig. S2). The difference in DLS values and zeta potential values between RBCM-cRGD, RAWM, HNP, HNP@RBCM-cRGD, and HNP@RAWM indicated that HNP was tightly loaded in the cell membrane. After the cell membrane encapsulated the HNP, SDS electrophoresis indicated that the cell membrane components were well preserved (Fig. 1D). The RBC marker-CD47 which could avoid macrophage phagocytosis and increase circulation time, was detectable both in the purified RBCM-cRGD and HNP@RBCM-cRGD (Fig. 1E). The macrophage marker-integrin 4α was also detected in the purified RAW 264.7, RAWM, and HNP@RAWM, which confirmed that the cell membrane proteins were still maintained after HNP encapsulation (Additional file 1: Figs. S3 and S4). To investigate whether the HNP@RBCM-cRGD would maintain the spectral property of HNP and RBCM-cRGD, we next examined the absorption of RBCM-cRGD, HNP, and HNP@RBCM-cRGD. The absorption peaks of HNP and HNP@RBCM-cRGD were both at 300 nm (0.1 mg/mL, Fig. 1F).
We next started to explore whether the photothermal properties of HNP were affected by membrane encapsulation. The aqueous dispersions of RBCM-cRGD, HNP, and HNP@RBCM-cRGD were irradiated by 808 nm laser at a power of 1.0 W/cm 2 and monitored by a bolometric imager (Fig. 1G). The temperature of RBCM-cRGD (0.25 mg/mL) and PBS remained slightly above room temperature. The maximum temperature of HNP gradually increased from 40 °C to 68 °C as the solution concentration increased from 0.2 mg/mL to 0.5 mg/mL. In addition, the coating of RBCM-cRGD outside HNP did not affect the photothermal properties of HNP (Fig. 1H). The photothermal conversion efficiency of (η) of HNP (calculated as 43.28%) was similar to that of HNP@RBCM-cRGD (calculated as 42.13%), indicating that HNP had decent photothermal conversion efficiency which was an essential factor for further applications. As we can see from the cyclic irradiation evaluation of HNP@RBCM-cRGD, the photothermal performance did not change significantly in the process of repeated heating and cooling (808 nm, 1.0 W/ cm 2 , 0.5 mg/mL, 3 cycles; Fig. 1I), which indicated that the photothermal stability of HNP@RBCM-cRGD was excellent.
In vitro tumor-targeting ability of HNP@RBCM-cRGD and PTT assays
To validate the in vitro toxicity, we incubated Hepa 1-6 with PBS, RBCM, HNP, and HNP@RBCM-cRGD at a concentration of 50 μg/mL HNP and irradiated them with 808 nm laser for 5 min (1.0 W/cm 2 ) and examined the cell viability by MTS assay. The cRGD peptides can bind to integrin receptors expressed on tumor cell membranes [27]. As different types of tumor have different levels of integrin receptors expression, it determines the affinity of RBCM-cRGD to tumor cells (Additional file 1: Fig. S5). As shown in Fig. 2A, Hepa 1-6 had the highest uptake of HNP@RBCM-cRGD compared with CT26 and PANC-2. The cytotoxicities of the materials were similar among groups during the 12, 24, and 48 h after treatment, which suggested that the biosafety profile of the material was considerable (Fig. 2B; Additional file 1: Fig. S6). Twenty-four hours after the laser irradiation, the PTT efficiency of HNP and HNP@RBCM-cRGD was higher than that in the PBS and RBCM groups, as the cell viability dropped from 92.43% to 43.38% and 32.89% compared with PBS group, respectively (Fig. 2B). In order to exclude the interference of heating caused by pure laser irradiation, we measured the growth of Hepa 1-6 cells at 0, 12, 24, and 48 h after laser irradiation, and there was no significant difference compared with the negative control group (Additional file 1: Fig. S7). In vitro experiments demonstrated that RBCM-cRGD encapsulated HNP enhanced the specific targeting ability and PTT efficiency of HNP in Hepa 1-6 cells compared with other tumor cells.
Tumor-targeting ability of HNP@RBCM-cRGD in Hepa 1-6 bearing mice
After clarifying the anticancer effect of HNP@RBCM-cRGD in vitro, we next studied the performance of HNP@RBCM-cRGD in Hepa 1-6 tumor-bearing mouse model. We firstly injected 200 μL aqueous dispersion of PBS, RBCM-cRGD, HNP, and HNP@RBCM-cRGD into tumor-bearing mice (1.5 mg/mL of HNP) via tail vein (Fig. 2C). Thermal observations of in vivo materials were performed 24 h after injection ( Figure S8). As shown in Fig. 2D, a significant temperature rise was observed in HNP and HNP@RBCM-cRGD up to 51.57 °C and 59.8 °C respectively, whereas the temperature of PBS and RBCM-cRGD groups were mildly increased. At the time of 2.5 min after the laser irradiation, the temperature of the HNP and HNP@RBCM-cRGD groups reached 45.6 °C and 48.4 °C, respectively (Fig. 2E). This indicated that the cRGD modification could help the materials concentrate around the tumor lesion and endow HNP with the ability to target tumors, which assisted the PTT effect of HNP. Also, after reaching the maximum temperature, HNP@ RBCM-cRGD maintained the maximum temperature until the end of the treatment, which could enhance the anti-tumor effect (Fig. 2D). To further investigate the materials distribution ex vivo in tumors after treatment, we linked HNP-NH 2 and ICG-COOH to generate stable HNP-ICG and HNP-ICG@RBCM-cRGD so as to monitor the brightness and distribution of ICG in the NIR-II region. Consistent with the infrared thermal images, we found that after 24 h of materials injection, the distribution of the material remained in the tumor regions in the HNP and HNP @RBCM-cRGD groups compared with the control group (Additional file 1: Fig. S9).
The mice were treated by laser only once before we evaluated the tumor volumes every 2 days. Among the laser-irradiated groups, the RBCM-cRGD could improve the tumor target ability of HNP, therefore, the tumor volume of HNP@RBCM-cRGD remained stable or shrank since the irradiation (Fig. 2F). Tumor growth was also significantly slower in the RBCM-cRGD coating HNP group in comparison with HNP without coating (P < 0.001; Fig. 2G). Moreover, the tumors were resected to be weighed after we sacrificed the mice on day 10. In accordance with the tumor volume, the non-laser-irradiated groups had minor differences in tumor weight between groups (P > 0.05) while the HNP@RBCM-cRGD + Laser group had a significant reduction in tumor weight in comparison with HNP + Laser, RBCM-cRGD + Laser groups (both P < 0.001; Fig. 2H). In vivo treatment also reflected good biosafety, as there was no statistical difference in animal body weight between groups (Fig. 2I).
Tumor cells can activate tumor-killing immune responses of NK cells after PTT
As shown in Fig. 3A, in the tumor histologic sections of HNP and HNP@RBCM-cRGD after laser irradiation, it could be found that there was obvious necrosis inside the tumor. To further explain the mechanism of PTT exerted by HNP to kill tumor cells, we performed an RNA sequence between HNP and HNP + Laser groups in Hepa 1-6 cells. The enrichment of the differential genes between the 2 groups mainly focused on immune response, such as "Complement and coagulation cascades", "IL-17 signaling pathway" and "Natural killer cell-mediated cytotoxicity" (Fig. 3B). Considering that NK (natural killer) cells could kill tumor cells without specific antigen stimulation, we proposed whether PTT along with photothermal material, HNP, could stimulate cytokines from HCC cells to activate the tumor cytotoxicity of NK cells. As illustrated in Fig. 3C, we firstly incubated 2 human liver cancer cell lines, LM3, and SK-Hep-1 cells, with HNP solution (0.1 mg/mL) for 4 h. Later, the LM3 and SK-Hep-1 cells were divided into control, HNP, and HNP + Laser groups (808 nm laser, 0.5 W/cm 2 , 5 min). Twelve hours after the PTT, the supernatant from the control group, HNP, and HNP + Laser groups was added to the NK cells for 24 h of cultivation, respectively. We found that treatment with HNP and HNP + Laser, could not only induce the upregulation of IL-12b in NK cells but also down-regulated the expression of IL-6 and IL-10, in comparison with the control groups (Fig. 3D). This suggested that the combination treatment of PTT and HNP could effectively damage tumor cells by enhancing the interactions between NK cells and HCC cells by modulating cytokines to provoke the cytotoxicity of NK cells to eliminate tumor cells.
In vitro heating ability and anti-infective ability of HNP@ RAWM
To elucidate that encapsulating RAWM could enhance the targeting and adhesion ability of HNP on S. aureus, we incubated the S. aureus suspensions with HNP and HNP@RAWM (0.4 mg/mL) at room temperature for 1 h in the dark before we irradiated the solution with 808 nm laser for 5 min (Fig. 4A). After the irradiation, we diluted the bacterial solution and applied it to the plate to compare the difference in colony growth (Fig. 4B). From the heating curves of HNP and HNP@ RAWM (Additional file 1: Fig. S10), we found after coating the RAWM, the solution reached the treatment temperature 50 °C faster compared with the HNP group. We hypothesized that RAWM were more likely to adhere to bacteria and enabled them to aggregate. The HNP and HNP@RAWM were precipitated by centrifugation (1000 rpm/2 min). Considering that a small amount of S. aureus would adhere to the HNP and HNP@RAWM, the bacterial liquid in the supernatant was removed after centrifugation, and the remaining precipitate was applied to the plate to clarify the pulling effect of RAWM on bacteria (Fig. 4C). As shown in Fig. 4D, the bacterial counts in HNP@RAWM were 3 times more than HNP group, suggesting that our hypothesis stood. The adhesion of HNP in S. aureus observed by SEM was consistent with the in vitro results, that the adhesion efficiency of pure HNP to S. aureus was low in comparison with HNP@RAWM (Fig. 4E).
In vivo anti-bacterial and promote healing capacity of HNP@RAWM in mice with DFIs
Mice diabetic wounds with S. aureus infection were used to assess the healing process of diabetic wound matrices. Full-thickness epidermal tissues were removed by biopsy equipment, and S. aureus infection was established 24 h before treatment. Suspensions of PBS, RAWM, HNP, and HNP@RAWM were dropped onto the infective wound inside the plastic ring. A 5-min 50 °C 808 nm laser treatment (Additional file 1: Fig. S11) was given an hour after the materials were applied. The wound-healing outcome was measured on day 0, 1, 4, and 7, and the bacteria in infected wounds were collected for culture and plating. The laser irradiation accelerated the healing of the wounds, and the effect of anti-bacteria and PTT appeared on day 1 and became more significant on day 4 and day 7 (Fig. 4F, I, J). On the pathological level, on day 7, it could be seen that the degree of inflammatory infiltration in the HNP@RAWM + Laser group was significantly less than that in the PBS + Laser, RAWM + Laser, and HNP + Laser group (Fig. 4H). In groups without laser irradiation, the differences between PBS, RAWM, and HNP in the aspect of inflammation were not obvious, while HNP@RAWM had mild inflammatory cell infiltration in the sections. There appeared a phenomenon of collagen regeneration in HNP@RAWM + Laser and HNP + Laser groups but not in PBS + Laser, RAWM + Laser groups, and groups without laser irradiation (Fig. 4H). In accordance with the wound healing process, the two-pronged treatment combined laser and HNP@RAWM was able to kill a large number of S. aureus on the first day of treatment (Fig. 4G, K). On day 7, compared with the simple laser and HNP group, the RAWM coated group achieve the effect of killing S. aureus more thoroughly (Fig. 4G, L). In the groups without laser treatment, there was no significant difference in colony growth among different groups (Fig. 4G, K, L). Moreover, as the infection persisted, the mice in the nonlaser control group developed hyperosmolar syndrome caused by consistently exacerbated infection and died of severe dehydration; in the laser group, there was no death occurred in RAWM, HNP, and HNP@RAWM groups; the PBS group also died on the first day, but considering that PTT still had a certain effect on bacterial killing, the number of days for subsequent death to occur was longer than that in the non-laser group (Additional file 1: Fig. S12). Combined with wound healing condition and survival rate, HNP@RAWM + Laser group had the best curative effect among all groups.
To better prove that HNP can well alleviate the dilemma of current clinical application, we took the local pus from patients with DFIs for culture and propagated the bacteria on diabetic ulcer model mice (Fig. 5A).
According to the clinical examination (Fig. 5B), the patient was infected with Proteus vulgaris, 1 of the top 3 bacteria that are susceptible to infection in patients with DFIs. The laboratory report indicated that the patient was sensitive to various antibiotics, but the infection of the patient did not improve significantly after several days of anti-infection treatment (Fig. 5B). Due to the inferior peripheral circulation in patients with DFI, the effective therapeutic concentration of antibiotics at the site of inflammation is difficult to meet the bactericidal concentration requirements despite the sensitivity to antibiotics. In terms of therapeutic effects, RAWM had equally good effects on Proteus-infected ulcers, promoting wound healing and promoting collagen regeneration at the microscopic level (Fig. 5C-E). In the aspect of the bacteria collection, the blood plates after laser with HNP and HNP@RAWM treatments had few bacterial colonies (Fig. 5C), which was 1.9% in the HNP group and 1.4% in the HNP group on day 1, and 17.1% in the HNP group and 1.4% in the HNP group on day 7 (Fig. 5F). It suggested that RAWM could adhere well to bacteria, regardless of the type of bacteria infected, and allow HNP to better attach to bacteria, indicating excellent photothermal action to inhibit bacteria.
Toxicity and biosafety of HNP@RBCM-cRGD in Hepa 1-6 tumor-bearing mice and HNP@RAWM in DFI mice model
In addition to the good therapeutic performance of natural materials in vivo, their toxicity and biocompatibility are also important evaluation items for preclinical research. Therefore, the mice were sacrificed 24 h and 30 days after the injection of different materials in the tumor study, and 7 days post the treatment of HNP@ RAWM on the ulcers. We collected the vital organs including the heart, liver, spleen, lung, kidney, and brain for histological analyses, as well as the blood serum for biochemistry analyses. We discovered no short or longterm damage to the liver and kidney function on day 1 and day 30 in all groups (Fig. 6A). Moreover, no histological damages were captured in the vital organs (Fig. 6B, C; Additional file 1: Fig. S13).
Discussions
HNP has a natural PTT effect for tumor treatment and anti-bacteria, while the precise delivery of HNP to target tumor sites and enhancement of the bacteria-killing efficiency were yet to be improved [16,17,28]. Herein, we combined the controllable targeting properties of cell membranes and the PTT properties of HNPs to strengthen the application value of natural HNP in the field of tumors and DFIs.
Although traditional NPs have the incomparable advantages of high permeability, long retention effect (EPR effect), long plasma half-life, slow-release, and intelligent response, they are subject to uncertain chemical structures, complex formulations, and relatively difficult metabolism. Due to the high cost of quality control and the difficulty in the quantitative determination of toxicology and pharmacokinetics, it is rarely possible to achieve clinical translation [29]. The use of inorganic NPs in vivo has certain risks, as David Tai Leong et al. found that SiO 2 , TiO 2, and other inorganic NPs could eventually lead to vascular endothelial leakage by binding to vascular endothelial cadherin (VE-cadherin) on the surface of epithelial cells, and caused tumor metastasis [30]. So is the dilemma in organic NPs, only 2% of the NPs were deposited in the tumor site, leaving 98% of the NPs injected into the mice engulfed by mononuclear phagocytes in the liver and spleen, which posed a great risk of biological toxicity [31]. Coating NPs with cell membranes, such as platelet membranes, can well adjust biological toxicity by mimicking NPs with platelet properties and selectively targeting damaged human and rodent vasculatures in tumor sites [32,33]. S. aureus can evade antibiotics that are unable to pass through mammalian cell membranes, and it is reported that antibacterial NPs packaged in RAWM can better kill S. aureus internalized by macrophages, thereby relieving peritoneal infection [34]. It suggests that cell membrane-coated NPs can well reduce the capture of NPs by the endothelial system, enhancing the local concentration and biosafety of NPs.
Biomaterials from human sources can well reduce toxicity and improve biocompatibility compared with inorganic and organic NPs. HNP in our study is humansourced, readily available, and accompanied by excellent photothermal properties. The previous study has shown that HNP-induced tumor cell death was mainly due to the photothermal conversion effect produced by melanin, resulting in tumor cell apoptosis [16]. In this study, we used cell membranes to coat HNP for the first time. Both HNP and cell membranes could be taken from humans and were pure biological materials with strong biocompatibility. Meanwhile, it exerted the great photothermal effect of HNP, and combined the characteristics that the cell membranes could prolong the circulation time in vivo and enhance the targeting ability of local diseases, to achieve a therapeutic effect of "1 + 1 > 2". To mimic the application of the cell membranes-encapsulated HNP nano-system in the human body, we took the pus from a DFI patient and dropped it into the wound of diabetic ulcer mice to imitate the patient's condition for the first time, and verified the HNP@RAWM has good bactericidal activity and wound healing effects, which would have a bright future clinical application. Keratin, one of the main components of HNP, might be involved in the regulation of tumor microenvironment. Hair keratin protein-KRT81, can downregulate inflammatory cytokine interleukin-8 to inhibit tumor progression [35]. Combination immunotherapy for cancer treatment is increasingly showing its superiority [36]. On one hand, combination immunotherapy can reduce the non-specificity of tumor-targeted binding and thereby reduce the manslaughter of normal cells. On the other hand, the combination can inhibit the negative immune regulation between tumor cells and immune cells, and improve the cytotoxicity of immune cells to tumor cells, thereby exerting a synergistic effect and can enhance the tumoricidal effect of targeted drugs. The representative immunotherapy regiment PD-L1 inhibitor atezolizumab, combined with the anti-angiogenic drug bevacizumab, has achieved success in the first-line treatment of advanced HCC [37]. However, these treatment regimens are mostly focused on T-cell immunity, which leads to an embarrassing situation when tumor cells turn off their major histocompatibility class 1 (MHC-I) expressions. In this case, the NK cells become irreplaceable as they can recognize tumor cells and mount a rapid immune response without antibodies or MHC [38]. Wenfeng Lin et al. reported that PLGA-ICG-R848 after laser irradiation can stimulate the increased number of NK cells and generate an anti-tumor immune response via secretion of chemokines and cytokines, but HNP as a PTT material on the activation of NK cells has not yet been reported [39,40]. In terms of mechanism, we also explored the effect of HNP on NK cells under 808 nm laser irradiation. Our study reported for the first time that HNP can activate the tumoricidal effect of NK cells under the excitation of an 808 nm laser. NK cells kill tumors mainly through killing mediators, including perforin, NK cytotoxic factor, and TNF. IL-6 is a pro-inflammatory cytokine, and excessive IL-6 may reduce the production of perforin and granzyme, thereby mediating impaired NK cell function [41]. Literature has shown that IL-6 can inhibit the immune-killing ability of NK cells to tumors by activating the JAK/STAT3 pathway, thereby promoting tumor progression [42,43]. Although IL-12 is a proinflammatory factor, due to its pro-inflammatory and immunomodulatory abilities, it can induce tumors to change from "cold" to "hot" [44]. The IL-12 family is the "third party" of NK cell activation, and IL-12b can activate NK cells and enhance the effector functions of NK cells, including IFN-γ production [45]. Along with the delivery system of the cell membrane encapsulation, it can play a triple role in tumors combining tumor-specific targeting, photothermal killing, and immune activation.
As for DFIs, standard treatment to promote the healing and protect the affected limbs include improving the basic medical condition (debridement + anti-infection + improvement of underlying diseases), and new dressings with growth factors or cytokines are encouraged if patients respond badly after 2 weeks [46,47]. Traditional dressings (bandages, sterile gauze, and cotton pads, etc.,) facilitate exudate drainage well but high adhesion to tissues can cause secondary injuries [48]. Wet dressings (hydrogels, hydrocolloids, alginates, etc.,) need less frequent replacement but still have not solved the problem of low local antibiotic concentration due to poor acral microcirculation [49]. So far, a variety of nanomaterials with broad-spectrum antibacterial properties have been developed, such as nano-silver, nano-copper, and nano-zinc oxide [50]. Despite the good antibacterial activity, the delivery of metal ions to wounds and surrounding tissue still causes high toxicity to the tissues, and the antibacterial activity is greatly reduced with the release of ions, making it a one-time use [51,52]. Our study focused on the physical destruction of bacteria by HNP through high-temperature damage. Considering that the wounds of diabetics enter the stage of chronic inflammation without the normal healing process, the RAWM used in our study can effectively identify the site of inflammation, so as to carry the HNP to the bacterial surface more efficiently, achieving a better PTT curative effect. As bacteria density in diabetic wounds is a nonneglectable factor to determine the rate and quality of healing, clinical healing of wounds is often accompanied by the elimination of wound bacteria (< 5 logs per gram of tissues) [53]. Our study demonstrated that the RAWM can align with the PTT effect of HNP to eradicate the bacteria number of tissues, which holds great potential
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2022-11-18T06:18:06.146Z
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2022-11-16T00:00:00.000Z
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253579583
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Human Behavior-Inspired Linchpin-Directed Catalysis for Traceless Precision Labeling of Lysine in Native Proteins.
The complex social ecosystem regulates the spectrum of human behavior. However, it becomes relatively easier to understand if we disintegrate the contributing factors, such as locality and interacting partners. Interestingly, it draws remarkable similarity with the behavior of a residue placed in a social setup of functional groups in a protein. Can it inspire principles for creating a unique environment for the precision engineering of proteins? We demonstrate that localization-regulated interacting partner(s) could render precise and traceless single-site modification of structurally diverse native proteins. The method targets a combination of high-frequency Lys residues through an array of reversible and irreversible reactions. However, excellent simultaneous control over chemoselectivity, site selectivity, and modularity ensures that the user-friendly protocol renders acyl group installation, including post-translational modifications (PTMs), on a single Lys. Besides, it offers a chemically orthogonal handle for the installation of probes. Also, a purification protocol integration delivers analytically pure single-site tagged protein bioconjugates. The precise labeling of a surface Lys residue ensures that the structure and enzymatic activities remain conserved post-bioconjugation. For example, the precise modification of insulin does not affect its uptake and downstream signaling pathway. Further, the method enables the synthesis of homogeneous antibody-fluorophore and antibody-drug conjugates (AFC and ADC; K183 and K249 labeling). The trastuzumab-rhodamine B conjugate displays excellent serum stability along with antigen-specific cellular imaging. Further, the trastuzumab-emtansine conjugate offers highly specific antiproliferative activity toward HER-2 positive SKBR-3 breast cancer cells. This work validates that disintegrate theory can create a comprehensive platform to enrich the chemical toolbox to meet the technological demands at the chemistry, biology, and medicine interface.
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253597928
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Elevation of circulating TNF receptor 2 in cancer: A systematic meta-analysis for its potential as a diagnostic cancer biomarker
High Tumor Necrosis Factor Receptor 2 (TNFR2) expression is characteristic of diverse malignant cells during tumorigenesis. The protein is also expressed by many immunosuppressive cells during cancer development, allowing cancer immune escape. A growing body of evidence further suggests a correlation between the circulating form of this protein and cancer development. Here we conducted a systematic meta-analysis of cancer studies published up until 1st October 2022, in which the circulating soluble TNFR2 (sTNFR2) concentrations in patients with cancers were recorded and their association with cancer risk was assessed. Of the 14,615 identified articles, 44 studies provided data on the correlation between cancer risk and the level of circulating sTNFR2. The pooled means comparison showed a consistently significant increase in the levels of sTNFR2 in diverse cancers when compared to healthy controls. These included colorectal cancer, ovarian cancer, breast cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, lung cancer, hepatocarcinoma, and glioblastoma. In a random-effect meta-analysis, the cancer-specific odd ratios (OR) showed significant correlations between increased circulating sTNFR2 levels and the risk of colorectal cancer, non-Hodgkin’s lymphoma, and hepatocarcinoma at 1.59 (95% CI:1.20-2.11), 1.98 (95% CI:1.49-2.64) and 4.32 (95% CI:2.25-8.31) respectively. The overall result showed an association between circulating levels of sTNFR2 and the risk of developing cancer at 1.76 (95% CI:1.53-2.02). This meta-analysis supports sTNFR2 as a potential diagnostic biomarker for cancer, albeit with different predictive strengths for different cancer types. This is consistent with a potential key role for TNFR2 involvement in cancer development.
Introduction
Cancer remains one of the most lethal diseases and is currently the world's second most common cause of death (1). According to the World Health Organization, approximately 9.6 million deaths occur annually because of cancer (1). Currently, the high mortality rate from cancers is mostly due to late diagnosis as many cancers can be efficiently treated if diagnosed early. The existing cancer diagnostics methods and techniques present various limitations. While diagnostic imaging techniques such as digital mammography, ultrasonography, computed tomography, and magnetic resonance imaging are non-invasive, they lack absolute sensitivity and specificity for the detection of different cancer types (2). Furthermore, these imaging techniques require expensive specialized equipment and highly trained medical personnel, which limits patient access due to high costs (3). On the other hand, while biopsy staining is useful for definitive cancer diagnosis, its invasive nature makes it unattractive for most patients and it is less sensitive to early-stage cancers. Thus, diagnostic biomarkers from a minimally invasive liquid biopsy such as blood that could identify the presence of specific cancers with high precision and at their early stage, are highly desired.
Tumor necrosis factor (TNF) is a cytokine implicated in inflammation and cancer development (4)(5)(6). In the tumor microenvironment, TNF via its receptor TNFR1 and TNFR2 plays a dual role to suppress or promote cancer proliferation and metastasis (7,8). TNFR1 can be expressed by nearly all cells, while TNFR2 can be highly expressed by tumor cells (9)(10)(11). In malignant cells, TNFR2 promotes tumor cell proliferation and is increasingly being considered as an oncogene as it is overexpressed in more than 20 types of cancer, including multiple myeloma, human renal cell carcinoma, breast, oesophageal, myeloma, colon cancer, ovarian cancer, and cutaneous T-cell lymphomas, among others (9)(10)(11). The presence of TNFR2 at cancer sites has prompted research on utilizing TNFR2 as a target for therapeutic agents.
TNFR2 is also highly expressed in immune cells, which could be associated with tumorigenesis and tumor growth or conversely tumor controls (10, 12,13). This protein however has been shown to be broadly expressed in the repertoire of immunosuppressive cells present on tumors and tumor microenvironments promoting pro-tumor activity (9). They include regulatory T cells (Tregs) (14, 15), natural killer cells (NK cells) (16), myeloid-derived suppressor cells (MDSCs) (17), mesenchymal stem cells (MSCs) (18, 19), endothelial progenitor cells (EPCs) (20), neural stem cells (NSCs) (21) and cancer-associated fibroblasts (CAFs) (22). The immunosuppressive cells are activated by the TNF-TNFR2 axis as well as the TNFR2 alone without its ligand, as TNFR2 can autoassociate in the absence of TNF and promote active signaling (23). The active immunosuppressive cells could then promote cancer immune evasion by suppressing the immune response against cancer. TNFR2 overexpression and TNF-TNFR2 signaling on Tregs results in their proliferation into a subpopulation of highly suppressive phenotype, promoting enhanced immunosuppressive activities within tumor microenvironments (24) which in turn promotes tumor cell proliferation (25). As an example, the expression of TNFR2 by Tregs in peripheral blood is strongly correlated with cancer development of the lymphatic system (lymph nodes), distant metastases, and advanced lung cancer disease (26). In NK cells, the TNF-TNFR2 axis acts as a checkpoint molecule, reducing NK cells' tumoricidal activity (16). In MDSCs, TNFR2 boosts differentiation capacity and immunosuppressive activity of these immunosuppressive cells as well as promoting the activation of Tregs (27). TNFR2 promotes MDSC survival by inhibiting the apoptosis processes of the cells, which in turn contributes to tumorigenesis (12,28). Similarly, in MSCs, EPCs, and NPCs, the TNF-TNFR2 axis promotes immunosuppression within the tumor microenvironment and induction of active Tregs (18, 20, 21, 29). In addition, the TNF-TNFR2 signaling on CAFs enhances the synthesis of immunosuppressive interleukin (IL)-33, which increases tumor cell migration and invasion (22). On the contrary, TNFR2 is an important costimulatory molecule to enhance the proliferation and activation of both CD4 + and CD8 + conventional effector T cells (Teffs) necessary to eliminate the neoplastic cells (14, 30, 31).
The levels of circulating form of TNFR2 (sTNFR2) has been shown to increase in chronic inflammatory conditions such as obesity and Type 2 diabetes (DM2), diabetic kidney disease characterized by increased albuminuria, and juvenile chronic arthritis (32-35), as well as in infectious diseases including severe malaria (36). The circulating sTNFR2 comes from membrane shedding or as a spliced variant, following immune cell activation (37-41). This soluble receptor is secreted in vivo by Tregs and consistently counteract the action of TNF (42), which in turn suppresses the active immune response exerted via TNFR1, and meanwhile, the membrane-bound TNFR2 on Tregs can independently promote immunosuppressive behavior of Tregs (14, 15). In vitro, some pathogens stimulate the secretion of sTNFR2 (43, 44). In vivo, TNFR2-overexpressing cancer cells promote the accumulation of TNFR2 + Tregs in the draining lymph nodes and increase the levels of sTNFR2 in the circulation (45). These studies suggest that elevated sTNFR2 could be an indicator of cancer (10, 12,13). Therefore, the circulating sTNFR2 could potentially lend itself as a novel diagnostic biomarker to detect the presence of cancer. Thus, we conducted a systematic review and meta-analysis to test the utility of sTNFR2 as a diagnostic biomarker for cancer.
Study design
The study was designed to evaluate the utility of sTNFR2 in plasma or serum as a diagnostic biomarker for various cancers and a prognostic biomarker to predict cancer outcome, by performing a meta-analysis on published studies. The literature research was conducted by the authors following the guideline set by the statements provided by "Preferred Reporting Items for Systemic Reviews and Meta-Analysis" (PRISMA) (46).
Search strategy
The literature was searched systematically in Medline, Embase, and Scopus databases, from inception to 1 st October 2022, for studies investigating the associations between circulating sTNFR2 and cancer. The text word search included (TNFR2, TNFR2-p75, TNFR2p75, TNFRp75, TNFR-p75, sTNFR2 or CD120b) and (cancer, cancers, carcinoma, tumor, neoplasm, malignant, or malignancy). Duplicates were removed using EndNote20 software (Clarivate Analytics, Boston, USA), and this software was further used to select articles.
Inclusion and exclusion criteria
The selection of articles for studies was based on defined inclusion and exclusion criteria. Titles, abstracts, and full articles were first screened independently by three authors (AK, EC, MR). Case studies, conference papers, animal and in vitro studies were excluded. Secondary source articles such as meta-analyses and reviews that do not provide the original data of a study were excluded. No restriction to time or age was applied. Additional search by scanning the reference lists from other related articles was also performed. Relevant articles were then independently reviewed by the three authors (AK, EC, MR) and selected based on the content of the articles, which includes: 1. the study is on cancer, 2. the biomarker of interest is soluble TNFR2 in serum or plasma. The collected articles were sorted and recorded using the PRISMA flow diagram (46).
Quality assessment
The article's quality was assured by noting the author, year, abstract, and number of citations. Importantly, the study fitness was assessed by two authors (AK, EC) in discussion, using the updated Strengthening the Reporting of Observational studies in Epidemiology (STROBE) checklist for diagnostic studies (47).
Data collection and extraction
Three reviewers (AK, EC, and MR) recorded data from all studies that met inclusion criteria using a standardized data collection procedure. Data collected was arranged in a table for all the studies (Supplementary Table 1). Study title, author's name, name of journal, year, cancer type, country of study, the biological liquid used, population ethnicity, age, and gender were recorded. The number of patients and healthy controls participating in the study were recorded. Furthermore, the reported values related to sTNFR2 levels in serum or plasma were recorded. Additionally, the odds ratios presented in the studies were recorded or calculated based on the provided supporting data available in the studies.
Data analysis and statistics
The changes in the levels of sTNFR2 levels in various cancers were interrogated by determining the pooled mean values of sTNFR2 concentrations in serum/plasma from the controls and cancer patients. The pooled mean value is the mean of the weighted means from the selected studies, obtained by logtransforming the means as the sTNFR2 is expected to display log beta distribution and adjusting the means with their corresponding sample sizes (48). We also constructed the 95% confidence interval (CI) following log-transformed data. When median values were recorded, we estimated the mean values following Cochrane recommendations and the handbook (49) with the calculation described by Wan et al. (2014) (50). Then, we applied the Tukey test to assess the significant difference of sTNFR2 levels in cancer in comparison to controls. We then back-transformed the variables that have been log transformed for reporting purposes.
To evaluate the diagnostic utility of sTNFR2, we extracted odd ratio values from the selected studies, as this style of metaanalysis is frequently used to quantify disease occurrence in populations to answer concerns about disease risk (51). Since there is variability in study populations, especially with differs cancer types, the methodology of sample handling, as well as the methodology of sTNFR2 measurements, we utilized a randomeffects meta-analysis model to evaluate the sTNFR2 correlation with the risk of cancer in our selected studies (52). We then generated the forest plot using Review Manager 5.4, London, UK, that calculated the overall odds ratios for each cancer type, and all cancers combined. An odds ratio of more than one with a P-value of <0.05 indicates that there is a likelihood of cancer occuring and a potential for sTNFR2 to indicate the presence of cancer. The between-study invariance in our random-effect meta-analysis was calculated using t 2 , as the estimated standard deviation of underlying effects across included studies (49). The Chi 2 and I 2 tests measure the heterogeneity between studies. I 2 above 50% could represent substantial heterogeneity depending on the magnitude and direction of the effects and the P-value of the Chi 2 test (49). Publication bias was assessed based on the presence of data asymmetry on the Funnel plot (49).
Study selection and characterization
A total of 18,502 relevant articles were identified from Medline, Embase and Scopus searches. Of the 18,502 articles, 3,887 were duplicates and removed, and 14,615 records were selected for screening. After excluding 2,787 animal studies, 4,089 in vitro studies, 2,642 secondary source articles, and 1,173 conference articles, 3,942 full-length articles were assessed for their eligibility. After applying the inclusion and exclusion criteria, 44 studies were included in this meta-analysis. PRISMA Flow chart (Figure 1) shows the number of studies searched and selected. The full list of the selected studies is listed in Supplementary Table 1.
STROBE checklists
All the articles were assessed for their fitness following the STROBE checklist criteria. This includes meeting the criteria of participant selection, an introduction describing the background of the study, study design, the methods of sample handling, the results, and the outcome of the study. Each criterion scores one, and all the studies selected here were scored six and thus regarded as good quality studies for the purpose of this metaanalysis (Supplementary Table 1).
sTNFR2 levels in the circulation of cancer patients
We investigated the evidence for differences in sTNFR2 levels in serum/plasma between healthy subjects and cancer patients, to explore its potential as a diagnostic biomarker. Here, we analyzed the cancer types that are observed by at least two studies (Table 1). In total, we extracted data from 28 studies, encompassing 7520 healthy and 5981 cancer participants. The pooled mean values of the healthy controls did not differ significantly across different cancers. On the contrary, we observed that the pooled mean of sTNFR2 in patients with colorectal cancer at 2.69 ng/mL (95% CI:2.45-2.95) was significantly higher (P-value of the difference <0.001) than that of the healthy controls at 2.51 ng/mL (95% CI:2.36-2.68) in the same studies. Similarly, the pooled mean of sTNFR2 in patients with ovarian cancer at 3.23 ng/mL (95% CI:2.28-4.59) was also significantly higher than that of the healthy controls (Pvalue of the difference <0.05) at 2.27 ng/mL (95% CI:2.15-2.40) in the same studies ( Table 1). The significant differences in the Selection of studies following PRISMA flowchart. Medline, Embase, and Scopus database searches resulted in the identification of 18,502 relevant articles. Following the exclusion and inclusion criteria, 44 articles were selected for the meta-analysis. levels of sTNFR2 in the serum/plasma between controls and cancer participants were also observed in several other cancers including non-Hodgkin's lymphoma, breast cancer, Hodgkin's lymphoma, lung cancer, hepatocarcinoma, and glioblastoma ( Figure 2, Table 1). This result suggests the potential involvement of circulating sTNFR2 concentrations in cancer and could prove its utility as a diagnostic biomarker for cancer.
sTNFR2 association with the risk of developing cancer
Here, we extracted data from 34 eligible articles with sufficient data on the odd ratios (OR) for sTNFR2 and the risk of developing cancer (Figure 3). We divided the studies based on the cancer types that are being investigated and performed the random effect The pooled weighted means +/-95% CI of sTNFR2 levels in serum/plasma from patients with the indicated cancers. The Tukey test was used to assess the significant difference of sTNFR2 levels, with *, **, and **** indicate P-values of <0.05, <0.01, <0.0001. (Figure 4). These studies thus showed that sTNFR2 potentially increases the risk of some cancers to various extent, significantly in colorectal cancer, non-Hodgkin's lymphoma, and hepatocarcinoma. This thus indicate sTNFR2 potential to be used as a circulating diagnostic biomarker for cancer. We further tested our selected studies comprising various cancer types using a funnel plot, as this plot could indicate study FIGURE 3 The forest plot showing overall OR of sTNFR2 and its correlation with cancer risk.
heterogeneity and reporting bias. The funnel plot ( Figure 4) shows a symmetrical feature which indicates the absence of reporting bias and that the random-effect model assumption used in this meta-analysis fits with the heterogeneity present in the selected studies (99).
Discussion
Overall, most cancers still present with a high mortality rate, due to late diagnosis. As such, there is a pressing need for the identification of reliable biomarkers facilitating early detection. It has been frequently proposed that inflammation, orchestrated by various cytokines may promote cancer formation and further its development (6,48). Cancer and immune cells may secrete immune proteins to control inflammation, such as sTNFR2, that may also mark cancer formation. Thus, in this systematic review, we investigated the correlation between increased circulating sTNFR2 levels with the risk of developing cancer. Finding the utility of sTNFR2 as a diagnostic marker could be useful to improve the effectiveness of current cancer diagnosis and provide a convenient detection approach for patients, especially with various technologies have been developed recently to facilitate circulating cytokine detection (100).
Based on the calculated pooled mean values, circulating sTNFR2 levels were found to be consistently reported as significantly higher in various cancers in comparison to the healthy controls ( Figure 5), suggesting sTNFR2 may be involved in cancer development. Using a random-effect OR meta-analysis, we observed significant correlations between sTNFR2 and several cancers, including colorectal cancers, non-Hodgkin's lymphoma, and hepatocarcinoma. This indicates the potential of sTNFR2 as a diagnostic biomarker. Indeed, sTNFR2 levels are correlated with lung cancer development even 6 years before diagnosis (84). However, in other cancers, including ovarian, breast, and glioblastoma, the correlation was not significant, suggesting that sTNFR2 levels may not be sufficient as an independent diagnostic biomarker, especially for these cancers. It has been previously suggested that the circulating inflammatory biomarkers such as sTNFR2 which are highly correlated with cancer risks, could be combined with the circulating cancer-specific biomarkers that otherwise would have low sensitivity and specificity to indicate the presence of cancer (100). Furthermore, in agreement with our findings, a previous meta-analysis of prospective studies also found no significant association (of diagnostic value) between circulating levels of sTNFR2 and the risk of ovarian cancer (101). However, some studies show that high expression of TNFR2 at cancer sites is correlated with cancer size, metastasis and progression in epithelial ovarian cancer (90, 102), non-small lung carcinoma (103), anal carcinoma (104), and esophageal carcinoma (105,106).
The increased levels of circulating sTNFR2 do not only correlate with cancer risk but also cancer outcomes such as overall survival and progression-free survival, as has been shown by several studies of various cancers (56, 65-67, 78, 88, 107-113). In patients with ovarian cancer for example high levels of TNFR2 + Tregs have been associated with poor OS, while ovarian tissue with strong expression of TNFR2 was associated with The funnel plot showing the OR of individuals studies against the standard error of the OR to detect bias and heterogeneity of between study.
longer PFS (14, 114). Additionally, a high pre-diagnosis plasma sTNFR2 level corelate with overall mortality in colorectal cancer patients (110). Moreover, several studies show that the effectiveness of anti-cancer drugs at reducing tumor size and improving survival is correlated with reduced levels of circulating sTNFR2 (115)(116)(117)(118)(119). This observation may thus extend the use of sTNFR2 not only as a minimally-invasive diagnostic biomarker to predict cancer outcome, but also to monitor therapy effectiveness during treatment (120).
In summary, our meta-analysis study confirms the correlation between increased circulating sTNFR2 levels and increased risk of cancers, albeit the extent of this association varies between different cancers. This indicates circulating sTNFR2 may have utility, perhaps combined with other blood accessible biomarkers, to aid in the diagnosis of cancer. We believe that the easy access to this biomarker through liquid biopsies makes it an ideal candidate to be used alone, or in combination with other markers, as a minimallyinvasive cancer screening method, potentially accelerating the implementation of point-of-care devices (100) for cancer diagnosis in clinical settings outside of central laboratories.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The summary schema of the elevated sTNFR2 levels in various cancers, and its potential as a biomarker for cancer detection. Figure was created using Microsoft power point.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The
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The Role of Phorbol Diesters in Mediating Human Placental Aromatase Cytochrome P450 Activity
Due to the aromatase enzyme’s involvement in estrogen biosynthesis, aromatase inhibitors have emerged as the preferred treatment for postmenopausal women with ER+ breast cancer. Using computational chemistry tools, we investigate how the human placental aromatase cytochrome P450 interacts with various phorbols with distinct chains at C-12, C-13, and C-20, as well as the well-known aromatase inhibitors anastrozole, exemestane, and letrozole. To identify phorbol-aromatase interactions, we performed a protein–ligand docking using the structures of our ligands and proteins using the Flare software (version 2.0, Cresset Software, Litlington, UK). These preliminary findings show that the phorbols considered (P-12,13-diAcPh, P-12,13-diiBu, P-12AcPh-13iBu, P-12Ang-13iBu, P-20Ac-12AcPh-13iBu and P-20Ac-12Ang-13iBu) had the highest binding energies in comparison with the commercially available aromatase inhibitors (anastrozole, letrozole, exemestane) used in this study. A subset of the previously described binding residues of testosterone (TST), the endogenous ligand, were also found to be responsible for the phorbol diesters’ binding to the aromatase enzyme, as demonstrated by the findings. This further suggests that the phorbol diesters can bind efficiently to CYP19A1 and may be able to alter its activity because they had higher binding energies than the commercially available drugs.
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Neoadjuvant nivolumab with chemotherapy: a miraculous new regime in treatment of resectable lung cancers
Madam, lung cancer is one of the commonly diagnosed causes of cancer mortality and morbidity worldwide, accounting for approximately 1.76 million deaths annually.1 In the past overall survival in patients with non-small cell lung cancer (NSCLC) remained uncertain. Many treatment regimens were under trial2 until, a breakthrough study incorporating neoadjuvant nivolumab with chemotherapy showed tremendous success in phase 3 CheckMate 816 trail3. Moreover, the FDA has recently approved this therapy for early-stage NSCLC.4 Nivolumab is an anti–programmed death 1 (PD-1) human antibody, which restores antitumor T cells activity. Whereas platinum-doublet chemotherapy improves antitumor immunity.3
In this trail, approximately 350 patients were randomly assigned in an equal ratio to receive nivolumab (360 mg) plus chemotherapy or chemotherapy alone for consecutive 3 weeks for three cycles before surgery. Two primary end points were established, event-free survival and complete pathological response. Median event-free survival was 31.6 months with nivolumab plus chemotherapy compared to 20.8 months with chemotherapy alone (hazard ratio for disease progression, recurrence, or death, 0.63; P=0.005). The pathological complete response was 24.0% in the nivolumab plus chemotherapy group and 2.2% in chemotherapy alone group (odds ratio, 13.94; P<0.001). Also, no surgery-related adverse events were observed with the addition of nivolumab to neoadjuvant chemotherapy.3
It concludes that nivolumab with chemotherapy showed promising results than chemotherapy alone in patients with resectable NSCLC. However, stage IIIA patients were majorly benefited than stage IB or II patients.3 Therefore, it is imperative that more clinical trials must be conducted to obtain maximum benefit at all possible stages and a proper follow up for data is needed to draw better conclusions.
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Unknown‑primary neuroendocrine neoplasms diagnosed by short‑acting somatostatin test: Case series in one institution
. Neuroendocrine neoplasms (NENs) are a rare heterogeneous group of neoplasms that arise from neuro‑ endocrine cells. Unknown‑primary NENs (UP‑NENs) are particularly challenging to diagnose and treat. Techniques such as immunohistochemical stains, functional imaging studies, and molecular cancer classifier assays may help clinicians identify the origin of a tumor. However, numerous medical facilities lack the necessary medical equipment, such as func‑ tional imaging scanning, to provide patients with a complete primary tumor survey. Even these tests are not enough to determine the original tumor in some cases. The present case series described the diagnosis and treatment outcomes of patients with UP‑NEN in a single institution. The medical records of four patients treated between November 2012 and January 2022 were retrospectively reviewed and clinical symptoms, diagnostic methods, image findings and treatment modalities were considered. All patients were diagnosed having functional UP‑NENs by using a short‑acting soma‑ tostatin test. These patients were treated with long‑acting release somatostatin analogs along with a positive result. Short‑acting somatostatin is an alternatively simple method to determine if a patient has UP‑NENs that are functional or expresses somatostatin receptors in the absence of imaging scanning.
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Combined surgical treatment of oncological patient with concomitant acute coronary syndrome with tumor lesions of the gastric stump in a multidisciplinary clinic
Patients with various forms of coronary artery disease often suffer from other comorbid conditions that affect the quality of life and make it difficult to select the optimal therapy. The most acute problem of comorbidity manifests itself in the need for surgical treatment, especially extensive surgical interventions. The key to successful surgical treatment in this category of patients is the work of a multidisciplinary team of specialists deciding on the operability, stages of surgical interventions, choice of methods revascularization and features of patient management in the postoperative period. The article presents a case of successful surgical treatment of the patient with unstable angina and gastric stump cancer, demonstrating the well-coordinated work of a multidisciplinary team of specialists.
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Broad-spectrum antibiotics associated gut microbiome disturbance impairs T cell immunity and promotes lung cancer metastasis: a retrospective study
Background Gut microbiome has been linked to a regulatory role in cancer progression. However, whether broad-spectrum antibiotics (ATB) associated gut microbiome dysbiosis contributes to an impaired T cell immune function, and ultimately promotes lung cancer metastasis is not well known. Methods In this study, a retrospective analysis was performed in a cohort of 263 patients initially diagnosed with non-small cell lung cancer (NSCLC) patients, including the ATB group (patients with broad-spectrum antibiotics treatment) (n = 124), and non-ATB group (n = 139) as control. ATB patients were prescribed ATB for over 5 days within 30 days prior to the collection of blood and fecal specimens and followed surgical treatment or first-line therapy. T cell immune function and metastasis-free survival (MFS) were evaluated between the two groups. Gut microbiota was evaluated by 16S rDNA sequencing. The predictive value of T cell immunity for MFS was evaluated by ROC analysis and Cox regression analysis. Results Our results suggest that broad-spectrum antibiotics (ATB) impair T cell immune function in patients with either early-stage or advanced NSCLC, which likely contribute to the promotion of lung cancer metastasis. Results of the survival analysis show that metastasis-free survival (MFS) is significantly shorter in the ATB patients than that in the non-ATB patients with stage III NSCLC. The 16S rDNA sequencing shows that ATB administration contributes to a significant dysbiosis of the composition and diversity of gut microbiota. Moreover, ROC analysis results of CD4 (AUC 0.642, p = 0.011), CD8 (AUC was 0.729, p < 0.001), CD16 + 56 + (AUC 0.643, p = 0.003), and the combination of CD4, CD8 and CD16 + 56+ (AUC 0.810, p < 0.001), or Cox regression analysis results of CD4 (HR 0.206, p < 0.001), CD8 (HR 0.555, p = 0.009), which is likely regulated by ATB administration, have significantly predictive values for MFS. Conclusion These results provide evidence of gut microbiome disturbance due to ATB administration is involved in the regulation of T cell immunity, and their predictive value for the tumor metastasis in lung cancer patients. Thus, gut microbiota may serve as a therapeutic target for lung cancer. Consequently, caution should be exercised before the long-term administration of broad-spectrum antibiotics in cancer patients.
Background
Human microecosystem is associated with the regulation of immune system. Studies have explored how lung microbiota influences cancer outcome [1][2][3][4][5], Abnormal gut microbiome composition may attribute to cancer progression [6][7][8][9][10][11]. For example, gut bacteria are involved in the regulation of tumor treatment responses [12][13][14][15]. Bifidobacterium administration contributes to the enhancement of anti-cancer immunity, and blocks the melanoma growth [13]. Moreover, a recent study reported that anti-cancer role of gut microbiota, such as the Clostridiales members, are associated with the activation of tumoral CD8+ T cells [16]. These results, either in preclinical murine models or human studies, have highlighted the importance of gut microbiota in the regulation of anti-cancer therapeutics, and thus help to develop better therapeutic strategies by modulating gut microbiota.
Cancer patients receive broad-spectrum antibiotics (ATB) generally for common indications (such as pneumonia or urinary tract infection) [17,18], or to exclude infectious diseases before the final diagnosis of cancer. However, studies have suggested that ATB represented a predictor of resistance to chemotherapy [6]. Antibiotics also inhibit the benefits of immunotherapy in patients with advanced cancer [14,19], In addition, ATB can alter the composition of gut microbiota [20][21][22]. Thus, maintaining a healthy gut microbiome may help patients combat cancer.
Accumulating evidence has indicated that gut microbiota is associated with cancer development. Probiotics can remodel the tumor microenvironment, including reducing inflammatory T helper cells and the differentiation of regulatory T cells (Treg cells) [23], or promoting the maturation of dendritic cells [24], and subsequently enhancing the response of antigen-specific cytotoxic T lymphocyte (CTL) and cancer immune surveillance. For example, Lactobacillus bacteria can improve the treatment response of cisplatin in murine cancer model [25]. Thus, it is possible to improve the therapeutic response by modulating the gut microbiome [26][27][28].
As a new hallmark of cancer, microbiota has caught a great attention in recent years [13]. In this regard, gut microbiota may have been considered as a potential biomarker for cancer diagnosis, treatment, and prognosis. However, it is largely unknown whether gut microbiota disturbance due to ATB contributes to an impaired T cell immune function, ultimately promoting lung cancer metastasis. In this study, we retrospectively analyzed the clinical data in a cohort of NSCLC patients with or without receiving ATB, and evaluated the effect of ATB on gut microbiota. We also performed T cell immune function, and ROC analysis and Cox regression analysis for the prediction of MFS.
Patients and clinical data collection
Data of patients diagnosed with lung cancer from Tongji Hospital of Tongji University in Shanghai China, from January 2016 to November 2021, were collected in this retrospective study. Individuals with any tumors other than lung cancer were excluded from this study. All patients were diagnosed by cytological and/or histological examination according to the WHO classification. The laboratory data of patients were collected. Total 332 lung cancer patients were included in this study, patients with Hematological diseases or with missing data were excluded. Finally, a cohort of 303 patients with initially diagnosed of lung cancer was included in the study, including 263 NSCLC patients (Fig. 1). Demographics and clinical characteristics, including age, gender, pathology, and clinical stage, were collected. Patient characteristics, such as the smoking status of patients included in this study were obtained. The smoking history of patients included in this study was obtained via in-patient history recording or interview using a questionnaire. All procedures performed in this study involving human participants were following the Declaration of Helsinki (as revised in 2013).
Among the cohort of 303 patients, 145 patients, including 124 NSCLC patients, were prescribed an intravenous infusion of broad-spectrum antibiotics (ATB). ATB administration was performed because of the diagnostic treatment to exclude infectious diseases in the suspected infection patients, or the infection patients due to common indications (combined with pneumonitis). ATB group of patients received ATB therapy for over 5 days within 30 days prior to the collection of blood and fecal specimens on admission and followed surgical treatment or first-line therapy. The other 158 lung cancer patients, including 139 NSCLC patients who did not receive antibiotics treatment as control. The blood and fecal specimens of all the patients were collected for the evaluation of T immune cells and gut microbiome prior to the microbiota may serve as a therapeutic target for lung cancer. Consequently, caution should be exercised before the long-term administration of broad-spectrum antibiotics in cancer patients.
Keywords: Gut microbiota, Lung cancer, Metastasis, T cell immunity, Broad-spectrum antibiotics (ATB) surgical treatment or first-line therapy. Kaplan-Meier estimates for metastasis-free survival (MFS) of patients with stage III lung cancer were performed. All the stage III NSCLC patients were initially diagnosed of lung cancer. The MFS evaluation of stage III patients was during the period from the initial treatment until the development of metastasis. Moreover, fecal specimens of 22 out of these 303 patients were collected for 16S rDNA sequencing.
Clinical data of patients were collected, including age, gender, ECOG value, tumor stage, pathological type of tumor, and smoking status et al. Data of laboratory tests including white blood cell count, neutrophil count, lymphocyte count, C-reactive protein, platelet count, D-dimer, and T cell series et al. were collected. This study was approved by the Ethics Committee of Tongji Hospital, Tongji University (No. K-KYSB-2020-189). Informed consent was signed by the participants or their authorized family members.
16S rDNA sequencing
DNA extraction and PCR amplification as described in our previous study [29]: Bacterial DNA was extracted using the E.Z.N.A. ® Soil DNA Kit (Omega Bio-Tek, Norcross, U.S.) from mouse feces specimens. We amplified the V4-V5 region of the bacteria 16S ribosomal RNA gene by PCR, and using primers 515F 5′-barcode-GTG CCA GCMGCC GCG G)-3′ and 907R 5′-CCG TCA ATTC-MTTT RAG TTT-3′. The PCR amplification conditions were:95 °C for 2 min, followed by 25 cycles at 95 °C for 30s, 55 °C for 30s, and 72 °C for 30s, and a final extension at 72 °C for 5 min. PCR reactions were performed as described previously [29]. Amplicons were extracted from 2% agarose gels and purified according to the manufacturer's instructions. Library Construction and Sequencing: The purified PCR products were quantified by Qubit ® 3.0 (Life Invitrogen). We used the pooled DNA product to construct the Illumina pair-end library by following the Illumina's genomic DNA library preparation procedure. Then this constructed amplicon library was paired-end sequenced (2 × 250) by an Illumina HiSeq platform (Shanghai BIOZERON Co., Ltd) as described previously [29], according to the standard protocols.
Statistical methods
According to the same type of study [30], the test efficiency is 0.8, the sample size included in this study meets the statistical requirements. Descriptive analyses were performed with either means ± standard deviation (continuous variables) to describe the patient's characteristics. Continuous variables were compared by rank-sum test and T-test. The receiver operating characteristic (ROC) curve was calculated from the logistic regression model. The area under the curve (AUC) was used to evaluate the strength of prediction. Using the ROC curve to analyze the levels of CD4+ T cells, CD8+ T cells, CD16 + 56+ T cells, and D-Dimer to predict the best truncation value of MFS in patients with stage III NSCLC [determined by Youden index, Yordan index = sensitivity + specificity-1, the best truncation value is taken at the maximum of Yoden index]. All statistical analyses were performed using SPSS (version 23.0). A two-sided p-value < 0.05 was considered as statistically significant.
Baseline characteristics of lung cancer patients
The clinical characteristics of 303 lung cancer patients enrolled in this study were presented in Table 1. Compared with 158 lung cancer patients without broadspectrum antibiotics (ATB) treatment, 145 lung cancer patients were prescribed ATB. Patients with NSCLC were treated by the standard lung cancer therapy scheme.
In the ATB patients, the mean age was 71.31 years, and 71.72% of the patients were male, 124 out of 145 ATB patients were NSCLC, and there was 23.44% for stage I-II and 76.55% for stage III-IV. In the non-ATB patients, the mean age was 69.35 years, and 75.32% of the patients were male, 145 out of 158 non-ATB patients were NSCLC, and there was 22.51% for stage I-II and 77.85% for stage III-IV. (Table 1).
Antibiotics administration associated with enhanced cancer metastasis
To determine the impact of ATB on patients with advanced NSCLC, we performed the analysis for a cohort of 143 patients with stage III NSCLC out of the above 303 lung cancer patients. Among them, 47 patients have prescribed an intravenous infusion of ATB (ATB group, n = 47), and the other 96 patients did not receive antibiotics treatment (non-ATB group, n = 96). The demographic and clinical characteristics of 143 lung cancer patients with stage III NSCLC are present in Table 2. After the initial diagnosis of lung cancer, the patients received standard anti-cancer therapy.
In this study, it was evident that ATB promoted lung cancer metastasis. Metastasis-free survival (MFS) was significantly shorter in the ATB group than that in the non-ATB group. (Fig. 2A). The influences of ATB on metastasis were further evaluated according to the pathological types (adenocarcinoma or squamous carcinoma), and the results showed that ATB administration significantly promotes tumor metastasis in either adenocarcinoma or squamous cell carcinoma of lung cancer ( Fig. 2B-C).
Evaluation of gut microbiota by 16S rDNA sequencing
To evaluate the taxonomic composition and microbial diversity of gut microbiome between the ATB and non-ATB lung cancer patients, which might influence tumor metastasis, alpha and beta diversity were analyzed. The results of alpha diversity (Chao and Shannon index), which reflect the species richness and diversity, were significantly higher in the non-ATB than that in the ATB group (Fig. 3A). To compare the composition of the microbial community between the two groups, we used beta diversity to generate the weighted UniFrac principal coordinates analysis (PCoA) and showed the clustering between non-ATB and the ATB patients, as shown in Fig. 3B.
To identify the specific microbial communities associated with ATB treatment, we analyzed the composition of the gut microbiota by using LEfSe analysis. A total of 37 discriminative taxa at all taxonomic levels from phylum to genus were identified (LDA > 3, p < 0.05). At the phylum level, the abundance of Bifidobacteriaceae, Actinobacteria, and Coriobacteriaceae were enriched in the non-ATB patients, whereas.
Gammaproteobacteria,
Enterobacteriaceae, and Corynebacteriales was enriched in the ATB group ( Fig. 3C-D). Moreover, as shown in the Venn diagram, 311 and 372 OTUs were detected in the ATB and non-ATB (control) groups, respectively, with 214 OTUs concurrent in the two groups (Fig. 4A). Bar plots of the class taxonomic levels in the two groups were shown in Fig. 4B. At the genus level, the abundance of Bifidobacterium, Faecalibacterium, and Agathobacter were significantly decreased in the ATB group, compared with the non-ATB patients (Fig. 4C). The 16S rDNA sequencing data have been deposited to the NCBI Sequence Read Archive (SRA) database (Accession Number: SRP226777).
Effects of broad-spectrum antibiotics on T cell immune function
The association of T cell subsets and the use of antibiotics is shown in Table 3. We firstly evaluated all the 303 lung cancer patients in this study, the results showed that CD3, CD4, CD8, and CD16 + 56+ T cells were significantly decreased in the ATB group (n = 145) than that in The composition and diversity of the gut microbiota of fecal specimens from stage III lung cancer patients treated with or without ATB. Alpha diversity (Chao, Shannon index) between the ATB and control (A). Principal coordinate analysis (PCoA) using weighted-UniFrad of beta diversity (B). Taxonomic Cladogram from LEfSe, depicting taxonomic association between microbiome communities from the two groups (C). LDA score computed from features differentially abundant between the two groups (D) the non-ATB group (n = 158) (p < 0.01). In addition, Lymphocyte ratio (L%), NLR, and D-dimer were also significantly altered between the two groups. However, there was no significant difference in CD19, C3, IgG, IgA, IgM, and C4 between the two groups.
Next, according to the 263 NSCLC patients out of the above 303 patients, including ATB patients (n = 124) and non-ATB patients (n = 139). Our result showed that CD4, CD8, and CD16 + 56+ T cells, and L% were significantly decreased in the ATB group than that in the non-ATB group (p < 0.05) ( Table 4). In the early stage of NSCLC patients, CD4, and CD8 T cells were significantly lower in the ATB group (n = 29) than that in the non-ATB group (n = 35) (p < 0.05) ( Table 5). In the advanced stage of NSCLC patients, CD3, CD4, CD8, and CD16 + 56+ T cells were significantly decreased, while D-Dimer was significantly increased in the ATB group (n = 95) than that in the non-ATB group (n = 104) (p < 0.05) ( Table 6). In ADC patients, CD3, CD4, CD8, and CD16 + 56+ T cells were significantly decreased, while D-Dimer was significantly elevated in the ATB group(n = 86) than in the non-ATB group (n = 94) (p < 0.05) ( Table 7). In SCC patients, CD3, CD4, CD8, and CD16 + 56+ T cells were significantly decreased, while D-Dimer was significantly elevated in the ATB group (n = 38) than in the non-ATB group (n = 45) (p < 0.05) ( Table 8).
Univariate and multivariate cox regression analysis of the risk factors on MFS in patients with stage III NSCLC
In this study, the Univariate analysis showed that age, ATB administration, CD4+, CD8+ and CD16 + 56+ T cell levels, but not sex, smoking status, tumor pathological type, or D-Dimer, are associated with MFS in patients with stage III NSCLC (Table 10). The risk factors (p < 0.05) in the univariate analysis were included in the subsequently multivariate Cox regression analysis. The Cox regression analysis showed that only CD4 T cells or CD8 T cells is significantly associated with MFS in the patients with stage III NSCLC (Table 11). These results suggest that CD4+ or CD8+, but not ATB administration itself, are the independent risk factors for MFS in patients with stage III NSCLC. Thus, the disturbance of gut microbiome due to ATB administration, but not antibiotic application itself, may be directly involved in the regulation of T cell immunity, and ultimately influence the metastasis-free survival. However, further studies are needed.
Effect of treatment line and typology on T cell immunity in patients with stage III NSCLC
In this study, we further evaluated CD4 T cells and CD8 T cells in patients who received first-line therapy or subsequent lines therapy, between the non-ATB and ATB groups. CD4 (p = 0.004) and CD8 T cells (p = 0.003) in patients who received first-line therapy, and CD4 (p = 0.035) and CD8 T cells (p = 0.181) in patients who received subsequent lines therapy were found between the non-ATB and ATB groups. We also evaluated CD4 T cells and CD8 T cells in patients who only received chemotherapy or patients who received other treatments (including Immunotherapy, targeted therapy or radiotherapy, which were used individually or in combination), between the non-ATB and ATB groups. CD4 (p = 0.002) and CD8 T cells (p = 0.171) in patients who only received chemotherapy therapy, and CD4 (p = 0.033) and CD8 T cells (p = 0.376) in patients who received other treatments were found between the non-ATB and ATB groups. (Table 12).
Influence of infection on the baseline immune function indexes
In order to determine and exclude the influence of infection on the baseline immune function indexes, 145 patients with ATB administration were divided into the non-infection group (n = 70) and the infection group (n = 75). Non-infection patients were treated with antibiotics only for their diagnostic needs, infection patients were prescribed antibiotics because of complications with pulmonary infection. Our results showed that there was no significant difference in the levels of CD3+, CD4+, CD8+, CD4/CD8, CD16 + 56+, CD19, IgM, and D-dimer between the two groups. However, there was a significant difference in L%, NLR, and CRP between the two groups. The results suggested that infection complications in the lung cancer patients enrolled in this study may affect the baseline L%, NLR, and CRP, but had no significant effects on T cell immunity (Table 13). So, this result provided the probability that broad-spectrum antibiotics associated with gut microbiome disturbance, but not infection itself may contribute to impaired T cell immunity.
Discussion
Recent studies [8,11,14,[31][32][33] have highlighted the key role of gut microbiota in mediating tumor responses to chemotherapies or immunotherapies. Gui et al. observed that mouse models of lung cancer treated with cisplatin and antibiotics had larger tumors and lower survival rates than those treated with cisplatin alone [25]. In contrast, mice given cisplatin in combination with Lactobacillus responded better to the treatment, which appears to be related to the enhancement of T-cell immunity mediated by commensal microbiota [34]. Overuse of antibiotics may alter the composition of the gut microbiota and have harmful effects on the host. Accumulating evidence has demonstrated that specific microorganisms or microbial disorders promote the progression of hepatic, biliary, and pancreatic tumors by damaging DNA, activating oncogenic signaling pathways, or producing tumor-promoting metabolites [34]. Studies [35,36] also have shown that the integrity of gut microbiota or Probiotics such as Bifidobacterium is favorable for anti-cancer. Our results have demonstrated that gut microbiota regulates tumor metastasis via non-coding RNA networks [29]. However, whether gut microbiota dysbiosis is involved in the regulation of cancer metastasis in clinical lung cancer patients remains largely unknown. The purpose of this article was to determine whether gut microbiota dysbiosis due to the administration of ATB impairs T cell immune function and ultimately promotes metastasis in lung patients. Our retrospective analysis showed a significantly shorter MFS in the ATB group compared to the non-ATB group. The influences of ATB were further evaluated according to pathological types such as adenocarcinoma or squamous carcinoma, and these analyses suggest that ATB significantly promotes tumor metastasis in both adenocarcinoma and squamous cell carcinoma of lung cancer. The 16S rDNA sequencing analysis revealed that Firmicutes abundance is significantly decreased along with increased Proteobacteria and decreased Actinomycetes in the ATB group compared to the non-ATB group. Thus, ATB administration may damage the integrity of gut microbiota including reduction of the probiotics, such as Bifidobacterium and Lactobacillus, which belong to the Actinomycetes or Firmicutes. These changes in turn may promote cancer metastasis.
We found that compared with the non-ATB group, CD3, CD4, CD8, and CD16/56 T cells in the ATB group were significantly decreased. The result of the ROC curve showed CD4, CD8, and CD16/56 have predictive values for MFS, but not D-Dimer, or IgM. These results suggest that long-term broad-spectrum antibiotic administration impairs the clinical benefits in lung cancer patients, either in early staged or advanced lung cancer, and the enhanced metastasis may be attributed to gut microbiome dysbiosis. Therefore, emerging strategies for microbiome control, such as the cautious use of long-term broad-spectrum antibiotics in cancer patients or the consideration of interventions for gut microbiome disorders [37], such as probiotics [38] during chemotherapy or immunotherapy might need to be considered.
In this study, a further stratification of treatment line and typology was performed. Between the non-ATB and ATB groups, our results suggest that there is a significant difference of CD4 T cells in patients who received either first-line therapy or subsequent lines therapy, while CD8 T cells was found to be significant different only in the patients with first-line therapy. Furthermore, CD4 cells but not CD8 T cells were found to be significant different in patients who received either chemotherapy therapy or other treatments. However, due to the limited sample size, for further evaluation and stratification of the treatment line and typology, more studies are needed.
Given that the performance of gut microbiota in cancer has surprised us, it is maybe the prime time to overcome the upcoming challenges in the cancer therapeutic field through more high-quality research. In order to translate the presented results into future clinical possibilities, more samples are needed for subgroup analysis. In addition to lung adenocarcinoma, more pathological types of lung cancer can be analyzed. It is also important to evaluate whether categories of antibiotics or combinations have different effect on cancer. Finally, our study hopefully can raise awareness of careful administration of antibiotics, which is currently a major problem in medicine, not only in associated oncology.
Conclusions
This study demonstrates a strong interaction between gut microbiota and cancer metastasis, and suggests a potential mechanism linking microbial dysbiosis to cancer progression. Thus, a gut microecological disorder caused by broad-spectrum antibiotics may lead to the imbalance of the human immune system, impair T cell immune function, and cause immune tolerance or immune escape, ultimately promoting cancer metastasis. Therefore, our data suggests the previously unrecognized regulatory potential of the gut microbiome in lung cancer metastasis.
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Enhanced immunogenic potential of cancer immunotherapy antibodies in human IgG1 transgenic mice
ABSTRACT Clinical anti-drug-antibody (ADA) responses represent a substantial obstacle to the development of efficacious therapeutic antibodies. The enhanced ADA production against the idiotype (Id) often displayed by cancer immunotherapy antibodies (CitAbs) can lead to exposure loss and subsequently affect anti-tumor efficacy and cause undesired effects on safety. Thus, ADA responses contribute to prolonged clinical development and high attrition rates. Most conventional therapeutic antibodies are now of human origin or humanized proteins, and are hence immunologically tolerized in most patients. In contrast, the contribution of additional factors, other than the protein sequence, to the higher rates of clinical ADA to certain CitAbs, remains poorly understood. Here, we used human immunoglobulin gamma 1 (IgG1) transgenic mice (named “hIgG1 transgenic mice” or “TG”), which are immunologically tolerant to human IgG1, to study the immunogenicity of 13 conventional antibodies and 2 CitAbs. We found that tolerance to non-germline encoded Ids is maintained in part by the function of neonatal Fc-receptor (FcRn). Additionally, the incorporation of T cell-engaging moieties like an interleukin 2 (IL-2)-based immunocytokine or a CD3ε-specific antigen-binding fragment (Fab) was sufficient to revert tolerance and trigger ADA production directed to the Id of these compounds. We postulate that T cell receptor or IL-2 receptor activation may result in activation of unresponsive T cells specific for the crystallizable fragment (Fc) that typically inactivate Id-specific B cells and mediate “linked-antigen tolerance”. Reversal of this unresponsiveness by the action of CitAbs on T cells may be the cause of undesired ADA responses. Abbreviations ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.
Introduction
The broad clinical use of therapeutic antibodies has revealed that in some cases this treatment results in unwanted immune responses and the production of anti-drug antibodies (ADA). In some cases, ADA can cause substantial exposure loss that affects efficacy, with the potential to provoke undesired adverse events. 1 Presently, it is unknown which factors, in addition to foreign immunogenic protein sequences, contribute to the onset of ADA production by some therapeutic antibodies and not by others. In a recent survey, the collected ADA data from 60 clinical antibodies revealed that the majority (76%) of these antibodies display an immunogenicity rate of less than 10% and the minority (10%) of the antibodies tested cause an ADA rate higher than 20%. 2 Thus, most human or humanized conventional therapeutic antibodies are weakly immunogenic in humans. In contrast, bispecific, immunomodulatory antibodies frequently used in cancer immunotherapy (CIT) -herein referred to as CitAbs -appear to cause ADA with a higher incidence than expected, given their generally human/humanized origin. 1,3 Therefore, it is crucial to better understand the mechanisms involved in breaking tolerance toward CitAbs, leading to ADA responses.
CitAbs are engineered bispecific antibodies bearing binding arms directed at multiple targets. 4 They can cross-link agonistic receptors like the T cell receptor (TCR) or interleukin 2 receptor (IL-2R) on immune cells and a tumor antigen or checkpoint inhibitor on the respective target cancer cells in order to induce tumor cell killing by the immune cell. 5 Here, we aimed to study the immunogenic properties of CitAbs by making use of a transgenic system that provides immune tolerance to a broad range of human antibodies of the immunoglobulin gamma 1 (IgG1) isotype, the hIgG1 transgenic mouse. 6 As this transgenic system has previously been shown to sense immunogenic modifications of an otherwise tolerated human therapeutic antibody, 7 we reasoned that it would also help with the study of the immunogenicity of CitAbs preclinically. To this end, we first conducted a study to validate this transgenic mouse system using 13 commercially available conventional therapeutic antibodies with known clinical immunogenicity rates. The study revealed that the immunogenicity in hIgG1 transgenic mice is substantially reduced as compared to control mice. Then, we tested the immunogenic potential of CEA-IL2v (cergutuzumab amunaleukin) and CEA-TCB (cibitasamab), two prototypical CitAbs, 8,9 and investigated possible mechanisms responsible for the enhanced ADA responses they elicit in the hIgG1 transgenic mice.
In current models of immune tolerance, early expression of germline-encoded self-antigens in the bone marrow and the thymus predisposes B and T lymphocytes, respectively, for a lifelong state of unresponsiveness toward most self-proteins. The case of antibodies is peculiar because, although germlineencoded, the amino acid composition of the antigen-binding site can be further altered during the process of gene rearrangement (to assemble the variable (V) region) and of somatic hypermutation (SHM), which increases the affinity to antigen during maturation of antibody responses. Thus, the resulting array of complementary-determining regions (CDR) of mature, high-affinity antibodies that bind defined antigens can diverge substantially from corresponding germlineencoded sequences. Nonetheless, the variant amino acid segments of secreted antibodies during immune responses normally do not provoke antibody responses to the newly formed CDR combinations or idiotypes (Id), even if they deviate from the tolerated V regions. Similarly, the iterative clinical infusion of large quantities of antibody-based therapeutics does not generally result in immunogenic ADA responses. In a simplified experimental system, we made use of the mouse immune system, transgenically rendered tolerant to human antibodies, and asked the two basic questions: 1) How is tolerance to unforeseen V region epitopes (idiotypes) maintained? and 2) What causes the enhanced immunogenic properties of CitAbs, as compared to conventional antibodies?
The results of our study revealed two regulatory pathways associated with the Fc region of antibodies that downmodulate anti-Id ADA responses. We propose a model to explain the predominant tolerance to non-germline Id determinants and its breakdown by CitAbs and discuss mitigation strategies derived thereof.
Immunogenicity of conventional antibodies
As described previously, hIgG1 transgenic mice are immunologically tolerant to a broad range of human IgG1 antibodies. 6 Here, we interrogated if this transgenic system could reproduce experimentally the clinical ADA incidence of 13 therapeutic antibodies of the IgG1 isotype surveyed by Dingman and Balu-Iyer. 2 We selected 5 antibodies with an immunogenicity rate higher than 10% and 8 antibodies with an immunogenicity rate lower than 10% (Table 1). According to regulatory documents and patents, cross-reactivity to the target is absent or only low in mice. Hence, ADA data in transgenic mice refer mostly to the intrinsic antigenicity, and not to the function-related immunogenic properties, of the antibodies analyzed. Table 1 Comparison of clinical ADA rates and experimental in vivo immunogenicity of 13 selected human IgG1 clinical mAbs. Table 1 lists the 13 clinical mAbs with reported clinical ADA rates and experimental in vivo immunogenicity (Incidence; IGF) observed in hIgG1 transgenic (TG) and wild-type (WT) mice. In addition, the target, format, and highest V gene identity to the closest VH and VL gene of the transgene or germline is indicated in percent.
IGF (2)
Incidence Clinical-grade preparations of the selected antibodies were tested in groups of 10 hIgG1 transgenic and 10 littermate wildtype mice except for avelumab, where only 9 wild-type mice were used. The mice were immunized following the established immunization protocol with seven consecutive subcutaneous applications of 10 μg IgG antibody without adjuvant over a three-week period. 6 Supplementary Figure 1 gives an overview on the general study outline of all in vivo immunogenicity experiments in this work. The results of the individual immunization experiments are shown in Supplementary Figure 2 and are summarized in Table 1. As indicated in Table 1, the ADA incidence in wild-type mice varies from 20% to 90% of treated mice. These differences probably reflect intrinsic immunogenic differences of the V region sequences used, since all compounds tested bear the same human constant region. Two of the three chimeric antibodies bear murine V regions with a high degree (over 90%) of sequence identity with endogenous mouse germline V gene sequences (brentuximab, cetuximab) for both the heavy (H) and the light (L) chain. The finding that two (infliximab, brentuximab) of the three antibodies displaying higher clinical ADA scores are chimeric human/mouse antibodies might suggest that the murine V sequences increase their immunogenic potential in humans.
In contrast, hIgG1 transgenic mice display a low ADA incidence that is generally reminiscent of that observed in humans with these compounds. The clinical ADA incidence reported for eight antibodies analyzed range between 0 and 10%, but is very variable for two of the remaining five antibodies studied (brentuximab and infliximab), ranging between 7% and 51% (Table 1). Compared with the large case numbers contributing to the clinical ADA rates of the compounds listed in Table 1, the ADA frequency in hIgG1 transgenic mice stems from experimental groups of 10 mice. This explains the better granularity of the clinical ADA rates, especially for the small numbers, as ADA incidence values between 0% and 10% effectively cannot be reproduced in hIgG1 transgenic mice (less than one mouse). In fact, all compounds with a clinical ADA incidence up to 13% cause an ADA rate between 0% and 10% in hIgG1 transgenic mice (Table 1). Thus, given the group size limitation, the hIgG1 transgenic mice consistently reproduce the range of clinical immunogenicity reported for the compounds tested here.
Tolerance to human antibodies does not depend on V sequence similarity to resident V gene repertoire
Given the observed fluctuation of the measured ADA titer values within immunization groups (Supplementary Figure 2), and in order to provide a tool reflecting both the incidence and the intensity of individual immune responses, we introduced the concept of the "Immunogenicity Factor" (IGF in Table 1). This value integrates the frequency of Figure 1. Low identity of antibody V genes to endogenous V genes does not correlate with increased immunogenicity. V gene identity is given as percent amino acid sequence identity of the variable (V) domain of each antibody listed with the best matching hIgG1 transgenic (TG, orange circles) or endogenous murine wild-type (WT, gray circles) V heavy (VH, left panel) and V light (VL, right panel) gene elements. The Immunogenicity Factor (IGF) as a degree of immunogenicity is correlated with the V gene identity for hIgG1 transgenic (orange lines) or wild-type mice (gray lines) and R 2 values, as well as p-values are displayed in the plot. A robust linear model was used, based on Huber's M-estimator, which downweighs the influence of single outlying data points, as well as deviations from non-normality. R 2 coefficients of determination were calculated using modified residual and total sums of squares, i.e., weighing the squared deviations with the respective weights assigned by the robust estimator. Compounds showing higher identity to endogenous V genes compared to transgenic V genes are labelled and in addition, chimeric compounds are labelled in blue.
responders and the respective titer values as described in the materials and methods section.
We determined the level of identity between the V sequence of the 13 tested antibodies and the endogenous murine or the five transgenic human V gene sequences for the H and the L chain in each case. These values are given in Table 1 for the H and the L chain V gene of each antibody as sequence identity (in %) to the closest resident transgenic or endogenous murine V genes. For example, for alirocumab, a maximum of amino acid identity of 89% with one of the five transgenic VH genes and of 66% with one of the two transgenic V kappa (Vκ) genes is indicated. Figure 1 displays the IGF of the responses to each compound as a function of the sequence identity of its V gene. Interestingly, this comparison in wild-type mice denotes a trend toward increased tolerance when V sequence identity increases ( Figure 1, gray symbols). However, this systematic effect only accounts for a few percent of the overall variability in the data, based on R2 values, therefore not constituting a pivotal factor determining the IGF. In hIgG1 transgenic mice, V sequence identity does not correlate at all with observed immunogenicity (Figure 1, orange symbols). The data suggest an "equalizing principle" in hIgG1 transgenic mice causing a general flattening of IGF irrespective of their V sequence identity to resident transgenic or endogenous V sequences. In summary, we conclude that tolerance to human antibodies has a broader coverage than expected by the five transgenic V genes expressed in hIgG1 transgenic mice. Therefore, we next asked if the common Fc domain has an influence in maintaining unresponsiveness to V sequences.
Fab fragments display enhanced ADA responses
We analyzed the immunogenic properties of purified antigenbinding fragments (Fab) lacking the crystallizable fragment (Fc) of three representative IgG1 antibodies: the therapeutic antibody bevacizumab, the scaffold antibody CEA-IgG containing the same sequence as the CitAbs CEA-IL2v and CEA-TCB, and the experimental antibody DP47-IgG bearing VH and Vκ regions encoded by the transgenic human VH3-23 and Vκ3-20 genes, respectively. The data in Figure 2A illustrate that the immunogenic potential of these antibodies observed in wild-type mice is strongly reduced or abolished in hIgG1 transgenic mice. In contrast, the Fab preparations of bevacizumab (beva.-Fab) and CEA-IgG (CEA-Fab), but not of DP47- Figure 2. Fc contributes to the unresponsiveness towards conventional mAbs. Murine IgG anti-drug antibody (ADA) titers in hIgG1 transgenic (orange, upper panels) and wild-type (gray, lower panels) are measured over time before (day 0) and after the first immunization. Groups of 10 hIgG1 transgenic and 10 wildtype mice were immunized with the full antibody and the respective Fab preparation in the same experiment. The immunization compound (I:) and the coating compound for the ELISA (C:) are displayed above each plot. ADA titers above the arbitrary threshold of 200 are considered as positive and hence used to calculate the ADA incidence displayed in percent and the Immunogenicity Factor (IGF). Panel A) displays ADA titers against the full IgG1 antibodies bevacizumab, CEA-IgG, DP47-IgG and panel B) shows ADA titers against the respective Fab fragments beva.-Fab, CEA-Fab, and DP47-Fab. Panel C) shows ADA titers against the Intravenous Immunoglobulin subclass 1 Fab fragment (IVIG1-Fab) induced by immunization with the full IVIG1 or IVIG1-Fab preparation.
IgG (DP47-Fab), display enhanced immunogenicity both in hIgG1 transgenic and wild-type mice ( Figure 2B). The results imply that Fab-specific B cells exist in hIgG1 transgenic mice that are silenced upon immunization with full antibody, but are unleashed when triggered with Fab preparations. Interestingly, the Fab of the antibody DP47-IgG fails to elicit this "reactivation" response ( Figure 2B). Given that the same human VH3-23, joining (J)H4, Vκ3-20, and Jκ1 genes of DP47 are also expressed in hIgG1 transgenic mice, 6 we interpret the absence of a corresponding antibody response to DP47-Fab as resulting from central tolerance-induced deletion of DP47-specific lymphocytes. Immunization with IgG1 fraction of intravenous immunoglobulin (IVIG1) and Fab preparations thereof (IVIG1-Fab) extend the validity of this assumption over the two cases with beva.-Fab and CEA-Fab. As shown in Figure 2C, the incidence and the intensity of the ADA response to IVIG1-Fab is enhanced when compared with IVIG1, both in hIgG1 transgenic and in wild-type mice.
Reitan and Hannestad 10 discovered that the immunogenicity of Id depends on the isotype of the antibody. To assess the influence of the constant region on immunogenicity of the CEA-binder, we generated antibody constructs composed of this particular human V region associated with the murine IgA constant region (CEA-mIgA). Immunization of hIgG1 transgenic and wild-type littermate mice demonstrates that the V region associated with the mIgA isotype elicits a similar ADA response as the corresponding CEA-Fab preparation ( Figure 3A). Further, the results in Figure 3A demonstrate that the observed ADA response to Fab is not caused by cryptic epitopes exposed in the Fab, but results from the absent tolerogenic effect of IgG-Fc.
IgG binding to FcRn is required for immune tolerance
One major difference between IgG and IgA is the binding capacity to the neonatal Fc receptor (FcRn). 11 To investigate the contribution of this receptor, we generated a variant of CEA-IgG bearing a murine IgG1 constant region (CEA-mIgG) and an AAA variant thereof (CEA-mIgG-AAA) containing a triple mutation (I253A, H310A, H435A) that abolishes binding to the FcRn. 12 The elevated ADA response of CEA-mIgG-AAA clearly links FcRn to the Fc-mediated unresponsiveness with CEA-mIgG ( Figure 3B).
Elevated ADA response in mice deficient for FcRn
Immunization experiments in mice deficient for the FcRn receptor (FcRn-KO) further corroborated this assumption. Because this mouse strain does not express human IgG1 as self-protein, it requires immunizations with the mouse surrogate CEA-mIgG antibody. In analogy to the enhanced ADA response found with CEA-mIgG-AAA, the CEA-mIgG antibody elicits a stronger ADA response in FcRn-KO mice as compared to wild-type mice ( Figure 3C), despite the expected strongly reduced exposure of the injected antibody (Supplementary Figure 3A) and the overall reduced endogenous IgG levels (Supplementary Figure 3B) in mutant mice due to the lack of FcRn-mediated recycling. The control immunization with the T cell-dependent antigen keyhole limpet hemocyanin (KLH) in this experiment demonstrates that FcRn-KO mice are capable to mount ADA responses, but the titers are generally reduced in comparison to wild-type mice ( Figure 3C). Taken together, we conclude that tolerance to idiotype components of antibodies is mediated by the Fc domain of IgG (but not by IgA isotype) through interaction with FcRn.
IL-2 immunocytokine and T-cell bispecific antibody display enhanced immunogenicity
The identification of Fc-associated mechanisms preserving unresponsiveness to conventional antibodies prompted us to address the widely observed strong immunogenic potential of bispecific therapeutic antibodies. 1 Thus, we extended our studies to two forms of CitAbs, based on the same CEA-specific scaffold antibody. The structure of CEA-based CitAbs used is shown in Figure 4A. The T cell bispecific (TCB) cibisatamab (CEA-hTCB) is composed of two Fab arms specific for CEA and one arm specific for the CD3ε chain of the TCR protein complex. 8, In our experiments, we also used a variant with a V domain of the hamster-anti mouse CD3ε clone 145-2C11 (CEA-mTCB), in the similar, though not identical, 2:1 format of clinical CEA-hTCB. The Fc-distal position of the CD3ε binder in CEA-mTCB, as compared to its Fc-proximal position in CEA-hTCB, results from protein expressibility and activity limitations of the murine version. The immunocytokine cergutuzumab amunaleukin (CEA-IL2v) is composed of the same CEA-binding scaffold antibody fused to a modified human IL-2 molecule at the carboxy terminal of one of the Fc moieties by a linker. 9 This IL-2 variant (IL2v) used in this immunocytokine contains mutations that abolish binding to the high-affinity IL-2Rα chain (CD25), but preserve binding to the intermediate affinity IL-2Rβγ heterodimer receptor. 9 Therefore, it preferentially stimulates CD25 dull effector T cells and NK cells, while avoiding activation of CD25 high regulatory T cells. 9 In addition, the CEA-IgG backbone bears the PGLALA modification within the Fc region that eliminates the Fc-gamma receptor (FcyR) and complement C1q binding, while retaining FcRn binding. 13 In the experiments presented here, the CEA-IgG scaffold antibody served as control of the baseline immunogenic potential. As shown in Figure 4A, CEA-IgG and CEA-hTCB are poorly immunogenic, whereas CEA-mTCB and CEA-IL2v elicit immune responses with high incidence and titer in hIgG1 transgenic mice. It is particularly significant that the fusion with the human CD3ε-specific Fab in CEA-hTCB does not raise the immunogenic potential of CEA-IgG, whereas addition of the mouse CD3ε-reactive Fab in CEA-mTCB and of the human IL2v protein in CEA-IL2v results in increased immunogenicity. As both CEA-mTCB and CEA-IL2v can engage the corresponding receptors in murine T cells (CD3ε and IL-2R), the data support a role for this interaction in reverting the preexisting unresponsiveness to CEA-IgG. Furthermore, Figure 4B shows that immunization with CEA-IL2v induces ADA responses directed against CEA-Fab, indicating the involvement of Fab-specific B cell clones. In contrast to wildtype, hIgG1 transgenic mice usually do not produce ADA against the IgG1 Fc portion, suggesting an elimination of Fcspecific B cells mediated by the expression of the transgene.
When similarly modified variants of the germline antibody DP47-IgG are tested for their immunogenicity, a different picture emerges. It is evident that no ADA against DP47 are elicited with DP47-IL2v ( Figure 4C) and DP47-mTCB ( Figure 4D) despite the addition of IL2v or mouse CD3ε binder, respectively. This demonstrates that the absence of anti-DP47 ADA reflects the lack of germline-specific B cell clones. Interestingly, the detection of mTCB-specific ADA in mice immunized with DP47-mTCB ( Figure 4D, lower panels) suggests the presence of T and B cell epitopes in the hamster-anti mouse CD3ε binder.
Enhanced immunogenicity of CitAbs does not correlate with dendritic cell uptake
It is known that pre-existing ADA, even below the detection limit, can contribute to the formation of immune complexes (ICs) with infused antibodies, thus increasing their immunogenicity. 14 The underlying mechanism could be enhanced uptake, processing, and presentation by dendritic cells (DC). 15,16 Concerns were raised that structural modifications of CEA-IgG, such as the fusion of IL2v or TCB and the knob-into-hole amino acid changes for engineering of bispecific antibodies, 17 could promote binding of pre-existing ADA, and hence increase immunogenicity or cause stimulation of DCs via other mechanisms. Thus, we generated large ICs formed by CEA-IgG and mouse IgG2a antibodies specific to the idiotype of CEA-IgG (CEA-IgG-IC) to investigate this hypothesis (Supplementary Figure 4A). Immunizing hIgG1 transgenic and wild-type mice with CEA-IgG-IC leads to a distribution of intact ICs in the serum (Supplementary Figure 4B) but causes only a slight increase of the ADA response against CEA-IgG ( Figure 5A). This slight increase caused us to question the contribution of DC internalization to the immunogenicity of CitAbs. To that end, we differentiated DCs from the bone marrow of three hIgG1 transgenic and three wild-type mice using recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF). Subsequently, we measured by flow cytometry the internalization rate of the compounds labeled with a pH sensitive fluorescent dye, which is brighter in the acidic environment of the lysosome. As expected, DCs internalized CEA-IgG-IC more efficiently than monomeric CEA-IgG ( Figure 5B), which could contribute to the slightly enhanced immunogenicity. The internalization rate of monomeric CEA-IL2v and CEA-mTCB is also in the range of CEA-IgG-IC, even though these compounds induce a stronger ADA response in hIgG1 transgenic mice ( Figure 4A). Interestingly, CEA-mIgG displays the highest DC internalization despite its low immunogenicity in hIgG1 transgenic and wild-type mice ( Figure 3B). Similarly, CEA-hTCB is not immunogenic in hIgG1 transgenic mice ( Figure 4A), but shows an intermediate DC internalization rate. From the combination of these data, we conclude that there is no simple correlation between the rate of uptake and internalization and the immunogenic attributes of the analyzed compounds. Thus, other explanations are needed to explain the enhanced immunogenicity of CitAbs.
The assumption that ADA responses to CEA-mTCB and CEA-IL2v are stimulated by activation of T cells targeted by their mTCB or the IL2v moieties is further supported by immunization experiments with mixtures of scaffold CEA-IgG with purified IL2v or a one-arm variant of the mCD3εspecific antibody (mCD3-OA). These immunization studies show an incremental difference in immunogenicity comparable to that found with the corresponding T cell engager antibodies (Supplementary Figure 5). From these results, we conclude that idiotype-specific B cell clones exist that are indirectly reactivated by the action of IL2v or CD3ε binder molecules. Given the T cell-activating nature of the IL2v and anti-CD3ε Fab, the question arises whether this reactivation is an antigen-cognate process, or the sole result of bystander responses mediated by nonspecific T cells.
ADA responses elicited by CitAbs require cognate T-B cell interaction
To address the question of cognate T-B cell activation, we studied the role of antigen-presentation by B cells in the formation of ADA. To do so, we used an experimental system of lethally irradiated mice reconstituted with a mix of bone marrow cells. The mix is composed of 80% bone marrow cells from mice homozygous for the Igh-J tm1Cgn targeted mutation (JhT −/− ), and thus lacking functional B cells, and of 20% bone marrow cells derived from MHC-II deficient mice (MHC-II −/− ) in order to create a system where only B cells are deficient for MHC-II expression. A mix containing 20% bone marrow cells from wildtype mice serves as a control. The generation of the bone marrow chimeric mice (BMC) is described in Figure 6A and in the materials and methods section (Mice and Immunization). The B cells in such reconstituted mice are MHC-II −/− , while all other immune cells bear the JhT −/− mutation but express MHC-II ( Figure 6, B and C). The system is apt to test if the CitAbs CEA-IL2v and CEA-mTCB were able to induce antigen presentation-independent, non-cognate T cell activation, and hence MHC-II-independent ADA production. As shown in Figure 6D, the experiment evinced that only mice reconstituted with the mix containing wild-type bone marrow cells (WT BMC) produced ADA to CEA-IL2v and CEA-mTCB upon immunization. In contrast, chimeric mice containing MHC-IIdeficient B cells (MHC-II KO BMC) were unable to respond to the immunization with CEA-IL2v and CEA-mTCB. Since the bone marrow reconstitution generated similar numbers of lymphocytes in the two groups of chimeras ( Figure 6C), we totally exclude a non-cognate type of T cell activation and ADA production as explanation for the immunogenic potential of CEA- Figure 6A), both groups can be considered equally immunocompetent. This was also evident from the comparable antibody response elicited by the T cell-independent antigen NP-Ficoll in both groups of chimeric mice (Supplementary Figure 6B). Therefore, reversal of unresponsiveness to CEA-IgG by the bispecific versions CEA-IL2v and CEA-mTCB requires both extracellular binding of IL-2 or TCB to their receptors on the T cell surface and intracellular processing and presentation by MHC-II.
Discussion
Despite intensive efforts in the development of bispecific antibody therapeutics specifically designed for cancer immunotherapy, few have reached the market. 1 Doubtless, antibody therapy based on CitAbs and related biotherapeutics has the potential to be highly efficacious in cancer treatment. 5,18 Given that immunogenicity represents one of the major liabilities of this class of anti-tumor drugs, the preclinical assessment of their immunogenic properties is the objective of intensive research efforts. [19][20][21][22] Methods are in place aiming to identify potential antigenic epitopes of defined biotherapeutic proteins in silico (e.g., EpiVax, netMHC-pan), in vitro (MAPPs), ex vivo (e.g., PBMC and DC-TC assays) or in vivo with transgenic mouse systems. 23,24 Yet, the basic question of the mechanism(s) causing break of tolerance to antibody proteins in general, and more specifically to CitAbs in particular, was often raised but insufficiently addressed experimentally. 1 Immunogenicity of antibodies is centered around anti-Id responses. [25][26][27] Anti-Id ADA responses have been studied in detail in experimental murine systems based on the transgenic expression of Id-specific B and T cell receptors. [28][29][30] These studies demonstrated that, upon administration of Id-bearing antibodies, Id-specific naive B and T cells collaborate in vivo to the formation of germinal centers and plasma cells, resulting in isotype-switched anti-Id ADA. This process occurs in the absence of adjuvants and does not require antigen presentation by DCs. 31 These mechanisms differ significantly from conventional antibody responses to foreign antigens and could also apply to Id-specific ADA responses to human antibodies in clinical trials. Using these transgenic tools, it became evident that self-reactive B and T cell clones specific for germline-encoded Id are deleted centrally. 28,30 However, lymphocytes with specificity for nongermline Ids can mature and persist in the periphery and could respond to challenge by idiotypic determinants arising during SHM of antibodies or appearing upon infusion of exogenous therapeutic antibodies.
Nonetheless, the accumulated clinical data of numerous marketed therapeutic antibodies suggest a low to moderate immunogenicity potential for most human therapeutic antibodies clinically tested so far (Table 1, ref. 2). In contrast, CitAbs tend to elicit ADA more frequently, often causing exposure and efficacy loss and even adverse events, which contribute to high attrition rates. 1 Here, we addressed the apparent broad tolerance to Ids encoded by non-germline V genes of exogenous conventional therapeutic antibodies and the comparably pronounced immunogenic attributes of CitAbs. We used the hIgG1 transgenic mouse previously shown to display immunological tolerance to a broad range of human IgG1 antibodies and to be sensitive to immunogenic modifications thereof. 6,7 Of course, this model cannot evaluate patient-related factors contributing to ADA occurrence in clinical trials, including cross-reacting pre-existing antibodies, dosing regimens, premedication, administration route, biodistribution, and density of the tumor target antigen(s). While all these factors, along with genetic factors, can influence the ADA outcome to a defined treatment with CitAbs, our study addresses fundamental immunological mechanisms governing immune tolerance and its reversal, that are shared by the human and mouse immune systems. In this study, we have not addressed other questions potentially adding to the immunogenic attributes, such as the affinity of CitAbs to the T cell target antigens or the different formats of CitAbs. However, given the tightly coordinated interaction between T and B cells, anti-CD3ε with higher affinities could in turn lead to stronger B cell help and thus be more immunogenic.
Our results confirm the previously reported broad range of tolerance to human IgG1 antibodies in the hIgG1 transgenic mice. 6 Nonetheless, this is somehow surprising considering that only five human V sequences are included in the germline of hIgG1 transgenic mice, in comparison to the marked diversity of V gene sequences of the human antibodies tested (Table 1 and Figure 1). The lack of correlation between immunogenicity and sequence identity to resident V sequences in hIgG1 transgenic mice suggests a mechanism ensuring tolerance to V sequences not encoded in the germline. We hypothesized that the IgG1 constant region plays this tolerizing role. Indeed, we found that Fc-devoid preparations of two prototypical antibodies (bevacizumab and CEA-IgG) and of IVIG1 elicit strong ADA responses in hIgG1 transgenic mice (Figure 2), indicating that Id-specific B and T cells exist in these mice, but are kept silent toward Fc-containing antibodies. Taken together, these results support the general tolerogenic role of the Fc moiety of IgG1 antibodies, but not of antibodies of other isotypes like IgA ( Figure 3A) in hIgG1 transgenic mice. Even considering that Fab are unnatural, artificial substances used here for experimental purposes, the results clearly suggest that the Fc has a role in modulating immune responses toward Id determinants of antibodies. Interestingly, the few published immunogenicity studies on bispecific T cell engagers (BiTEs) have revealed the immunogenic character of this class of small therapeutic antibodies composed of two different single-chain variable fragment (scFv) binding one tumor antigen and an activating receptor on T cells. 32,33 According to our findings, it should be expected that appendage of an Fc moiety would abrogate or mitigate the immunogenic properties of BiTEs in humans.
Using FcRn-deficient mice we demonstrated that the tolerogenic role of Fc is related to the function of FcRn (Figure 3). Based on these data, we propose that FcRn-directed recycling diverts internalized antibodies away from the proteasomal degradation pathway to peptide presentation and immune activation. 12 Similar observations have been made for murine anti-Id ADA responses. Indeed, certain murine Id-bearing antibodies proved to be very immunogenic and efficient in inducing anti-Id ADA responses even when administered adjuvant-free in syngeneic mice. 34 In that study, Reitan et al. showed that the immunogenicity of these mouse idiotypes depends on the associated isotype. Indeed, the same idiotype is tolerized or can elicit vigorous anti-Id responses when borne by IgG isotypes or by IgM, IgE and IgA isotypes, respectively. Furthermore, this IgG-associated tolerogenic Id becomes immunogenic when administered Fc-free, as Fab fragments. 10 Here, using hIgG1 transgenic mice, we demonstrate that germline encoded V regions of human antibodies promote central elimination of corresponding Id-specific lymphocytes, as exemplified by the antibody DP47-IgG, while unresponsiveness to non-germline human antibodies (bevacizumab, CEA-IgG) is aided by a recycling pathway mediated by FcRn. Assuming efficient binding of bevacizumab and CEA-IgG to the murine FcRn 35 and the lack of binding to murine FcyRs of the CEA-Fc with the PGLALA mutations, 13 we conclude that FcRn, but not FcyR, substantially contributes to the observed unresponsiveness to infused antibodies. Upon B cell receptor (BCR) ligation, antigen-specific B cells are also able to present BCR-derived Id determinants and receive help from Id-specific T cells. 29 However, naive B cells initially bind antigen with low affinity via non-mutated germline BCR. These B cells are not likely to encounter T cells specific for such germline-encoded Id, as they are clonally deleted in the thymus. 28 In contrast, whenever Id-bearing antibodies are generated through SHM (or are exogenously added) and capable of ligating a corresponding anti-Id BCR, ADA can result 29 from direct Id- specific B-T cell interaction. 31 Here, we describe that this process is down-modulated by the action of FcRn (Figure 3) and by an additional suppressive and unresponsive state conveyed by putative Fc-specific, suppressor, or anergic T cells.
In contrast to the poorly immunogenic scaffold antibody CEA-IgG, the derivatives CEA-IL2v and CEA-mTCB elicit vigorous anti-Id ADA responses in hIgG1 transgenic mice ( Figure 4A). This fundamental finding identifies the IL2v and TCB moieties as responsible for the immunogenicity increment. This assumption is further supported by the finding that the fusion of a human CD3ε-binding Fab, which does not cross-react with mouse CD3ε, does not cause an increase in immunogenicity ( Figure 4A). The fact that germline V-based DP47-IL2v was immunologically inert ( Figure 4C) and DP47-mTCB elicited exclusively an anti-mTCB ADA response ( Figure 4D) further substantiates the deletion of DP47-Id B cell clones during central tolerance. The absence of ADA against DP47 excludes the formation of new epitopes in CEA-IL2v and CEA-mTCB as the primary reason for the onset of immunogenicity.
The possible formation of IC composed of CEA-IL2v or CEA-mTCB and pre-existing low levels of ADA was simulated with antibodies directed against the Id of CEA-IgG, yet the immunogenicity rate of this preparation failed to explain the typical enhancement of immunogenicity of the CitAbs. The measurement of antibody internalization in antigen-presenting DCs has been proposed as a tool for immunogenicity risk assessment because a higher degree of internalization generally correlates with higher clinical immunogenicity. 36 However, in a similar assay, the internalization rate of these compounds in murine DC revealed no simple correlation with their corresponding immunogenic potential in hIgG1 transgenic mice. Finally, the artificial mixture of CEA-IgG with soluble IL2v or a one-armed antibody specific for mCD3ε confirmed T cell activation as the mechanism causing the ADA responses of CEA-IL2v and CEA-mTCB, respectively (Figure 4 and Figure 5 and Supplementary Figure 5).
Taken together, these results add another level of control of anti-Id responses in addition to FcRn that is sensitive to T cell engagement. Binding of IL-2R or TCR-CD3 by CEA-IL2v and CEA-TCB, respectively, seemingly causes the reversal of a state of unresponsiveness not compensable by the recycling pathway driven through FcRn. This unresponsiveness has features reminiscent of T cell anergy, as it is sensitive to T cell stimulation by activating molecules like IL-2 or TCR-binding moieties of certain CitAbs (Figure 4). Indeed, [37][38][39] FcRn relates to a salvage pathway of internalized antibody while reversal of unresponsiveness by CitAbs like CEA-IL2v and CEA-TCB occurs through the extracellular triggering of T cell activation surface receptors like IL-2R and TCR-CD3. The immunomodulatory property of the Fc region discussed before seems to be overruled by direct engagement of surface T cell activators. Thus, the questions arise, if the reversal of the unresponsive state requires extracellular ligation of the affected T cell surface receptors alone, or if cognate antigen presentation by the Idspecific B cell is also needed. We answered these questions with bone marrow chimeric mice designed to lack expression of MHC-II in B cells, but not in other antigen-presenting cells like 40 DCs ( Figure 6A-C). These animals are apt to discern the need for antigen presentation by B cells in the process of breaking tolerance to CEA-IgG by immunization with CEA-IL2v and CEA-mTCB. The experiment clearly demonstrated the need for MHC-II-dependent antigen presentation by B cells (Figure 6D).
In summary, we conclude that tolerance to antibodies, in addition to clonal deletion, is also the result of a peripheral state of T cell unresponsiveness that can be broken if the CitAb includes additional T cell-activating entities like the IL2v or TCR-CD3-binding moieties. This could lead to the activation of hitherto inactive Id-specific B cells, and to ADA production. We postulate that immunomodulatory T cells specific for common "public" epitope(s) of the Fc region (T Fc cells), do maintain this unresponsive state on B cells of different "private" Id specificities. Hence, reversal of the unresponsive status of T Fc cells with CitAbs will result in activation of all possible Id-specific B cell clones (B Id ) ligated by the corresponding CitAb. This type of linked-antigen presentation would then lead to unresponsiveness rather than to activation, and hence it is termed "linked-antigen tolerance". Interestingly, the immunogenicity study of commercial antibodies shown in Table 1 and Figure 1 additionally supports the idea that expression of the human Fc in hIgG1 transgenic mice creates an immunological resort ensuring tolerance to associated V sequences independent of their degree of identity to (immune tolerized) resident V sequences. (Figure7, Panel 3). Such a mechanism of linkedantigen tolerance would explain the extended low immunogenicity of conventional therapeutic antibodies in humans and hIgG1 transgenic mice (Table 1). In contrast, the ligation of the BCR of B Id cells by CitAbs is accompanied by the triggering of activating surface receptors (IL-2R, TCR-CD3) on T Fc cells (Figure 7, Panel 4). The ensuing reversal of unresponsiveness and re-activation of T Fc cells would set in motion the program of helping the cognate B Id cells for anti-Id ADA production. This model could explain the high rates of ADA observed with many of the CitAbs. 1 If confirmed, the hypothesis proposed here of breakage of linked-antigen tolerance to CitAbs represents an intrinsic hurdle asking for specific mitigation strategies. Confirmation of the linked-antigen tolerance hypothesis requires identification of the putative T Fc cells and of the predicted Fc epitopes involved in unresponsiveness. If verified, the model would open a door for mitigation strategies through elimination of the putative Fc epitopes recognized by the unresponsive T Fc cells, such as to cancel their cognate reactivation by CitAbs. This Fc amino acid modification(s) would result in mitigation of all immunogenic CitAbs. In essence, this Fc mutation would restrict anti-Id ADA production to that resulting from interaction of rare B Id with rare T Id cells, to an extent possibly comparable to other, low immunogenic antibodies (Table 1), thus reducing their high attrition rate.
Antibodies and compounds
All marketed therapeutic antibodies (Table 1) are clinicalgrade products purchased from pharmacies. All other therapeutic antibodies (overview in Supplementary Table 1) were transiently expressed in HEK293 cells or produced in stable CHO clones and subsequently purified using Protein A affinity chromatography and size exclusion chromatography. All Fabs were prepared by enzymatic digestion from IgG fractions and subsequently purified using Protein A affinity chromatography or Kappa select capture resin, followed by size exclusion chromatography. The IVIG1 and enzymatically digested IVIG1-Fab preparations were obtained from Athens Research & Technology. Fab preparations of bevacizumab were generated at Genscript by papain digestion, Protein A/G Fc removal, and subsequent endotoxin removal. All compounds were assessed for purity and endotoxin after manufacturing.
Immune complexes were prepared and characterized as described previously. 41 In short, a mixture containing 2 mg/ ml CEA-IgG and 3 mg/ml monoclonal mIgG2a antiidiotype antibody was prepared in histidine buffer (20 mM histidine, 140 mM NaCl, pH 6.0) and incubated for 1 h at room temperature on a shaker at 500 rpm. The ICs were characterized as previously described. 41 In brief, a Waters XBridge Protein BEH SEC Guard Column, 450 Å, 3.5 μm, 7.8 mm × 30 mm and a XBridge Protein BEH SEC Column, 450 Å, 3.5 μm, 7.8 mm × 300 mm were used in a Dionax UltiMate 3000 system from Thermo Fisher Scientific GmbH (ultraviolet (UV) detector MWD-3000, auto sampler, automated fraction collector). 20 μl of centrifuged dosing solutions were injected for analysis. Phosphate-buffered saline (PBS) with 5% ethanol (v/v) was used as running buffer with a flow rate of 0.5 ml/ min. Online UV detection was performed at 280 nm. Antibodies for the DC internalization assay were labeled using the SiteClick Antibody Azido Modification Kit (Invitrogen; Cat: S20026) according to manufacturer's instructions. Briefly, antibodies were labeled with a molar dye excess of 3.5, which was subsequently removed using the Amicon Ultra-2 Centrifugal Filter with a MWCO of 50 kD and rebuffered in 20 mM histidine 140 mM NaCl (pH 5.5). The dyeto-antibody ratio is calculated from the absorbance at 280 nm and 532 nm, the extraction coefficient of the dye and a correction factor of 0.36. ICs for DC internalization were generated as described above using labeled CEA-IgG and unlabeled anti-idiotype antibody.
Mice and immunization
Animal experiments were conducted in AAALAC certified facilities of F. Hoffmann-La Roche in Basel or at the Department for Biomedicine of the University of Basel (Switzerland) in accordance with local rules and regulations of the veterinary authorities under cantonal licence number 2634 and 3125. Throughout the studies, mice were kept under specific-pathogen-free conditions with continuous health monitoring.
To generate B cell-specific MHC-II deficient bone marrow chimeras, lethally irradiated JhT −/− mice (B6.129P2-Igh-Jtm1Cgn/J) were reconstituted with 80% bone marrow of JhT −/− mice and 20% bone marrow of MHC-II −/− mice (B6.129S2-H2dlAb1-Ea/J). Control mice were reconstituted with 20% bone marrow of wild-type C57BL/6 mice instead of MHC-II KO mice. One day prior to reconstitution, recipient mice were irradiated twice in an interval of 4 h with 475 cGy using a Gammacell 40 137Cs irradiator. The bone marrow of donor mice was extracted by crushing femur and tibia, depleted from red blood cells using Lysing Buffer (BD Biosciences; Cat: 555899), depleted from T cells using CD90.2 beads on LD columns (Miltenyi; Cat: 130-121-278) and mixed in the previously mentioned ratio prior to intravenous injection of 10 × 10 6 cells per recipient. All mice used for studies employing bone marrow chimeric mice were bred and maintained in an animal facility at the Department of Biomedicine, University of Basel.
Unless otherwise mentioned, five female and five male mice between 6 and 15 weeks of age were carefully assigned to treatment groups in order to balance relevant factors such as litter, sex, body weight and exact age at the time of the experiment, reducing the potential danger of confounding the treatment effect with these factors. Mice were immunized two times per week with a total of seven subcutaneous injections into the abdominal region, alternating between the left and right sides. A dose of 10 µg full IgG molecule or the equimolar equivalent in the case of antibody fragments or conjugates was used per injection. The dose of ICs was adjusted to 10 µg CEA-IgG per mouse and the KLH subunits (Thermo Scientific; Cat: 77649) were injected twice on day 0 and 7 at a dose of 125 μg/mouse. The test items were freshly diluted in a total volume of 100 µl DPBS (Gibco; Cat: 12559069) or histidine buffer (20 mM histidine, 140 mM NaCl, pH 6.0) at dosing dates prior to administration.
Blood was collected prior to dosing on day 0 (naive), and weekly on day 7, 14, 21, and 28 by tail vein sampling into serum gel micro-sample tubes (Sarstedt; Cat: 41.1500.005). After clotting, the samples were centrifuged as indicated and stored at -20°C until analysis by enzyme-linked immunosorbent assay (ELISA). In case drug exposure was assessed, additional blood was sampled 7 and 24 h after the first injection.
The drug exposure and endogenous IgG concentrations were measured similarly by coating with anti-Id antibody (in house production) or goat anti-mIgG light chain specific (Jackson ImmunoResearch; Cat: 115-005-174) antibody, and detection with alkaline phosphatase-conjugated goat anti-mIgG Fcy fragment-specific antibody.
The baseline for titer determination was calculated as the mean OD value at 1:50 dilution of all naive samples plus six times the standard deviation of those values (6 sigma cutoff). OD values greater than the upper quartile plus 1.5×IQR (interquartile range) were classified as outliers and excluded from baseline calculation. For a few cases, due to high background signals, cut-off values were adjusted manually.
The titration curve was fitted using the 5 th degree polynomial and intersected with the baseline to obtain titer values. If no intersection occurred within the range of applied dilutions and the titration curve stayed consistently below baseline, we fixed the titer at the value corresponding to an intersection at the lowest dilution (1:50). If the intersection was not reached until the highest dilution, we allowed extrapolation of the titration curve up to a dilution of 1:328050, i.e., three times as high as the actual highest one used, applying a more stable 2 nd degree polynomial fit. In any case, the titer was bounded by a maximum value corresponding to a 1:328050 dilution, and a flag was retained to indicate whether the result was obtained by proper interpolation or whether extrapolation/bounding was applied instead. Titers above the arbitrary threshold of 200 are considered ADA positive results.
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Transcription Properties of Beta-HPV8 and HPV38 Genomes in Human Keratinocytes
High-risk HPV, from the genus alpha, can cause anogenital or oropharyngeal malignancies. The oncogenic properties of high-risk HPV are important for their differentiation-dependent replication in human keratinocytes, the natural target cell for HPV. ABSTRACT Persistent infections with high-risk human papillomaviruses (HR-HPV) from the genus alpha are established risk factors for the development of anogenital and oropharyngeal cancers. In contrast, HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer (cSCC) in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Keratinocytes are the in vivo target cells for HPV, but keratinocyte models to investigate the replication and oncogenic activities of beta-HPV genomes have not been established. A recent study revealed, that beta-HPV49 immortalizes normal human keratinocytes (NHK) only, when the viral E8^E2 repressor (E8−) is inactivated (T. M. Rehm, E. Straub, T. Iftner, and F. Stubenrauch, Proc Natl Acad Sci U S A 119:e2118930119, 2022, https://doi.org/10.1073/pnas.2118930119). We now demonstrate that beta-HPV8 and HPV38 wild-type or E8− genomes are unable to immortalize NHK. Nevertheless, HPV8 and HPV38 express E6 and E7 oncogenes and other transcripts in transfected NHK. Inactivation of the conserved E1 and E2 replication genes reduces viral transcription, whereas E8− genomes display enhanced viral transcription, suggesting that beta-HPV genomes replicate in NHK. Furthermore, growth of HPV8- or HPV38-transfected NHK in organotypic cultures, which are routinely used to analyze the productive replication cycle of HR-HPV, induces transcripts encoding the L1 capsid gene, suggesting that the productive cycle is initiated. In addition, transcription patterns in HPV8 organotypic cultures and in an HPV8-positive lesion from an EV patient show similarities. Taken together, these data indicate that NHK are a suitable system to analyze beta-HPV8 and HPV38 replication. IMPORTANCE High-risk HPV, from the genus alpha, can cause anogenital or oropharyngeal malignancies. The oncogenic properties of high-risk HPV are important for their differentiation-dependent replication in human keratinocytes, the natural target cell for HPV. HPV from the genus beta have been implicated in the development of cutaneous squamous cell cancer in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. Currently, the replication cycle of beta-HPV has not been studied in human keratinocytes. We now provide evidence that beta-HPV8 and 38 are transcriptionally active in human keratinocytes. Inactivation of the viral E8^E2 repressor protein greatly increases genome replication and transcription of the E6 and E7 oncogenes, but surprisingly, this does not result in immortalization of keratinocytes. Differentiation of HPV8- or HPV38-transfected keratinocytes in organotypic cultures induces transcripts encoding the L1 capsid gene, suggesting that productive replication is initiated. This indicates that human keratinocytes are suited as a model to investigate beta-HPV replication.
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Pharmacotherapeutic options for pancreatic ductal adenocarcinoma
ABSTRACT Introduction Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy projected to be the 2nd leading cause of cancer related death in the USA by 2030. This manuscript discusses current and evolving treatment approaches in patients with pancreatic cancer. Areas covered PDAC is classified as: a) resectable, b) borderline resectable, c) unresectable (locally advanced and metastatic). The standard of care for patients who present with resectable pancreatic adenocarcinoma is six months of adjuvant modified (m) FOLFIRINOX, gemcitabine plus capecitabine, or single agent gemcitabine. For many reasons, there has been a paradigm shift to employing neoadjuvant chemotherapy. For resectable and borderline resectable patients, we generally start with systemic therapy and reevaluate resectability with subsequent scans specifically when the tumor is located in the head or body of the pancreas. Combined chemoradiation therapy can be employed in select patients. The standard of care for metastatic PDAC is FOLFIRINOX or gemcitabine and nab-paclitaxel. Germline and somatic genomic profiling should be obtained in all patients. Patients with a germline BRCA mutation can receive upfront gemcitabine and cisplatin. Expert opinion Thorough understanding of molecular pathogenesis in PDAC has opened various therapeutic avenues. We remain optimistic that future treatment modalities such as targeted therapies, cellular therapies and immunotherapy will further improve survival in PDAC.
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Molecularly defined subsets of Ewing Sarcoma tumors differ in their responses to IGF1R and WEE1 inhibition.
PURPOSE
Targeted cancer therapeutics have not significantly benefited Ewing sarcoma patients with metastatic or relapsed disease. Understanding the molecular underpinnings of drug-resistance can lead to biomarker-driven treatment selection.
EXPERIMENTAL DESIGN
Receptor tyrosine kinase (RTK) pathway activation was analyzed in tumor cells derived from a panel of Ewing sarcoma tumors, including primary and metastatic tumors from the same patient. Phospho-RTK arrays, Western blots and immunohistochemistry were used. Protein localization and the levels of key markers were determined using immunofluorescence. DNA damage tolerance was measured through PCNA ubiquitination levels and the DNA fiber assay. Effects of pharmacological inhibition were assessed in-vitro and key results validated in-vivo using patient-derived xenografts.
RESULTS
Ewing sarcoma tumors fell into two groups. In one IGF1R was predominantly nuclear (nIGF1R), DNA damage tolerance pathway was upregulated, cells had low replication stress and RRM2B levels, and high levels of WEE1 and RAD21. These tumors were relatively insensitive to IGF1R inhibition. The second group had high replication stress and RRM2B, low levels of WEE1 and RAD21, membrane-associated IGF1R (mIGF1R) signaling, and sensitivity to IGF1R or WEE1-targeted inhibitors. Moreover, the matched primary and metastatic tumors differed in IGF1R localization, levels of replication stress, and inhibitor sensitivity. In all instances combined IGF1R and WEE1 inhibition led to tumor regression.
CONCLUSIONS
IGF1R signaling mechanisms and replication stress levels can vary among Ewing sarcoma tumors (including in the same patient) influencing the effects of IGF1R and WEE1-treatment. These findings make the case for using biopsy-derived predictive biomarkers at multiple stages of Ewing sarcoma disease-management.
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2022-11-18T07:05:22.769Z
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Differentially Infiltrated Identification of Novel Diagnostic Biomarkers Associated with Immune Infiltration in Nasopharyngeal Carcinoma
Background The prognostic value of tumor-infiltrating immune cells has been widely studied in nasopharyngeal carcinoma (NPC). However, the role of tumor-infiltrating immune cells in the diagnosis of NPC has not been fully elucidated. Thus, tumor-infiltrating immune cell-related biomarkers in the diagnosis of NPC patients were explored in the current study. Methods Gene expression profiles of NPC patients were downloaded from the Gene Expression Omnibus (GEO) database. Differentially infiltrating immune cells (DDICs) between NPC and control samples were analyzed by the CIBERSORT algorithm. Weighted gene coexpression network analysis (WGCNA) was performed to screen hub genes significantly correlated with DDIC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of hub genes were performed with R package clusterProfiler. The diagnostic value of hub genes was evaluated by receiver operating characteristic (ROC) curves. RT-qPCR was conducted to validate the expression patterns of diagnostic markers in NPC and adjacent control tissues. The correlations between diagnostic markers and immunomodulators were analyzed using the TISIDB. The protein-protein interaction (PPI) network based on immunomodulators significantly associated with diagnostic biomarkers was constructed and visualized by STRING. The functional enrichment analysis of genes in the PPI network was analyzed by the WebGestalt online tool. Results The abundances of memory B cells, plasma cells, follicular helper T cells, activated NK cells, M0 macrophages, M1 macrophages, M2 macrophages, resting mast cells, and activated mast cells were significantly different between NPC and control samples. Dark orange was identified as the hub module, with a total of 371 genes associated with memory B cells, plasma cells, and M0 and M1 macrophages defined as hub genes, which were enriched into immune-related biological processes and pathways. FCER2, KHDRBS2, and IGSF9 were considered diagnostic biomarkers with areas under ROC curves as 0.985, 0.978, and 0.975, respectively. Moreover, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) suggested that the expression patterns of FCER2, KHDRBS2, and IGSF9 were consistent with the results in GEO datasets. TISIDB analysis revealed that FCER2, KHDRBS2, and IGSF9 had a strong association with 8 immunoinhibitors (BTLA, CD160, CD96, LAG3, PDCD1, TIGIT, CD244, and TGFB1) and 11 immunostimulators (CD27, CD28, CD40LG, CD48, ICOS, KLRC1, KLRK1, TMIGD2, TNFRSF13C, CXCR4, and C10 or f54). The PPI network implied that these 19 immunomodulators had interactions with other 50 genes. WebGestalt analysis demonstrated that 69 genes in the PPI network were enriched into cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, and pathways in cancer. Conclusion Our study identified novel diagnostic biomarkers and revealed potential immune-related mechanisms in NPC. These findings enlighten the investigation of the molecular mechanisms of tumor-infiltrating immune cells regulating NPC.
Introduction
Nasopharyngeal carcinoma (NPC) is a malignant tumor caused by epithelial cells of the nasopharynx. It is one of the most common head and neck tumors, the global geographical distribution of which is extremely uneven that more than 70% of new cases occur in East and Southeast Asia [1]. Common clinical manifestations of NPC contain nasal congestion, epistaxis, ear blockage, hearing loss, diplopia, and headache. Radiotherapy is only the main treatment for early diseases, while chemotherapy combined with radiotherapy is an essential progress in the treatment of locally advanced diseases [2]. However, the mortality rate of NPC remains high, making the detection of preclinical specific markers for nasopharyngeal carcinoma particularly important for the early diagnosis and treatment of NPC [3]. There are still significant differences in outcomes among patients receiving similar treatments at the same stage, though the tumor-lymph node metastasis (TNM) cancer staging system provides useful criteria for recommending treatment strategies or assessing patient outcomes. Therefore, it is of great significance to explore novel diagnostic biomarkers for NPC patients to assess the NPC status or guide treatment.
The malignant phenotype of cancer is also defined by immune cells activated in the tumor microenvironment (TME) [4]. TME consists of immune cells, endothelial cells, mesenchymal cells, inflammatory mediators, and extracellular matrix molecules [5]. In TME, immune cells are a major type of nontumor component and can be applied to tumor diagnosis and prognosis assessment [6][7][8][9]. Besides, the tumor-infiltrating immune cells (TIICs) have a significant prognostic value in NPC, such as T cells, macrophages, dendritic cells, and mast cells [10,11].
However, the diagnostic value and molecular mechanism of immune infiltration-related genes in NPC remain to be elucidated. Therefore, this study is aimed at exploring the possibility of TIIC-related genes as diagnostic markers for NPC, analyzing the pathways of their involvement in NPC immune regulation, and thus laying a theoretical foundation for the value of TIICs in NPC diagnosis.
Materials and Methods
2.1. Ethical Statement. From June 1, 2021, to November 1, 2021, 10 patients with NPC were selected from the throat and head and neck surgery at the First Affiliated Hospital of Zhengzhou University. All patients were confirmed as nasopharyngeal carcinoma by histology at the primary tumor site. All cases were nonkeratinizing squamous cell carcinoma. The patient did not receive radiotherapy or chemotherapy before the operation. Patients with World Health Organization (WHO) type II NPC were excluded. Fresh living cancer tissues and adjacent tissues of 10 nasopharyngeal carcinoma patients were collected during the operation. The basic clinical information of all patients is collected in Table 1. The study was approved by the central institutional review committee and ethics committee of the First Affiliated Hospital of Zhengzhou University (ethical approval number: 2021-KY-1024-002). This study was conducted following the provisions of the Declaration of Helsinki.
Data Source.
Gene expression profiles were downloaded from GSE53819 and GSE12452 datasets. GSE53819 contained 18 NPC and 18 control noncancerous nasopharyngeal tissue (NCNT) samples and was used to identify diagnostic biomarkers. GSE12452 included 31 NPC and 10 NCNT samples and was applied to external validation of the expressions of diagnostic biomarkers between NPC and NCNT samples. 2.4. WGCNA Analysis. A sample clustering tree map was first constructed to detect and eliminate outliers. Then, WGCNA was performed based on the gene expression profiles from the GSE53819 dataset and sample traits (differentially infiltrating immune cells between NPC and NCNT samples) [13]. Besides, β from 1 to 30 was calculated using the pick soft threshold function of WGCNA to select the best soft threshold. Based on the selected soft threshold, the adjacency matrix was converted to a topological overlap matrix to construct the network, and the gene dendrogram and module color were established with the degree of dissimilarity. Additionally, the initial module was further divided by dynamic tree cutting, and similar modules were merged. The Pearson correlation coefficient between the module eigengenes and sample traits was calculated to reveal the most relevant module (hub module) associated with sample traits [14].
Identification of Hub Genes Associated with Immune
Infiltration. The genes in the hub module were further screened using the module membership ðMMÞ > 0:8 and jgene signif i cance ðGSÞj > 0:5. Hub genes were identified by overlapping genes associated with differentially infiltrating immune cells using Venn software online (http://bioinformatics.psb.ugent .be/webtools/Venn/) [15]. Next, functional enrichment analyses including GO pathway analysis and KEGG pathway analysis were performed with R package clusterProfiler [16]. The top five GO terms and the top three KEGG pathways were visualized in a circle plot through the R package GO plot, and p adjust value > 0.5 was considered significant difference.
2.6. Identification of Diagnostic Markers in NPC. R package limma [17] was applied to identify differentially expressed hub genes between NPC and NCNT samples with jlog 2 ð fold changeÞj > 2 and FDR < 0:05. DEGs were visualized in the volcano plot. The diagnostic value of differentially expressed hub genes was evaluated using ROC curves. Hub 2 Disease Markers genes with the areas under the ROC curves > 0:7 were identified as diagnostic biomarkers with high accuracy in NPC. The correlations between diagnostic markers and immunomodulators (immunoinhibitors and immunostimulators) were calculated using the TISIDB (http://cis.hku.hk/ TISIDB/) to better understand the relationship between diagnostic markers and immune response [18]. A PPI network based on diagnostic biomarker-related immunomodulators was constructed using the STRING database [19]. Moreover, the functional enrichment analysis of genes in the network was analyzed by the WebGestalt online tool [20].
The Real-Time Reverse Transcriptase-Polymerase Chain
Reaction (RT-qPCR). Total RNA of NPC (N = 10) and adjacent control tissues (N = 10) were extracted by Nuclezol LS RNA Isolation Reagent (ABP Biosciences Inc., China). After the concentration and purity of RNA were detected, qualified RNA was used for reverse transcription with Sure-Script-First-strand-cDNA-synthesis-kit (GeneCopoeia, USA). Then, qPCR on a CFX96 real-time PCR system (Bio-Rad, USA) was performed using BlazeTaq™ SYBR® Green qPCR Mix 2.0 (GeneCopoeia, USA) under the thermal cycling conditions: 40 cycles at 95°C for 30 s, 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s. Besides, gene expressions were calculated with the 2 -△△Ct method [21]. The primer sequences used in the current study are listed in Table 2.
2.8. Statistical Analysis. All data were analyzed by R (version 4.0.0). Comparisons between the two groups were calculated using the Wilcoxon test. p value < 0.05 indicated statistical significance unless specified.
Results
3.1. Nine Immune Cells Were Differential Infiltration between NPC and NCNT. The composition of infiltrating immune cells in NPC and NCNT was first detected and compared. It was revealed that the top two most abundant immune cells were memory B cells and follicular helper T cells in NCNT (Figure 1(a)), while the proportions of M0 and M1 macrophage infiltration were the highest in NPC (Figure 1(b)). The comparison suggested that the infiltration levels of memory B cells, resting memory CD4 T cells, follicular helper T cells, resting NK cells, M2 macrophages, and resting mast cells were significantly higher in NCNT, while the abundances of plasma cells, activated NK cells, M0 macrophages, M1 macrophages, resting dendritic cells, activated mast cells, and neutrophil infiltration in NPC were significantly boosted (Figure 1(c)). Memory B cells, follicular helper T cells, M0 macrophages, M1 macrophages, M2 macrophages, resting mast cells, activated mast cells, plasma cells, and activated NK cells with extremely significant differences between NPC and NCNT (p < 0:01, Figure 1(c)) were selected for the following WGCNA analysis.
WGCNA Identifies the Dark Orange Module as the Hub
Immune Cell-Related Module. WGCNA was performed to screen the most relevant module associated with infiltrating immune cells. According to the sample clustering result, two outlier samples were detected and eliminated (Figure 2(a)), and then, the sample dendrogram and trait heatmap were established (Figure 2(b)). It was indicated by the pick soft threshold function of WGCNA that the optimal soft threshold power was 5, in which R 2 was about 0.9 (Figure 2(c)). After similar modules were merged, ten modules from the coexpression network were identified (Figure 2(d)). As 1 Male 39 T1 N1 M0 II 2 Female 69 T3 N3 M0 IVa 3 Male 42 T2 N2 M0 III 4 Male 57 T1 N2 M0 III 5 Male 47 T2 N1 M0 II 6 Female 15 N2 N2 M0 III 7 Male 43 T1 N2 M0 III 8 Male 49 T1 N2 M0 III 9 Male 72 T1 N1 M0 II 10 Male 54 T1 N0 M0 I Disease Markers Sample clustering to detect outliers and was identified as hub module related to infiltrating immune cells.
Identification and Functional Enrichment Analysis of 371
Immune Cell-Related Hub Genes. Thereafter, MM > 0:8 and jGSj > 0:5 were used to further screen hub genes in the dark orange module. A total of 810, 600, 615, and 637 genes were discovered to be correlated with memory B cells, plasma cells, and M0 and M1 macrophages, respectively (Figures 3(a)-3(d)). By overlapping these genes, 371 genes were obtained and identified as hub genes associated with immune cell infiltration (Figure 3(e)). GO and KEGG pathway analyses were conducted to investigate the biological function of hub genes. A total of 114 biological processes (BP), 6 cellular components (CC), 14 molecular functions (MF), and 18 KEGG pathways were significantly enriched (Table S1 and S2). As illustrated in Figures 4(a) and 4(b), the top five GO terms were B cell activation, lymphocyte differentiation, immune response-activating cell surface receptor signaling pathway, immune response-activating signal transduction, and B cell differentiation; the corresponding genes involved in these GO terms were visualized in the circle plot. The top three KEGG pathways were B cell receptor signaling pathway, natural killer cellmediated cytotoxicity, primary immunodeficiency, and hub genes involved in the three pathways, as displayed in the circle plot (Figures 4(c) and 4(d)).
Identification of Diagnostic Biomarkers Associated with
Immunomodulators. Next, the expressions of 371 hub genes between NPC and NCNT cells were compared to identify hub genes associated with NPC. A total of 50 differentially expressed hub genes were identified, including 7 upregulated and 43 downregulated hub genes in NPC samples related to NCNT ones ( Figure 5(a)). ROC curves identified FCER2, KHDRBS2, and IGSF9 with high diagnostic accuracy that the areas under the ROC curves for FCER2, KHDRBS2, and IGSF9 were 0.985, 0.978, and 0.975, respectively ( Figure 5(b)). Thus, FCER2, KHDRBS2, and IGSF9 were identified as diagnostic biomarkers in NPC. The expression of IGSF9 was significantly higher, while the expressions of FCER2 and KHDRBS2 were significantly lower in NPC samples compared with NCNT ones (Figure 5(c)). Consistent expression results were obtained in the external validation dataset GSE12452 (Figure 5(d)). Moreover, the expressions of FCER2, KHDRBS2, and IGSF9 in vivo were detected by RT-qPCR. Similarly, the expression of IGSF9 was significantly higher, while the expressions of FCER2 and KHDRBS2 were significantly lower in NPC samples compared with adjacent control ones ( Figure 5(e)).
Discussion
The growth of NPC tumor cells is regulated by surrounding tumor cells, various immune cells, fibroblasts, and endothelial cells [22]. It is of great clinical significance for early diagnosis, treatment, and prognosis to study the distribution and affinity of immune cells in the tumor microenvironment. Therefore, in this study, the landscape of infiltrating immune cells in NPC was detected, and FCER2, KHDRBS2, and IGSF9 associated with immune were identified as diagnostic biomarkers of NPC.
In this study, memory B cells, follicular helper T cells, M0 macrophages, M1 macrophages, M2 macrophages, resting mast cells, activated mast cells, plasma cells, and activated NK cells were revealed to be differentially infiltrating between NPC and NCNT samples. Tumor-infiltrating B cells (B cells and memory B cells) participate in the genesis and development of NPC [22]. Gong et al. applied singlecell RNA sequencing of 66,627 cells from 14 patients with NPC and integrated clonotype identification on T and B cells. The findings suggested that the severe infiltration of dysfunctional and immunosuppressive T cells remarkably affected T cell immunity against NPC [23]. Liu et al. evaluated the potential prognostic value of tumor-infiltrating macrophages (TIMs) in patients with NPC [24]. TIMs can be divided into M1 and M2 subtypes following their phenotypes and functions. CD68 is expressed by all TIMs, whereas CD163 is a marker of the M2-like subpopulation. Additionally, CD163+ TIMs are predominantly correlated with NPC's poor prognosis, while total CD68+ TIMs are not associated with survival [25].
Deng et al. believed that tumor-related macrophages (TAMs) are a new target for the combined treatment of NPC to improve the efficiency of ICBs [26]. Our study verified that follicular helper T cells are one of the most abundant immune cells in NCNT. The proportion of infiltrating M0 and M1 macrophages was the highest in NPC. High expression of T cells in NCNT can be ascribed to low [27].
In addition to mast cells, NK cells are cytotoxic natural immune cells, which are specially employed to defend against tumor and virus-infected cells. NK cells can quickly recognize and dissolve mutant cells without antibody participation and early sensitization through the perforin-/granzyme-mediated cytotoxicity pathway or Fas/FasL-mediated apoptosis pathway [28]. Furthermore, the complex interaction between NK cells and the tumor microenvironment of NPC contributes to the prognosis of NPC [29]. Liao et al. discovered that the presence of CTL, Treg cells, neutrophils, and mast cells was associated with a poor prognosis of NPC, while a considerable number of tumor-infiltrating NK cells were correlated with a good prognosis of NPC. Meanwhile, the number of NK cells combined with mast cells can be used as a biomarker to predict the recurrence or distant metastasis of NPC [20]. Although Asians have a high content of resting mast cells and a good prognosis in lung adenocarcinoma [30], there are few studies on nasopharyngeal carcinoma. In this study, NK cells were activated, M0 macrophages, M1 macrophages, dendritic cells rested, and activated mast cells and neutrophils infiltrated in NPC. It was suggested that these cells are related to the occurrence and development of NPC, consistent with the view of Lu et al. [27].
The WGCNA analysis was conducted to find the genes related to these immune cells. A total of 371 key genes were revealed to be significantly associated with memory B cells, plasma cells, M0 macrophages, and M1 macrophages. Functional enrichment of those key genes demonstrated that they were involved in the B cell receptor signaling pathway. CD40 and B cell receptors (BCRs) are simulated by LMP1 and LMP2, respectively [31], and are tumor necrosis factor (TNF) receptors and key costimulatory receptors for B cells [32]. Kim et al. [33] reported that LMP1 is required for EBVmediated B lymphocyte transformation in EB Epstein-Barr virus-related NPC. LMP1 can activate PI3K/AKT and HIF-1α signaling pathways in EBV-positive NPC cells and function in chemokine ligand 5-(CCL5-) mediated tumor angiogenesis [34]. In our study, it was hypothesized that the B cell receptor signaling pathway may be involved in the development and metastasis of EB virus-related NPC. Next, we will explore a lot of evidence about how these genes participate in cell signal transduction in NPC.
The ROC and RT-qPCR analyses revealed that FCER2, KHDRBS2, and IGSF9 may play as potential biomarkers for NPC. There is little research on the pathogenesis of FCER2 in NPC. In B cells, FCER2 is a polypeptide gene encoded by CD23 FCER2 microsatellite [35]. The single nucleotide polymorphism of FCER2 (19p13 locus) indicates that the genetic changes of this gene may also impact the level of IgE [36,37]. IgE receptor IIFC fragment (FCER2) is expressed in macrophages, eosinophils, B cells, and platelets. FCER2 is involved in the regulation of IgE response, the growth and differentiation of T and B cells, cell adhesion, and antigen presentation [38][39][40]. As mentioned previously, memory B cells and M2 macrophages infiltrate better in NCNT than in NPC. Concurrently, the expression of FCER2 in NPC samples was significantly lower than that in adjacent tissues, reflecting that FCER2 is a downregulated gene of NPC. This would correspond to the degree of infiltration of memory B cells and M2 macrophages.
KHDRBS2 encodes an RNA binding protein involved in regulating alternative splicing. It can function as an adaptor protein during mitosis and interact with the product of the EBV early gene BSLF2/BMLF1 [41]. A comprehensive study measured EBV copy numbers in an array of lymphoblastoid cell lines derived from more than 1700 individuals and identified multiple genetic variants pointing to putatively relevant genes related to EBV infection, such as KHDRBS2 [42]. Our study demonstrated that the expression of KHDRBS2 significantly decreased in NPC samples than that in NCNT samples. It was previously reported that KHDRBS2 is associated with the coding gene of the EBV. Thus, KHDRBS2 may be involved in cell cycle control and transcription involving cell signaling pathways associated with the occurrence of EBV-related NPC. Moreover, the low expression of KHDRBS2 promotes the occurrence and development of NPC.
IGSF9 belongs to the immunoglobulin superfamily and plays a key role in inhibiting synaptic development by regulating calmodulin-like activity. Huang et al. confirmed that IGSF9 promoted the proliferation, migration, and invasion of NPC cells in vitro. IGSF9 may be a prognostic gene promoting the invasion and metastasis of NPC cells. The expression of IGSF9 in NPC cells may be affected by hypoxia [43]. Our results suggested that IGSF9 was highly expressed in NPC tissues compared with NCNT. Meanwhile, IGSF9 was strongly negatively correlated with immunostimulators C10 or f54 and strongly negatively correlated with immunosuppressants CD244, PDCD1, and TGFB1. This deepened the understanding of the signal pathway related to IGSF9 in NPC and confirmed that IGSF9 can be used as a suitable diagnostic and prognostic gene for the prognosis of NPC.
Identifying central genes and key pathways can highlight the development of NPC at the molecular level and can be used for diagnosis, prediction, and treatment research. The potential impact of these three marker genes on immunotherapy was explored, namely, the correlation analysis with immunomodulatory factors. The results demonstrated that it was significantly correlated with 8 immunosuppressive factors and 11 immune activators. In other words, these three diagnostic markers are closely related to the immune response and immunotherapy of patients with NPC. Thus, the exact role of those 3 biomarkers in the immunomodulatory signaling pathways should be further explored in the next step, so as to help develop a novel strategy for the application of those biomarkers in the immunotherapy of NPC.
Disease Markers
There are several limitations to this study. First, the data source of this study is single, and the amount of data included is not large, leading to some deviation in the analysis results. Second, our study is a retrospective study, and more prospective studies are required to verify the prognostic function of immune microenvironment-related signals. Third, although these three NPC-related genes are involved in many immune-related biological processes and signaling pathways, further functional tests should be performed to complement and clarify their specific roles in cellular signaling regulation.
In conclusion, the potential molecular mechanism of immune cell infiltration-related genes regulating NPC was explored; FCER2, KHDRBS2, and IGSF9 were identified as diagnostic markers related to immune cell infiltration in NPC for the first time; their relationship with immune regulatory factors was analyzed. Our findings would facilitate diagnosis and guide the treatment of NPC.
Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Ethical Approval
The Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) has approved this study. The processing of clinical tissue samples had been in strict compliance with the ethical standards of the Declaration of Helsinki.
Consent
All patients signed a written informed consent. Consent for publication was provided by all participants.
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Comorbidities of Chronic Urticaria: A glimpse into a complex relationship
Chronic Urticaria (CU) is a chronic inflammatory, predominantly mast cell-driven disease, characterized by the development of wheals and/or angioedema for more than 6 weeks. It affects approximately 1%–5% of the total population worldwide and imposes a substantial burden on health-related quality of life, significantly affecting patients' daily life. The economic impact on the health system is also not negligible, with an estimated cost per patient per year of approximately 2.000 $ in the United States. Although the underlying pathophysiology is not fully explored, autoimmune mechanisms have been proposed, including type I (“autoallergy” by means of autoantibodies to self-antigens) and type IIb (autoimmunity). Atopic, autoimmune, and psychiatric disorders are prevalent comorbidities in both children and adults with Chronic Spontaneous Urticaria (CSU). Although malignancies, cardiovascular diseases and other comorbidities have also been reported as associated diseases in patients with CSU, data remain scarce. It is still unknown whether the aforementioned comorbidities share common pathophysiological mechanisms with specific endotypes of CSU. The current review aims to overview current data on comorbidities of CU, and furthermore to comment on the potential linked pathways underlying these diseases.
Graphical abstract Introduction
Chronic Urticaria (CU) is a predominantly mast cell-driven disease presenting with recurrent wheals, angioedema, or both for more than six consecutive weeks (1,2). The disease is further classified into Chronic Inducible Urticaria (CIndU) and Chronic Spontaneous Urticaria (CSU), based on the presence or absence of specific causative triggers respectively (2), while 10%-30% of the patients with CU present both the spontaneous and inducible type (3).
While CU affects all age groups, it is more frequent in patients aged 30-50 years (10), and thus influences mostly young and middle-aged women (11), compromising not only the quality of life but also work productivity and emotional well-being (12,13). The socioeconomic burden is also substantial with an estimated cost per patient per year of 2,047$ in the United States and total direct and indirect costs accounting for 244$ million per year (14).
CSU is considered a chronic inflammatory skin disease and mast cells (MC) are undoubtedly the key effector cells, while various other cells and mediators are involved (15). The crucial role of basophils in CSU has recently been explored, revealing new aspects of CSU pathomechanisms (16). Blood basophil counts in patients with CSU inversely correlate with urticaria severity, and basopenia per se is linked with poor response to omalizumab treatment (17)(18)(19). Moreover, basophil infiltration has been detected in urticarial skin lesions, indicating a possible migration of these cells to the skin (20). Omalizumab administration has been associated with increased blood basophil counts and surface activation markers (21,22). Based on this observation and omalizumab kinetics regarding rapid downregulation of FcϵRI on the surface of basophils, Takimoto-Ito et al. hypothesized that activated basophils in CSU patients migrate to the skin. In contrast, inactive ones remain in the bloodstream. Upon omalizumab administration and urticaria resolution, levels of activated basophils increase in the blood, further highlighting basophils' role in CSU (16).
Although the underlying mechanisms of CSU remain largely unclear an autoimmune basis was first proposed in 1962 (23) and during the last decade two different endotypes have been described and classified as type I and Type IIb autoimmune mechanisms (24-27). In type I autoimmunity or "autoallergy", activation of mast cells is driven by an IgE mediated reaction against an endogenous allergen (autoantigen) such as thyroid peroxidase (TPO), interleukin-24, double-stranded DNA, tissue factor, thyroglobulin etc (28)(29)(30)(31). In type IIb, IgG autoantibodies, and to a less extent IgM and IgA autoantibodies, are directed against IgE or its high affinity receptor (FcϵRI) resulting in activation of MCs (28,(32)(33)(34)(35). The presence of MC activating autoantibodies can be identified by the autologous serum skin test (ASST), basophil tests (BTs) and immunoassays (32). Low total IgE levels and elevated IgG against TPO are present in type IIb autoimmune CU and are inversely correlated in patients belonging to this endotype (32) Coexistence of IgG and IgE autoantibodies against the same endogenous antigen has also been reported (36). Multiple other triggers can activate MCs resulting in different, yet unexplored, non-autoimmune endotypes of CU (37). Apart from high (FcϵRI) and low affinity (FcϵRII) IgE receptors in the surface of MCs, numerous other receptors are capable of activating MC, such as Mas-Related GPR family member X2 (MRGPRX2) for substance P, eosinophilic peroxidase and major basic protein, C5a receptor for anaphylatoxins, CRTh2 for Prostaglandin D2(PGD2), cKit for stem cell factor (SCF), cytokine receptors like IL-4Rα, IL5R, and TSLP-R, Toll-Like Receptors (TLRs) for pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Moreover, inhibitory receptors like Siglec-8 and CD200R exist on the surface of MCs (38-40). Endothelial cells and the coagulation system have also been implicated in CU pathogenesis (40), as well as the dysregulation of intracellular signals within mast cells and basophils (37, 41). Moreover, aggregation and stacking of highly lipophilic IgE molecules can result in crosslinking of FcϵRI in the absence of antigen binding (42).
Pruritus, pain and burning sensation of wheals and angioedema can result in anxiety, stress, sleeplessness, poor self-esteem, shyness, anger, and social isolation (43,44). Furthermore, patients' quality of life is further compromised by the coexistence of CU with a broad spectrum of comorbidities, such as sleep disorders, anxiety, depression, other psychiatric disorders, autoimmune diseases, atopic diseases, cardiovascular disorders, and less frequently malignancies (9, 45-47) ( Figure 1).
The above-mentioned data on CU pathophysiology and the underlying immune pathways has raised the interest for a more holistic approach of CU; to this end, both epidemiological data and possible common pathophysiological mechanisms linked to CU comorbidities are of major interest. In the present review we aim to overview data on the complex interplay between CSU and associated comorbidities, apart from CIndUs, and comment on their potential relationships in terms of underlying mechanisms.
CSU and autoimmunity
CSU as an autoimmune-autoreactive skin disorder per se, often coexists with a variety of other autoimmune diseases (37). Overall, approximately 30% of CSU patients present with at least one autoimmune disorder, while 2% may have two or more autoimmune disorders, with Hashimoto's disease and vitiligo presenting more frequently as co-existent diseases (48).
It has been recently acknowledged that the type IIb autoimmune CSU, as assessed by positive ASST, BHRA and/or BAT and identification of specific IgG antibodies against FcϵRI/IgE, is highly related with other autoimmune diseases (48).
Thyroid diseases
IgG autoantibodies against thyroid peroxidase (TPO) have been identified in up to 50% of CSU patients, with 5-to-7-fold increased risk of presenting anti-TPO antibodies in CSU patients compared to controls, while increased levels of IgE antibodies against TPO have also been detected in those subjects (49,54). Thyroid dysfunction disorders, such as hypothyroidism and Hashimoto's thyroiditis, are also reported more significantly in CSU adult patients than healthy controls (49).
First, Rumbyrt et al. suggested that the inflammation in the thyroid gland can lead to a generalized inflammatory response with a subsequent complement activation along with activation of mast cells, mainly through anaphylatoxins receptors (55). Moreover, the recognition of IgE antibodies against TPO as a cause of Type I autoimmune CSU has further enhanced the link between thyroid dysfunction and CSU (25, 56). In line, although a causative role of IgG antithyroid autoantibodies on the occurrence of CSU has not been demonstrated (57-59), IgE antithyroid autoantibodies have been implicated in the formation of immune complexes, and activation of complement system, potentially facilitating activation of MCs and subsequent clinical expression of CSU (49).
Although conflicting evidence exists, especially in euthyroid patients with CSU, data support the efficacy of levothyroxine or other thyroid drugs on CSU morbidity, potentially by reducing inflammatory thyroid pathways mediating mast cell activation (49).
Other autoimmune diseases
In a large registry-study from Denmark including more than 12.000 CU patients, rheumatoid arthritis was reported as the most prevalent autoimmune comorbidity (1.7%), while thyroiditis (0.3%), vitiligo (0.1%) and Systemic Lupus Erythematosus (SLE) (0.3%) were also identified, although to a lower extend. Of note, it cannot be excluded that the high prevalence of RA might be attributed to the high prevalence of the disease per se, in relation to the other autoimmune diseases. At the day of the diagnosis, rates of vitiligo and SLE were significantly higher than in the control group (OR = 5. Additionally, it is well known that Urticarial rash is common in patients with Systemic Lupus Erythematosus (SLE), ranging from 0.4%-27.5% in adults and in 4.5%-12% in children as shown in the meta-analysis by Kolkhir et al. Data on the vice versa relationship is scarce. It has been proposed that the underlying pathogenetic mechanism associating both diseases might include the activated complement and coagulation system, linking inflammation and autoimmunity (60).
Autoimmune diseases in paediatric population
The prevalence of autoimmune diseases in children with CU is diverse, ranging from 0%-16% (61, 62). A prospective study in Canada, evaluating the prevalence of autoimmune diseases in children with CSU, demonstrated an increased prevalence of autoimmune diseases, such as hypothyroidism, lupus, juvenile rheumatoid arthritis, and type I diabetes compared to the general paediatric population (2.10% vs.0.13%, 0.52% vs.0.005%, 1.05% vs. 0.053% and 1.57 vs. 0.19% respectively) (63). Nevertheless, the overall prevalence of autoimmune diseases in children with CSU was relatively low (<5%), thus evaluation for autoimmune diseases is proposed only when a suggestive clinical history and/or laboratory findings are present (63). Moreover, autoimmune hypothyroidism was observed in older children with CSU and with increased CD63 levels, a well-established marker of IgG-mediated autoimmunity, potentially attributed to the impact of epigenetic changes, due to environmental factors, on the development of inflammation and autoimmunity with increasing age (63).
In respect to the prevalence of atopic diseases in children with CSU, studies have shown an increased occurrence compared to autoimmune diseases while in adults respective rates are either similar or even lower (49,51,63,64). Moreover, in agreement with recent finding linking autoimmune type IIb endotype with higher prevalence of other coexisting autoimmune diseases in adults, elevated levels of CD63, may propose such a relationship in children as well (48,63,65).
A systematic review reported that positive ASST, identifiable antinuclear antibodies (ANA) and thyroid biological abnormalities were present in 36.8%, 6.4% and 10.4% of children <12 years with CSU respectively (66), supporting further the presence of a type IIb autoimmune endotype in children. The lower rates of thyroid function abnormalities are in line with the observation that autoimmune mechanisms are evolving and may manifest several years after the initial diagnosis (66). However, whether children with positive ASST and ANA need to be screened for autoimmune diseases is a matter of debate (67, 68).
The importance of identifying autoimmune comorbidities in patients with CU Specific endotypes of CSU are linked to comorbid autoimmune diseases, and thus early diagnosis and therapeutic intervention of associated diseases may be beneficial in the multidisciplinary therapeutic approach as suggested by EAACI/GA 2 LEN/ EuroGuiDerm/APAAACI Guidelines (2, 48). In the era of precision medicine, knowledge of a patient's profile, shaped not only by CU per se but also by the various coexisting diseases, may lead to targeted, personalized interventions (69,70). As new therapeutic options are developing, identifying the presence of comorbid autoimmune diseases is of importance, since they can interfere with CSU activity, duration, natural course, and response to treatment (69,71). Thus, in the updated CU 2022 guidelines the measurement of IgG anti-TPO and total IgE in all CSU patients is strongly supported to identify autoimmune thyroiditis and to untangle the underlying endotype (2, 32).
CU and atopic diseases
Atopic diseases have been commonly reported in CU patients. The results from the Scandinavian arm of the AWARE study, showed that atopic diseases are the most frequent comorbidities in a cohort of 158 adult patients with CU. In specific, asthma was reported in 19.6% of the patients, allergic rhinitis in 16.5%, atopic dermatitis in 6.3% and food allergy in 8.2% (11). Higher rates of sensitization -approximately 40%-to at least one inhalant or food allergen have been reported by Zuberbier et al. in a general German population with CU (72), while allergic rhinitis and asthma were among the five most common comorbidities among CU patients in a large Korean study (73). In agreement, Ghazanfar et al. found that atopic diseases like rhinoconjuctivitis and atopic dermatitis are overrepresented among CU patients with an increased risk of developing atopic diseases following CU diagnosis (HR = 3.09, CI 2.0-4.8 for atopic dermatitis and HR = 1.4, 0.75-2.55 for rhinoconjuctivitis) (51).
With regards to the pediatric population, a personal history of atopic dermatitis in children has identified as a risk factor for subsequent CSU development, (OR 2.92, 95% CI 1.64-5.18, p < 0,001) in a pediatric population (74). In addition, in a recent systematic review evaluating comorbidities and interventions in children younger than 12 years with CSU, including 522 patients with CU (or CSU), atopic diseases were found in 28.1% of the population with a reported prevalence of 15.4% for asthma, 13.8% for allergic rhinitis and 9.4% for atopic dermatitis respectively (66). In agreement, Lachover-Roth et al. in a retrospective study of 250 children with CSU showed that atopic diseases were significantly more prevalent in children with CSU than in the general paediatric population, with one out of three children suffering an atopic comorbidity (17.2% atopic dermatitis, 16% allergic rhinitis, 13.2% asthma and 3.2% food allergy) (75). Allergic sensitization, as assessed by total IgE has been identified in almost 30% of children with CU, irrespective of relevant clinical symptoms (76). Moreover, 24 out of 77 children with CU were described as atopic with presence of allergen specific-IgE to at least one allergen. Importantly, total levels of IgE were positively associated with disease duration. (r = 0.262, p = 0.021) (77). In CU adults, high IgE levels correlated with disease severity and duration, but not the clinical course of the disease (64,78).
Despite the robust epidemiologic association between atopic diseases and CU, both in adults and children, no causal relationship has been established so far, thus therapeutic interventions for allergy-associated symptoms have no effect on the natural course or severity of CSU and vice versa (75, 79). Nevertheless, a TH2 endotype in CSU patients, especially children, with atopic diseases along with high IgE levels, which in turn are associated with type I autoimmunity or "autoallergy" and IgE autoantibodies detected in CSU patients, has been suggested (26,34,42,75).
CU and psychiatric disorders
Psychiatric and mental disorders are quite frequently reported among CU patients, in the literature (80)(81)(82)(83). A recent systematic review and meta-analysis reported that almost one out of three CU patients have at least one underlying psychiatric disorder (84). Sleep-wake disorders, followed by anxiety and mood disorders, including depression are frequently identified (pooled prevalence 36.7%, 30.6% and 29.4% respectively). Trauma and stressor related disorders, somatic symptom and related disorders, obsessive-compulsive and related disorders and substance-related and addictive disorders were also reported. Regarding CU severity, duration, and mental functioning, no association has been demonstrated. Konstantinou et al. conclude that none of the studies included in the systematic review clearly stated whether psychiatric disorders pre-existed or follows CU diagnosis (84).
Data from the Danish National Patient Registry (n = 12.185 CU patients) found that CU patients were at increased risk of presenting depression, while a marginally increased risk for presenting psychosis was observed over time [HR adjusted = 1.38 (0.99-1.93) in CU patients] (51). Affective disorders (27.0%) were frequently in adults with CU in a cross-sectional study in Germany; of interest, in pediatric CU patients somatoform disorders were the most frequently reported comorbidities (7.7%), following rhinitis (24.7%) and asthma (20.2%) (9). Recently, Lachover-Roth et al. found a prevalence of 2.8% with respect to psychiatric disorders in a retrospective study of children with CSU (n = 380); depression, anxiety, bipolar disorders, and schizophrenia were identified (75).
Anxiety disorders are also prevalent in CSU patients compared to healthy controls (9.6% vs. 5.7%, p < 0.001), with a strongest association observed between anxiety, younger and higher socioeconomic status subjects (85). Moreover, anxiety can negatively correlated with social functioning (86).
Both anxiety and depression were negatively correlated with Quality of Life assessed by Chronic Urticaria Quality of Life Questionnaires (CU-QoL) (87).
Although a number of studies reports increased frequencies of depression and anxiety among CU patients (48,1% and 38% respectively) other reports show lower levels (11); discrepancies are potentially attributed to selection bias, heterogenous population and diagnostic criteria regarding diagnosis of psychiatric disorders (11).
Suicidal ideation is also reported in patients with CU (84 The underlying pathogenetic mechanisms are unclear, although a potential interplay between the immune and central nervous system has been reported (91). A "brain-skin connection" may contribute to inflammatory skin diseases like CU, with stress causing aggravation of urticaria (92,93). Moreover, a causal relationship between stress and inflammatory disorders, including CU, has been reported (94, 95). It has been postulated that chronic inflammation can dysregulate the immune and the central nervous system, resulting in mental disorders (96). The role of substance P, through neurogenic inflammation in acute stress has been described (97). Substance P is produced by a variety of inflammatory cells and is implicated in the release of histamine and serotonin from mast cells (98). In accordance, in a study evaluating patients with CSU and depression levels of Substance P were higher in CSU with depression than those without, but no dissimilarity was observed between CSU and healthy controls (99).
As CU has a debilitating effect on quality of life and productivity, data are inconclusive on whether psychiatric disorders affect or are affected by CU (84). Albeit case series have reported that pharmacological interventions with antidepressants and anti-anxiety drugs may have a beneficial impact on CU (100,101).
It is advised that CU patients be evaluated for phycological disorders and be treated accordingly.
CU and malignancies
The association between CU and malignancies remains controversial (37). The first implication of a causal relationship between CU and cancer was described in 1942, when the removal of a rectal carcinoma in a 70-year-old male was associated with CU remission (102). Since then anecdotal cases of urticaria linked to malignancies have been reported in the literature (103).
Neoplasms have been reported to promote both chronic spontaneous and inducible urticaria in a systematic review, suggesting a linkage. The most frequently reported cancers in CSU patients are carcinomas (68%) with 24% of all cases being papillary carcinomas of the thyroid gland (103). In agreement, Napolitano et al., in a retrospective populationbased study of 1,493 patients with CU, reported that CU was associated with cancer in 0,007% of the population, while CSU in those patients is (a) antihistamine resistant, (b) resolves after chemotherapy, or tumor removal, (c) can reoccur upon cancer relapse and (d) presents 2 to 8 months before malignancy diagnosis (103, 104). In accordance, a large registry study from Taiwan reported an increased risk of cancer in patients with CU (standardized incidence ratio 2.2; 95% CI 2.0-2.3). The risk was even higher for hematologic malignant tumors (SIR = 4.1, 95% CI, 3.1-5.4) and non-Hodgkin lymphomas (SIR = 4.4,95% CI, 3.0-6.1) (105). Moreover, two additional cases of urticaria remission after colorectal cancer removal are also reported in the literature, suggesting that urticarial lessons may manifest as a paraneoplastic phenomenon (106, 107). The incidence rates of CSU were statistically significantly higher for neoplasms (adjusted HR 1.14, 95% CI 1.02-1.27) in a population-based study in Italy (108). Non hematological neoplasms were among the most common comorbidities in a large Korean population-based study with the likelihood of occurrence 1.37 higher than in patients without CU. Stomach, thyroid and liver cancer were the most common neoplasms in CU patients while thyroid, liver and prostate in the CSU subgroup (73). In contrast, data from a Swedish registry showed no association between cancer and CU (109).
As urticaria and cancer are common diseases in the general population, they can incidentally coexist, although the immediate CU resolution following cancer remission and the reoccurrence upon relapse suggests causality (104). Neoplasms may induce immune dysregulation and activate coagulation and complement system, while the release of tumor-derived antigens detected by IgE can cause cross-linking of highaffinity IgE receptors in mast cells' surface, inducing degranulation (110-113).
Despite the reported cases in the literature, the overall rate is quite low among CSU patients and hence, the international EAACI/GA 2 LEN/ EuroGuiDerm /APAAACI guidelines suggest not to routinely screen for malignancies as potential underlying causes of CU (2, 114).
A careful clinical examination and history are essential for this rare relationship to be exposed in a cost -effective way.
CU and hypertension, hyperlipidemia, metabolic syndromes, and cardiovascular disorders
The relationship between CU and cardiovascular diseases is unclear. A retrospective population-based cohort study in Denmark found no association between CU and cardiovascular diseases (115). On the contrary, a prospective study showed that systemic hypertension was associated with urticaria persistence (hazard ratio, 0.71; 95% CI 0.53-0.95; p = 0.02) (110), while hypertensive and lipoprotein metabolic disorders were among the more frequent reported comorbidities (43.5% and 32.1% of CU adult population respectively) in a recently published cross-sectional German study (9), and in a Swedish registry based-study (12% and 17% respectively) (116).
Metabolic syndrome was reported in 29.8% of patients with CU compared to 17.8% in a matched control group (p = 0,001) in a Korean cohort study and was independently correlated with uncontrolled urticaria, as assessed by total urticaria activity score. Larger waist-circumference, as a marker of obesity, was more prevalent in subjects with CU, and significantly associated with IgE, Eosinophilic Cationic Protein (ECP) and Tumor Necrosis Factor-a (TNF-a) levels (117), while a postive association between CU and obesity was shown in a large population-based Italian study (adjusted HR 1.40,95% CI 1.17-1.67) (108). Moreover, hyperlipidemia has been identified as a risk factor for CU development (OR 1.97 95% CI: 1.85-2.09) (118).
The Scandinavian arm of the AWARE study also reported a prevalence of obesity and hypertension at 7% and 1.9%, respectively, among an adult CU population with half of the patients being overweight (BMI > 25) (11).
Similarly, a pediatric cohort with CU from Spain, Italy, Germany, France, and the UK manifested significantly higher BMI compared to the control group (119).
CU is a chronic inflammatory disease presenting with low grade systemic inflammation (37). Hence, although an increased ratio of cardiovascular diseases derived from atherosclerosis could be partially explained by the inflammation stage in CU patients, data by Egeberg et al. report otherwise (115). The relatively short duration of CU may not be sufficient to increase the risk of presenting cardiovascular diseases (8,120). However, alterations in lipid metabolism and co-occurrence of obesity can result in immune system dysregulation and presentation of autoimmune diseases (121, 122), with a subsequent activation of mast cells resulting in CU clinical presentation. Nevertheless, this hypothesis is far from well-established and further studies are needed to unravel the potential relationship between urticaria, hyperlipidemia, obesity, and cardiovascular diseases.
CU and other comorbidities
Although less common, a variety of other associated diseases have been reported in patients with CU.
Osteoporosis and diabetes mellitus were found in 2.9% and 2.3% of 12.185 CU patients respectively (51). It is speculated that corticosteroid use plays a significant role as, despite current guidelines recommending against their use, they are still prescribed by physicians (2, 123, 124). The same study reported increased risk of having or achieving mastocytosis and anaphylaxis in the CSU group. However, the adjusted HR decreased when the diagnosis of these diseases within the first year were excluded, supporting a possible misdiagnosis before patients were referred to specialized centres (51). Drug allergy has also been identified to co-occur with CU with a likelihood of 4.68 times higher than in patients without CU (73).
Inflammatory diseases were the most prevalent comorbidities identified in a population-based study in Taiwan, with peptic ulcer (4.83%), hepatitis B or C (1.64%) and periodontitis (2.82%) presenting more frequently. In patients with persist CU, an increasing prevalence of inflammatory diseases was observed, indicating a possible link between inflammation and endurance of CU (125).
Back pain, acute upper respiratory infections, noninflammatory disorders of the vagina, spondylosis, and gastritis were among other rare disorders detected by using the anonymized research database of the Institute for Applied Health Research in Berlin, including insured individuals with a diagnosis of CU (9).
Additionally, a systematic review assessing the relationship between CSU and Vitamin D levels revealed that Vitamin D levels in 12 out of 14 included studies were significantly lower in CSU patients compared to controls (34.3%-89.7% of CSU patients and 0%-68.9% in controls). No causal relationship was identified, although supplementation of vitamin D for 1-3 months might have a beneficial effect in CU course (126). In accordance, a systematic review assessing comorbidities in children with CU found low vitamin D levels in 69.1% of the children (66); however data from other studies are not confirmatory (64).
Conclusion
CU presents with a wide range of associated comorbidities. Autoimmune, psychiatric, and atopic diseases are the most frequently reported associated diseases among CSU patients. Although the link between specific comorbidities and CU is solid, the potential interplay, regarding the nature of cooccurrence, is a recently explored era. The existing data cannot provide evidence in order to elucidate whether those diseases circling CU coexist independently with it or if a causal relationship, deriving from shared pathogenetic mechanisms, exists. Besides, if this is the case, a further unanswered question would be whether therapeutic interventions regarding comorbidities could interfere with CU's clinical course and vice versa. Therefore, prospective well-designed studies addressing the impact of various comorbidities on CU course and severity, as well as the impact of therapeutic interventions of comorbidities in both CU activity and natural course, are of urgent need. As we are marching into the era of personalized medicine, patients with CU should be recognized as a multimorbid group, and management should involve recognizing and treating any comorbid disorders in addition to urticaria management.
Author contributions
All authors contributed equally to the manuscript preparation. All authors contributed to the article and approved the submitted version.
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Opportunities and challenges in interpretable deep learning for drug sensitivity prediction of cancer cells
In precision oncology, therapy stratification is done based on the patients’ tumor molecular profile. Modeling and prediction of the drug response for a given tumor molecular type will further improve therapeutic decision-making for cancer patients. Indeed, deep learning methods hold great potential for drug sensitivity prediction, but a major problem is that these models are black box algorithms and do not clarify the mechanisms of action. This puts a limitation on their clinical implementation. To address this concern, many recent studies attempt to overcome these issues by developing interpretable deep learning methods that facilitate the understanding of the logic behind the drug response prediction. In this review, we discuss strengths and limitations of recent approaches, and suggest future directions that could guide further improvement of interpretable deep learning in drug sensitivity prediction in cancer research.
Introduction
Cancer is a disease with multiple levels of complexity and the leading cause of mortality in EU countries after cardiovascular diseases (Nagai and Kim, 2017). First, for a given tumor entity, there is significant molecular variability across patients, which is referred to as inter-patient tumoral heterogeneity (Sanchez-Vega et al., 2018). Second, variability can also be observed within tumors, also termed as intra-tumoral heterogeneity (Marusyk et al., 2012). Third, under therapeutic pressure, tumors adapt through (epi-) genetic changes, which is defined as temporal heterogeneity (Venkatesan et al., 2017;Yu et al., 2021). With this knowledge in mind, therapy has gradually shifted from a one-sizefits-all approach towards precision oncology. The latter involves analysis of (epi)genome, transcriptome and proteome biomarkers that inform clinicians about the molecular characteristics of the tumor before and after therapy and aids in an improved diagnosis, prognosis, therapy stratification, and monitoring of the patient (Schwaederle et al., 2015).
However, an important challenge in precision oncology is how to predict the drug sensitivity of a certain tumor based on its molecular make-up.
As it is ethically and practically unfeasible to compare the sensitivity of a panel of drugs on human cancer patient, different types of patient-derived cancer models such as cell lines, organoids, and patient-derived xenografts (PDX) are used instead. Cancer cell lines are the easiest to handle, and in general they also recapitulate the (epi)genetic and transcriptomic alterations as observed in the actual tumors (Kinker et al., 2020). These features make cell lines a widely used platform for drug screening (Tables 1 Tables 2 Tables 3). Large panels of cell lines that include a wide range of cancer types have been characterized at different omics levels (Barretina et al., 2012;Ghandi et al., 2019;Nusinow et al., 2020). Also, drug response profiles have been determined on these cell lines for a broad range of drugs (NCI60 (Shoemaker, 2006), CCLE (Barretina et al., 2012), GDSC (Garnett et al., 2012;Yang et al., 2012), CTRP (Rees et al., 2016), gCSI (Haverty et al., 2016)). These rich data resources can be harnessed to associate the phenotype (drug response) with the genotype of the tumors. Tables 1 Tables 2 Tables 3 provides a systematic view of the data resources available in pharmacogenomics that are supportive for predictive modeling of cell lines for drug sensitivity.
To train computational prediction models for drug sensitivity, both omics and drug sensitivity data of cell lines are used. The trained models can afterwards be applied on omics data of human tumors to predict drug vulnerabilities for individual patients (Lee et al., 2007). Various computational strategies are used to develop drug sensitivity prediction models, mainly machine learning based methods which include matrix factorization, support vector machines, random forests, and deep learning (Dong et al., 2015;Kim et al., 2019;Lind and Anderson, 2019). These methods generally use molecular information (omics data for a certain panel of genes) of the cell lines with structural and molecular information of the drug as input features, and the drug sensitivity as output/label for training (illustrated in Figure 1 for deep learning) (Menden et al., 2013).
Deep learning (DL) is a subset of machine learning that is based on artificial neural networks (ANN) (Goodfellow et al., 2016). The name and structure of ANN are inspired by the brain, simulating the way the biological neural network system works to process stimuli. An ANN is composed of an input layer, one or more hidden layers, and an output layer. When the ANN is composed of many hidden layers, it is referred to as a Deep Neural Network (DNN). One of the advantages of DNNs is that they can handle high-dimensional and noisy data. Furthermore, they learn input-output relationships incrementally through their hidden layers by transforming low-level features (raw data) to high-level features (embeddings), capturing nonlinear and complex relationships, which then are useful for output prediction. These capabilities give them superior predictive performance compared to many conventional machine learning algorithms. However, in most DL approaches, the data transformation inside the neural network is very complex and lacks interpretability. Therefore, the DL models are black box models, where the logic behind the predictions is hidden.
In this article, we review the recently published interpretable deep learning methods for drug sensitivity prediction that can be applied in precision oncology. We present how the interpretability of DL models could be an added value in rendering biological insights. We briefly introduce interpretability techniques and also describe and comment on interpretable DL methods for drug sensitivity prediction which include DrugCell (Kuenzi et al., 2020), HiDRA (Jin and Nam, 2021), PathDNN (Deng et al., 2020), PaccMann (Manica et al., 2019), consDeepSignaling , DEERS (Koras et al., 2021), ParsVNN (Huang et al., 2021), DNN (Sakellaropoulos et al., 2019) and SWnet (Zuo et al., 2021). We address the factors that account for both strengths and limitations of these methods and discuss suggestions for further improvement.
Interpretable deep learning
Despite the superior predictive performance of DL models, the lack of interpretability is an important shortcoming compared to other machine learning models. The model interpretability can be an essential added value. Firstly, it can explain how the model processes the input data to make the prediction. In addition, it can indicate if the model is paying proper attention to crucial input features which potentially influence the output prediction. Lastly, in case of prediction errors, it can explain why and how a model has malfunctioned and point to biases or artifacts present in the input data (Explainable, 2019). These interpretability features, in case of a drug sensitivity prediction model, can provide mechanistic insights to explain the drug mechanism of action (MOA) and the confidence in the prediction system. Hence, it will help the clinician with risk management for designing clinical trials, based on consistency between their expert knowledge and the model interpretability.
In recent years, many efforts have been made to develop strategies and techniques to interpret black box DL models. Three commonly used strategies are probing, perturbation, and surrogation (Azodi et al., 2020). The probing of a model is meant to discern the logic that the model has learned during the training. The perturbing strategy involves removing an input feature to see the effect on the output. The surrogating strategy is based on using an inherently interpretable model (e.g. a linear model) to approximate a black box model. Based on the intrinsic properties of a model, the interpretation can be performed at different levels, i.e. global, semi-global and local, and at different stages i.e. ad hoc and post hoc (see Table 4 for definitions).
Nearly all the interpretable DL based drug sensitivity prediction studies available are based on the probing strategy. Since it is easier to describe the interpretability strategy using example methods, we only focus on the probing strategy in this review. The reason why probing is widely applied is that it can explain how the input data is processed inside the neural network, what input features got more attention to predict a specific output, and that it can justify the relevance of transformed data at each node/layer of the neural network (explained in Figure 2). There are mainly three techniques used in the probing strategy i.e. embeddings, weights, and gradients, which we will address in detail in the next section.
Three classes of probing based interpretable deep learning Embeddings
The embedding at a neuron can be regarded as the representation of inputs of the neuron in the form of its output. Therefore, the embedding at neuron can be represented by the neuron output. The neuron embedding sometimes, is also referred to as the neuron state or the activation level (Azodi et al., 2020;Deng et al., 2020;Kuenzi et al., 2020;Lin and Lichtarge, 2021). The output of the neurons can be used as a means to interpret the model and this is useful when the complete or a part of the ANN architecture is based on some constrained connections between the neurons e.g. some form of biological organization where a neuron node or neurons in a layer represent an actual pathway or biological process (illustrated in Figure 2). Kuenzi et al. developed Drugcell for predicting drug sensitivity in the context of cancer by adopting principles of the previously published method DCell which predicts yeast cell growth from its gene deletion genotype (Ma et al., 2018). The DrugCell model is developed to fulfil two objectives. The first objective is to predict drug response for a given drug on a given cell line using mutational profiles of the cell line and the chemical structure of the drug as features. The second objective is to use the trained model to explain the drug MOA, suggesting important pathways implicated in the drug response. The DrugCell model architecture consists of two ANNs, one for the genotype and another for the drug structure that are combined to predict the drug sensitivity. The ANN for the drug structure is made up of fully connected layers (i.e. not constrained). The ANN for the genotype is structured in such a way that it mirrors the hierarchy of biological processes inside a cell, by constraining the connections inside the ANN using the Gene Ontology hierarchical information. The constrained connections help to represent the hidden layers as actual biological processes and render them biologically interpretable.
DrugCell
After the model is trained on the whole dataset, a post hoc interpretability analysis is performed by computing the Relative Local Improvement in Predictive Power (RLIPP) score for each of the GO biological process term layers in the genotype ANN. The RLIPP scoring system gives a semi-global interpretation, in which it ranks a set of GO terms as important signatures for a specific drug across all cell lines (cancer types). However, this
FIGURE 2
An illustration to show how embeddings at neurons of a biologically constrained ANN can be used for interpretation. In the left part, the densely connected ANN (black box model) is very complex, which makes it difficult to understand how the input is processed to arrive at the output. In the right part, the sparsely connected ANN based on some biological hierarchy of genes, pathways and biological processes makes the data processing visible and also explains the relevance of each neuron and its output. For example, the node of Gene 3 has a high activation level, and the signal only from the Gene 3 node is passed to the Pathway 2 node. The latter has a low activation level which can be interpreted that the pathway is inactive, and Gene 3 has a suppressing effect on Pathway 2.
Frontiers in Bioinformatics frontiersin.org RLIPP scoring may not be a good way to rank GO biological processes in all cases because scores of biological processes may not be comparable among each other in order to identify the top processes (illustrated in Figure 3). The authors validated the model interpretability using external RNA-seq data of the 25 most sensitive versus the 25 most resistant cell lines against the drug docetaxel. For this, they performed a differential gene expression analysis and GO Biological Process enrichment using DAVID (Huang et al., 2009a;Huang et al., 2009b). The obtained list of overrepresented pathways was then compared with the list of pathways obtained from the model post hoc analysis and notably, they were found to be distinct. However, the authors claimed that the experimental validation was convincing. As the model suggested "Response to cAMP" as a top pathway, they treated A427 cells with three different treatments-paclitaxel, the glycolysis inhibitor 2-deoxyglucose, or a combination of the two, and found that the combination was substantially more effective than either individual compound.
HiDRA
Jin et al. introduced a DL model called Hierarchical network for Drug Response prediction with Attention (HiDRA) to predict drug sensitivity of cancer cell lines (Jin and Nam, 2021). In the ANN architecture, they employed a hierarchical attention network that showed highly attended biological pathways and their member genes related to the drug response. The hierarchical attention here means while passing input data from gene-level to pathway-level and then from pathway-level to output in the neural network, the attention mechanism figures out which part of the data is more important than others at the respective levels for the prediction. The model consists of four ANNs: a drug network, a gene-level network, a pathway-level network, and a response prediction network. The gene-level network and pathway-level network have an attention module for calculating the importance of genes and pathways, respectively. The model uses gene expression profiles as features to represent the cell lines and the drug structure in the form of hashed Morgan fingerprint as features to represent the drugs.
FIGURE 3
An illustration of how the RLIPP score can be non-informative in a given setting. The GO biological processes BP4 and BP5 are at the same hierarchical level and their respective RLIPP scores are computed. Although the predictive power of BP4 is greater than BP5, BP5 has a greater RLIPP score than BP4. Therefore, a high RLIPP score for a biological process does not guarantee that it will be more informative and important for the prediction, rather it will only say how much the biological process has more or less predictive power than its children processes. The color scheme of the nodes in the neural network represents the flow of signals from the children biological processes to the parent biological process and the color fusion symbolizes the fusion of signals from the children to the parent node. Each node here represents a layer in the genotype ANN and the name, and the structure of the layers are based on GO hierarchy of biological processes.
Frontiers in Bioinformatics frontiersin.org
HiDRA suggests a set of pathways and the pathway member genes that are important behind a specific cell line-drug response prediction: hence this approach performs interpretation at the local level. This is realized by looking at the attention scores of pathways and their member genes derived from the pathwaylevel network and the gene-level network, respectively.
The validation of the interpretability was done by performing a case study with Gefitinib and Rapamycin on the LB2241-RCC cell line. The log fold-change of the genes' attention score was computed for Gefitinib in comparison to Rapamycin. It was found that the target genes of Gefitinib had high positive log foldchange, whereas the target genes of Rapamycin had high negative log fold-change, inferring that the respective target genes received more attention and are important for the respective predictions. The case study only showed a gene-level analysis, skipping pathway level analysis. Moreover, the attention scores were not directly used (contrary to the claim), rather their relative change between two drugs was used to identify the important genes behind the drug sensitivity prediction.
PathDNN
Deng et al. developed a Pathway-guided Deep Neural Network (PathDNN) to predict the drug sensitivity in cancer cell lines (Deng et al., 2020). The ANN architecture is structured by constraining the connections between the gene layer (input layer) and the pathway layer (first hidden layer) based on genepathways relations obtained from the KEGG pathway database. The model input layer is divided into two parts. The first part takes gene expression data of a certain set of genes, representing the cell line features. The second part takes binary data of a certain set of genes indicating whether they are drug targets or not, to represent drug features. The drug targets were retrieved from the STITCH database (Kuhn et al., 2007).
The model interpretation is based on post hoc analysis of the neuron output of the pathway nodes in the pathway layer. A comparison is made between the output of the pathway nodes with drug treatment and those without drug treatment and quantified in terms of log2 fold-change. The no-drug treatment setting is made by setting drug features equal to zero. For a given cell line-drug pair, the log 2 fold-change is computed for all pathway nodes and the pathways with the highest log 2 fold-change are considered as important pathways responsible for drug response prediction. The model is locally interpretable as it can explain a single cell line-drug pair.
The authors validated the interpretability performance of their method with a case study of eight rhabdomyosarcoma cell lines treated with the CTK7H7014 drug. One important remark is that the specified drug cannot be found back in any drug database. Moreover, no gene target information is available for the drug and therefore the mode of action is not known. In the case study, the log 2 fold-change (drug vs. no-drug) of the neuron output of 323 pathways was computed for each of the eight cell lines. This analysis revealed that the Hsa05202 pathway, which is related to rhabdomyosarcoma, frequently occurred among the top pathways across the eight cell lines that were treated with the drug. However, the other top pathways were not further discussed in the study.
PaccMann
Manica et al. adopted their previous work on the Prediction of AntiCancer Compound sensitivity with Multimodal Attention-based Neural Networks (PaccMann) and modified it with a novel architecture that uses attention based multiscale convolution encoders (Manica et al., 2019). The model uses two encoders, a gene expression encoder and a SMILES encoder (Weininger, 1988). The input to the gene expression encoder is gene expression profiles of a set of most informative genes. These genes are selected using a network propagation scheme carried out on the STRING PPI network. For each drug, the weights initialized (=1) to the reported drug targets are diffused over the STRING network, resulting in an importance distribution over the genes. The important genes obtained for each of the drugs are merged to form a set of most informative genes. The SMILES encoder takes SMILES embeddings plus the output embeddings from the gene expression encoder as input. The output from both the encoders are further connected to a feedforward layer to predict drug sensitivity. As a result of using the output of the gene expression encoder into the SMILES encoder, for a given cell line across all the drugs, the gene attention values remain constant and the attention values over SMILES encoding change.
The model interpretability is based on a post hoc analysis of gene attention values from the gene expression encoder. The genes that are frequently attended across all cell lines are used for pathway enrichment analysis to investigate which pathways are induced by the drugs. The analysis identified that the apoptosis signaling pathway is elicited in general by all anti-cancer drugs present in the dataset. Since this interpretation assumes that every drug has the same drug MOA across all cell lines, it gives a global interpretation.
The authors presented a case study to validate the method based on the sensitivity of the MEG-01 cell line for Imatinib and Masitinib. They showed how the model predicted differently between two different drugs on the same cell line. Based on attention values over the SMILES encoding, they determined what molecular substructures of the drug were important for the sensitivity prediction. They also aimed to show what genes were important for the prediction for a given cell line-drug pair. However, this may be misleading because for a given cell line the model will show the same attended genes across all drugs, so it will not be possible to distinguish drug-specific MOA among different drugs.
Gradient
The feature importance score based on gradient is determined by calculating the change in the predicted Frontiers in Bioinformatics frontiersin.org 07 output upon a small change in an input feature, using partial derivatives (illustrated in Figure 4). The gradient-based approach has the limitation that it is not useful when small changes in the feature value have no effect on the output prediction. Moreover, individual features may sometimes not have any effect on the output but may have an effect when combined. Therefore, this approach only explains the individual feature relationship to the output, which is a second limitation. However, this technique is applicable to all kinds of ANN architecture and hence it is a flexible approach.
consDeepSignaling Zhang et al. developed a constrained neural network architecture guided by signaling pathways, called consDeepSignaling for performing drug response prediction on cancer cell lines . The ANN is structured by constraining the connections between the input layer and the pathway layer (first hidden layer) based on genepathways relations obtained for all available 46 signaling pathways from the KEGG pathway database. The model uses a set of genes related to the pathways as features, where each gene is represented by its gene expression, copy number variation, and drug target-gene binary call.
The post hoc interpretability analysis of the model is based on investigating the importance of signaling pathways behind the drug response prediction using the SmoothGrad model in the "iNNvestigate" python package (Alber et al., 2018). This tool is used to extract the gradients of the pathway layer in the trained model to score the importance for each of the pathways. The model interpretability does not explain the drug MOA but rather reported what signaling pathways are important across all predictions, thus providing a global interpretation.
The publication of Zhang et al. did not attempt to include validation for the interpretability performance of the model.
DEERS
Koras et al. developed a neural network recommender system for kinase inhibitor sensitivity prediction, called Drug Efficacy Estimation Recommender System (DEERS) (Koras et al., 2021). The ANN architecture contains two autoencoders to represent cell line and drug features into low dimensional representation and a feed forward ANN to combine them for drug response prediction. The model uses gene expression, binary mutation calls, and tissue type information as features to represent the cell lines, and binding strength of the drug across 294 protein kinases obtained from HMS LINCS KINOMEscan data resource as features to represent the drugs (HMS LINCS KINOMEscan data). This method can be considered a hybrid of both gradient and embeddings based interpretation.
The post hoc analysis for interpretation is done at the feature and the biological process level. At the feature level, for a given drug, the input features' attribution towards the final output are computed for each cell line separately using the Integrated Gradients method (Sundararajan et al., 2017). The attribution scores are then averaged across all cell lines. The averaged attribution gives a semi-global interpretation as it cannot explain at a specific cell line level, but instead explains for all cell lines together for a given drug. At the pathway level, each dimension of the low dimensional representation from the cell line autoencoder is assigned to biological processes by correlating it with the expression of each gene across all cell lines, followed by GSEA on the ranked correlation values. The same dimensions are also correlated with the sensitivity for a given drug across all cell lines, to associate drug response with biological processes.
The authors used a case study with three drugs (PHA-793887, XMD14-99, and Dabrafenib) to validate the interpretability of the method. They computed the averaged feature attribution to show the top cell line and drug features important for the prediction for a given drug. They observed that the drug target gene was present among the top drug features for all three drugs, but the top cell line features did not include relevant genes (cancer or tissue type related gene), except for Dabrafenib. They also showed which biological processes best represented the drug sensitivity across all cell lines for a given drug, by performing a correlation analysis between the predicted drug sensitivity and hidden dimensions embedding. Almost the same biological processes (hidden dimensions) are associated with the drug sensitivity for the three drugs. However, the authors did not explain how these biological processes are related to the drug sensitivities for each of the drugs based on literature evidence.
Weights
The connection weights between an input layer neuron, representing a specific cell line or drug feature, and the neurons of the first hidden layer can be used to quantify the importance of this feature by summing over the learned weights between them (illustrated in Figure 5). Therefore, the first hidden layer is interpretable based on weights and the remaining part of the ANN has no role in the interpretability. The features with high weights could be interpreted as the more important components for the prediction. The feature importance scores based on weights can be misleading when features are on different scales, when positive and negative connection weights cancel each other out, or when a connection has a large weight but is rarely activated.
ParsVNN
Huang et al. introduced parsimony visible neural network (ParsVNN) for cancer type specific drug sensitivity Frontiers in Bioinformatics frontiersin.org prediction and interpretation (Huang et al., 2021). The authors believed that the biological hierarchy used in DrugCell is agnostic to the downstream prediction task, as some of the functional components in the biological hierarchy are not involved in the biological process related to the drug sensitivity phenotype. The conventional learning algorithm used in DrugCell also does not distinguish the redundant and irrelevant functional components which may permit them to make contribution towards the prediction. Therefore, such redundant and uninformative functional components in the visible neural network architecture can lead to overfitting and also result in misleading interpretations. To address this problem, the authors have built cancer-type specific model by pruning the redundant and irrelevant components for that cancer-type. To do this, they introduced sparse inducing penalty terms to the loss function and employed proximal alternative linearized minimization (PALM) algorithm as an optimization method to minimize the loss function and learn the model parameters.
The penalty terms help to prune the components with less important weights. The cancer-type specific model is achieved by training the model with the cancer-type specific training
FIGURE 4
An illustration to show how the gradient-based approach scores the input features which are then used for making the interpretation. Here [x1, x2, x3] and [x4, x5, x6] represent cell line and drug features respectively. Y represents drug sensitivity. F represents a function in the form of a neural network that takes [x1, x2, x3, x4, x5, x6] as input and returns Y as an output. The gradient of the model with respect to each of the input features is computed to find the attribution of the features towards the output. ∇F(xi) represents the gradient of the function at xi which is also equal to the partial derivative of the function with respect to xi. The computed attribution score for a feature tells us how much the feature contributes to a prediction.
FIGURE 5
An illustration of how connection weights between the neurons in an ANN can be used for interpretation. In this figure, the neuron weights between the input layer and the first hidden layer are used to identify important features determining the prediction.
Frontiers in Bioinformatics frontiersin.org data. The architecture and features used in ParsVNN are exactly the same as in DrugCell, which includes the same genes and subsystems (biological process GO terms) to build the visible neural network. The interpretability for a given cancer-type specific model is based on post-hoc analysis to identify the non-zero connection weights between the components. This reveals the components i.e. the gene nodes and the subsystem nodes that remain in the parsimonious architecture which contribute to the drug sensitivity prediction. Since this approach explains the predictions for a given cancer entity across a panel of drugs, it gives a semi-global interpretation.
The authors validated the interpretability for five different cancer types (stomach, breast, pancreatic, kidney and liver cancer) at both gene level and subsystem level. The genes and subsystems nodes remained in a cancer-type specific parsimonious model were hypothesized to be the driver genes and the prognostic biological processes for that cancer type, respectively. The authors validated the first part of the hypothesis by checking the degree of overlap between the identified genes and the cancerspecific driver genes reported by IntOGen pipeline (Martínez-Jiménez et al., 2020). The second part was validated by analyzing each of the leaf subsystems (GO terms) in cBioPortal's survival analysis with the cancer-type specific samples. The samples were divided into two groups, where one group had the samples that did not contain any gene mutated among the member genes of the GO term, and the other contained at least one gene mutated among the member genes of the GO term. The authors found that some of the GO terms that had its member gene(s) mutated significantly influence the clinical outcome.
DNN
Sakellaropoulos et al. developed a conventional deep neural network model (DNN) using gene expression data for a panel of highly variable genes as features to predict drug response (Sakellaropoulos et al., 2019). For each drug, the model training is performed on cancer cell lines, and the prediction is done on cancer patients.
The interpretability based on post hoc analysis of the model assigns biological meaning to the nodes of the first hidden layer. The weights are extracted from the nodes of the first hidden layer that are connected to gene nodes in the input layer. The weights are used to perform gene set enrichment analysis and the normalized enrichment score of every node is calculated against every pathway in the Reactome database. The normalized enrichment score for each significant pathway across the nodes of the first hidden layer is plotted as a heatmap. The nodes are clustered into subgroups, where each subgroup shows its signature of enriched pathways, suggesting possible drug mechanisms. The training and interpretability analysis is done for each drug separately. The interpretation is qualitative because the drug signature pathways are inferred from cluster patterns. The authors claimed that the pathways inferred from the analysis for cisplatin, paclitaxel, and bortezomib, are consistent with the literature evidence.
SWnet
Zuo et al. developed a DL predictive model called Selfattention gene Weight layer Network (Swnet), which uses gene expression, gene mutation, and drug structure data to predict drug sensitivity (Zuo et al., 2021). The model consists of a gene branch and a drug branch. The drug branch uses a graph neural network to convert the 2D representations of chemicals into embeddings in the latent space. The gene branch uses a gene weight layer in the form of a weight matrix to integrate the information of gene expression and gene mutation. The weight matrix is based on self-attention formed on the chemical similarity between all drugs, and this accounts for the heterogeneity of the gene-drug relationship. The integrated information is then processed using a convolution neural network leading to transformation into embeddings in the latent space. The embeddings from the drug and genomic branches are integrated and processed by another CNN to transform it into a unified output.
The model interpretation is based on post hoc analysis of the gene weight layer, where the genes with a specific weight = 1 are identified for each drug. The authors expected that the proteins encoded by these genes would interact with the drug targets and consequently a protein-protein interaction database can be used to validate the existence of the interaction.
The authors performed a validation of the approach with case studies on BRAF and BCL2 inhibitors. The genes identified in these case studies (BRAF and BCLS) were shown to be only two edges away from the drug target gene in the PPI network, which was interpreted as a validation by the authors. As shown in supplementary data, most of the genes identified for all other drugs were found not to be related to the drugs or the targets.
Discussion
While DL methods have proven their value in precision oncology applications (Coudray et al., 2018;Campanella et al., 2019;Chen et al., 2020), lack of interpretation of these black box models makes it difficult to implement them in clinical practice. In the past years, research has shifted focus toward interpretable DL and several methods have been developed to predict drug sensitivity. Table 5 gives an overview of the different published approaches. The methods cover different techniques for interpretation of the models, and each of them has certain advantages and limitations that we will discuss in this section. However, it is important to mention that it is not possible to precisely compare the methods based on their predictive performance, as they use different metrics and cross validation schemes for performance evaluation.
Frontiers in Bioinformatics frontiersin.org The comparison of the different methods indicates that the important ingredients of the interpretable DL models for drug sensitivity prediction are the input data, the ANN architecture, and the ontological information on genes and pathways.
The quantity and quality of the training/testing datasets influence the predictive performance. Xia et al. showed that machine learning models trained on the CTRP dataset (887 cell lines and 544 drugs) showed more accurate predictions than models trained on GDSC (cell lines 1075 and 249 drugs) (Xia et al., 2022). First, GDSC shows more replicate variability in drug response assay than CTRP. Second, CTRP contains a larger number of drugs and cell lines, allowing the models to better capture a large diversity of relationships between cell processes and drugs. Moreover, multiple omics data are available for cell lines, and this can be beneficial in the learning and improving the model performance (Malik et al., 2021). However, not all omics and all drug sensitivity assays are characterized for all cell lines, which could be a slight limitation for not having muti-omics training data as large as single-omics training data.
The ANN architecture determines whether the interpretation is done in a post hoc or ad hoc manner and what resolution of interpretation can be achieved. For example, HiDRA has the ad hoc ability to render interpretation at the time of prediction, at both pathway and gene-level resolution. The design of an ANN architecture also depends on the objective of the study. For example in PaccMann, the architecture is designed to explain which drug substructure is responsible for drug sensitivity, whereas in other methods the architecture is designed to explain what pathways or genes are responsible for drug sensitivity.
The gene and pathway ontological information in the form of pathways gene set and pathways hierarchical relationships that are used to design the ANN determines the quantity of information i.e. number of features and the depth and the type of interpretability. For example, HiDRA uses the highest number of genes among all the methods, which could provide more information to the model for a deeper resolution of the interpretation. Another example is consDeepSignalling, where the authors wanted to explain the prediction in terms of signaling pathways, therefore they used signaling-pathway gene sets to design the ANN.
Among the three probing techniques discussed, the embeddings based approach is the most promising. It avoids the pitfalls of the gradient and the weight based approach. Moreover, the embeddings based approach can explain MOA at gene, pathway, and biological process level behind a drug response by allowing visible data processing inside the ANN. The gradient and the weight based technique can only reveal the important features behind a prediction. Moreover, the comparison of the discussed DL methods also shows what different levels of interpretation the three probing techniques can confer. The embeddings based technique can offer local, semi-global and global interpretation, using ad hoc and post hoc analysis. It is important to note that ad hoc analysis can only provide local interpretation. The gradient based technique can also offer interpretation at all three levels, but it can only be achieved through post hoc analysis. However, it can be applied on both constrained and fully connected ANN. The weight based approach can only offer global and semi-global interpretation using post hoc analysis.
There are many issues regarding the validity of the interpretability observed across the methods. Quantitative methods for testing the accuracy of the predicted biological interpretability are lacking. The interpretability remains mainly at a qualitative level, checked with anecdotal evidence from the literature. Moreover, genes or pathways in the top list that had an agreement with the literature are prioritized, and other genes or pathways are completely ignored. Another issue is that the majority of the methods offer a semi-global interpretation to explain the drug MOA, picturizing that all cancer types show the same signature pathways for a given drug. Similarly, the methods that had global interpretation showed that all cancer types across all drugs will have the same signature drug pathways.
The difficulty with the validation of interpretability is that there is no good source of ground truth available. The L1000 study can be considered a potential source for ground truth, where it provides the drug signature pathways at different drug concentrations and time points for a given cell line (Subramanian et al., 2017). In the case of DL methods, binarized labels or IC50/AUC values are used as a measure of drug sensitivity for training and prediction. These drug sensitivity metrics represent an overall effect of the drug, therefore the interpretability will also explain the overall MOA. Since the L1000 explains concentration and time point specific drug MOA, it complicates its use as ground truth.
In one study, interpretable DL was already successfully implemented in vitro studies, where its mechanistic insights are used to design and test drug combination therapy and drug repositioning (Kuenzi et al., 2020). As a proof of concept, DrugCell used its interpretability to discover a synergistic drug combination of Paclitaxel and 2-DG on A427 cells and discovered in in vitro studies that the combination was indeed more effective than either individual compound.
Although there are already many studies on interpretable DL in cancer research, there is still room for improvement. In precision oncology, patient-specific treatment is the ultimate goal; therefore, focusing on locally interpretable methods to predict drug sensitivity at a personalized level is very relevant. Moreover, to address drug resistance in the context of intratumoral heterogeneity, interpretable models at the single cell level would allow to gain ultraprecise mechanistic Frontiers in Bioinformatics frontiersin.org insights that could help in designing patient-specific drug combinations. Last but not least, it is noteworthy to mention that the developments of interpretable techniques in cancer research could also be useful in other diseases which we have not discussed in this review.
Author contributions
BS: Conceptualized and designed this review, analyzed and interpreted the literature, and wrote the manuscript. JL: Reviewed the manuscript and provided feedback. VV: Conceptualized and designed this review and wrote the manuscript. KD: Conceptualized and designed this review and wrote the manuscript.
Funding
The work was supported by grant from the Kom op tegen Kanker (Stand up against Cancer).
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Study of the anticancer effect of new quinazolinone hydrazine derivatives as receptor tyrosine kinase inhibitors
The advent of novel receptor tyrosine kinase inhibitors has provided an important therapeutic tool for cancer patients. In this study, a series of quinazolinone hydrazide triazole derivatives were designed and synthesized as novel MET (c-MET) receptor tyrosine kinase inhibitors. The antiproliferative effect of the synthesized compounds was examined against EBC-1, A549, HT-29 and U-87MG cells by MTT assay. MET kinase inhibitory effect was tested by a Homogenous Time Resolved Fluorescence (HTRF) assay. The antiproliferative effect of compounds in a three-dimensional spheroid culture was studied by acid phosphatase (APH) assay, while apoptosis induction was examined by Hoechst 33258 staining. We found that compound CM9 bearing p-bromo benzyl pendant inhibited MET kinase activity at the concentrations of 10–50 μM (% Inhibition = 37.1–66.3%). Compound CM9 showed antiproliferative effect against cancer cells, in particular lung cancer cells with MET amplification (EBC-1) with an IC50 value of 8.6 μM. Moreover, this derivative inhibited cell growth in spheroid cultures in a dose-dependent manner and induced apoptosis in cancer cells. Assessment of inhibitory effect of CM9 against a panel of 18 different protein kinases demonstrated that this compound also inhibits ALK, AXL, FGFR1, FLT1 (VEGFR1) and FLT4 (VEGFR3) more than 50% at 25 μM. Finally, molecular docking and dynamics simulation corroborated the experimental findings and showed critical structural features for the interactions between CM9 and target kinases. The findings of this study present quinazolinone hydrazide triazole derivatives as kinase inhibitors with considerable anticancer effects.
Introduction
Cancer is the first or second leading cause of death before the age of 70 in 112 countries, causing 10 million deaths in 2020 worldwide (Sung et al., 2021). Various studies have focused on cancer treatment approaches targeting the signaling pathways in cancer cells and, in particular, protein kinases (Bhullar et al., 2018). It is well established that genetic aberrations in receptor tyrosine kinases (RTKs), such as MET (mesenchymal-epithelial transfer factor tyrosine or c-MET), VEGFR-1/2/3 (vascular endothelial growth factor receptor 1/ 2/3) (Qin et al., 2020), EGFR (epidermal growth factor receptor) (Ayati et al., 2020), RET (rearranged during transfection), FLT3 (FMS-like tyrosine kinase receptor 3) (Cilibrasi et al., 2022) and AXL (AXL receptor tyrosine kinase) (Okura et al., 2020), among others, contribute to tumorigenesis, disease aggressiveness, and drug resistance in different malignancies (Cohen et al., 2021) making RTKs promising therapeutic targets. Hence, substantial efforts have been devoted to the search for novel anticancer agents to pharmacologically modulate RTK signaling pathways, representing itself in the FDA approval of many inhibitors for clinical management of cancer patients (Zhao et al., 2021). MET receptor is an important oncogenic RTK that has received much attention as a promising drug target in various malignancies (Fogli et al., 2022). This receptor, also named as hepatocyte growth factor receptor (HGFR), is activated by binding to its natural ligand, HGF/scatter factor (Organ and Tsao, 2011). The aberrant activation of HGF/MET signaling pathway arises from overexpression, MET gene amplification or activating mutations, as well as excessive autocrine or paracrine HGF secretion, which has been reported to be associated with the development and progression of many types of cancers including lung, renal, gastrointestinal, thyroid, and breast cancers as well as glioblastoma among others (Zhang et al., 2018;Fu et al., 2021).
Considering the important oncogenic role of this receptor, many small molecule kinase inhibitors are being studied with the aim of targeting HGF/MET signaling pathway in different tumors. These agents can be divided into ATP competitive (type I and II) and non-competitive (type III) inhibitors based on their binding modes and selectivity profiles (Figure 1) (Merchant et al., 2013;Zhang et al., 2015). More selective inhibitors of MET kinase include type I agents such as capmatinib with a U-shaped structure (Dhillon, 2020), while type II agents are high molecular weight compounds that occupy the hydrophobic back pocket of MET, leading to increased interactions with the hydrophobic site (Yuan et al., 2020). Multiple kinases are typically inhibited by type II MET inhibitors. Examples include foretinib, cabozantinib, and BMS777607, which have been approved by the FDA or are currently in clinical trials across a broad range of cancer types (Underiner et al., 2010;Yakes et al., 2011).
The general structural characteristics of Type II MET inhibitors consist of four distinct parts of A, B, C and D as shown in Figure 2A. Structure-activity relationship (SAR) studies of type II MET inhibitors have suggested that fragment A usually consists of nitrogen containing heterocyclic moieties such as quinolines, quinazolinone, and pyridines which have a great potential for hydrogen bonding interactions with the amino acid residues of the kinase domain (Li et al., 2013;Damghani et al., 2019). Moreover, quinazolinone derivatives such as compound I and II have been reported to have potent cytotoxic activities, and also, they were used as an important core in the structure of kinase inhibitors ( Figure 2B) (Baek et al., 2004;Raffa et al., 2004;Mirgany et al., 2021). On the other hand, B and D fragments are usually a phenyl or substituted phenyl ring in the more promising compounds. The C fragment provides the fiveatom linker with the capability of establishing H-bond interactions with the active site (Li et al., 2013;Tang et al., 2016). We and other investigators have previously employed 1,2,3-triazole as C linker for design of MET kinase inhibitors; which is a privileged heterocycle extensively used for the design of potent cytotoxic agents such as carboxyamidotriazole (Duan et al., 2013;Mareddy et al., 2017;Gholampour et al., 2019;Xu et al., 2019;Damghani et al., 2021) (Figure 2B).
We focused on the design of new antiproliferative agents with potential interactions with MET active site as type II inhibitors by applying the principles of molecular hybridization and bioisosteric replacement. Therefore, quinazolinone core was employed as part A, hydrazide moiety was selected as a linker between A and B fragments to enhance the hydrogen bonding interactions with the active site and the phenyl ring was applied as a central aryl ring (part B). The nitrogen atoms in 1,2,3-triazole and oxygen atoms in the linker were applied as potential H-bond acceptor or donor interacting moieties to provide favorable H-bond interactions with the kinase active site. Finally, different substituted benzyl derivatives and heteroaromatic pendants were utilized as part D in order to assess the structure-activity relationship of designed analogues ( Figure 2C).
Upon the design and synthesis of anticancer agents with MET kinase inhibitory potential, the antiproliferative effect of compounds against cancer cell lines was evaluated in monolayer and three-dimensional (3D) cell cultures and the kinase inhibitory activity of synthesized derivatives was determined.
In silico studies were also performed in order to investigate the binding interactions of the most promising compounds with the active site of target kinases.
Chemistry
Melting points were determined with a Thermo Scientific Electrothermal digital apparatus (Thermo Fisher Scientific Inc.). 1 H NMR (300 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker 300 Fourier transform spectrometer; the chemical shifts are expressed in d (ppm) downfield from tetramethylsilane. Infrared (IR) spectra were recorded on the Perkin Elmer Spectrum RXI FTIR spectrophotometer in the KBr phase. Mass spectra were carried out using Agilent 7000 triple quadrupole mass spectrometer at an electron impact mode with an ionization voltage of 70 eV.Chemicals used were supplied from Sigma-Aldrich, Fluka, and Merck chemical companies. TLC was performed on the glass-backed silica gel sheets (Silica Gel 60 GF254) and visualized under UV light (254 nm).
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was applied to evaluate the antiproliferative effects of synthetic compounds against cancer and normal cell lines as previously described (Damghani et al., 2021). The MTT assay is based on the reduction function of NAD(P)H-dependent cellular oxidoreductase enzymes on yellow tetrazolium dye MTT to its insoluble formazan, which has a purple color and reflects the number of viable cells. Cancer and non-cancer cells were seeded in 96-well flat bottom plates at different densities of 1.5 × 10 4 (HT-29), 3 × 10 4 (A549 and U-87MG), 4 × 10 4 (EBC-1). Synthetic compounds were first dissolved in DMSO and then diluted in growth medium. After 24 h the attached cells were exposed to 100 µL of 3-4 different concentrations of synthesized derivatives in triplicate manner and incubated for an additional 72 h at 37°C. In addition, 3-4 different concentrations of reference compounds were added to wells. To perform the MTT assay the media was discarded from each well and MTT solution in phosphate-buffered saline at a concentration of 0.5 mg/ml was added to all wells. Then, plates were incubated at 37°C for 4 h to allow formation of formazan crystals. Afterwards, crystals were dissolved in 200 μL DMSO, shake on an orbital shaker for 30 min and absorbance was measured at a wavelength of 570 nm with background correction at 650 nm using a Bio-Tek microplate reader (Model Synergy HTX). Finally, IC 50 values for each compound was calculated with CurveExpert software version 1.4 for windows. Each experiment was repeated 3-5 times.
In vitro enzymatic assays
MET kinase inhibitory activity of synthesized derivatives was determined by Homogeneous Time Resolved Fluorescence (HTRF) assay (Damghani et al., 2021). In this method, the phosphorylation level of a biotinylated tyrosine kinase substrate peptide (TK substrate) is measured. The HTRF ® KinEASE ™ TK kit and MET kinase were purchased from Cisbio and Millipore, respectively. The optimal conditions were established for enzyme, substrate, ATP concentrations, and enzymatic reaction time. Test compounds were first dissolved in DMSO and then diluted in the reaction buffer containing 50 mM HEPES pH 7.0, 0.1 mM sodium orthovanadate, 0.01% BSA, 0.02% NaN 3 , 10 mM MgCl 2 , 5 mM MnCl 2 , 2 mM DTT. Briefly, 4 μL of test compound at different concentrations (10, 25, 50 and 100 μM final concentrations) were loaded in a white 384-well plate (Cisbio Cat Number: 6007299). Then, 2 μL of MET kinase diluted in kinase buffer (0.25 ng/μL) were preincubated with 4 μL of the test derivatives for 10 min. In the next step, in order to initiate the reaction, 2 μL of TK substrate (1 μM final concentration), and 2 μL ATP dissolved in kinase buffer (25 μM final concentration) were sequentially added. After the incubation of the reaction mixture for 50 min at room temperature, the phosphorylated peptide substrate was detected by adding 10 μL mixed detection solution containing 5 μL Europium 3+ -conjugated antibody and 5 μL Steptavidin-XL665 (125 nM final concentration). Finally, the Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) signal was determined after an hour of incubation at room temperature. The plate was read at excitation of 337 nm and dual emissions of 665 and 620 nm using a Bio-Tek multimode plate reader (Model Cytation 3). The following equations were used to determine the inhibition rate (%):
Measurement of the anticancer effect in three-dimensional spheroid assay
An agarose-based liquid overlay method was implemented to perform three-dimensional spheroid cell cultures assays. To this purpose, agarose solution in RPMI (1.5% w/v) was prepared and high-pressure sterilized. The wells of a 96well sterile flat bottom plate were coated with 50 µL sterilized agarose solution and allowed to become gelatinous at room temperature for at least 2 h. A suspension of EBC-1 cells in RPMI medium containing 10% FBS at a density of 2 × 10 5 cells/ml were added to each well (125 µL per well). Then, In order to allow the formation of a single spheroid in each well, the plate was centrifuged at 700 g for 5 min and incubated under standard culture conditions for 48 h. Afterwards, formed spheroids were exposed to 3-4 different concentrations of active derivatives diluted in a fresh medium containing 10% FBS. After 72 h incubation period, the images of spheroids were recorded by a bright field microscope (Nikon model DS-Ri2) and Nikon NIS-Elements AR imaging software for Windows version 4.30. The obtained images were analysed with ImageJ software for windows. In addition, cell viability was measured by a colorimetric acid phosphatase (APH) assay. The APH assay used p-nitrophenyl phosphate (pNPP) as substrate which dephosphorylates and converts to yellow p-nitrophenol by intracellular acid phosphatases present in viable cells. Briefly, 160 µL of the medium was disposed and 200 µL of APH solution containing 2 mg/ml pNPP dissolved in 0.1 M sodium acetate at pH 4.8 were added to each well and incubated for 120 min. Afterwards, 10 µL of 1 M NaOH was added to each well as the stop solution. Finally, the absorbance was recorded at 405 nm within 10 min by a Bio-Tek microplate reader (Model Synergy HTX).
Assessment of apoptosis induction by Hoechst 33258 staining
In order to evaluate the apoptosis induction effects of test compounds on cancer cells, Hoechst 33258 staining was applied. Hoechst dye is a member of blue fluorescent nuclear specific dyes which binds to DNA and stains nuclei in live and fixed cells. EBC-1 cells were cultured in 6-well plates with a density of 5 × 104 cells/ml (2 ml per well). After 24 h incubation, the whole culture medium from all wells was removed and 2-3 different concentrations of active derivatives were added to each well and incubated for 72 h. Then, the whole culture medium was replaced with 1 ml of 4% cold freshly prepared paraformaldehyde (PFA) and cells were incubated for 20 min at room temperature allow cells were fixed. Afterward, the cells were washed two times with PBS and incubated with 1 ml Hoechst 33258 2.5 μg/ml for 30 min at room temperature in the dark place. Finally, the cells were washed with PBS and imaged with a fluorescence microscope (Nikon model DS-Ri2).
Docking analysis
Molecular docking analysis was carried out against MET and FLT4 (VEGFR3) to investigate the binding modes and the critical molecular interactions between the synthetic compounds and the binding site of the targets, using the smina molecular docking software (Koes et al., 2013). smina was developed based on Auto-Dock Vina to enhance scoring function development and energy minimization. The protein structure was prepared using adding hydrogens removing water molecules and native ligands. Then, the Kollmann charges were assigned to the receptor. All compounds were sketched using the Marvin Sketch (http:// www.chemaxon.com), and assigned gasteiger charges and energy optimization of synthetic compounds using the steepest descent algorithm carried out by Open Babel (O'Boyle et al., 2011). The enzyme's binding site for the docking process was determined automatically using the coordinates of co-crystallized native ligand foretinib with MET kinase. Then, smina was applied to predict the interaction and binding modes of the ligands inside the enzyme active site. The computational docking approach was evaluated based on the root-mean-square deviation (RMSD) value from re-docking of the co-crystalized ligand foretinib back into the active pocket site of the receptor (Gohlke et al., 2000).
Molecular dynamics simulation
The molecular dynamics simulation (MD) was carried out using GROMACS package version 2019.1 on a GPU Linux server to Frontiers in Chemistry frontiersin.org 07 simulate the interaction of MET and FLT4 (VEGFR3) with CM9. The Amber99sb force field was chosen to carry out the MD simulations at a mean temperature of 300 K and physiological pH 7. Chimera software (Pettersen et al., 2004) was applied to adding the AM1 partial charges to prepare the topology, and the algorithm acpype (Da Silva and Vranken, 2012) was used for the creation of force filed parameters (Da Silva and Vranken, 2012). A dodecahedral solute box was defined around the solute such that there is the shortest distance between any two periodic images of a protein complex and the edge of the box. TIP3P water types were used to fill the protein complex box. Then, 0.15 mol/lit sodium chloride was added to the system for neutralization by replacing the equal number of water molecules. System energy optimization was carried out using the steepest descent algorithm through a 100 ps run. In the next step, the macromolecule and ligand's atom positions were restrained using a force constant of 1000 kJ mol −1 nm −2 during a 500 ps NVTperiod. During the NVT stage, the temperature was set to 300 K using V-rescale thermostats. Then, the pressure of the system was stabilized over the period of 500 ps equilibration step during the NPT step.
Generally, MD simulations need to be adequately long to draw logistic conclusions from the studies. The production MD simulation was completed during 100 ns for both complexes under a well-equilibrated system with a desired temperature and pressure. The long-ranged electrostatic contributions were calculated by the particle-mesh Ewald (PME) algorithm, and lengths of covalent bonds were restrained by applying the LINCS constraint algorithm, three to four times faster than the SHAKE algorithm. Upon terminating the MD run, the complex was centered by returning the protein to the box center, and the trajectory was corrected in the point of periodic boundary condition.
The RMSD values were determined over the entire run for the alignment of the protein backbone atoms of each snapshot against the first frame as the reference to determine the equilibrium time range further. Moreover, cluster analysis of the trajectory during the equilibrium time range was applied by gromos method.
Binding free energy calculations using MM-PBSA
The binding free energy of complexes of potent compound CM9 with MET and FLT4 (VEGFR3) was determined using MM-PBSA approach. MM-PBSA analyses were performed using the g_mmpbsa tool provided by Kumari et al. (Baker et al., 2001;Kumari et al., 2014). MM-PBSA estimates the binding free energies using the combination of molecular mechanics and continuum solvent models. Besides the calculation of binding energy components, it can also report the individual energy contributions of amino acids. In this study, the last 1 ns of the equilibrium time range elucidated by the RMSD graph of all complexes was applied for precise MM-PBSA estimation. The adaptive Poisson-Boltzmann Solver (APBS) approach calculated the electrostatic energy, VDW energy, and polar solvation energy contributions. In contrast, the non-polar contributions of solvation energy were estimated by the Solvent-accessible surface area (SASA) approach. The grid spacing of 0.5 Å and probe radius of 1.4 Å were used for SASA estimate with a solvent dielectric constant value of 80, and solute dielectric constant value of 2. The binding energy of the complexes and energy contribution of ligand were specified at the end.
Chemistry
All target compounds CM1-CM10 were synthesized according to the procedure shown in Scheme 1. At first, 2mercapto-3-methyl quinazoline-4(3H)-one (Bhullar et al., 2018) was prepared via the reaction of anthranilic acid 1 and methyl SCHEME isothiocyanate in the presence of triethylamine in refluxing ethanol. In the next step, the reaction of compound 2 with hydrazine hydrate under reflux condition in BuOH afforded 2-hydrazinyl-3-methyl quinazoline-4(3H)-one (Qin et al., 2020). Then, 4-hydroxy aldehyde was set to react with 3-bromoprop-1yne in the presence of K 2 CO 3 in dry acetone/reflux to produce 4-(prop-2-yn-1-yloxy) benzaldehyde (Cilibrasi et al., 2022). Furthermore, different benzyl azides (Cohen et al., 2021) were prepared from the reaction of desired benzyl halide and sodium azide in a mixture of H 2 O/isopropanol at room temperature. The intermediate 4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy) benzaldehyde (Zhao et al., 2021) was prepared via the reaction of compounds 5 and 7 in the presences of CuSO 4 and sodium ascorbate in a mixture of H 2 O/isopropanol at room temperature. Finally, different derivatives (CM1-CM10) were synthesized through the reaction of compounds 8 and 3 in the presence of acetic acid in the absolute ethanol at room temperature (Scheme 1). All structures, characteristic chemical and physical properties of the compounds are demonstrated in Table 1.
Antiproliferative effect against cancer cells
We employed the MTT assay in order to investigate the antiproliferative effects of the synthetic compounds against four different cancer cell lines, including lung cancer cell lines EBC-1 (with MET amplification) and A549, the human colorectal cancer cells HT-29, as well as the human glioblastoma cell line U-87MG. CM7, CM8, CM9 and CM10 were the most effective compounds at inhibiting the growth of the tested cancer cell lines, particularly EBC-1 cells with IC 50 values ranging from 8.6 ± 1.9 to 22.9 ± 4.6 µM. Compound CM9 (bearing para-bromophenyl moiety) displayed the highest growth inhibitory effect against the EBC-1 cell line harboring amplified MET gene. In addition to being effective against EBC-1 cells, the CM9 and CM10 derivatives exhibited good growth
MET kinase inhibitory effect
The MET inhibitory activities of synthesized compounds (CM1-CM10) were determined by a Homogeneous Time Resolved Fluorescence (HTRF) assay. The phosphorylation of a TK substrate triggered by MET kinase is measured in this method. Table 3 summarizes the inhibition results for CM1-CM10 at three concentrations of 10, 25, and 50 μM. CM9 significantly inhibited MET kinase with percent activities of 53.0 and 66.3% at 25 and 50 μM, respectively, while CM7, CM8, and CM10 showed weaker inhibitory effects. In contrast, CM1-CM6 were devoid of any effect against MET. Cabozantinib was also evaluated as a positive control with an IC 50 value of 24.4 nM. Furthermore, the concentration-effect curve of CM9 was also determined by HTRF assay and an IC 50 value of 22.8 ± 3.9 µM was calculated (Figure 3).
Antiproliferative effect against cancer cells grown in three-dimensional cultures
The growth inhibitory effects of active derivatives CM9 and CM10 were assessed in three-dimensional (3D) spheroid models. After formation of EBC-1 single spheroids with liquid overlay technique, treatment with different concentrations of CM9 and CM10 was performed. The growth inhibitory effects of selected derivatives was measured by acid phosphatase (APH) assay. There was a significant reduction in cell viability of spheroids treated with CM9 and CM10 derivatives compared to control in a dose-dependent manner (Figures 4A,B). Moreover, two spheroid indices including circulatory and solidity were also analyzed. The spheroid circulatory was dose dependently decreased, although this effect was less prominent in solidity analysis (Figures 4C,D).
Apoptosis induction measured by Hoechst 33258 staining
Considering the results obtained by the MTT assay, compounds CM9 and CM10 were selected for further investigation by Hoechst 33258 staining. Hoechst 33258 is a fluorescent DNA stain for apoptosis studies; Live cell nuclei are detected with a uniformly light blue emission, while apoptotic cell nuclei exhibit signs of apoptosis. As shown in Figure 5, EBC-1 cells exposed to 10 and 25 µM of test compounds exhibited the characteristic morphological changes of apoptotic nuclei, such as chromatin condensation, nuclear fragmentation and shrinkage in contrast to control cells with no apparent morphological changes. The results indicated that these agents induced apoptosis and might be considered as promising anticancer agents.
Kinase selectivity profile
In order to investigate the activity of compound CM9, as the most promising agent in this series, against other oncogenic kinases, we further assessed this derivative against a panel of 18 other protein kinases using a radiometric assay at the concentrations of 10 and/or 25 μM (Table 4).
Consistent with the HTRF results, CM9 showed inhibitory activity against MET kinase also in this assay. Interestingly, this compound also demonstrated a considerable inhibitory potential against FLT4 (VEGFR3) by 86% at 10 μM. The concentrationeffect curve was determined against this kinase, and the results are shown in Figure 6. As demonstrated in Table 4, CM9 also exhibited inhibitory effect against ALK (Anaplastic lymphoma kinase), AXL, FGFR1 (Fibroblast growth factor receptors 1), and FLT1 (VEGFR1) with the inhibitory activities of 51%, 65%, 66%, and 82% at 25 μM, respectively. These results suggest that compound CM9 is a promising multitarget RTK inhibitor.
3.7
In silico studies 3.7.1 Molecular docking studies Molecular docking analysis was carried out in an attempt to evaluate the ability of synthesized compounds to interact with MET and FLT4 (VEGFR3) kinase using smina docking. The cocrystallized structure of MET (PDB code: 3LQ8) in complex with foretinib and the established FLT4 (VEGFR3) homology model were utilized for the docking study. The smina docking algorithm was evaluated using the re-docking of foretinib into the active site of MET kinase. The root-mean-square deviation (RMSD) value was 1.5 Å which is lower than the tolerable marginal value of 2 Å. As shown in Figure 7A, compound CM9 made one hydrogen bond interaction with Lys1110 in the kinase
FIGURE 4
Growth inhibitory effects of synthesized compounds against cancer cells grown in three-dimensional spheroid cultures. Spheroids of EBC-1 cells were formed by agarose-based liquid overlay technique in 96-well plates. (A) Representative images of spheroids treated with CM9 and CM10 at 10, 25, 50, and 100 µM are shown. The images were captured with Nikon NIS-Elements imaging software. (B) Growth inhibitory effects of compounds against EBC-1 spheroids was measured by acid phosphatase (APH) assay. (C) Circularity and (D) solidity of 3D spheroids after drug treatment were measured by ImageJ software. Cabozantinib was also tested as a positive control. Data are presented as mean ± S.E.M. of 3-6 independent experiments. The difference with control was statistically significant at * (p < 0.05), ** (p < 0.01).
FIGURE 5
Apoptosis induction measurement by Hoechst 33258 staining assay. EBC-1 cells were seeded in 6-well plates and treated with different concentrations of test compounds for 72 h. After fixation with 4% cold paraformaldehyde (PFA) the cells were stained with 2.5 μg/ml Hoechst 33258 and imaged with a fluorescence microscope (Mag: 100X and 200X). A representative image is shown for each compound. Red arrows show apoptotic cells.
On the other hand, examining CM9 binding mode with (FLT4) VEGFR3 demonstrated two hydrogen bond interactions with Asp1067 and Asn934, through NH of hydrazine and oxygen of methoxy linkers, respectively. Triazole and phenyl rings participated in the pi-pi interaction with Phe982, and Phe929, respectively. Moreover, quinazoline, triazole, and phenyl rings made pialkyl interactions with Val1069, Val850, Leu851, Ala877, Val910, Cys930, Leu1044, Cys1054, Val859, and Val927 ( Figure 7B). Frontiers in Chemistry frontiersin.org 15 3.7.2 Molecular dynamics simulation of CM9 with MET and VEGFR3 receptors MD simulation was carried out for MET and FLT4 (VEGFR3) kinases in complex with the most potent compound CM9. To evaluate the conformational stability of the enzyme, the RMSD values were measured based on the alignment of backbone atoms of each frame versus the first frame against time over the whole course of simulations. The regular RMSD profile showed that CM9 reached the equilibrium phase at 45 ns for MET and 60 ns for FLT4 (VEGFR3) complexes ( Figure 8).
In this study, we calculated the number of hydrogen bonds formed as a function of time between the CM9 and amino acid residues in the active site of MET and FLT4 (VEGFR3) during the equilibrium time range of MD simulations. According to the obtained results, CM9 made at least one hydrogen bond 95.82% of the time with MET kinase and one and two hydrogen bonds in 91.84% and 54.41% of the times with FLT4 (VEGFR3) kinase, respectively. Furthermore, a maximum of three and six hydrogen bonds were formed for MET and FLT4 (VEGFR3) kinase, respectively. Moreover, the stability of hydrogen bond interactions between the ligand CM9 and promising residues inside the active sites of MET and FLT4 in the equilibrium time range was shown in Figure 9.
The clustering analyses of complex CM9 with both targets were carried out, and the result showed that the percent of the population in cluster 1 was 97.64% with MET and 97.25% with FLT4 (VEGFR3) kinase. The representative frame of cluster 1 of each complex is shown in Figure 10. The interaction patterns of representative frames of all the clusters have been shown in the Supplementary Material.
Analysis of interaction of CM9 with MET kinase showed two critical hydrogen bonds from carbonyl of quinazoline ring with Met1160 and a hydrogen bond from NH of the hydrazide linker with Asp1222. Also, two important pi-pi interactions were made between the triazole ring with Phe1200 and Ala1221. Moreover, CM9 made hydrophobic interactions with Ile1084, Ala1108, Met1211, Leu1157, Val1220, Met1131 and Ala1221, see Figure 10A.
Moreover, as shown in Figure 10B, the complex of CM9 with FLT4 (VEGFR3) kinase showed two hydrogen bonds from N atom of quinazoline with Arg1070 and N atom of hydrazine linker with Tyr1068. Phenyl rings participated in pi-pi stacked interaction with Phe929, and methoxyphenyl ring made amide pi-pi stacked interaction with Leu851. Furthermore, there was hydrophobic interaction from the common substructure of the compound with Arg1070, Lys1064, Val1069, Leu851, and Leu1044.
MM-PBSA analysis
MM-PBSA approach was utilized for the calculation of the binding energies and ligand contributions of MET and FLT4 (VEGFR3) kinases in complex with the most potent compound CM9. The potent compound inside the mentioned proteins kJ/mol for FLT4, respectively. However, the energy analysis of CM9 complex in both proteins has been shown in Table 5.
Physicochemical properties
The SwissADME web tool was applied to predict pharmacokinetics properties and drug-likeness of all compounds (Daina et al., 2017). Most compounds presented acceptable molecular properties (Table 6).
Discussion
In this study, 10 novel quinazolinone hydrazine triazole derivatives were synthesized and evaluated for MET inhibitory effect in cell-free and cell-based assays. Among them, compound CM9 bearing p-bromo benzyl pendant on the triazole ring exhibited the highest MET inhibitory activity and antiproliferative effects towards MET-amplified lung cancer cells. This derivative together with CM10 bearing phetalimidemoiety also exhibited growth inhibitory effects against threedimensional spheroid cultures of cancer cells and induced characteristic morphological changes of apoptosis. Data from a kinase selectivity profile assessment showed that compound CM9 is able to inhibit other important oncogenic kinases such as FLT4 (VEGFR3). Computational studies supported our experimental observations and showed critical interactions between synthesized derivatives and target kinases.
In an attempt to evaluate the anticancer potential of synthesized compounds, antiproliferative effects were assessed against four cancer cell lines, including lung (EBC-1 and A549), colorectal (HT-29) and glioblastoma (U-87MG) cells measured using MTT assay in monolayer cell cultures. The lowest IC 50 value was observed for compound CM9 (8.6 µM) against the MET amplified EBC-1 cell line. It is interesting to note that EBC-1 are dependent on MET oncogene for proliferation and survival and are considered a good model for testing the MET inhibitory potential of different compounds (proteinatlas.org, 2022; cansarblack.icr.ac.uk, 2022). Moreover, another derivative, CM10, displayed moderate growth inhibitory effects in the EBC-1 and HT-29 cells with IC 50 values of 18.0 ± 5.6 and 18.4 ± 2.3 µM, respectively.
According to the results of MET kinase inhibition and MTT assay, CM9 bearing para-bromophenyl moiety seems to be the most promising agent. As for the structure-activity relationships (SAR), there seems to be a strong relationship between the nature and position of the phenyl substitution linked to triazole ring and MET kinase inhibition; the introduction of bulky electron withdrawing groups (EWG) at para position of benzyl linked to triazole ring enhances the activity of compounds as represented in compound CM9 bearing p-bromo benzyl pendant. In addition, compounds with two-EWG at meta-and para-positions of benzyl moiety were better than mono-EWGs substituted derivatives; compound CM8 with two chlorine groups at 3, 4-position of benzyl pendant demonstrated intermediate inhibitory activity, while its para-chlorinated counterpart CM5 was almost inactive. Finally, the presence of bulky lipophilic groups like phetalimide on the triazole ring showed modest influence on MET inhibitory potential of compounds. For instance, CM10 and CM7 showed intermediate inhibitory activities.
It has been recently recognized that three-dimensional cellular models are powerful tools offering reliable platforms for in vitro drug screenings. Compared to conventional twodimensional cultures, three-dimensional models can more closely mimic features of in vivo human solid tumors, such as their gene expression patterns, complexity, heterogeneity, as well Frontiers in Chemistry frontiersin.org as drug resistance . In this context, the growth inhibitory effect of the most promising antiproliferative agents, CM9 and CM10, was evaluated in EBC-1 cells grown in 3D cultures. It was observed that the spheroid viability as well as their physical properties, including circularity and solidity, were decreased after both CM9 and CM10 exposure in a dosedependent manner. Moreover, the morphological studies performed using DNA staining with Hoechst 33258 confirmed the apoptotic induction effect of CM9 and CM10 on EBC-1 cells. These compounds displayed typical apoptotic features such as nuclear shrinkage and fragmentation.
The inhibitory potentials of the most promising agent, CM9, was assessed against a panel of 18 well-known oncogenic kinases. This derivative showed considerable inhibitory activity against FLT4 (VEGFR3) receptor tyrosine kinase, in addition to MET kinase. Targeting the members of VEGFR families, noticeably VEGFR1, VEGFR2 and VEGFR3, have been evaluated as potential antiangiogenic therapies. In particular, VEGFR-3 plays a vital role in the progression of lymphangiogenesis, in addition to angiogenesis, promoting tumor cell invasion and metastasis. Therefore, the development of drugs targeting the FLT4 (VEGFR3) signaling pathway may be therapeutically beneficial in cancer management (Hsu et al., 2019). CM9 also exhibited considerable potency against ALK (Anaplastic lymphoma kinase), AXL, FGFR1 (Fibroblast growth factor receptors 1), and FLT1 (VEGFR1). Based on our findings, CM9 represents a promising kinase inhibitor. Clearly, due to a high degree of sequence and structural similarity in the kinase domain of RTKs, a large numbers of kinase inhibitors have an expected cross-reactivity within the kinase family (Pottier et al., 2020). Until now, 55 small molecule compounds targeting kinase proteins, especially RTKs, have received FDA approval for indications in oncology, and among them, at least 25 agents are multitargeted, inhibiting several protein kinases (Roskoski, 2020). Cabozantinib, as an approved RTK inhibitor, for example, is effective in targeting a broad range of RTKs, including MET, VEGFR-1/2/3, RET (Rearranged during transfection), FLT3, AXL, and c-KIT (Viola et al., 2013).
Another important point to consider about multi-target agents is the drug resistance issue that appears to be a more severe problem for single-target drugs. In this regard, multitarget drugs have generally shown higher efficacy compared to single-target drugs in overcoming drug resistance (Raghavendra et al., 2018;Desai and Small, 2019). The interactions of compound CM9 was also evaluated with MET and FLT4 (VEGFR3) kinases by docking analysis. CM9 in the active site of MET kinase made an important hydrogen bond interaction with Lys1110 through triazole ring and a key pi-pi stacked interactions with Phe1223 from the phenyl ring of methoxyphenyl linker. Moreover, hydrophobic interactions through phenyl and triazole rings with residues in hydrophobic pocket may cause the higher inhibitory potential of CM9. In addition, docking studies of CM9 with VEGFR3 demonstrated two hydrogen bond interactions with Asp1067 and Asn934 and two pi-pi interaction with Phe982, and Phe929. In order to better understand the stability of the interactions of compound CM9 with the active site residues, an MD simulation was also performed. It was observed that CM9 makes two critical hydrogen bonds with Met1160 and Asp1221 in the kinase domain of MET. Also, two important pi-pi interactions were made between the triazole ring with Phe1200 and Ala1221, while hydrophobic interactions were made with hydrophobic residues. Moreover, CM9 with FLT4 (VEGFR3) kinase showed two hydrogen bonds with Arg1070 and Tyr1068 and two pi-pi interactions with Phe929 and Leu851 and broad hydrophobic interactions with the active site of FLT4 (VEGFR3).
Hence, the findings of the computational analysis showed the formed important interactions from different part of CM9 structure with key residues in the active site of targets may justify the inhibitory activities of this agent against MET and FLT4 (VEGFR3) kinases.
Finally, the evaluation of drug-like properties such as log P, molecular weight, number of hydrogen bond donors and acceptors, and TPSA were also performed on the basis of Lipinski's rule of five. According to the predictions, all synthesized molecules were in accordance with Lipinski's rule of five with no violations.
In this study we provide cell based and cell free assays as well as in silico studies to evaluate the MET inhibitory effects of 10 novel quinazolinone hydrazine triazole derivatives. The considerable antiproliferative effect of CM9 and CM10 derivatives against cancer cells was confirmed. The In vitro enzymatic assays results suggest CM9 bearing p-bromo benzyl pendant on the triazole ring as a promising tyrosine kinase inhibitor especially against MET and FLT4 (VEGFR3). Eventually, important structural features for the interactions of CM9 with MET and FLT4 (VEGFR3) kinases verified by Molecular docking and molecular dynamics simulation studies. These novel quinazolinone derivatives present promising anticancer agents with kinase targeting potential.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding authors.
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Radiation pneumonitis prediction after stereotactic body radiation therapy based on 3D dose distribution: dosiomics and/or deep learning-based radiomics features
Background This study was designed to establish radiation pneumonitis (RP) prediction models using dosiomics and/or deep learning-based radiomics (DLR) features based on 3D dose distribution. Methods A total of 140 patients with non-small cell lung cancer who received stereotactic body radiation therapy (SBRT) were retrospectively included in this study. These patients were randomly divided into the training (n = 112) and test (n = 28) sets. Besides, 107 dosiomics features were extracted by Pyradiomics, and 1316 DLR features were extracted by ResNet50. Feature visualization was performed based on Spearman’s correlation coefficients, and feature selection was performed based on the least absolute shrinkage and selection operator. Three different models were constructed based on random forest, including (1) a dosiomics model (a model constructed based on dosiomics features), (2) a DLR model (a model constructed based on DLR features), and (3) a hybrid model (a model constructed based on dosiomics and DLR features). Subsequently, the performance of these three models was compared with receiver operating characteristic curves. Finally, these dosiomics and DLR features were analyzed with Spearman’s correlation coefficients. Results In the training set, the area under the curve (AUC) of the dosiomics, DLR, and hybrid models was 0.9986, 0.9992, and 0.9993, respectively; the accuracy of these three models was 0.9643, 0.9464, and 0.9642, respectively. In the test set, the AUC of these three models was 0.8462, 0.8750, and 0.9000, respectively; the accuracy of these three models was 0.8214, 0.7857, and 0.8571, respectively. The hybrid model based on dosiomics and DLR features outperformed other two models. Correlation analysis between dosiomics features and DLR features showed weak correlations. The dosiomics features that correlated DLR features with the Spearman’s rho |ρ| ≥ 0.8 were all first-order features. Conclusion The hybrid features based on dosiomics and DLR features from 3D dose distribution could improve the performance of RP prediction after SBRT.
Background
Toxicity assessment is a very important step in radiotherapy. Radiation pneumonitis (RP) is the main complication of stereotactic body radiation therapy (SBRT) in patients with lung cancer. As per some studies, the incidence of RP ranges from 9 to 49% [1][2][3][4][5]. For the fact that patients treated with SBRT are prone to a fragile condition, RP may impair their quality of life and subsequently increase hospitalization and mortality rates [6][7][8]. Therefore, it is necessary to establish a model for predicting RP during the initial evaluation and therapeutic regimens.
Recently, the advancement in machine learning (ML) and radiomics has provided new methods for RP prediction. Quantitative medical imaging features can be extracted for computed tomography (CT) images to predict RP [9][10][11]. Kraf et al. proposed a predictive model for RP toxicity using pretreatment CT-based radiomics features extracted from the whole-lung volume [9], Kawahara et al. [10] and Hirose et al. [11] used radiomics features from dosimetric-based segmentation to predict the occurrence of RP. However, the occurrence of RP is affected by radiation dose, all the above prediction models for RP using radiomics features on pretreatment planning CT images rather than dose distribution. Some researchers have established RP prediction models based on some dose volume histogram (DVH) parameters, such as V5, V10, and mean lung dose (MLD) of the radiotherapy plan [12,13]. However, it can only summarize the two-dimensional dose distribution in the target from DVH parameters, and the spatial dose distribution or organ architecture cannot be obtained from DVH parameters [14]. RP can be clinically controlled by limiting the dose to the lungs. However, dose limitation does not always prevent serious toxicities in some patients. It has been demonstrated in some studies that the voxel dose is related to the risk of tumor response, lung injury and other complications [15], and hence features extracted from the dose distribution may be of predictive significance. Thus, radiomics based on 3D dose distribution has become a more effective way to explore the toxicity induced by the radiation dose [16].
In some studies, dose-based radiomics based on 3D dose distribution is also known as dosiomics features [17][18][19][20][21]. Liang [20] and Adachi [21] extracted dosiomics from 3D dose distribution for RP prediction. These models for predicting RP expand the application of ML in the field of radiotherapy and promote the development of RP prediction. To the best of our knowledge, RP after SBRT has not been predicted by DLR features based on 3D dose distribution.
In this study, dosiomics and DLR features were extracted from 3D dose distribution of normal lung patients with lung cancer, and three prediction models were constructed based on random forest, including (1) a model constructed based on dosiomics features, (2) a model constructed based on DLR features, and (3) a hybrid model constructed based on dosiomics and DLR features. Besides, the correlation between dosiomics features and DLR features from 3D dose distribution was analyzed. The establishment of an accurate prediction model for RP is expected to realize the dose increase for low-risk patients or the treatment optimization for highrisk patients. This would further minimize the incidence of RP and significantly benefit cancer patients receiving radiation therapy.
Study cohort
A total of 140 patients who were admitted to our hospital from 2019 to 2021 were included for retrospective analysis. All patients provided written informed consent before enrollment. Patients were performed with 4-dimensional computed tomography (4D-CT). The gross tumor volume (GTV) was delineated on ten respiratory phasesorted 4D-CT datasets. The internal target volume (ITV) was generated by performing the union of the 10-phase sorted GTVs. All patients were treated using an ITVbased strategy with an additional ITV-to-planning target volume (PTV) margin of 5 mm. The entire lung, excluding the ITV (Lung-ITV), was regarded as a normal lung. The dose distribution was calculated by Collapsed cone Convolution Superposition (CCCs) algorithm on the Pinnacle treatment planning system (TPS), with the grid size being 2.5 mm × 2.5 mm× 2.5 mm. An example image of a dose distribution was shown in Fig. 1. The patients were treated with 6 MV X-rays; the prescribed dose was 50 or 60 Gy in 4-8 fractions at an isocenter, with 95% volume of PTV was covered by the prescription dose.
Patients were followed up every month after treatment completion until 6 months, and every 3 months thereafter. Each patient was performed by chest X-ray or CT at each follow-up visit. During routine follow-up, the cases were evaluated in terms of RP based on clinical findings (e.g., dyspnea, cough, pain, and low-grade fever) and radiological findings. Once diagnosed, RP was further graded by at least two radiation oncologists according to the Keywords: Radiation pneumonitis prediction, 3D dose distribution, Dosiomics, Deep learning-based radiomics, Random forest Common Toxicity Criteria for Adverse Events (CTCAE) version 5.0 [22]. Grade 1: RP with symptoms or radiographic features without the need for steroids; Grade 2: RP requiring steroids or with symptoms that interfered with daily activities; Grade 3: RP requiring oxygen and steroids; Grade 4: RP requiring intubation. A diagnosis of RP grade ≥ 2 was defined as the primary end point. CT image examples of a CT without/with radiation pneumonitis were shown in Fig. 2. Patients with Grade 2 or higher (Grade ≥ 2) were labeled as having developed RP. A total of 40 patients were assessed as having RP with Grade ≥ 2. These 140 patients were randomly divided into the training set (n = 112, including 34 RP cases) and the test set (n = 28, including 6 RP cases). The design flow of this study is shown in Fig. 3.
Feature selection
First, redundant features were eliminated through Spearman's correlation coefficient (CC) analysis. Normalization may reflect the difference of prescribed dose. Here, as there was no significant difference in prescribed dose between the RP and non-RP groups, the normalized z-score was used for feature selection and RP classification in this study. Subsequently, Spearman's CCs were calculated. One of the two features that were highly correlated with the other remaining features would be eliminated if the CC between two kinds of features was ≥ 0.9 [25]. Next, least absolute shrinkage and selection [26] was employed to select a subset of features with predictive significance for each of the three binary classification models.
Model construction and performance
A random forest model was selected as the classifier that was widely used in radiomics and achieved good performance in many studies [27]. The area under the curve (AUC) score was used to test the performance of the prediction model. The optimal cut-off value by Youden index was calculated in the process of model construction and integrated into the calculation of the accuracy, sensitivity, and specificity.
Dosiomics and DLR feature correlation
In this study, we correlated the dosiomics features and DLR features through Spearman's rank CCs. Besides, the correlation analysis was visualized by the Circos software (http:// circos. ca) [28]. The feature sets with a correlation coefficient larger than 0.8 were selected to avoid over-cluttering during visualization.
Statistical analysis
The Spearman's correlation, LASSO regression, random forest classifier, and ROC curve analysis (evaluating the performance of binary classifiers) were conducted by the "sklearn" package, and the DLR features were extracted by the "PyTorch" package. The differences in clinical characteristics between patients with RP and without RP were evaluated by the t-test and Chisquare test. P value < 0.05 was considered statistically significant.
Plan and clinical characteristics
A total of 140 patients (102 males and 38 females; median age: 65.5) were included in this study, including 40 RP patients (27 males and 13 females; median age: 67) Grade ≥ 2. There was no significant difference in age, gender, tumor location, ITV volume, dose fractionations, V5, V10, V20, and MLD between RP and Non-RP. The plan and clinical characteristics of these patients are listed in Table 1.
Model performance
There were 22, 10 and 12 features in the dosiomics model, DLR model and hybrid model, respectively. The optimal cut-off value of the dosiomics, DLR, and hybrid models was 0.60, 040, and 0.50, respectively, in the training set, while that of dosiomics, DLR, and hybrid models in the test set was 0.60, 0.40, and 0.60, respectively. The ROC curve of different models in the training and test sets are presented in Fig. 4. The AUC of three models was larger than 0.99 in the training set; While, the AUC of the dosiomics, DLR, and hybrid models was 0.8462, 0.8750, and 0.900, respectively, in the test set. The accuracy, AUC, sensitivity, and specificity of dosiomics, DLR, and hybrid models in the training and test sets are listed in Table 2. The accuracy of dosiomics, DLR, and hybrid models in the training set was 0.9643, 0.9464, and 0.9642, respectively; While that of dosiomics, DLR, and hybrid models in the test set was 0.8214, 0.7857, and 0.8571, respectively. This indicated that combining dosiomics and DLR features could improve the model performance of RP prediction.
Correlation between dosiomics features and DLR features
The results obtained from correlation analysis based on the Spearman's correlation (represented by ρ) are listed in Table 3. For a quantification purpose, the number of ρ with an absolute value > 0.8 was counted. Group A was the Spearman's rho |ρ| ≥ 0.8 between dosiomics features and DLR features. Group B was the Spearman's rho 0.5 ≤ |ρ| < 0.8 between dosiomics features and DLR features. Besides, the ratio of the number of correlated feature In order to avoid over-cluttering, the correlation density in Group A was visualized by Circos (as shown in Fig. 5). The width of the link represents the correlation between the two kinds of features. The wider the link, the greater the absolute correlation. The positive correlation was represented in red color, while the negative correlation was represented in blue. All of the dosiomics Table 3 Correlation analyses between the dosiomics and DLR features using the Spearman's rho a Setting A: the Spearman's rho |ρ| ≥ 0.8 between the dosiomics and DLR features and B: the Spearman's rho 0.5 ≤ |ρ| < 0.8 between the dosiomics and DLR features b Format (l, m, n): l is the total number of feature pairs that were correlated, m is the number of dosiomics features correlated with DLR features, and n is the number of DLR features correlated with dosiomics features c Format (r, r r , r c ): r = number of correlations /total number of feature pairs, r r = number of dosiomics features correlated with DLR features/total number of radiomics features, and r c = number of DLR features correlated with dosiomics features /total number of DLR features used
Discussions
In this study, the RP prediction model for patients with lung cancer after SBRT was established based on dosiomics features and DLR features from 3D dose distribution of normal lung. The AUC of the dosiomics, DLR, and hybrid models was 0.8462, 0.8750, and 0.900. Both the dosiomics and DLR features could be used to predict the occurrence of RP after SBRT. Importantly, combining dosiomics features and DLR features could further improve the accuracy of the prediction model. The hybrid model is feasible in clinical scenarios. The dosiomics features and DLR features can be extracted from 3D dose distribution of normal lung, and the occurrence of RP can be predicted based on the previously established model within a few minutes after the completion of the radiotherapy plan. Interestingly, the prediction model does not depend on any clinical characteristic data apart from 3D dose distribution.
SBRT is the standard therapy for NSCLC patients who cannot receive surgery and could achieve favorable clinical outcomes [29]. Given that most patients receiving SBRT have severe comorbidities or are in a vulnerable state, RP should be prevented and/or actively managed. It is necessary to predict the occurrence of RP for the reason that it may reduce the benefits of SBRT. RP is directly related to dose information. Most clinical prediction models for RP only rely on clinical factors and DVH parameters. However, DVH cannot effectively explain spatial dose distribution or organ structure. Buettner et al. proved the importance of dose distribution relative to DVH in predicting the toxicity in patients with advanced rectal cancer, and the specific information provided by 3D dose distribution can better explain the relationship between dose information and toxicity [30]. Dosiomics features are statistical, geometric, or textural measures and they can provide quantitative measurements of the intensity, shape, or heterogeneity of a given volume of interest (VOI) in medical images [31]. When applied to dose distribution, these features may be related to the inhomogeneity of dose distribution [32]. Normalization may reflect the difference of prescribed dose. Here, as there was no significant difference between the RP and non-RP groups, the normalized to z-score was used for feature selection and RP classification in this study. DLR features have been applied to disease diagnosis and prediction [33,34]. The results of these studies have confirmed the potential of DLR features combined with dosiomics features in predicting RP.
The ML-or DL-based prediction models are highly dependent on datasets, and hence it is difficult to make a direct comparison between different studies due to different data sets. AUC can be used to compare the prediction performance of different models from different studies. For instance, the AUC of a model established by Liu et al. based on the clinical and DVH parameters was 0.76 [35]. In a study of RP prediction based on 3D dose distribution, Adachi et al. obtained an AUC of 0.837 ± 0.054 based on dosiomics features [21], which was at the same level of accuracy as the AUC of 0.846 in our study based on dosiomics features only. In this study, the DLR model outperformed the dosiomics model, and the hybrid model achieved the best performance. This indicated that combining dosiomics features and DLR features based on 3D dose distribution can improve the accuracy of RP prediction.
To the best of our knowledge, this is the first study to extract DLR features from 3D dose distribution to predict RP after SBRT. The correlation analysis was conducted between dosiomics features and DLR features. In this study, 0.8 and 0.5 were selected as the cutoff value, and there was a low correlation between dosiomics features and DLR features. There was little overlap in the RP-discriminative information expressed by these two groups of features. For the Spearman's rho |ρ| ≥ 0.8, the dosiomics features that correlated with the DLR features were all identified as the first-order features. Among them, original_firstorder_Median was applied to model established. The DLR features that correlated the origi-nal_firstorder_Median with the Spearman's rho |ρ| ≥ 0.8 included DLR 156, DLR676, DLR 483, and DLR 888, and they were not applied to model training. Different from dosiomics features, these DLR features have better performance in predicting RP.
However, there is a lack of an external testing cohort in this study. Nevertheless, the dosiomics and DLR features from 3D dose distributions can still be demonstrated to have benefits to RP prediction. Currently, collecting additional data from new patients represents a significant challenge, while it is an essential task for obtaining an even greater clinically relevant accuracy in predicting RP. Thus, data sharing collaboration and distributed learning suggested by Lambin et al. may play a key role in radiation oncology [36]. It is possible to establish an accurate prediction model for RP after SBRT based on sufficient multi-center data.
Conclusion
In this study, an ML model based on dosiomics and DLR features could effectively predict RP after SBRT, which indicates that hybrid radiomics is expected to be applied to RP prediction.
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Editorial: Molecular chaperones and human disease
Protein dysregulation is a hallmark of several human diseases. As such, molecular chaperones (being custodians of cellular proteostasis) are largely implicated in several diseases, including cancers and neurodegenerative disorders. The goal of this topic was to provide an update on the recent progress made in understanding the roles of molecular chaperones in the progression of model diseases with the prospects of targeting molecular chaperones toward the identification of novel drug therapies. Heat shock proteins (Hsps) are some of the most studied molecular chaperones that are associated with several chaperonopathies. They particularly play an important role in the pathogenesis of malaria, which is caused by the parasite Plasmodium falciparum. P. falciparum exhibits a very complex life cycle where its development partly occurs in a poikilothermic mosquito and a homeothermic human host. Hsps, therefore, play an integral role in ensuring that proteostasis is maintained throughout the parasite’s life cycle and may thus be exciting antimalarial drug targets. Despite several efforts in the development of novel antimalarial therapies, drug resistance to front-line malarial treatments presents an urgent need for the development of novel therapies that are more reliable as highlighted by (Mrozek et al.). Using in vitro and cell-based assays, Muthelo et al. reported that the anti-cancer drug 2-Phenylthynesulfonamide (PES) exhibits antiplasmodial activity and capability to inhibit the functions of the P. falciparum cytosol-localized chaperones PfHsp70-1 and PfHop (Muthelo et al.). Barth et al. reviewed the current state of knowledge about the Hsp70 family of chaperones focusing on the suitability of these proteins and interactions for drug development (Barth et al.). Together with the Hsp90s, Hsp70 proteins have been widely studied in several disease models though, to date, there have not been many FDA-approved Hsp70 drugs. A review by Daniyan et al. looked into the roles of the exported parasite chaperone PfHsp70x in the pathophysiology of cerebral malaria. The article also explored the possible links between host-parasite chaperones, and neurotransmitters, in relation to other molecular signalling components in the development of cerebral malaria (Daniyan et al.). This OPEN ACCESS
Editorial on the Research Topic Molecular chaperones and human disease
Protein dysregulation is a hallmark of several human diseases. As such, molecular chaperones (being custodians of cellular proteostasis) are largely implicated in several diseases, including cancers and neurodegenerative disorders. The goal of this topic was to provide an update on the recent progress made in understanding the roles of molecular chaperones in the progression of model diseases with the prospects of targeting molecular chaperones toward the identification of novel drug therapies.
Heat shock proteins (Hsps) are some of the most studied molecular chaperones that are associated with several chaperonopathies. They particularly play an important role in the pathogenesis of malaria, which is caused by the parasite Plasmodium falciparum. P. falciparum exhibits a very complex life cycle where its development partly occurs in a poikilothermic mosquito and a homeothermic human host. Hsps, therefore, play an integral role in ensuring that proteostasis is maintained throughout the parasite's life cycle and may thus be exciting antimalarial drug targets. Despite several efforts in the development of novel antimalarial therapies, drug resistance to front-line malarial treatments presents an urgent need for the development of novel therapies that are more reliable as highlighted by (Mrozek et al.). Using in vitro and cell-based assays, Muthelo et al. reported that the anti-cancer drug 2-Phenylthynesulfonamide (PES) exhibits antiplasmodial activity and capability to inhibit the functions of the P. . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Frontiers in Molecular Biosciences
frontiersin.org signalling pathway may provide further insights in antimalarial drug discovery. Collectively, the findings from these studies may contribute to the ongoing efforts in identifying novel antimalarial therapies, especially in the wake of growing parasite resistance against currently used drugs. Chaperone/co-chaperone inhibition is being explored as a potential therapeutic target for several diseases. A review by Caillet et al. looks into the potential roles of molecular chaperones in mediating cell and systemic stress in COVID-19. The article discusses the roles of the host stress response as a convergent point for COVID-19 and several non-communicable diseases while further assessing the merits of targeting the host stress response to manage the clinical outcomes of COVID-19. It sets an interesting argument on the possible roles Hsps might play in COVID-19 and the potential of targeting Hsps in novel COVID-19 therapy. Using an in silico approach, Jamabo et al. conducted a structural analysis of the Trypanosoma brucei (T. brucei) Hsp90 variants in relation to human and other trypanosomal species. T. brucei is responsible for African trypanosomiasis which is a neglected tropical disease mostly endemic to sub-Saharan Africa. The parasite is spread by insects (tsetse fly). Similar to P. falciparum, the trypanosome relies on heat shock proteins for survival in the insect vector and mammalian host. In their analysis, Jamabo et al. identified a total of eighteen putative T. brucei Hsp90 co-chaperones with one notable absence being cell division cycle 37 (Cdc37) (Jamabo et al.). Their findings provide an updated framework for approaching Hsp90 and its interactions as drug targets in the African trypanosome (Jamabo et al.).
Hsp90 has previously attracted a lot of attention in drug discovery research and several Hsp90-targeting drugs have gone through various stages of clinical trials in the development of treatments for cancers and cardiovascular diseases. A study by Scalia et al. reported the reduced Hsp90 expression levels observed in a mutant version of the CCT5 subunit from a patient with distal motor neuropathy. This indicates that the imbalance of the chaperone has a negative impact which potentially triggers the development of distal motor neuropathy. Follow-up studies could provide further information on how Hspdysregulation triggers neurophysiological disorders. The role of Hsp90 in tumor progression and prognosis was also investigated in the development of small cell lung cancer (Huang et al.). Upon analyzing the relationship between eHSP90α expression and clinicopathological features, eHSP90α and NSE were found to be positively correlated in patients with small cell lung cancer (Huang et al.). This study provided new evidence for the efficacy response and prognostic assessment of SCLC with eHSP90α being suggested to be a potential SCLC biomarker.
Altogether, the articles included in this topic highlight the new advances that have been made in the application of molecular chaperones in translational medicine. The section also reported on the successes and potential use of Hsps in novel drug therapies and biomarkers for several disease models. As there has been rapid development in the field of chaperone biology, we envision that novel, groundbreaking findings will further contribute to the development of applicable solutions in drug and biomarker discovery.
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Comparative analysis of dosimetry and predictive somatotype parameters of prone and supine whole-breast irradiation among Chinese women after breast-conserving surgery
Purpose Finding a better treatment position (prone or supine) for whole-breast irradiation for Chinese female patients diagnosed with breast cancer by identify the associations between predictive somatotype parameters and dosimetric gains. Materials and methods Two volumetric-modulated arc therapy (VMAT) plans were deployed for whole-breast irradiation in supine and prone position with a total dose of 50 Gy in 25 fractions. Dose-volume parameters were compared and analysed both in the target volume and organs at risk, and equivalent uniform dose-based figure-of-merit (fEUD) models were further used to quantitatively evaluate the overall merits of the two plans. Body shape parameters, including body mass index (BMI), body surface area (BSA), breast shape, cup size, bust size and chest size, were collected. Anatomic features such as the central heart distance (CHD) were measured on supine CT. Spearman’s correlation analysis, receiver operating characteristic (ROC) curve analysis, and the linear regression models were conducted. Results Doses to the heart and left anterior descending coronary artery (LADCA) are greater in left-sided breast cancer (BC) patients in the prone position than in the supine position, and the opposite was true for right-sided BC patients (p<0.001). 19 of 63 patients (5 left-sided and 14 right-sided BC) achieved greater benefit from the prone position according to the fEUD score. Right-sided BC patients with a bust size ≥92.25 cm, drop-type breasts and cup size ≥B are very likely to benefit from prone-position radiotherapy. The CHD is significantly positively associated with △fEUD among right-sided BC patients (rho=0.506, p=0.004). Using a cut-off point of 2.215, the CHD had 71.4% sensitivity and 81.2% specificity in predicting a successful prone plan. Conclusions Right-sided BC patients had better dosimetric gain in the prone position than left-sided BC patients. The CHD is an especially good and novel predictor that could help to select prone-benefitting right-sided BC patients.
Introduction
Breast cancer (BC) is the most commonly diagnosed cancer both in China and the whole world (1,2). Given the increased prevalence of cancer screening, the proportion of early BC diagnoses has significantly increased. Breast-conserving surgery (BCS) is the standard surgical treatment for operable, early-stage BC. The percentage of patients undergoing BCS increased from 10.83% to 30.83% between 2006 and 2015 in China (3). Adjuvant radiotherapy (RT) after BCS for early-stage BC can effectively improve the survival rate and reduce the risk of recurrence (4,5) while providing satisfactory cosmetic results as well as psychological support. As such, postoperative RT is considered the standard treatment for early-stage BC.
Generally, the supine position has been widely used for clinical RT in BC, as it is more comfortable and reproducible for patients than the prone position. However, irradiation for BC patients in the prone position could achieve better dose distributions and spare more normal tissue than the supine position (6)(7)(8), especially those with large breasts. Two randomized trials focused on the 2-year and 5-year whole breast irradiation outcomes in the prone versus supine positions among large-breasted women (9, 10) demonstrated better cosmetic outcomes and lower rates of late toxicity in the prone position.
Consistent criteria have yet to be established for selecting patients who would benefit most from prone RT. Studies on prone positioning for BC treatment have mainly been conducted in American and European countries. One South Korean study (11) suggested that patients with small breast volumes (such as those with a clinical target volume (CTV) of approximately 100 cm 3 ) could also benefit from the prone position.
Therefore, we conducted this study comparing the prone position with the supine position for delivering volumetricmodulated arc therapy (VMAT) to Chinese BC patients. The purpose was to assess the effects of the prone position on the dose distribution and determine differences in normal organ sparing between VMAT in the two positions. We further attempted to identify that body shape characteristics associated with prone position-benefitting breast RT among Chinese women to provide a reference basis for the rational, clinical use of the radiotherapy position.
Patients and treatment simulations
The inclusion criteria were as follows: age between 18 and 70 years, pathologically confirmed stage 0-II BC (Tis-T2) after BCS, and Eastern Cooperative Oncology Group performance status 0 or 1. Patients were excluded if they needed irradiation of the locoregional lymph node area and had prosthetic implants, supraclavicular/internal mammary nodes, bilateral BC, previous irradiation or other malignancies. All patients were asked to provide their written informed consent before being registered in the study, and the present study was approved by the ethics committee of Tumor Hospital of Yunnan Province (approval number of Institutional Review Board: KYLX2022025).
Enrolled patients underwent two computed tomography (CT) simulations in the supine and prone positions. First, patients were imaged on a conventional supine breast board (R610-DCF, Klarity Medical & Equipment Co. Ltd. Guangzhou, China) with arms above the head to adequately expose the breast ( Figure 1A). Then, they were repositioned on a prone board (R62-BCF4, Klarity Medical & Equipment Co. Ltd. Guangzhou, China) with a removable right and left aperture to allow the index breast tissue to hang away from the chest wall ( Figure 1B). The borders of the breast tissue and midline of the chest were marked for each patient with radio-opaque wires before CT acquisition. For both setups, free-breathing CT scans were performed using a large-aperture CT system (SOMATOM Sensation Open 24, Siemens, Germany) without contrast, starting below the mandible and caudally ending below the lower edge of the liver with a slice thickness of 3.0 mm. The CT scan images were transferred to the Treatment Planning System (Monaco version 5.11, Elekta AB, Stockholm, Sweden) of the department.
Radiotherapy planning and evaluation
CTVs and organs at risk (OARs) were contoured manually according to the Radiation Therapy Oncology Group (RTOG) breast cancer atlas (12) (Figures 2A, B). The breast CTV was contoured up to the inferior margin of the clavicular heads (cranially), to the farthest visible breast contour, at approximately the level of apex disappearance (caudally), to the perforating mammary vessels or to the edge of the sternum (medially), to the anterior edge of the latissimus dorsi (laterally), to the junction of the breast tissue and the pectoralis muscles (posteriorly), and up to 5 mm under the skin surface (anteriorly). The CTV was delineated based on the glandular breast tissue visible on the CT images. Planning target volumes (PTVs) were generated by the addition of threedimensional, 5-mm margins to the CTV up to 5 mm from the skin. The whole heart was delineated in accordance with the guidelines proposed by Feng et al. (13). The left-anterior descending coronary artery (LADCA) does not include the left main trunk, which was delineated down to the apical level. Considering the planned volume of the heart while beating, the uniform diameter of the LADCA is 1 cm. OARs such as lungs, spinal cord, esophagus and liver were delineated according to the RTOG 1106 atlas (14). In detail, all inflated and collapsed, fibrotic, and emphysematic lungs were contoured with inclusion of small vessels extending beyond the hilar regions, excluding the proximal bronchial tree. The contralateral breast was delineated up to 5 mm under the skin surface. The spinal cord was delineated starting at the same cranial level as the esophagus to the bottom of L2 or at the level in which the cord ended. The oesophagus was delineated starting cranially from the inferior margin of the cricoid and ending inferiorly at the gastroesophageal junction. The whole liver was delineated along the outer edge of the liver, excluding the gallbladder. The CTV and OARs were delineated on CT slices by one radiation oncologist and verified by two other senior experienced radiation oncologists. The RT plans were generated for a Versa HD linear accelerator (Elekta Medical Systems Co., Stockholm, Sweden) with 6 MV photon energy. Previous studies have showed that (15), VMAT could achieve better target conformability and uniformity compared to intensity-modulated radiation therapy (IMRT). Considering the further comparison of dosimetric differences between important normal organs, such as the heart and lung, on the basis of ensuring adequate target coverage, the VMAT irradiation technology being commonly used in our institutions and in this study. Referring to the correlational researches (15, 16), we used a continuous VMAT (cVMAT) treatment plan with one dual arc of (140.0 ± 10.0)∼(320.0 ± 10.0)°for the supine position ( Figure 2A). The prone plans consisted of tangential VMAT (tVMAT) plans with two tangential dual arcs of (140.0 ± 10.0)∼(120.0 ± 10.0)°and (340.0 ± 10.0)∼(310.0 ± 10.0)°rotations, accounting for the limitations of the machine boom rotation ( Figure 2B). A prescription dose of 50 Gy in 25 fractions was delivered to the whole breast according to the ICRU report 83 (17), with the prescribed dose covering ≥95% of the PTV and ≤7% receiving 105% of the prescribed dose. And according to the relevant research (11) and institutional experience, we constrainted OARs were as follow: V20 < 30% for contralateral lung; mean heart dose < 6 Gy (left and right), and maximum dose of spinal cord <40Gy in the supine position; V20 < 20% for contralateral lung; mean heart dose < 8 Gy (left) or 6Gy (right), and maximum dose of spinal cord < 40 Gy in the prone position. A radiotherapy planning consensus for both sets was achieved by the agreement of more than two physicists. Only the supine treatment plan was used for real-world clinical daily RT.
All plans were compared according to the planning target volume coverage, dose-volume histogram and other dosimetric parameters of normal tissues. For target coverage, we recorded the minimum, maximum and mean doses to the PTV (Dmin, Dmax, Dmean), V95%, V105%, V100%, homogeneity index (HI) (18), and conformity index (CI) (19). The CI and HI were calculated using the following equations: 1) CI=(TV95/ TV) × (TV95/V95), where V95 is the total volume receiving 95% of the prescription dose, TV is the target volume, and TV95 is the target volume receiving 95% of the prescription dose, with values closer to 1 indicating optimal conformation; 2) HI= (D2% -D98%)/D50%, where D2%, D50% and D98% are the doses covering 2%, 50% and 98% of the volume of the PTV, with lower values indicating administration of a more homogeneous dose to the target volume. For normal organs, such as the heart and ipsilateral and contralateral lung, we compared Dmax, Dmean, and the percentage of the volume that received more than 5, 10, 20, 30, and 40 Gy (V5, V10, V20, 30, and V40).
Anthropometric body shape parameters
Body shape parameters, including height, weight, body mass index (BMI), body surface area (BSA), bust size and chest size were collected. BMI=weight(kg)/height(m) 2 . BSA=0.0073×/ height(m)+0.0127×weight(kg)-0.2106. Bust size was measured as the circumference around the chest at the plane of the nipple. Chest size was measured as the circumference around the chest under the fold of the breasts. We also collected general information, including the breast shape ( Figure 3) and cup size of all patients.
Supine anatomic feature measurements
Song et al. (20) reported that breast separation (BS) was positively correlated with the mean skin dose and was an important parameter for the selection of electronic tissue compensation radiotherapy. BS was defined as the distance between the entry points of two opposing beams on the central plane. In addition, the central lung distance (CLD) has been said to provide a close estimation of the volumetric lung dose; when the CLD is greater than 3.0 cm, the reduction in the dose delivered to the ipsilateral lung was found to be remarkable when using the medial breast technique (21). The CLD was defined as the perpendicular distance from the chest wall to the posterior border of the tangential fields. Type of breast shape.
Since the BS and the CLD could only be recorded after RT planning, we choose the modified breast separation (mBS) and modified central lung distance (mCLD) as alternative indicators which could be measured on routine chest CT. The mBS was defined as the distance from the border of the sternum and the anterior border of the latissimus dorsi extending to the skin. The mCLD was defined as the maximum perpendicular distance from the mBS to the posterior part of the anterior chest wall. Both parameters were measured on the central plane (similar to the central PTV plane) from the lower edge of the clavicular head to the cardiac apex on supine CT ( Figure 4).
Additionally, we creatively assessed a new concept, the central heart distance (CHD), as a predictive parameter for the heart doses. The CHD is the perpendicular distance from the centre point of the heart to the midline on the central heart plane on supine CT ( Figure 5). The central heart plane is the middle CT slice from the bifurcation of the pulmonary trunk (superior border) to the last slice containing cardiac tissue (inferior border). The midline was measured from the sternum centre to the posterior margin of the spinous process. The centre point of the heart was automatically computed as a threedimensional point by Monaco ® TPS 5.11.
EUD and fEUD models for plan comparison
The equivalent uniform dose (EUD), defined as the uniform dose giving the same biological effect as a given nonuniform dose distribution, was generalized to normal structures and tumours by Niemierko in 1999 (22). The generalized EUD (gEUD) was calculated based on the power-law dependence of the dose response for the tumour and the OARs with the following where vi is the fraction of the reference volume irradiated with dose Di, and a is a free structure-specific parameter that is usually positive for OARs and negative for tumours. Base on the article by a previously published article by Boughalia et al. (23), we set a(PTV)=-6, a(heart)=2, a (ipsilateral lung)=2, a(contralateral lung)=5, a(LADCA)=5, a (contralateral breast)=5, and a(liver)=5. The vi and di values in the prone and supine position plans of each patient were derived from the Monaco TPS and substituted into the EUD formula to calculate the EUD values of the target areas and OARs in the two plans.
Qi et al. (24) created an EUD-based figure-of-merit (fEUD) to quantify the overall plan quality when attempting to use the EUD model to optimize the target and OAR doses. The results showed that the fEUD model can effectively evaluate plans for brain, head and neck, lung, pancreas and prostate tumours. In our previous study, the fEUD model was successfully applied to evaluate the quality of the physical scheme in cervical cancer. The fEUD is computed according to the following equation: where n and m are the numbers of OARs and targets, respectively, wi and wj are the corresponding weighting factors, and k is the relative importance factor between the weighted sums of the EUDs for all targets and the OARs. We set wi, wj and k to 1 in this study. The fEUD value ranges from 0 to 1, with greater values indicating superior plan quality. Then, the FIGURE 4 Anatomic parameters in the supine CT. The central plane from the low edge of clavicular head to the cardiac apex in the supine CT. The breast separation (BS) is the distance between entry points of two opposing beams on the central plane. The central lung distance (CLD) is the perpendicular distance from chest wall to the posterior boarder of the tangential fields.The modified breast separation (mBS) is the distance from the border of the sternum and the anterior border of latissimus dorsi then extending to skin.The modified central lung distance (mCLD) is the maximum perpendicular distance from BS to the posterior part of the anterior chest wall.
EUD value is substituted into the fEUD formula to calculate the fEUD value of the prone position and supine position. Finally, we calculated fEUD (prone-supine) to compare the overall quality of the two plans. A positive value of fEUD (prone-supine) indicates that the prone position plan is better, and a negative value indicates that the supine position plan is better.
Statistical analysis
Dosimetric parameters were examined by the paired t test or Wilcoxon signed-rank test. Correlations were measured using Spearman's correlation coefficient (rho). Receiver operating characteristic (ROC) curve analyses were used to examine the predictive validity of the somatotype parameters. Linear regression models were used to explore more conveniently measurable predictors. All statistical analyses were conducted by SPSS Statistics software for Windows ver. 25.0 (IBM Corp., Armonk, NY). Differences were considered significant at p values < 0.05.
Dosimetric analyses
Between June 2020 and June 2021, 160 female patients underwent whole-breast RT after BCS were randomly chosen for this study. Of these patients, 58 did not meet the inclusion criteria, and 39 did not give consent and were excluded. Finally, a total of 63 patients were enrolled (33 with left-sided and 30 right-sided breast cancer). The baseline patient characteristics are shown in Table 1.
We performed comparisons between the prone and supine positions for the entire patient cohort, and the results are summarized in Table 2. For all patients, the prone position reduced the doses to lungs but increased the average volume of the breast and ipsilateral lung and the Dmean of the contralateral breast relative to the supine position (p< 0.05). For left-sided BC, compared with those of the supine position, all dose values (Dmean and V5-V40) of the heart and the Dmax and Dmean of LADCA were higher in the prone position (p ≤ 0.001). For rightsided BC, the Dmax and Dmean of the LADCA was lower in the prone position than in the supine position (p< 0.001). The Dmean of the heart was lower in the prone position, although the difference was not significant.
Overall plan figure-of-merit (fEUD) Table 3 shows the fEUD values for the prone and supine VMAT plans. We found that 19 patients (5 with left-sided and 14 with right-sided BC) benefitted from the prone position according to this quality score. The mean, minimum, maximum volume of the CTV for these 19 patients were found to be 686.45cm 3 , 396.98cm 3 , 1512.25cm 3 , respectively.
Correlation analysis
According to the comparison between the two setups' fEUD values, we used "△fEUD" to assess whether the prone plan was better than the supine plan; if so, the patient was given a value of 1, and otherwise. Correlations between various analysed parameters were calculated using the Pearson test or Spearman rank test, depending on the normality of the distribution. If the FIGURE 5 CHD in the supine CT. The central heart plane. The central heart distance (CHD) is the perpendicular distance from centre point of heart to the midline. assumption of normality was not fulfilled, we calculated the Spearman correlation coefficients. So Spearman's correlation analysis was conducted between the △fEUD value and the values of the different somatotype parameters ( Figure 6). Figure 6 shows the correlation between somatotype parameters and the △fEUD value; for example, the value in the BS grid indicates that the Spearman correlation coefficient (rho) between BS and △fEUD is 0.368, and the corresponding p value is 0.003. We found a weak, positive correlation between BS and △fEUD, and the p value indicates statistical significance. In other words, a longer BS indicates a greater likelihood that the prone position will be better than the supine position. △fEUD was weakly negatively correlated with breast side, bust size, BS and CTV (rho=0.276~0.368, p< 0.05). Subsequently, a multiindex ROC curve was drawn to evaluate the accuracy of these predictors. As shown in Figure 7A, the AUC values for supine CTV, BS, bust size and breast side were 0.702, 0.731, 0.673 and 0.687, respectively; this indicated that supine CTV≥495.996 cm 3 (68.4% sensitivity, 68.2% specificity), BS≥21.735 cm (57.9% sensitivity, 84.1% specificity), bust size≥92.25 cm (84.2% sensitivity, 59.1% specificity) and breast side=right (73.7% sensitivity, 63.6% specificity) could predict a benefit from the prone position.
The above results potentially suggest that right-sided breast cancer patients with a CTV≥495.996 cm 3 , BS≥21.735 cm and bust size≥92.25 cm were very likely to benefit from prone RT. However, the CTV and BS values were not available directly from routine chest CT images. Therefore, we attempted to explore the relationship between BS and CTV and other directly measurable somatotype parameters. Positive correlations were identified between BS and breast shape (rho=0.468, p< 0.001) and between CTV and cup size (rho=0.452, p< 0.001), according to the Spearman correlation analysis. Analysis of the linear models (Table 4) demonstrated Lower doses were delivered to the heart, LADCA and both lungs for right-sided breast cancer patients, and the fEUD model scored 14/30 right-sided breast cancer patients as the "prone beneficial group", as previously described. Based on these data, we found that the CHD was significantly and positively associated with △fEUD among right-sided breast cancer patients (rho=0.506, p =0.004), and ROC curve analyses showed an AUC of 0.792 ( Figure 7B). When using 2.215 cm as the cut-off value, the CHD index achieved a sensitivity of Values are presented as mean±standard deviation. CTV, clinical target volume; LADCA, left anterior descending coronary artery; Dmin, minimum dose; Dmax, maximum dose; Dmean, mean dose; CI, conformity index; HI, homogeneity index; OARs, organs at risk; V X , percentage of the volume that receives more than X Gy.
FIGURE 6
Color map of rho between "△fEUD" and somatotype parameters. "△fEUD", whether the prone plan is better than the supine, yes=1, no=0. 19/63 cases were determined as prone-position benefited according to fEUD scores' comparison. The higher the fEUD value, the better the overall quality of plans.Supine-CTV,clinical target volume in supine computed tomography.
71.4% and a specificity of 81.2% in predicting a successful response to prone RT for right-sided breast cancer patients.
The CHD was originally designed as a cardiac dose predictor; Spearman's correlation analysis showed that the CHD was negatively correlated with DHeart V10 (prone-supine) among right-sided BC patients (rho=-0.441, p< 0.05) but was not correlated with the heart dose values among left-sided BC patients.
Discussion
Prone-position breast RT has previously been confirmed to be more beneficial for women with pendulous or large breasts of volumes ≥750 or 920.3 cm 3 than the supine position (6,8) because it elongates the treated breast away from the chest wall, which could help to prevent acute skin toxicity, especially along the inframammary fold. This study is one of few about prone breast RT that focus specifically on patients of Eastern ethnicities, such as Chinese, Korean and Japanese, who usually have a smaller breast size and body size than Western women.
Our results suggest that right-sided BC patients with a bust size≥92.25 cm, drop-type breasts and cup size≥ B are highly likely to benefit from prone positioning, while left-sided BC patients conversely are unsuitable for prone RT because of their higher heart and LADCA doses than in the supine position. According to relevant previous studies, the reasons for this phenomenon may include the following. 1) The heart could fall anteriorly towards the chest wall due to gravity in the prone position, moving it closer to the breast target volume and increasing the area that receives higher doses. 2) The average breast size was 549.24 cm 3 (in the supine position) in this research, generally smaller than the recommended pronebeneficial breast volume of 750 cm 3 in some studies (6). Taking the motion of the heart into account, if the breast is not sufficiently large and pendulous enough to be pulled away from the chest wall, the cardiac dose is likely to increase. 3) The RT technique used in this study is VMAT. Compared with IMRT, which was used in the majority of previous proneposition breast RT studies, the VMAT technique has been shown to improve the target dose homogeneity and conformity but inferior in terms of cardiac protection (15, 25). Our institution has been using the VMAT technique for many (11) also showed a dosimetric advantage in prone breast RT for patients with a small breast size (approximately 100 cm 3 ). When exploring the relationship between body shape and dosimetry, we chose two methods to collect somatotype parameters, i.e., anthropometric and image CT measurements. Moreover, the fEUD model, proposed by Qi et al. (24) was used to score the prone and supine plans for a quantitative assessment of overall quality. The OARs in the formula do not include the skin, spinal cord, or oesophagus, which are less irradiated within the treatment field. Correlation and ROC curve analyses showed that the possibility of a benefit from the prone position increased for a CHD≥2.215 cm for right-sided BC patients.
Several studies (20, 21,26) have demonstrated that the maximum heart distance (MHD) is a good predictor of the mean heart dose. The MHD was measured as the maximum width of the heart in the tangent fields. Nonetheless, considering the following limitations of the MHD, we did not use it in this study. 1) The MHD needs to be recorded on beam's eye view of the simulation CT, not on a routine physical examination CT. 2) BC can be either left or right-sided, the MHD in this study was not always a positive value but could also be 0 or negative. Therefore, it cannot be comprehensively and efficiently measured and analysed. 3) The central level of the heart is the distance to the level where the MHD is located, and there is no clear relationship between the two (27). In addition, although it has been demonstrated that other CT lines, such as BS, CLD, mBS and mCLD, are related to cardiopulmonary sparing, they do not yield an obvious prediction.
Therefore, we creatively defined the CHD, which is longer in the prone position than in the supine position because of the leftanterior motion caused by gravity. Logically, if a left-sided BC patient has a longer CHD in the supine position, it means the heart is closer to the target area, and the irradiated volume and dose to the heart will increase when changing from the supine to the prone position. In contrast, the longer the CHD is, the more cardioprotective it is for right-sided BC patients. Consistent with the above hypothesis, our results indicate that the CHD was a good predictive parameter that could be measured on routine chest CT to help select patients with right-sided BC who may benefit from prone-position radiotherapy.
The clinical application and popularization of prone breast RT are mainly restricted for the daily repeatability and stability. Some patients can not tolerate RT in the prone position, especially those with lumbar spine diseases or thoracic malformations. In studies concerning prone BC RT, multiple institutions have modified their prone setups to improve comfort and reduce errors (28). At present, there is no standardized prone-treatment board for breast RT. The prone boards from Orfit, Bionix, and especially Civco have been described in related studies (11,29). Our prone board was provided by Klarity, and the tendency of the heart to move left anteriorly was less obvious, but the separation of the contralateral breast from the tangential field was not as notably protective as with the board from Civco. No comparison related to comfort and stability could be made.
We first raised the conception of CHD in this study to compare prone vs. supine whole breast radiotherapy for Chinese women, whose somatotype is relatively smaller than that of Western women. We sought to determine whether the smaller body figures and breast size of the Chinese population could benefit from prone radiotherapy. Additionally, we attempted to identify that anatomical characteristics could potentially indicate the benefit of normal tissue, further select the dominant treatment position without two CT simulations,which means more costs for the patients and more workload for physicians and physicists. We also used fEUD models in a innovative and prudent manner to quantitatively evaluate the overall merits of the two plans and the CHD and other geometric lines to explore their correlation with dosimetry.
However, we are aware that the relative small number of cases might increases the contingency of our analysis and some associations might be underestimated. Further studies in a wider cohort are needed to validate our existing results in a greater depth.
Conclusions
For whole-breast irradiation after breast-conserving surgery, compared with the supine position, the prone position resulted in lower heart and ipsilateral lung doses for right-sided BC patients, while higher heart and LADCA doses were observed for patients with left BC. The prone benefit was more prominent for right-sided BC patients with drop-type breasts, greater bust and cup sizes, and, notably, longer CHD.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
All patients were asked to provide their written informed consent before being registered in the study, and the present study was approved by the ethics committee of Tumor Hospital of Yunnan Province (approval number of Institutional Review Board: KYLX2022025). The patients/participants provided their written informed consent to participate in this study.
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2022-11-18T15:24:45.456Z
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2022-11-17T00:00:00.000Z
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253598496
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s2orc/train
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Multi-parameter ultrasonography-based predictive model for breast cancer diagnosis
Objectives To develop, validate, and evaluate a predictive model for breast cancer diagnosis using conventional ultrasonography (US), shear wave elastography (SWE), and contrast-enhanced US (CEUS). Materials and methods This retrospective study included 674 patients with 674 breast lesions. The data, a main and an independent datasets, were divided into three cohorts. Cohort 1 (80% of the main dataset; n = 448) was analyzed by logistic regression analysis to identify risk factors and establish the predictive model. The area under the receiver operating characteristic curve (AUC) was analyzed in Cohort 2 (20% of the main dataset; n = 119) to validate and in Cohort 3 (the independent dataset; n = 107) to evaluate the predictive model. Results Multivariable regression analysis revealed nine independent breast cancer risk factors, including age > 40 years; ill-defined margin, heterogeneity, rich blood flow, and abnormal axillary lymph nodes on US; enhanced area enlargement, contrast agent retention, and irregular shape on CEUS; mean SWE higher than the cutoff value (P < 0.05 for all). The diagnostic performance of the model was good, with AUC values of 0.847, 0.857, and 0.774 for Cohorts 1, 2, and 3, respectively. The model increased the diagnostic specificity (from 31% to 81.3% and 7.3% to 73.1% in cohorts 2 and 3, respectively) without a significant loss in sensitivity (from 100.0% to 90.1% and 100.0% to 81.8% in cohorts 2 and 3, respectively). Conclusion The multi-parameter US-based model showed good performance in breast cancer diagnosis, improving specificity without a significant loss in sensitivity. Using the model could reduce unnecessary biopsies and guide clinical diagnosis and treatment.
Introduction
According to Global Cancer Statistics 2020, breast cancer has surpassed lung cancer as the leading cause of global cancer incidence, and it ranked first for incidence and mortality in the vast majority of countries among women (1). Compared with the traditional management of breast cancer, the current therapy shifts from surgical approach to precise and individualized treatment. Especially in recent years, with the development of molecular biology, a number of new biological markers for the prognosis of breast cancer, represented by WDR34 mRNA, provide novel target for the diagnosis and treatment of breast cancer (2). However, the satisfactory treatment effect depends not only on the change of treatment methods and prognosis judgment, but also on accurate preoperative diagnosis.
Ultrasound (US) is the most used modality for breast cancer detection and diagnosis among Chinese women, whose breasts are usually more denser compared to Caucasian women (3). With the advantages of convenient, non-ionizing, non-invasive, inexpensive, and provides real-time imaging, conventional US can provide useful information about breast lesions and the surrounding tissue (4). Unfortunately, although US has relatively high sensitivity, its moderate specificity, due to the small lesion size or atypical features, often leads to false positive findings and many unnecessary biopsies. Therefore, new US technologies were developed to supplement the conventional US, including shear wave elastography (SWE) and contrastenhanced US (CEUS) (5,6). SWE can be used to estimate the stiffness of lesion qualitatively or quantitatively. The use of SWE, especially in combination with conventional US, has increased the diagnostic accuracy, compared to single mode US (7,8). The stiffness can be assessed qualitatively by analyzing a color-scaled image and/or quantitatively by determining the mean and maximum elasticity values (kPa) as well as the ratio of maximum elasticity to adipose tissue. In this way, the color closer to red and the higher elasticity value or ratio indicate malignant lesions.
CEUS has been used in clinical practice to provide more information regarding tumor blood supply to differentiate benign from malignant breast lesions (9,10). Abnormal blood perfusion or blood vessel filing patterns observed on CEUS images and videos could reveal perfusion characteristics associated with malignant tumors. Studies on the role of CEUS in the past decade have shown that CEUS could increase the specificity of conventional US (11)(12)(13).
Compared to the single mode US with obtain limited diagnostic information, multi-parameter US is considered to provide more systematic and comprehensive information. The diagnostic performance of SWE or CEUS combined with conventional US had been reported, but these studies only employed two of the three US modes (14,15). Only a few studies have combined all three modes, whereas it did not include all breast lesions categories of BI-RADS 3 to 5, nor did it include quantitative analysis of SWE (16)(17)(18). Therefore, we aimed to develop, validate, and evaluate a diagnostic predictive model for breast lesion diagnosis (BI-RADS 3 to 5) using multiparameter US (conventional US, SWE, and CEUS), comparing it to diagnosis by conventional US alone. The purpose of this study is to assess the value of multi-parameter US in the diagnosis of breast cancer, to invest whether it can improve the diagnostic efficiency, and reduce unnecessary breast biopsies.
Patients
This study retrospectively analyzed 674 consecutive patients (mean age, 47.26 ± 14.53; range, 18-94 years) with 674 pathologically-confirmed breast lesions treated at Shanghai General Hospital (Shanghai, China) from June 2018 to December 2020. The inclusion criteria were as follows: aged over 18 years; underwent conventional US, SWE, and CEUS examinations performed by the same sonographer with the same US machine, as was usually done when evaluating patients with a breast mass before surgery; available pathology results for each lesion after surgery or core needle biopsy. Thus, the study totally included 680 breast lesions in the main dataset and 200 breast lesions in the independent dataset.The exclusion criteria were incomplete or unsatisfactory images, treatment before surgery, pregnancy or breastfeeding, and past breast implant surgery. As a result, the main dataset finally included 567 breast lesions and the independent dataset finally included 107 breast lesions, as shown in Figure 1. The most suspicious or largest lesion was chosen in patients with multiple pathologically-confirmed lesions. This retrospective study was approved by the institutional ethics committee of Shanghai General Hospital, the patients signed informed consent forms before CEUS was performed, and all participating researchers were blinded.
The data were divided into three cohorts according to the two campuses of the hospital and in chronological order. The main dataset was divided into Cohorts 1 and 2. It included examinations conducted by two sonographers from the South Campus, both with over five years of experience in breast US. The independent dataset used for Cohort 3 included examinations conducted in the North Campus by a third sonographer with over five years of experience in breast US. Cohort
Conventional US, SWE, and CEUS examination
All three US techniques were performed using the same US machine (APlio 500, TOSHIBA Medical Systems, Minato Ward, Tokyo, Japan) following the American Institute of Ultrasound Medicine guidelines. All patients were positioned with their breasts fully exposed. Conventional US was performed using a linear transducer (7-12 MHz), noting the lesion location, size, length-to-width ratio, margin (well-defined, ill-defined), shape (oval, irregular), internal echo (hypoechoic, isoechoic, hyperechoic), posterior echo (with or without attenuation), peripheral tissue distortion (with or without), microcalcification (with or without), blood flow (Adler grades II and III were defined as rich, Adler grades 0 and I were defined as non-rich), and axillary lymph node status (normal, abnormal). The images were stored.
Subsequently, SWE was performed using the same linear array transducer. A region of interest (ROI) that included the entire lesion and a small amount of surrounding tissue was drawn. The hand holding the probe was as light as possible to ensure accurate results. The differential diagnosis of benign from malignant breast lesions considered both qualitative and quantitative SWE aspects. The analysis was based on color, with red representing stiff and blue representing soft tissue. Five SWE images were recorded for the qualitative analysis.
Finally, CEUS was performed using a linear transducer (4-9 MHz). The target section selected for CEUS was based on the plane with the richest blood supply as visualized on conventional US. The most suspicious plane was selected if no plane with abundant blood supply was detected, e.g., the plane with the maximal diameter or one with an irregular shape. CEUS was performed in the dual-image mode to ensure accuracy of the results, and the mechanical index was set to 0.06. Sulfur hexafluoride microbubbles (4.8 mL; SonoVue ® , Bracco Imaging S.p.A., Milan, Italy) were injected through the antecubital vein, followed by injection of 5-10 mL of saline. The videos and images were recorded for 180 s starting immediately after injection.
Image analysis
Two skilled sonographers, different from the above and blinded to the pathological results and each other's findings, analyzed all US images. Both had over five years of experience in conventional US, SWE, and CEUS for breast cancer diagnosis. Disagreements were resolved by discussion to reach a consensus. Lesions in the conventional US and CEUS images were classified following the Breast Imaging Reporting and Data System (BI-RADS) guidelines into categories 0, 1, 2, 3, 4a, 4b, 4c, 5, and 6. Lesions in Category 3 were considered benign, whereas those in categories 4a, 4b, 4c, and 5 were considered malignant. The suspicious sonographic features of malignancy were as follows: irregular shape, ill-defined margins (spiculated or angular), heterogeneity, microcalcification, posterior echo attenuation, length-to-width ratio >1, blood flow grades II-III, and abnormal axillary lymph nodes.
In the qualitative SWE analysis, lesions showing a maximal red color were referred to as stiff and those showing maximal blue color as soft. Quantitative SWE analysis was based on measurements performed on each SWE image and included the mean value of the entire lesion (SWEmean), maximum value (SWEmax, the ROI placed on the stiffest area), surrounding fat tissue (SWEfat, preferably at the same depth as the lesion), and lesion-to-fat velocity ratio (R, calculated using the acquired SWEmax and SWEfat). All five SWE images were analyzed, and the average values for SWEmean, SWEmax, and R were recorded.
CEUS analysis was based on our clinical experience and previous studies (19, 20). The following parameters were recorded: enhancement intensity (no, hypo-, iso-, or hyperenhancement compared to the surrounding breast tissue), time (synchronous, earlier, or later enhancement compared to the surrounding breast tissue), direction (from the periphery inward, from the inside to the periphery, or all simultaneously), and pattern (presence or absence of ring and crab claw-like patters), and internal homogeneity, perfusion defect, contrast agent retention, and penetrating vessel. The following parameters were measured at the peak enhancement inside the lesion: enhanced area enlargement and the lesion's size, margin, and shape. The CEUS BI-RADS scores were determined using the five-score system proposed by Xiao et al. (5). The following CEUS features were considered: enhancement homogeneity (heterogeneous, homogeneous), enhancement margin (not circumscribed, circumscribed), perfusion defect (present, absent), early hyperenhancement (present, absent), penetrating vessel (present, absent), and enhanced area enlargement (yes, no).
Histopathological examination
All patients underwent breast coarse-needle biopsy or surgery within three days of performing the multi-parameter US examinations. Typical sections were processed and stained with hematoxylin and eosin for histopathology examinations. For patients who underwent both breast needle biopsy and surgery, the histopathology results after surgery were used as the final diagnosis of the lesions. The tissue sections were examined by experienced pathologists who were blinded to the clinical information. The histopathology results were considered the reference standard for the lesion.
Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics for Window, Version 26.0 (IBM Corp., Armonk, NY, USA). Quantitative data (i.e., patient age and lesion size) are expressed as mean ± standard deviation and were compared by the Student's t-test. The chi-squared test compared categorical variables. Univariate and multivariable logistic regression analyses were used successively to determine predictors for malignancy using Cohort 1. Once the predictive model was established, the regression coefficient (b), standard error (SE), the results of the hypothesis test commonly used for the regression coefficient (Wald c 2 ), and odds ratios (ORs) with their 95% confidence intervals (CIs) were recorded. The diagnostic performance of the predictive model and conventional US BI-RADS were assessed by plotting receiver operating characteristic (ROC) curves and assessing the areas under them (AUC). Sensitivity and specificity were calculated using ROC analysis. The best cutoff values were obtained using the Youden index (maximum sensitivity + specificity -1). P values < 0.05 were considered statistically significant.
Based on the data in Table 2 If one of the indexes is positive, it will be defined as 1. Otherwise, it will be defined as 0. The final result p greater than 0 indicates a benign lesion, while p less than 0 indicates a malignant lesion.
Validation and evaluation of the predictive model performance
The model's performance was analyzed in terms of accuracy, sensitivity, specificity, and AUC. The respective values for Cohort 2 were 86.6%, 90.1%, 81.3%, and 0.857. The AUC values for Cohorts 1 and 3 were 0.847 and 0.774, respectively (Figures 2A-C), indicating that the model had a favorable diagnostic value. This was further confirmed in two cases (Figures 3 and 4) in which the conventional US was wrong while the model was correct.
Reduction of unnecessary biopsies by the predictive model
Cases of unnecessary biopsies were those with BI-RADS > 3 based on conventional US but pathologically proven benign. The number of lesions with conventional US BI-RADS > 3 in Cohort 1 that were correctly downgraded by the predictive model was 73
Discussion
Conventional US, SWE, and CEUS play distinct and important roles in breast cancer diagnosis. However, the diagnostic value of each single approach is insufficient to accurately diagnose malignant breast lesions. Multi-parameter US can assess the morphology, elasticity, and blood supply of these lesions. This study aimed to establish, validate, and evaluate a predictive model based on multi-parameter US in clinical breast cancer diagnosis, exploring whether it can improve the diagnostic efficiency and reduce unnecessary breast biopsies. We first built a model analyzing data of 448 patients by univariate and multivariable analyses. Subsequently, we validated the model in Cohort 2 (119 patients) and evaluated it in Cohort 3 (107 patients). The model's diagnostic value was compared to conventional US. The results of Cohorts 2 and 3 showed that the model had a great potential for use in clinical practice.
As shown in Table 2, nine indicators were included in the model: age over 40 years; conventional US findings, including illdefined margin, tumor heterogeneity, rich blood flow, and abnormal axillary lymph nodes; CEUS findings, including enhanced area enlargement, contrast agent retention, and irregular shape; SWEmean larger than the cutoff value. Patients with malignant lesions were shown to be older than those with benign lesions (17,21), consistent with our finding. Ill-defined margins can be attributed to the invasion of malignant tumors into the surrounding breast tissue (22)(23)(24), confirmed by our findings. Similarly, Zhang et al. (25) reported that the appearance of heterogeneity on US images indicated faster proliferation and worse prognosis, consistent with our findings. Our study also showed similar findings related to disordered and disseminated blood flow. Rich blood flow is an indicator of the faster growth and higher metabolism of malignant tumors over benign tumors (26,27). Abnormal lymph nodes on US images, a possible indicator of high tumor aggressiveness, could also help detect breast cancers, as previously reported (28,29).
Among the findings indicating malignancy on CEUS images, enhanced area enlargement is widely recognized (17,30). Although the area measured by CEUS for malignant lesions was reportedly closer to the pathological findings than conventional US (31), the measurements included the breast lesions and blood-rich areas around them. As normal tissue blood flow cannot maintain tumor growth, the surrounding tissues stimulate angiogenesis to support tumor growth (32). Contrast agent retention revealed that the disordered blood vessel distribution inside the tumor led to poor venous return. The irregular shape shown on CEUS images was also found in other studies (30,33), and attributed to the abundant and disorganized blood flow in malignant tumors and their infiltration into surrounding breast tissue. The SWE value refers to the lesion's stiffness. Malignant lesions tend to be stiffer than benign lesions and show higher SWE values, as previously confirmed (7,8,34).
The diagnostic performance of the predictive model in Cohorts 1, 2, and 3 was greater than by conventional US. Previous studies also compared multi-parameter US models to conventional US and achieved similar results (16)(17)(18). The sensitivity of the conventional US was 100.0% in Cohorts 2 and 3, but the specificity was rather low (31.0% and 7.3%, respectively). Low specificity might lead to many unnecessary biopsies. Therefore, it is important to maintain a balance between sensitivity and specificity in clinical practice. After combining data from conventional US, CEUS, and SWE, the specificity of the predictive model significantly improved to 81.3% and 73.1% in Cohorts 2 and 3, respectively, without a significant loss in sensitivity. These improvements could greatly B C A FIGURE 2 Receiver operating characteristic (ROC) curves of the predictive model in Cohorts 1 (A), 2 (B), and 3 (C). This study had several limitations. First, the malignancy rate was relatively high, possibly because most benign lesions were followed up. This may have led to some mistakes. Second, this A 33-year-old patient with pathologically confirmed breast adenosis showing a hypoechoic solid nodule at 4 o'clock in the right breast with well-defined margins, irregular shape, and poor blood flow on conventional US (A), indicating a malignant lesion with a BI-RADS 4b score. On CEUS, the tumor appears as a nodule without blood perfusion (B), indicating a benign lesion with a BI-RADS 3 score. The nodule was soft based on shear wave elastography, indicating that the lesion was benign (C). The nodule was assessed as benign by the multi-parameter predictive model. US, ultrasonography; CEUS, contrast-enhanced ultrasonography; BI-RADS, Breast Imaging Reporting and Data System. was a retrospective, single-center study, so the number of the cases was limited. A larger number of patients from multiple centers is needed to confirm our results. Finally, this study did not assess the repeatability of quantitative parameters such as the SWE measurements, which should be explored in future studies.
Conclusion
The multi-parameter US model showed good performance in diagnosing breast cancer. The model established in this study could improve the diagnostic specificity without a significant A 47-year-old patient with pathologically confirmed invasive ductal carcinoma showing an isoechoic solid nodule at 1 o'clock in the left breast with well-defined margins and a regular shape on conventional US (A), indicating a benign lesion with a BI-RADS 3 score. CEUS showed early hyperenhancement, enhanced area enlargement, contrast agent retention, clear margins, and irregular shape (B), indicating a malignant lesion with a BI-RADS 4b score. SWE also indicated that the lesion was malignant (C). The nodule was assessed as malignant by the multi-parameter predictive model. US, ultrasonography; CEUS, contrast-enhanced ultrasonography; BI-RADS, Breast Imaging Reporting and Data System; SWE, shear wave elastography. loss in sensitivity, helping reduce unnecessary biopsies and guide clinical diagnosis and treatment.
Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.
Ethics statement
The studies involving human participants were reviewed and approved by Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
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2022-11-18T15:29:06.903Z
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2022-11-17T00:00:00.000Z
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253599075
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s2orc/train
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Immune checkpoint inhibitors for unresectable or metastatic pleomorphic dermal sarcomas
Pleomorphic dermal sarcomas (PDS) are rare neoplasms of the skin that occur in UV-exposed sites in the elderly, but represent the most common cutaneous sarcomas. Although the majority of PDS can be surgically removed, local recurrences occur in up to 28%, usually occurring within the first two years after primary excision. Metastases are diagnosed in up to 20% of cases, mainly observed in the skin, lymph nodes and lungs, preferentially affecting patients with underlying hemato-oncologic diseases. Similar to other UV-induced tumors, PDS are inflammatory and immunogenic tumors (with a high number of CD4+/CD8+ tumor-infiltrating lymphocytes (TILs) and checkpoint molecule expression such as PD-L1, LAG-3, TIGIT) with a very high mutational burden. The most common genetic alterations include UV-induced TP53 loss of function mutations, followed by alterations in the CDKN2A/B gene. Rarely, targetable genetic alterations can be detected. Compelling experimental data and clinical reports about PD-1/PD-L1-blocking antibodies in patients with PDS suggest its use as first line treatment in unresectable or metastatic tumor stages. However, individual („off-line”) patient management should be discussed in an interdisciplinary tumor board based on molecular genetic testing, mutational burden, PD-L1 expression, and evidence of tumor-infiltrating lymphocytes in addition to comorbities of the individual patient.
Introduction
Pleomorphic dermal sarcomas (PDS) are rare neoplasms of the skin with a mesenchymal (fibroblastic) lineage differentiation, arising in UV-exposed locations, typically diagnosed in elderly male individuals (1)(2)(3). Although accurate incidence data do not exist, they represent the most common cutaneous sarcomas with increasing incidence due to demographic changes. Helbig This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Given the similarities in clinic, histology as well as molecular genetics and epigenetics, atypical fibroxanthoma (AFX) and PDS are now considered a spectrum of one entity, but differ in terms of therapy and prognosis (4)(5)(6)(7)(8)(9)(10). Histomorphologically, AFX and PDS show similar features. The main difference is that AFX are confined to the dermis whereas PDS involves distinct portions of the subcutis and/or have necrotic tumor portions and/or perineural or lympho-vascular invasion. In AFX, the local recurrence rate after R0 resection is less than 5% (3,11,12). While the majority of PDS can be treated by curative excisions, local recurrences occur in up to 28% of patients. Metastases are observed in up to 20%, mainly in the skin, lymph nodes and lungs, preferentially affecting patients with underlying hematooncologic diseases (3,10,11,13,14).
In the last years, it could be shown that PDS are inflammatory and immunogenic tumors with a very high mutational burden. Experimental data and clinical reports indicate that PD-1/PD-L1-blocking antibodies are highly effective in patients with unresectable or metastatic PDS and suggest its use as first line treatment in these advanced tumor stages (2, [15][16][17].
Genetic alterations
Both, AFX and PDS have a very high mutational burden with UV signature, which is even higher than that of other UVinduced skin tumors such as cutaneous squamous cell carcinomas (cSCC) and malignant melanomas (2, 18-20). Based on very similar gene mutations, gene expressions, copy number variations as well as DNA methylation profiles, AFX and PDS are now accepted to represent a spectrum of the same tumor entity (5,(7)(8)(9). In PDS, it has been shown that the tumors exhibit the UV-induced mutation signatures 7a and 7b in almost equal proportions. In other UV-induced tumors such as cSCC, basal cell carcinoma, and melanoma, signature 7a is typically detected, whereas signature 7b is rarely detected. Signature 44, which has been associated with defective DNA mismatch repair (MMR), has been detected in a small number of our investigated PDS (3 of 28); however, is much more common in cSCC ( Figure 1) (2).
Gene mutation and gene expression analyses revealed that AFX/PDS have the highest similarity to cSCC. The most frequent genetic alterations are TP53 loss of function mutations which can be detected in all PDS, followed by genetic alterations in the CDKN2A/B gene (CDKN2A/B mutations in 68%, deletions in 71%, and both in 46%, while 7% showed even a biallelic loss.) ( Figure 1) (2). Other common mutations include DNHD1, GNAS, RTN1, RTL1, ZBTB7A, NCKAP5L, FAM200A, NOTCH1/2, FAT1, and TERT promoter mutations (2, 5,6,8,20,21). In contrast, cSCC, basal cell carcinomas and malignant melanomas show a significantly lower mutation frequency of these frequently mutated genes (2). In a small proportion of PDS, amplifications of PDGFRA leading to a PDGFRA expression on protein level and mutations within the kinase domain of KIT could be detected.
Immunophenotyping
Immunohistochemical as well as mRNA expression analyses of the immune "microenvironment" have shown that the majority of PDS represent inflammatory and immunogenic tumors with a high number of CD8+ tumor-infiltrating lymphocytes (TILs) and expression of diverse checkpoint molecules such as PD-L1, TIGIT, LAG-3, and CTLA-4 (2, 15,16).
When we classified a series of PDS tumors into immunologically hot and cold tumors (high versus low amounts of both CD4/CD8+ cells and high versus low PD-L1 expression), we did not detect a significant difference of tumor mutational burden (TMB). Nevertheless, the TMB is usually high in almost all PDS cases. Differential gene expression analysis between these immunologically hot and cold tumors revealed upregulation of TIGIT in the immunologically hot tumors (2).
In general, elevated levels of immune-related cytokines such as IL1A, IL2, as well as markers that were very recently linked to enhanced response of immunotherapy in malignant melanoma, including CD27, and CD40L have been detected in PDS tumors (22, 23). Moreover, the majority of PDS showed strong MHC-I expression and upregulated HLA class I molecules (HLA-A, HLA-B, HLA-C and HLA-E, corresponding in humans to the MHC class I) that are involved in tumor neoantigen presentation to tumor-specific CD8+ T lymphocytes leading to tumor cell apoptosis (24)(25)(26)(27)(28).
In CD8+high PDS cases (defined as cases with CD8 levels above median), genes such as CD74, LYZ and HLA-B were found to be differentially expressed while the remaining cases revealed enhanced levels of immunosuppressive cytokines including CXCL14 (29). In addition, the majority of PDS was infiltrated by PD-L1-, PD-1-and LAG-3-expressing immune cells and showed strong MHC-I expression on tumor cells (15).
These results imply that PDS in general, but especially those with a lot of infiltrating CD8+ and/or PD-L1-and LAG-3expressing TILs as well as MHC-I expression, induce an adequate anti-tumor immune response, which could be enhanced by immune checkpoint inhibitors. Only a small proportion of tumors appear to develop "immune escape" mechanisms, such as downregulation of MHC-I molecules (2, 15, 16).
Treatment of localized stage PDS
Radical excision followed by histopathologic workup is usually performed with curative intent as initial treatment for PDS. An appropriate safety margin should be maintained, as the risk of local recurrence or metastasis can be reduced by wide local excision (3,12,13,30,31). If this is not possible, a microscopically controlled excision should be performed.
Although there are no published data on the radiation sensitivity of AFX/PDS, radiation of the tumor area may be considered if complete tumor excision is not possible. The efficacy of adjuvant radiation with respect to the prognosis of completely excised PDS has not been conclusively established. In an evaluation of a few patients who had received adjuvant postradiation, a positive tendency (fewer local recurrences and/or metastases) of this postradiation could be elicited (3).
Treatment of advanced stage PDS including immune checkpoint inhibition
In case of advanced stage PDS, therapy recommendations should be always discussed and issued in the context of an interdisciplinary tumor board because there is no proven standard therapy. Here, molecular genetic testing, mutational burden, PD-L1 expression, and evidence of tumor-infiltrating lymphocytes (TILs) should be incorporated into individual treatment recommendations.
Since PDS harbor a high mutational burden and mostly exhibit an inflamed, proimmunogenic tumor microenvironment, susceptibility to immune checkpoint inhibition by programmed cell death 1 (PD1)/programmed cell death ligand 1 (PD-L1) inhibitors, (e.g. pembrolizumab, nivolumab) or the anti-CTLA-4 antibody (ipilimumab), or a combination of these agents was presumed in reference to other highly mutated and immunogenic tumors including other skin tumors such as malignant melanoma and cSCC (18,32,33). In the meantime, the exceptionally high efficacy of the anti-PD-1 inhibitor pembrolizumab has been described in case reports or small case series (see Table 1) (2, [15][16][17]. Until now, there are no case reports describing the use of other CPI in PDS patients. Nevertheless, larger clinical studies are needed to investigate tumor response to CPI in PDS patients. For PDS cases with low levels of CD8+ TILs, interventions to increase the infiltration of these inflammatory cells in general need to be explored as a future direction for successful treatment with CPI. As shown in other tumor entities, a dual blockade of CTLA-4 and PD-1 or PD-1/PDL-1 and LAG-3 could probably enhance the efficacy of CPI monotherapies, also by rescuing CD8+ T cells more vigorously from exhaustion than single signaling blockade (34, 35). In case of contraindications for a CPI treatment and if oncogenic alterations are detected, targeted therapies should be discussed, although there is no experience with targeted therapies in PDS to date (2,7,8). In relation to this, rarely detected PDGFRA or KIT amplifications/mutations could be of interest as several drugs have proven to induce long-term remissions in PDGFR-expressing cancers, such as gastrointestinal stromal tumors, dermatofibrosarcoma protuberans, or myeloid malignancies (36)(37)(38)(39)(40). Furthermore, it has been shown that tumors with a loss of CDKN2A/B may benefit from CDK4/6 inhibitors, such as palbociclib, abemaciclib or ribociclib, all approved for the treatment of metastasized breast cancer (41)(42)(43)(44).
In patients with advanced stage PDS treated with CPI, further investigation of predictors is still needed. However, all existing studies suggest a high efficacy of immune checkpoint blockade in inoperable or metastatic PDS patients.
Author contributions
All authors contributed to the article and approved the submitted version.
Funding
We acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The reviewer GL declared a shared affiliation with the author SK to the handling editor at the time of review.
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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2022-11-19T05:14:32.549Z
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Age, race, insurance type, and digital divide index are associated with video visit completion for patients seen for oncologic care in a large hospital system during the COVID-19 pandemic
Introduction The COVID-19 pandemic drove rapid adoption of telehealth across oncologic specialties. This revealed barriers to telehealth access and telehealth-related disparities. We explored disparities in telehealth access in patients with cancer accessing oncologic care. Materials/Methods Data for all unique patient visits at a large academic medical center were acquired pre- and intra-pandemic (7/1/2019-12/31/2020), including visit type (in-person, video, audio only), age, race, ethnicity, rural/urban (per zip code by Federal Office of Rural Health Policy), distance from medical facility, insurance, and Digital Divide Index (DDI; incorporates technology/internet access, age, disability, and educational attainment metrics by geographic area). Pandemic phases were identified based on visit dynamics. Multivariable logistic regression models were used to examine associations of these variables with successful video visit completion. Results Data were available for 2,398,633 visits for 516,428 patients across all specialties. Among these, there were 253,880 visits from 62,172 patients seen in any oncology clinic. Dramatic increases in telehealth usage were seen during the pandemic (after 3/16/2020). In multivariable analyses, patient age [OR: 0.964, (95% CI 0.961, 0.966) P<0.0001], rural zip code [OR: 0.814 (95% CI 0.733, 0.904) P = 0.0001], Medicaid enrollment [OR: 0.464 (95% CI 0.410, 0.525) P<0.0001], Medicare enrollment [OR: 0.822 (95% CI 0.761, 0.888) P = 0.0053], higher DDI [OR: 0.903 (95% CI 0.877, 0.930) P<0.0001], distance from the facility [OR: 1.028 (95% CI 1.021, 1.035) P<0.0001], black race [OR: 0.663 (95% CI 0.584, 0.753) P<0.0001], and Asian race [OR: 1.229 (95% CI 1.022, 1.479) P<0.0001] were associated with video visit completion early in the pandemic. Factors related to video visit completion later in the pandemic and within sub-specialties of oncology were also explored. Conclusions Patients from older age groups, those with minority backgrounds, and individuals from areas with less access to technology (high DDI) as well as those with Medicare or Medicaid insurance were less likely to use video visits. With greater experience through the pandemic, disparities were not mitigated. Further efforts are required to optimize telehealth to benefit all patients and avoid increasing disparities in care delivery.
Introduction
During the COVID-19 pandemic, telehealth use increased around the world to provide remote options for the management of medical conditions [1]. This increase in telehealth use occurred to balance efforts to manage new and chronic medical needs of individual patients against public health imperatives such as COVID-19 transmission risk reduction, in-person visit capacity limitations, and preservation of limited stocks of medical supplies for inpatient acute care needs [2]. Broad utilization of telehealth revealed important disparities deserving of further exploration [3].
Patients with cancer constitute a unique subset of all patients with telehealth-associated needs given the demand for complex discussions regarding plan of care for new malignancies, manifold workup steps, in-person treatment administration, and long term follow up [4]. Patients with cancer also tend to be older, may require care delivery at great distance from their residence, and represent a diverse sociodemographic group [5,6]. Very early in the COVID-19 pandemic, many thought leaders offered guidance on the care of these complex patients [7][8][9][10][11][12][13][14]. This guidance was used by many departments to balance COVID-19-associated risks with cancer-associated risks using approaches that universally involved the use of telehealth. Available data suggested that older or minority patients may be less likely to access telehealth than general patient populations [3], and practitioners have noted that telemedicine has a long term role in delivery of care beyond the pandemic [15][16][17][18]. Therefore, much work is needed to understand and address factors that might limit access and worsen health inequity that is known to exist [19].
Telehealth provides patients with a means for accessing care when in-person care options are limited. However, barriers to telehealth access that may exist for patients receiving oncologic care have not been examined in detail across the oncology space to date. As policies for oncology practices are set for the future, it will be important to carefully consider the impact that telehealth policy and practice may have on equity in access to care. The COVID-19 pandemic provides a valuable opportunity to assess what these impacts might be so that appropriate countermeasures can be developed. We examined all patient visit data from a large hospital system before and during the COVID-19 pandemic with the goal of identifying factors associated with reduced telehealth access.
Ethics statement
This retrospective study was conducted with the approval of the University of Michigan Medicine Institutional Review Board, who waived the requirement for informed consent for analysis of deidentified data.
Dataset construction
Data for all unique patient visits at a large academic medical center from 7/1/2019 to 12/31/ 2020 were retrospectively gathered, including visit date, visit type (in-person, video, audio), age, gender, race, ethnicity, rural/urban home address (per zip code assessment by Federal Office of Rural Health Policy), insurance type, Digital Divide Index (DDI), provider, and clinic. DDI, which ranges from 0 to 100, is the sum of the infrastructure/adoption (INFA) and the socioeconomic (SE) score, which were calculated as described previously [20]. Together, the SE and INFA incorporate metrics of technology and internet access, age, disability, and educational attainment within a geographic area [20]. Lower scores are seen in areas with better access to technology and higher socioeconomic status. Data were abstracted from billing and scheduling databases. Distance from patient residence to the clinic site was approximated using zip code. The initial dataset contained all encounter types, including wellness visits, post-operative visits, evaluation, and management visits. The following visit types were excluded: infusion, radiation treatment, visits from patients who had 20+ visits, and visits from patients whose zip code of residence was outside of the United States. Pandemic phases were defined as described in the results section.
Identification of visit specialty
Patient visits that took place in oncology practices were identified initially based on the department where each visit took place. This categorization was then edited as appropriate based on provider-associated oncologic subspecialties on a provider-by-provider basis. Next, a listing of visit provider by oncologic subspecialty was used to sort visits into the following groups: radiation oncology, surgical oncology, and medical oncology. Visits included in any of these three subspecialty groups (radiation oncology, surgical oncology, or medical oncology) were included in the group "all oncology" when analyses of all oncology visits in aggregate were conducted.
Pandemic-associated changes in practice
Prior to March 2020, it was noted that there had been an increase in COVID-19 cases in the state of Michigan. As a result of the pandemic, there were changes in practice at Michigan Medicine in Ann Arbor, MI. By early March, those in oncology specialties had been instructed to categorize "patients according to urgency of care need: 1) those with clinical problems that require urgent and in-person evaluation and treatment, 2) patients with new or ongoing health problems of lesser urgency for whom temporary deferral of care or provision of care by virtual means will be safe, and 3) patients with routine, maintenance or non-health-compromising clinical problems for whom postponement of evaluation and treatment is safe." In this context, many oncology visits began to occur virtually to limit exposure of patients and staff to the virus while preserving personal protective equipment, which was quite limiting at the time.
Outcome
The primary outcome of this study was video visit completion. This was considered somewhat differently in the visit level analyses vs the subsequent patient level analyses. In the visit level analysis, all visits were considered based upon their visit type. In the initial patient level analyses, patients were categorized regarding the completion of a video visit. If a video visit was completed, then they were assumed to have the capacity to complete video visits. If they never completed a video visit but did complete audio visits, then it was assumed that they did not have the capability to complete video visits because video visits were encouraged throughout the hospital system from the first days of the pandemic, and audio visits were always discouraged. We will note that patients were scheduled for audio visits in cases where the patient made staff aware of their inability to engage in a video visit at the time of scheduling. Video visits were also converted to audio visits in cases where an inability to conduct video visits was identified at the time of the patient visit. For logistic regression models, the proportion of successfully completed video visits out of the total number of video and audio visits per patient was used to evaluate associations with patient level factors.
Statistical analysis
Initially, data were summarized on a visit level. Next, data were summarized on a patient level. Next, bivariate analyses were conducted on a visit level and a patient level. Lastly, patient level multivariable logistic regression models were used to evaluate associations of patient factors with successful video visit completion, considering the proportional representation of audio and video visits completed by a given patient. Therefore, an audio visit was considered a failed video visit, but an in-person visit was not considered to be a failed video visit in this analysis. The following pre-specified covariates were included in all multivariable models: age, gender, race, ethnicity, rural residence, Digital Divide Index (DDI), insurance plan type (Medicaid, Medicare, private insurance, other insurance), interpreter need, and distance. No variable selection was performed. Distance was taken to be the average across the patient's visits if the distance from the visit site varied. Separate models were fit for each of the two phases of the pandemic for all of oncology and for each of the three subspecialties: surgical, radiation, and medical oncology. Wald tests were used to assess the statistical significance of each covariate in each of these models by comparing two-sided p-values to alpha = 0.05. Odds ratio estimates and 95% confidence intervals from these models were used for construction of forest plots. All analyses were performed in SAS version 9.4 (SAS Institute Inc., Cary, North Carolina).
Pandemic phases
The full dataset included 2,398,633 visits from 7/1/2019 to 12/31/2020 in 29 departments with 4,031 providers. Visit numbers by week were graphically depicted for all visits and all of oncology to understand changes in visit distribution with time (Fig 1). Graphical representations highlight changes in visit representation, which can also be seen in Table 1 (described in detail below). There was a period pre-pandemic with little use of telehealth. With the beginning of the pandemic response, visit representation changed dramatically. This was followed by another more subtle shift in visit makeup several months after the pandemic response began; therefore, there appear to be two distinct phases of the pandemic (Fig 1). Phase 1 began on March 16, 2020 and corresponded to the first period of the pandemic response. During this phase, there was a large decrease in care delivered face-to-face with a rapid rise in telehealth use such that video and phone visits accounted for nearly two-thirds of visits. Phase 2 began on July 6, 2020; this date was selected because distinctly different telehealth usage characteristics were observed after this date compared to either Phase 1 or the pre-pandemic period. During this period, there was a persistent use of telehealth, though proportional representation changed to again favor in-person visits. There was a large decline in the proportion of visits completed by phone between Phase 1 and Phase 2 while video visits made up approximately one in five visits during this period. Visit patterns in radiation oncology, medical oncology, and surgical oncology were similar (Fig 2).
Patient level data summary
Patient level summary statistics with demographic and patient characteristics are provided for all of oncology (Table 2) and for the oncologic specialties, including, radiation, surgical, and medical oncology during pandemic Phase 1 and Phase 2 (S1 appendix). Patients seen in any oncologic specialty clinic had a median age of 61, were 50.84% female, and 87.21% Caucasian. In the presentation shown in Table 2, a patient was considered a video visit user if they completed at least one video visit. A patient was considered a phone visit user if they completed at least one phone but no video visits. A patient was considered a non-telehealth user if they did not complete any video or audio visits (only in-person visits). P values for bivariate patient level analyses are presented, and gender, age, race, ethnicity, need for interpreter, insurance (Medicaid, Medicare, private insurance, and other insurance), and residency impacted the distribution of patients among user groups ( Table 2).
Patient level multivariable analyses of factors related to video visit completion
Having established the phases of the pandemic and generally assessed in a simple patient level analysis factors related to visit type distributions, we next used multivariable analyses of patient level data to examine factors that were associated with completion of video visits in hopes of identifying potential barriers to telehealth access during each phase across oncology and within radiation oncology, surgical oncology, and medical oncology (S1 Appendix (Fig 3; S1 Appendix). Gender, ethnicity, and interpreter usage did not impact ability to complete video visits. Despite the difference between the pandemic telehealth phases with regard to visit type distribution, similar patterns of association were seen for all of oncology during Phase 2 of the pandemic to those seen in Phase 1 save that Medicare enrollment was no longer associated with lack of video visit completion (Fig 3; S1 Appendix).
After examining predictors of video visit completion among all oncology visits in aggregate, we next explored similar analyses for each oncologic subspecialty (radiation oncology, surgical oncology, and medical oncology). Generally, similar trends were noted in each of the subspecialties to the findings from the analysis of all oncology visits together. In radiation oncology, we noted that older age [OR: 0.980 (95% CI 0.970, 0.990) P<0.0001], Medicaid enrollment [OR: 0.366 (95% CI 0.217, 0.617) P = 0.0097], and higher DDI [OR: 0.874 (95% CI 0.803, 0.952) P = 0.0020] were associated with lack of video visit completion (S1 Appendix). Increasing distance from the facility (P = 0.0065) was protective of a patient's ability to complete video visits. Findings were similar in Phase 2 with age, DDI, Medicaid enrollment, and distance from facility significantly associated with video visit completion. However, females were less likely to complete video visits [OR: 0.620 (95% CI 0.462, 0.832) P = 0.0015]. The impact of race was less notable in radiation oncology than seen when all oncology patients were examined in aggregate (S1 Appendix). Findings from surgical oncology and medical oncology were similar to those seen in radiation oncology, with increased age, Medicaid enrollment, and higher DDI associated with lack of video visit completion (S1 Appendix). Distance remained protective in surgical and medical oncology for both phases of the pandemic. Race was significant for medical oncology in both phases but not for surgical oncology or radiation oncology in either phase. Details are included in the (S1 Appendix).
Discussion
Through analysis of all patient visits in a large academic medical center, we noted dramatic shifts in telehealth utilization during the early days of the COVID-19 pandemic. Among those seeking cancer care, we found that older individuals, African Americans, those utilizing Medicaid, and those from areas with higher DDI were less likely to complete video visits. These factors support a number of considerations for future telehealth applications across oncologic specialties. First, older individuals who make up the bulk of oncology patients may need additional help in accessing telehealth. Second, racial inequities in cancer care may be exacerbated through telehealth, suggesting the need for effective means to mitigate telehealth-related disparities in the post-COVID-19 world. Third, those from rural areas may require additional support in order to access telehealth resources. Fourth, Digital Divide Index is related to telehealth use, suggesting that investments in broader access to the internet and quality education may have important positive implications for oncologic care access. There is great interest in preserving telehealth advances to facilitate care delivery in the future. Our analyses supports the findings of others who have suggested that addressing barriers and inequality concerns will be critical to ensure full access for all oncology patients [15,18,21].
Visit dynamics during the pandemic
Institutional guidance borne out of efforts to protect patients and providers from COVID-19 led to initial shifts in visit types during the pandemic in the general patient population and among patients seen by oncologic specialties at our institution. Others have shown dramatic shifts in visit type in response to COVID-19 as visit dynamics have been reported extensively in the growing COVID-19 pandemic literature [22,23]. Across the US and around the world, guidance came at slightly different timepoints in the pandemic, accounting for locoregional variation in viral case numbers and resources. While generalizations across the United States with regard to the precise date selections that we have made in Fig 1 are not possible, it is likely that review of data from many institutions in the United States would demonstrate large shifts in visit composition at timepoints specific to individual healthcare systems as they responded to the needs of their patient populations. Though large shifts occurred universally, the nature of these shifts was likely dictated by many features of individual institutions such as technological resources and telehealth expertise as well as other contextual factors beyond individual health systems. Generally, the timing seen at our institution in March 2020 is consistent with timing reported at other institutions [23]. Outside of health system and institutional decisions made in the context of an understanding of the pandemic locally, there are other factors that should be considered at different levels that impact whether an individual patient might complete a video visit. These might vary across the pandemic period. Payor practices have guided telehealth use and enabled dramatic increases in telehealth use [24], highlighting the fact that any approach to telehealth in oncology will require collaboration between medical institutions and payors [25]. Additionally, patients can be taught, so it is likely that some patients might have gained the capacity to complete telehealth visits during the pandemic. Providers and institutions might also have varied skill sets and resources to enable patients' efforts to engage in telehealth. Patient and provider skills as well as payor guidance are all in flux along with the pace of the pandemic itself. It will be important for institutions, payors, providers, and patients themselves to have a voice in the process of refining telehealth as a component of cancer care delivery. Additionally, it will be important to consider all of the many factors that impact telehealth use in future studies designed to optimize its fair and ethical application.
Predictors of telehealth access in oncology
Older individuals. As age increased, patients were less likely to use video visits. It is known that many older adults lack access to the technology and expertise required to participate fully in telehealth [26]. Younger patients with cancer are more likely to prefer telehealth visits than older patients with cancer [27]. Outside of oncology, analyses across primary and specialty practices have shown that older adults are less likely to participate in video visits [22,28]. A study of patients at another cancer center similarly found that older individuals were less likely to use video visits [29]. In a previous non-oncology focused analysis from our institution during a shorter time frame than that outlined in our manuscript, the mean ages for video visit users, phone visit users, and non-telehealth users were 42, 56, and 41, respectively [28]. However, in our analysis, the mean ages for video visit users, phone visit users, and non-telehealth users were 54, 63, and 56, respectively. Because the oncology patient population is older than the general population, any age-related burdens or barriers will negatively impact patients with cancer to a greater degree. Patients seen in radiation oncology were the oldest of the three oncologic specialties, suggesting that age-related telehealth access difficulties may be most challenging in radiation oncology, among oncologic providers. Our work agrees with the findings of others who have noted that special attention must be paid to older patients to facilitate access to telehealth [30,31].
Race. People of color have been shown to have greater difficulty accessing telehealth in multiple prior studies [31][32][33]. In the data described in this report, the impact of race on video visit completion varied somewhat depending on whether the analysis included all oncology patients or various subspecialties that make up oncology. It is likely that these differences are the result of variable patient numbers and power to obtain statistical significance given the magnitude of odds ratios shown in Fig 3 (also S1 Appendix). Our findings in the aggregate analysis of all oncology visits resulted in similar findings to those of Shao et al who studied a population from an NCI designated cancer center in the state of Alabama (in a population with greater minority representation than that seen in our study population) and found that patients of color were less likely to use video visits [29]. This supports the concern that telehealth might exacerbate existing inequities in healthcare and cancer care specifically if countermeasures are not developed [19].
Gender. Some have found an impact of gender on video visit completion, specifically male gender [29]. We did not see an impact of gender in our aggregate analysis of oncology visits. However, an effect of gender was noted variably across pandemic phases and specialties such that it is difficult to draw strong conclusions on the impact of gender using our data (S1 Appendix). Females were less likely to complete video visits in Phase 2 within radiation oncology and Phase 1 within surgical oncology. Further exploration of the impact of gender is warranted as efforts to optimize telehealth access proceed.
DDI. The Digital Divide Index predicted video visit completion in the present study. This metric is complex in that it incorporates access to broadband internet, computing devices, download speed, upload speed, age, education, poverty rate, and disability within a geographical area. It was highly significant in all analyses in models containing age. Therefore, it is likely that the effect is driven by other components of the score. Many of the components relate to available infrastructure. Further enhancing access to broadband is likely to increase access to telehealth. Likewise, the significance of DDI may also point to the potential for telehealth to worsen educational attainment-related disparities in health outcomes in oncology. Patients from areas with lower income have been shown to be less likely to complete video visits by others [29]. Regional socioeconomic status and DDI are also clearly quite closely entwined. DDI could be easily adapted to guide interventions to address barriers to telehealth access.
Insurance. In our study, those with Medicare or Medicaid were less likely than those with private insurance to engage in video visits. This is similar to the results of an examination of patients at another cancer center, where patients with public insurance were less likely to engage in video visits [29].
Rural zip code. Those from rural zip codes were less likely to complete video visits. These findings generally parallel findings of previous research on factors that are related to lower rates of patient portal usage [31]. Efforts to improve oncologic care delivery through the use of telehealth are being studied; these ongoing efforts will need to carefully account for telehealth access-related concerns of rural patients with cancer [34].
Distance. Increasing distance from the medical facility was associated with increased ability to complete video visits. This was somewhat unexpected and may have been a product of a greater perceived incentive in the form of avoiding a lengthy trip to receive care. For example, those patients at greater distance from healthcare facilities might be more likely to organize approaches to take part in telehealth and to plan ahead prior to virtual visits with providers in hopes of avoiding the extra cost and effort required to attend visits in person. It is also possible that individuals living in remote areas were more likely to have engaged in video visits with another system prior to their encounter in our system. These previous visits might then have served as practice sessions, allowing patients time to train themselves prior to their initial encounters with our center. A better understanding of factors that motivate patient engagement with telehealth will be important to the further adoption of telehealth in the future.
The future of telehealth in oncologic care
Dramatic changes in oncology department operation occurred during the COVID-19 pandemic [13,35]. Great efforts were undertaken in radiation oncology practices toward reducing the number of patients under treatment [18,36]. Changes were suggested for infusion protocols in medical oncology practices [13]. Surgeons were encouraged to consider non-operative management where possible [14]. As oncologic care has reached a new steady state, physicians express high levels of satisfaction with the use of telehealth [16,37,38]. These providers report that they will continue offering telehealth visits, and practice guidelines for telehealth have been developed [15,[39][40][41].
Despite this, support is not uniform; skeptics caution that telehealth may lead to lower quality patient care [18] or exacerbate care disparities. Additionally, patients with positive impressions of telehealth still recognized the importance of in-person physical exams for detection of cancer recurrence [42]. This dialog supports discussion of approaches to balance in-person and telehealth visits to achieve patient and provider goals. Some have envisioned ways that telehealth could help reduce disparities [17,21]. As elements of telehealth continue beyond the COVID-19 pandemic in the area of on treatment monitoring in radiation oncology, long term follow up in all specialties, and in selected pre-treatment settings [17,25,43], developing models of telehealth that balance in-person and remote patient care priorities while addressing disparities will be necessary prerequisites for quality care delivery across oncologic specialties.
Were improvements observed over the course of the pandemic?
At our institution, two distinct pandemic phases were noted. Though many have examined telehealth during the pandemic and found inequities early in the pandemic, we show that these inequities persisted beyond the initial phase of the pandemic. There is little difference in significance of many key features from Phase 1 and Phase 2. Specifically, the odds ratios for the impact of race, DDI, Medicaid enrollment status, and age are largely unchanged between Phase 1 and Phase 2. This illustrates that there is still much work to be done to improve access to telehealth-based oncologic care.
Study limitations
This study uses data from a single large academic institution in a single area of the United States. Therefore, the dynamics of visit type distributions may not be generalizable to all clinical settings but may apply to similarly sized academic and non-academic centers with large catchment areas. It is important to note that people who were not Caucasian made up 18.87% of the population studied. Further study in more diverse populations will be helpful in the future. We recognize that there may be competing reasons why an individual might not complete a video visit. For example, the nature of their care might necessitate that they be seen in person for clinical reasons. It is also not possible to assess the impact of a family member with appropriate skills or other resources that might help patients complete virtual visits. Because our institution was strongly encouraging video visits whenever possible, we feel that it is reasonable to make the assumption that the great majority of patients would have been subject to a request to complete a video visit during the relevant period. It is likely that many other factors would bias toward not away from the null hypothesis when considering the impact of other factors.
Conclusions
The COVID-19 pandemic provides a valuable opportunity to study telehealth and more specifically barriers to access and disparities. Patients, providers, and regulators have seen benefits from telehealth, and as a result, features of telehealth-based care delivery will be preserved in the future. Therefore, it is imperative that steps be taken to ensure access to high quality telehealth for those less likely able to fully participate, including older patients, those from rural areas, those with lower socioeconomic status, and those with lower levels of infrastructural and past educational support.
Supporting information S1 Appendix. Analysis of factors related to video visit completion in each oncologic specialty alone (radiation oncology, surgical oncology, and medical oncology) and in the aggregate of all of oncology. Data are presented in figure and tabular form. (DOCX)
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Diagnostic yield, complications, pathology and anatomical features in CT-guided percutaneous needle biopsy of mediastinal tumours
Objectives This study presents the experiences of percutaneous CT-guided needle biopsy at a university hospital in Norway. Methods A retrospective examination of all mediastinal biopsy procedures between April 2015 and August 2019 was performed at Akershus University Hospital in Norway. We registered patient and procedure characteristics, along with lesion pathology and characteristics including localization according to anatomical and Felson mediastinal compartments. Results The study included 48 procedures, conducted in 45 patients (29 men and 16 women) with a mean age of 60,5 years. Pneumothorax occurred in 12 procedures (60% of the transpulmonary procedures) and pneumomediastinum in 18 procedures (38%). Pneumothorax was only seen in procedures with transpulmonal access. Four of the pneumothorax cases required pleural drainage. Diagnostic yield was 96%. We found significant (p = 0,006), moderate to high association between anatomical compartment localization and histopathological diagnosis (Cramér’s V = 0,49) for tumours selected for CT-guided percutaneous biopsy. Felson’s compartment division on the other hand, did not show any significant associations. Conclusion We found CT-guided percutaneous needle biopsy of mediastinal tumours to be an effective and safe procedure with a diagnostic yield of 96%. The main complications were pneumothorax and pnumomediastinum, with a relatively low chest drainage rate. Anatomical mediastinum compartment showed a significant, moderate to high association with the histopathological diagnosis for tumours selected for percutaneous CT-guided biopsies, where most malignancies were seen in the anterior compartment.
Introduction
Mediastinal tumours are rare and represent only 3% of tumours in the chest [1]. In patients with lesions in the mediastinum, precise pathological diagnosis is required to determine the appropriate treatment strategy [2,3]. Techniques to obtain tissue from the mediastinal tumours for pathological examination include approaches using needle biopsy, such as endoscopic ultrasound-guided fine needle aspiration and transbronchial needle biopsy. Furthermore, surgical techniques, such as anterior mediastinotomy, cervical mediastinoscopy, video assisted thoracoscopy and thoracotomy, can be utilized [3]. Ultrasound and computed tomography (CT) are the usual guiding methods of percutaneous needle biopsies, as these modalities enable accurate localization of the targeted lesion and visualization of the needle (4). In addition, image-guided percutaneous transthoracic needle biopsy enables access to practically all regions of the mediastinum [4]. CT is the preferred guiding method for mediastinal biopsy [5].
Because of essential vessels and organs in the mediastinum, biopsy may be challenging [6]. The mediastinum is limited by the thoracic inlet superiorly, the diaphragm inferiorly, the sternum anteriorly, the vertebral column posteriorly and the mediastinal pleura laterally [7]. According to the classical anatomical division system, the mediastinum is divided into a superior compartment and an inferior compartment by a plane extending from the fourth thoracic vertebra to the lower manubrium. The pericardium further subdivides the inferior compartment into an anterior, middle, and a posterior compartment [7], where the heart lies in the middle compartment.
Another classification system is the Felson classification which divides the mediastinum into an anterior, a middle and a posterior compartment. The lateral chest radiography is the basis for Felson's classification [8]. The posterior boundary of the anterior mediastinum is a line that runs from the thoracic inlet to the diaphragm in front of the trachea and behind the heart [8]. The posterior compartment is bordered anteriorly from the middle compartment by a line 1 cm behind the frontal borders of the vertebral column. The middle mediastinal compartment is localized between the anterior and posterior compartments [9].
Felson's anterior compartment is much larger than the anatomical anterior inferior compartment. The anatomical anterior inferior, middle inferior and part of the superior compartments are all included in Felson's anterior compartment [10].
The scope of this study are the tumours referred for CT-guided percutaneous biopsy. Endobronchial ultrasound (EBUS) can be used to obtain access to subcarinal, paratracheal, and paraesophageal lymph nodes [4]. At Akershus University hospital, mediastinoscopies is a rarely performed procedure and it has mostly been replaced by (EBUS) and CT guided biopsies. Mediastinoscopy requires general anesthesia and not all tumours of the mediastinum are accessible by this procedure [4]. CT guided percutaneous biopsy only require local anaesthesia [7]. At Akershus University hospital, if a mediastinal tumour is inaccessible by EBUS, a CT guided percutaneous biopsy will be performed. CT guided procedure is also preferred over EBUS when there is a need for larger biopsy size which often is necessary to make tumor marker analysis.
The purpose of this paper is to present our observations in regards to CT-guided percutaneous needle biopsy of mediastinal lesions with focus on complications and anatomical location, at a major university hospital in Norway.
Sample and study design
We performed a retrospective analysis of 48 CT-guided percutaneous needle biopsies of mediastinal tumours, performed between April 2015 and August 2019, in total 53 months, at Akershus University Hospital.
To find the procedures to be included in the study, the local picture archiving and communication system was utilized to search for procedures containing the words "mediastinum" and "biopsy" in their procedure description. The resulting procedures were manually examined. Percutaneous biopsy procedures of lesions located in the mediastinum were included in the study.
The study was ethically approved by the regional ethical committee for medical and health research ethics (REK), with waived written consent because of the retrospective design of the study.
CT-guided mediastinal biopsy procedure
Prior to the biopsy procedure, an antitussive and a mild sedative drug (5 mg dihydrocodeine and 5 mg diazepam orally), was administered. Most procedures were performed with Philips Brilliance 64 or Ingenuity 128 Core scanners, both with a tiltable gantry. While some procedures used Philips ICT 256 with a fixed gantry (Philips, Amsterdam, Netherlands). With the patient in the appropriate position in regards to the most safe and optimal trajectory path to the mediastinal lesion, a low-dose CT-scan was performed immediately prior to the biopsy procedure.
During the procedure a 17-G coaxial needle was inserted and usually positioned at the peripheral margin of the targeted lesion. This was done using local anaesthesia, intermittent CT fluoroscopy guidance and a stepwise technique. In some procedures, to access the lesion, the CT-gantry was tilted. Depending on the size of the lesion, respiratory movements and available scanner options, single CT slices with a thickness of 5, 7.5 or 10 mm were used. Usually, three or four needle insertions were performed. A reusable core (cutting) biopsy system with a 17G coaxial needle and an 18G biopsy needle (Bard1Magnum1, Bard Medical, Covington GA, USA) was used. The stroke length of the needle was set to either 15 or 22 mm, with the optimal stroke length determined for each individual case.
Immediately after the procedure, a full volume low-dose CT-scan was performed to detect any possible complications such as pneumothorax or lung haemorrhage. During the four hours of observation after the procedure, the patient was not allowed to drink or eat and had to lie flat. An upright, frontal projection chest x-ray was performed approximately two hours after the procedure. If a pneumothorax was symptomatic, if it evolved on the chest x-ray two hours after the procedure or if it occupied more than approximately 20-30% of the hemithorax, the radiologist performed a chest drainage using a pigtail catheter. In some cases, a thoracic surgeon inserted a Tru-Close Thoracic Vent 1 (UreSil, Skokie IL, USA) or a chest tube.
Target characteristics
The individual lesions were examined on a contrast-enhanced prebiopsy CT-scan. Their localisation within the mediastinum, both according to the anatomical division and Felson's division, was determined by a thoracic radiologist with more than 10 years experience. If the lesion was located in more than one compartment it was localised according to the compartment were the majority of the lesion was located. Each lesions morphology was decided. The size of the lesion was determined in the axial plane by measuring the two largest diameters of the lesion perpendicular to each other. In the craniocaudal direction, the distance between the superior and inferior boarder of the (continuous) lesion was registered. The three resulting measures were multiplied and divided by two to estimate the lesions volume.
The obtained tissue was sent to our hospitals Department for Pathology for histological examination. The final diagnosis was retrieved from the pathologists written reports. The histology of the biopsies was subdivided into following categories: benign, carcinoma, thymoma, lymphoma and other malignancies.
Registration of complications
The biopsy procedure description and the post-biopsy CT-scan and x-ray were manually examined to register complications. Pneumothorax, pneumomediastinum, chest wall emphysema and haemothorax on CT and/or chest X-ray were registered. In case of pneumothorax, it was noted if chest drainage was performed. Complications like parenchymal lung haemorrhage and mediastinal haemorrhage were registered from the post procedure CTs. Even the smallest observable signs of air-leak or haemorrhage on CT was registered as a complication.
Statistical analysis
The results were analysed using SPSS statistical software version 26 with a level of significance set to 5%. Associations between nominal variables such as mediastinal compartments and pathology groups were analysed using chi square Cramér's V-test.
Results
48 biopsy-procedures were performed. There were 45 patients, 29 men and 16 women, with an age range from 25 years to 92 years, a median age of 58 years and an average age of 60,5 years. For the men the age ranged between 25 years and 83 years; with the median age of 58 years. The women had a median age of 60,5 years and an age range from 35 years to 92 years.
According to the traditional anatomical mediastinal division, 11 lesions were found in the superior mediastinum, 30 of the lesions were in the inferior anterior mediastinum, and only 4 lesions were in the inferior posterior mediastinal region. No lesions were found in the inferior middle mediastinum. However, when sorting the lesion by Felson's mediastinal division criteria, 40 lesions were situated in the anterior compartment, 4 lesions in the middle compartment and 1 lesion was localized in the posterior compartment ( Table 1).
The estimated volume of the lesions ranged from 1.4 cm 3 to 2195 cm 3 with a median volume of 26.5 cm3 and an average volume of 154 cm 3 .
On CT the morphology of the lesions was homogeneous in 40% (18/45) of the cases and heterogeneous in the remaining 60% (27/45). The border was smooth in 42% (19/45) and irregular in 58% (26/45) of the lesions. Calcification was seen on CT in 13% (6/45) of the lesions. These features along with volume of the tumour could not distinguish whether the tumours were malignant or benign in multivariate analysis all with p values above 0.132.
The parasternal approach was used in most of the biopsy procedures (29/48) (Fig 1). Paravertebral approach was chosen in three procedures and transsternal approach was used in two procedures. A transpulmonary approach, which involves penetration of the lung and visceral pleura by the needle, was performed in 20 of the procedures. Five procedures were both parasternal and transpulmonal, and one procedure was both paravertebral and transpulmonal.
Pneumothorax only occurred when transpulmonal access was performed 20 transpulmonal procedures were performed and 12 of them resulted in pneumothorax, Thus, pneumothorax
PLOS ONE
Diagnostic yield, complications, pathology and anatomy of CT-guided biopsy of mediastinal tumours
PLOS ONE
Diagnostic yield, complications, pathology and anatomy of CT-guided biopsy of mediastinal tumours occurred in 60% (12/20) of the transpulmonal procedures. Nine of the pneumothoraxes were still visible on x-ray two hours after the procedure. Four of the pneumothoraxes required chest drainage, this accounts for 20% of the transpulmonary procedures (4/20). Pneumomediastinum was detected with 18 of the procedures (37,5% (18/48)), but did not result in any additional complications ( Table 2). Chest wall emphysema was seen after two procedures. Six procedures led to lung haemorrhage. Mediastinal haemorrhage was observed after nine procedures and haemothorax after one procedure (Table 2). These complications were minor and did not require further treatment or follow-up. There were no fatalities.
Adequate tissue material, i.e. enough material for the pathologists to make a diagnostic conclusion, was obtained in 46 out of 48 procedures. The remaining two biopsies were inconclusive. As shown in Table 3, the histology revealed that 80% (36/45) of the lesions were malignant and 20% (9/45) were benign. Thymomas, carcinomas (primary and metastasis) and lymphomas accounted for most of the malignant lesions.
Four patients underwent a repeated biopsy procedure. Two of these patients had a diagnostic first biopsy, but since the pathological diagnosis (normal thymus tissue) and the anticipated diagnosis did not correspond, a second biopsy was conducted to confirm that the lesions were benign. Two procedures were initially inconclusive and had to be re-biopsied. One of these rebiopsy procedures is included in this study, and the diagnosis turned out to be thymoma. The other re-biopsy procedure however is not included in this study because it was out of the timeframe of the study. The diagnosis of which was lymphoma. Hence, the study includes 45 patients with a total of 48 procedures. Tables 4 and 5 show the correlation between the tissue diagnosis of the 45 lesions and their localisation, according to the anatomical division and Felson's division respectively. The rebiopsied lesions are registered only once. Table 4 shows the tissue diagnosis in relation to anatomical compartments. All thymomas are in the anatomical inferior anterior compartment and account for 47% of the lesions in this compartment. The anatomical division shows a varying distribution of benign lesions, with 18,2% of the lesions in the superior compartment, 17% in the anterior compartment and 50% in the inferior posterior compartment. Table 5 shows the relation between tissue diagnosis and Felson's compartments. With this division 89% of the lesions, both benign and malignant, lie in the anterior compartment.
PLOS ONE
Diagnostic yield, complications, pathology and anatomy of CT-guided biopsy of mediastinal tumours
Discussion
Adequate tissue material was obtained in 46 procedures, which gives a diagnostic yield of 96%. The diagnostic yield is comparable with observations in other studies [3,13,14,16]. Hence, percutaneous CT-guided mediastinal biopsy is an effective procedure. This study has a relatively high complication rate compared to other studies [3,9,[13][14][15]. This is most likely explained by our definition of complications, as we defined even the smallest observable signs of air-leak or haemorrhage on CT as a complication.
As CT is a highly sensitive imaging technique, a lot of non-significant complications have been registered even though they had no clinical relevance.
Pneumothorax was observed in 60% of the transpulmonary procedures, one fourth of the total procedures. We report a higher pneumothorax -rate than the rates in similar studies [3,9,10,15]. This is again likely explained by our definition of complications. Relatively few patients needed chest drainage. In accordance with previously published material (10), pneumothorax only occurred when transpulmonal approach was utilized. As many as 60% of the transpulmonal approaches resulted in pneumothorax. Most of the pneumothoraxes were self-resolving, with chest drainage performed in only 4 out of 12 cases, 33% of the pneumothoraxes and approx. 8% of the total number of procedures. Despite the substantial risk of pneumothorax when transpulmonary access is chosen, the percutaneous CT-guided mediastinal biopsy is considered a safe procedure. Other complications were minor and demanded no further follow up or treatment. None of the procedures had a fatal outcome. Based on the dataset, it can therefore be concluded that percutaneous CT-guided mediastinal biopsy is a safe procedure.
The scope of this study is to analyse the tumours referred for CT-guided percutaneous biopsy. As mentioned, at Akershus University hospital tumours inaccessible by EBUS or tumours accessible by EBUS, but where there is need for a larger tissue sample, are referred for CT-guided percutaneous biopsy. Our data suggests that when a tumour has been selected for CT-guided percutaneous needle biopsy, the likelihood of a malignant diagnosis is higher, the further anteriorly the lesion is located within the mediastinum. In the anterior inferior compartment, as many as 83% of the lesions were malignant. All the 14 thymomas in our study were found in the anterior inferior compartment, where they commonly occur [7] and accounted for 56% of the malignant lesions in this compartment. There were 82% malignant lesions in the superior compartment, of which carcinomas accounted for 64%. Half of the relatively few lesions in the posterior compartment were malignant.
There was no significant association between tissue diagnosis and Felson's division system. Almost all the lesions were located in the Felson anterior compartment. The anatomical anterior inferior, middle inferior and part of the superior compartments are all included in Felson's anterior compartment. Hence, Felson's mediastinal compartments does not have any applicable value in regard to predicting whether a mediastinal tumors selected for CT-guided needle biopsy is benign or malignant We therefore recommend the anatomical compartment system for assessment and characterization of mediastinal tumours.
For lesions accessed by CT-guided needle biopsy, the data shows a significant, moderate to high association between tissue diagnosis and the classical anatomical division. The data suggests that for mediastinal tumours selected for CT guided biopsy, the lesions anatomical compartment can indicate the probability of a benign or malignant diagnosis. Whether the association between the anatomical localisation and the likelihood of a malignant diagnosis also is applicable for mediastinal tumours not selected for CT-guided percutaneous biopsy, is uncertain and it requires further research to elaborate this hypothesis. CT morphology features along with tumour volume could not distinguish between malignant and benign tumours in multivariate statistical analysis. Hence, «tissue is the issue».
A limitation of this study is the relatively small number of biopsy procedures and the retrospective design.
Conclusion
Our study found that CT guided mediastinal biopsy is a safe procedure with a high diagnostic yield of 96%. We therefore recommend CT guided biopsy for diagnostic workup of mediastinal tumours. Pneumothorax (60% of the transpulmonary procedures), pneumomediastinum (38%) and chest drainage (8%) were the main complications. We had no fatal outcome from the mediastinal biopsies.
We recommend use of anatomical compartment division of mediastinum compared to Felson's division-we found significant association between anatomical compartment and tissue diagnosis for tumours selected for CT-guided percutaneous needle biopsy. The likelihood of a malignant diagnosis is higher when the lesion is located in the anterior part of the mediastinum. Using anatomical mediastinal compartments most carcinomas were in the superior compartment, whereas all thymomas were found in the inferior anterior compartment.
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v2
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2022-11-19T06:16:17.961Z
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2022-11-17T00:00:00.000Z
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253627734
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s2ag/train
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Avelumab Plus Talazoparib in Patients With BRCA1/2- or ATM-Altered Advanced Solid Tumors: Results From JAVELIN BRCA/ATM, an Open-Label, Multicenter, Phase 2b, Tumor-Agnostic Trial.
Importance
Nonclinical studies suggest that the combination of poly(ADP-ribose) polymerase and programmed cell death 1/programmed cell death-ligand 1 inhibitors has enhanced antitumor activity; however, the patient populations that may benefit from this combination have not been identified.
Objective
To evaluate whether the combination of avelumab and talazoparib is effective in patients with pathogenic BRCA1/2 or ATM alterations, regardless of tumor type.
Design, Setting, and Participants
In this pan-cancer tumor-agnostic phase 2b nonrandomized controlled trial, patients with advanced BRCA1/2-altered or ATM-altered solid tumors were enrolled into 2 respective parallel cohorts. The study was conducted from July 2, 2018, to April 12, 2020, at 42 institutions in 9 countries.
Interventions
Patients received 800 mg of avelumab every 2 weeks and 1 mg of talazoparib once daily.
Main Outcomes and Measures
The primary end point was confirmed objective response (OR) per RECIST 1.1 by blinded independent central review.
Results
A total of 200 patients (median [range] age, 59.0 [26.0-89.0] years; 132 [66.0%] women; 15 [7.5%] Asian, 11 [5.5%] African American, and 154 [77.0%] White participants) were enrolled: 159 (79.5%) in the BRCA1/2 cohort and 41 (20.5%) in the ATM cohort. The confirmed OR rate was 26.4% (42 patients, including 9 complete responses [5.7%]) in the BRCA1/2 cohort and 4.9% (2 patients) in the ATM cohort. In the BRCA1/2 cohort, responses were more frequent (OR rate, 30.3%; 95% CI, 22.2%-39.3%, including 8 complete responses [6.7%]) and more durable (median duration of response: 10.9 months [95% CI, 6.2 months to not estimable]) in tumor types associated with increased heritable cancer risk (ie, BRCA1/2-associated cancer types, such as ovarian, breast, prostate, and pancreatic cancers) and in uterine leiomyosarcoma (objective response in 3 of 3 patients and with ongoing responses greater than 24 months) compared with non-BRCA-associated cancer types. Responses in the BRCA1/2 cohort were numerically higher for patients with tumor mutational burden of 10 or more mutations per megabase (mut/Mb) vs less than 10 mut/Mb. The combination was well tolerated, with no new safety signals identified.
Conclusions and Relevance
In this phase 2b nonrandomized controlled trial, neither the BRCA1/2 nor ATM cohort met the prespecified OR rate of 40%. Antitumor activity for the combination of avelumab and talazoparib in patients with BRCA1/2 alterations was observed in some patients with BRCA1/2-associated tumor types and uterine leiomyosarcoma; benefit was minimal in non-BRCA-associated cancer types.
Trial Registration
ClinicalTrials.gov Identifier: NCT03565991.
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v2
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2022-11-19T06:16:17.966Z
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2022-11-17T00:00:00.000Z
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253626774
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s2ag/train
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Perioperative Immune Checkpoint Inhibition in Early-Stage Non-Small Cell Lung Cancer: A Review.
Importance
Although cancer-related mortality continues to decline, lung cancer remains the No. 1 cause of cancer deaths in the US. Almost half of the patients with non-small cell lung cancer (NSCLC) are diagnosed with early-stage, local or regional disease and are at high risk of recurrence within 5 years of diagnosis.
Observations
Immune checkpoint inhibitors (ICIs) have improved outcomes for patients with metastatic NSCLC and have recently been tested in multiple clinical trials to determine their efficacy in the neoadjuvant or adjuvant setting for patients with local or regional disease. The landscape for perioperative ICIs in lung cancer is evolving rapidly, with recently reported and soon to mature clinical trials; however, the recent data highlight the potential of ICIs to increase response rates and decrease rates of relapse in early stages of lung cancer. Concurrently, novel applications of cell-free DNA may guide perioperative management strategies.
Conclusions and Relevance
This article reviews the various approaches of incorporating perioperative use of immunotherapeutic agents for the treatment of early stages of NSCLC.
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v2
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2022-11-19T06:16:18.426Z
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2022-11-17T00:00:00.000Z
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253626644
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s2ag/train
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Screening for Latent Infections Among Users of High-Risk Immunosuppressants: A Cross-Sectional Analysis From the Veterans Health Administration Healthcare System
Objectives Guidelines recommend screening for latent hepatitis B virus (HBV), hepatitis C virus (HCV), and tuberculosis (TB) before initiating biologics or targeted synthetic disease-modifying antirheumatic drugs (b/ts DMARDs) to avoid reactivation of life-threatening infections. The extent to which such screening occurs in the national Veterans Health Administration (VA) healthcare system is unknown. Methods Using data from the Veterans Affairs’ (VA) Corporate Data Warehouse, we performed a cross-sectional analysis of veterans receiving b/ts DMARDs between October 1, 2017, and September 30, 2019. We calculated the proportion of patients with screening completed for latent HBV, HCV, and TB between October 1, 1999 and September 30, 2019. Patient characteristics associated with complete screening were evaluated using mixed-effects multivariate logistic regression models. We also examined facility-level factors associated with high versus lower performance. Results A total of 51,764 unique patients from 129 VA facilities received b/ts DMARDs from 2017 to 2019. Of these, 63% had complete screening. Among the 11,006 patients identified as new users, 64% had complete screening. Higher screening rates were observed among Hispanic/Latinx and Black/African American patients, users of B-cell therapies, and patients who had seen oncology subspecialists. Substantial variation was observed across facilities, with complete screening ranging from 13% to 98% of patients. Higher screening rates were associated with highly complex, urban, and higher-volume facilities. Conclusions Approximately two-thirds of veterans taking b/ts DMARDs have received guideline-recommended screening for HBV, HCV, and TB, but substantial facility variation was observed. Performance measures, robust multidisciplinary workflows, and electronic health record–based tools to feed information back to providers may improve screening rates for low-performing facilities.
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v2
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2022-11-19T06:16:19.412Z
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2022-11-17T00:00:00.000Z
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253626463
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s2ag/train
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Effect of primary tumor resection on survival in patients with asymptomatic unresectable metastatic colorectal cancer: a systematic review and meta-analysis.
OBJECTIVE
It remains controversial whether primary tumor resection (PTR) improves survival in patients with asymptomatic, unresectable metastatic colorectal cancer (mCRC). Therefore, we conducted a meta-analysis to assess the latest evidence on clinical outcomes.
MATERIALS AND METHODS
We systematically searched PubMed, Web of Science, Cochrane Library, and Embase databases for eligible studies published between database inception and May 2022. RevMan 5.4 and Stata 16.0 were used for the meta-analysis.
RESULTS
A total of nine studies were included, including four randomized controlled trials (RCTs) and five retrospective cohort studies. Meta-analysis showed that overall survival (OS) [HR = 0.89, 95%CI (0.74, 1.06), P = 0.19] and progression-free survival (PFS) [HR = 0.87, 95%CI (0.71, 1.06), P = 0.17] were not significantly different between the PTR and non-PTR groups. In the subgroup analysis, all subgroups showed no significant difference in OS between the two groups.
CONCLUSION
PTR may not provide additional survival benefits over chemotherapy in asymptomatic, unresectable mCRC patients. However, in view of the limitations of this study, more well-designed RCTs are needed to validate our conclusions.
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v2
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2022-11-19T06:16:19.431Z
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2022-11-17T00:00:00.000Z
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253626772
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s2ag/train
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Early skeletal muscle deconditioning and reduced exercise capacity during (neo)adjuvant chemotherapy in patients with breast cancer
Fatigue is a hallmark of breast cancer and is associated with skeletal muscle deconditioning. If cancer‐related fatigue occurs early during chemotherapy (CT), the development of skeletal muscle deconditioning and its effect on exercise capacity remain unclear. The aim of this study was to investigate the evolution of skeletal muscle deconditioning and exercise capacity in patients with early‐stage breast cancer during CT.
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v2
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2022-11-19T06:16:20.252Z
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2022-11-17T00:00:00.000Z
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253626160
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s2ag/train
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[HER2-low breast cancer - A new entity that could expand possibilities of getting treatment].
Breast cancer is the most common form of cancer among women in Sweden. Several decades ago it was recognized that the Human epidermal growth factor receptor 2 (HER2) is involved in a critical growth system for breast cancer cells. Overexpression of HER2 (immunohistochemistry [IHC] 2+/3+, in situ hybridization [ISH] positive) is present in 15 percent of all breast cancers. HER2-low breast cancer has been discovered as a separate entity; the most commonly used definition so far is IHC 1+/2+ and ISH negative, but general consensus is still lacking. Clinical studies with the HER2 antibody drug conjugate trastuzumab deruxtecan (Enhertu) have shown impressive antitumor activity among women with advanced HER2-low breast cancer and this is expected to become part of routine treatment in the near future. Research is needed to establish refined ways to define HER2-low breast cancer, and a possible role lies in new imaging methods such as HER2 positron emission tomography (PET) with a [68Ga]Ga-ABY-025 tracer.
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v2
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2022-11-19T16:01:57.153Z
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2022-11-17T00:00:00.000Z
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253663640
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s2orc/train
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Methylation of CpG island promoters at ZNF625, LONRF2, SDC2 and WDR17 in a patient with numerous non-granular type laterally spreading tumors and colorectal cancer: A case report
Patients with adenomatous polyposis syndromes such as familial adenomatous polyposis are at higher risk of colorectal cancer, hence continuous management is necessary. However, little is known about the etiology of patients with numerous laterally spreading tumors (LSTs), or how genetic alterations uniquely influence LSTs in colorectal carcinogenesis. The present case report investigated a woman with >150 non-granular type LSTs (LST-NG) and one sigmoid colon cancer. After subtotal colectomy via ileorectal anastomosis, genetic and epigenetic analyses were conducted by comparing the profiles of the patient's normal colonic mucosa, four LST-NG lesions and a cancer lesion. Using customized multigene panel testing, no pathogenic germline mutations were detected, including APC regulator of WNT signaling pathway, but identified a somatic pathogenic variant of APC in one LST-NG lesion, and both TP53 and F-box and WD repeat domain containing 7 somatic mutations in the cancer. Comprehensive genome-wide methylation analysis showed that CpG island promoters at zinc finger protein 625, LON peptidase N-terminal domain and ring finger 2, WD repeat domain 17 and syndecan 2 were methylated in both LST-NG and cancer, which may contribute to colorectal tumorigenesis as early as the LST-NG phase.
Introduction
There are various types of polyposis syndrome including familial adenomatous polyposis (FAP), serrated polyposis syndrome, Peutz-Jeghers syndrome, juvenile polyposis syndrome, and PTEN-hamartoma tumor syndrome (1). Patients with these syndromes are at higher risk of developing colorectal cancer. Therefore, appropriate management via genetic testing and endoscopic surveillance is essential for the treatment and surgical prophylaxis of patients with colorectal polyposis. FAP is an autosomal dominant colorectal tumor syndrome caused by an APC pathogenic germline variant, and is characterized by the formation of numerous adenomatous polyps throughout the entire colon (2). Since 15-20% of cases are de novo without clinical or genetic evidence of FAP (3), it is important for clinical diagnosis to check for more than 100 colorectal adenomas via colonoscopy, regardless of family history of colorectal adenomatous polyposis (4). Each adenoma is typically polypoid in shape in patients with FAP. Therefore, it is difficult to judge whether a phenotype with vast numbers of an alternate morphological type of adenoma is associated to FAP.
Although the main colorectal tumorigenesis pathway is via protruded adenomas through the adenoma-carcinoma sequence, some colorectal cancers (CRCs) develop from these flat lesions via a de novo pathway (5,6). Kudo et al first called these flat, early lesions laterally spreading tumors (LSTs), and classified the horizontal growth lesions into two subtypes: the LST-granular type (LST-G), with granules and nodules on the tumor surface; the LST-nongranular type (LST-NG), with flat, smooth surfaces (7,8). In terms of histopathology, most LST-G and LST-NG lesions comprise tubular or tubulovillous adenomas, although the molecular characteristics of the two subtypes differ. Hiraoka et al demonstrated that LST-G lesions have KRAS mutations and are intermediate with regard to hypermethylation, whereas LST-NG lesions exhibit little pathogenic variation (9). Sakai et al reported that a TP53 mutation Methylation of CpG island promoters at ZNF625, LONRF2, SDC2 and WDR17 in a patient with numerous non-granular type laterally spreading tumors and colorectal cancer: A case report occurred during the development of cancer in both LST-NG and LST-G lesions (10,11). Although the authors reported the molecular characteristics of tumor progression from LST to CRC, the collected tumor samples were obtained from patients subjected to various environmental factors, including different lifestyles and microbiomes, as well as various genetic germline backgrounds. Therefore, ideal molecular analysis should be performed using normal colonic mucosa, LSTs, and CRCs from patients with identical backgrounds.
Herein, we investigate a patient with >150 LST-NG lesions in the entire large intestine and one adenocarcinoma in the sigmoid colon, which has not been previously reported. The lesions appeared to have the FAP phenotype, but otherwise atypical in that all the adenomatous lesions were LST-NG. The cancer lesion of this case might have been developed by somatic mutation of FBXW7 and/or TP53, differently with the tumorigenesis by germline APC mutation followed by acquired APC disfunction in patients with FAP. We further demonstrate the genomic/epigenomic difference among the normal colonic mucosa, LST-NGs, and cancer lesions of the patient as tumor progression occurs via accumulation of epigenetic alterations as well as pathogenic mutations of tumor suppressor genes. In this study, for the first time, we report that the hypermethylation of ZNF625, LONRF2, SDC2, and WDR17 as well as somatic mutation of FBXW7 and/or TP53 contribute to tumorigenesis from LST-NG.
Case report
A 72-year-old female presented at our hospital after a positive fecal occult blood test without any change to her bowel habits. She had no family history of colorectal polyposis or colorectal cancer. A complete colonoscopy revealed more than 150 amelanotic, flat, elevated lesions in the entire large intestine with melanotic background mucosa owing to prolonged self-administration of Sennoside A+B calcium (Fig. 1A). All the flat lesions were LST-NG, and the biopsy samples obtained from the lesions were pathologically diagnosed as tubular adenomas. Esophagogastroduodenoscopy was also performed and that no fundic gland polyposis was observed. After 4 years of annual endoscopic surveillance, one of the LST-NG lesions had developed into an adenocarcinoma in the sigmoid colon (Fig. 1A). Since LST-NG is a pre-cancerous condition, a subtotal colectomy by ileorectal anastomosis (IRA) was performed. The postoperative histopathology results demonstrated the presence of a tubular adenocarcinoma and multiple tubular adenomas ( Fig. 1B and C). No cases of both multiple LST-NG lesions and CRC have been previously reported; we hypothesize that the tumorigenesis might have had a genetic/epigenetic cause. The patient provided written informed consent, and the study was approved by the Institutional Review Board of the Hamamatsu University School of Medicine (approval no. . For genetic analysis, we first performed multigene panel testing using MiSeq sequencer (Illumina) and collected one normal colonic mucosa (N), four LST-NG lesions (L1-L4), and one cancer sample (C) from the patient immediately following IRA, extracted the DNA, and froze the fresh samples for storage. The extracted DNA was quantified using a Qubit dsDNA BR Assay Kit (Q32850; Thermo Fisher Scientific) on a Qubit 2.0 Fluorometer (Q33216, Thermo Fisher Scientific), and was prepared for shearing according to the SureSelect XT HS Target Enrichment System Manual (Agilent Technologies). Custom capture probes were designed using SureDesign (Agilent Technologies) covering the exons and boundary regions of 96 genes, including APC. For library preparation, a SureSelect XT HS Reagent Kit (G9702A, Agilent Technologies) was used according to the manufacturer's instructions. In brief, pre-enriched adapter-ligated libraries were prepared. Quality and quantity of libraries were determined by 4150 TapeStation System (G2992AA, Agilent Technologies) using D1000 ScreenTape (5067-5582, Agilent Technologies). 3.8 pmol of each library was used for hybridization. Subsequently, custom capture probes were hybridized to target sequences to enable sequence enrichment using streptavidin beads. Post-enrichment, libraries were quantified, pooled, and sequenced using MiSeq Reagent Kit v3 (MS-102-3001, Illumina) on a MiSeq sequencer. SureCall v4.0.1.46 (Agilent Technologies) and VariantStudio software (Illumina) were used for data analysis and alignment. GRCh37 was used as the reference genome.
All detected variants were validated using Integrative Genomics Viewer 2.9.2 (Broad Institute, Cambridge, MA, USA). We detected no pathogenic variants, including APC, in the normal mucosa. Of the four LST-NG lesions, only one had a pathogenic variant of APC (NM_000038.5: c.2396_2397delAT, NP_000029.2: p.Tyr799CysfsTer3), which was a somatic change because there was no mutation of APC in the normal colonic mucosa. In contrast, pathogenic mutations in both TP53 (NM_000546.5: c.499_500delCA, NP_000537.3: p.Gln167AlafsTer13) and FBXW7 (NM_033632.3: c.1513C>T, NP_361014.1: p.Arg505Cys) were detected in the cancer (Table I). These results suggest that the patient had no possibility of developing FAP because there was no APC mutation in the normal mucosa, and the somatic mutation of FBXW7 and/or TP53 contributed to tumorigenesis.
As tumor progression generally occurs by accumulation of epigenetic alterations as well as pathogenic mutations of tumor suppressor genes, it is important to understand the role of DNA methylation in tumorigenesis. Therefore, we next conducted a comprehensive genome-wide analysis using an Infinium MethylationEPIC BeadChip Kit (Illumina) according to the manufacturer's recommendations. Briefly, bisulfite-treated DNA was subjected to whole-genome amplification before fragmentation and precipitation. The resuspended DNA was subsequently hybridized to probes attached to the BeadChips (Illumina), which contained >850,000 CpG sites, and the nonhybridized DNA was removed. The attached probes were then subjected to single-base extension and stained. The BeadChips were scanned using the iScan™ system (Illumina) according to the manufacturer's recommendations. The red and green signals from the iScan™ system were converted into unmethylated and methylated signals. For each CpG site in the CpG island gene region, a DiffScore value of >100 between the normal mucosa and the cancer was defined as the absolute DMS value of the sample and calculated using GenomeStudio FrameWork v2011.1 software (Illumina) and the R statistical environment (version 3.1.3). Among the 766 detected DMSs, we selected one methylated CpG site from each of the cancer and normal mucosa samples, and performed clustering analysis of the normal mucosa sample, four LST-NG lesions, and cancer sample, as shown in Fig. 2A. In most DMSs, the methylation levels of the LST-NG lesions were the same as those in the normal mucosa (Group a), as previously demonstrated using sets of patients with LST (9-11). We next focused on the minority group (Group b) in which the DMS levels of the LST-NG lesions were as high as those in the cancer sample (Fig. 2B). DMS occurred at the CpG islands of the following nine genes: ZNF625, LONRF2, MSC, OPLAH, PCDHGA4, GSG1L, BEND5, SDC2, and WDR17. We further checked the methylation values at the CpG islands including the DMS site detected in Group b (Fig. 3). Among the regions, the CpG sites of ZNF625, LONRF2, MSC, and OPLAH were methylated in all four LST-NG lesions as in the cancer sample, and SDC2 and WDR17 were methylated in three of the LST-NG lesions. In the CpG islands in GSG1L, BEND5, and PCDHGA4, the CpG sites were not methylated as in the cancer lesions. When we observed on the gene regions of these CpG sites, we noticed that ZNF625, LONRF2, SDC2, and WDR17 might have been methylated at the promoter regions in both the LST-NG lesions and the cancer sample, because these methylated CpG sites were located 200 bases upstream of the transcriptional start site, or 5' untranslated region. This suggests that methylation-silenced ZNF625, LONRF2, SDC2, and WDR17 play roles in tumorigenesis as early as the LST-NG phase.
Discussion
The diagnosis and management of patients with polyposis syndromes is constantly evolving, owing to new scientific and technological advances with respect to the identification of causative genes, and the increased sophistication of endoscopic treatments for polyps. However, we were uncertain as to how to categorize the patient in the present study who had numerous LST-NG lesions, among the various colorectal polyposis syndromes, as the present work is the first report of a patient with >150 LST-NG lesions that developed a CRC during endoscopic surveillance. Our genomic and epigenomic analyses showed that: (1) no germline APC pathogenic variant was detectable via the multigene panel testing; (2) there was only one somatic APC frameshift mutant site in one of the four LST-NG lesions; (3) the somatic mutations of TP53 and FBXW7 were only present in the cancer sample; (4) there was methylation of the promoter CpG islands in ZNF625, LONRF2, SDC2, and WDR17 in most of the LST-NG lesions as well as the cancer lesion.
FAP is clinically diagnosed when approximately ≥100 adenomatous polyposis are detected in the large intestine, irrespective of the presence or absence of a family history of FAP, as 15-20% of FAP cases are de novo (3,4,12). Therefore, we first suspected the indexed patient had sporadic FAP since there were >100 adenomatous lesions throughout her large intestine and she had no family history of FAP-associated lesions.
The patient's adenomatous lesions were LST-NG, which are histologically the same as in protruding adenomatous polyposes (tubular adenomas) but differ morphologically to the naked eye. Moreover, no cases have been reported of FAP with multiple LST-NG lesions. Therefore, we explored the germline pathogenic variants using the normal colonic mucosa and customized multigene panel test and found no pathogenic variants, including APC, suggesting that the patient had no genetic evidence of FAP. The limitation of our analysis was that we could not completely exclude hereditary polyposis syndrome since the multigene panel did not include polyposis-related genes such as MUTYH and BMPR1A. Therefore, it is necessary to conduct whole exome/genome sequencing to detect any unknown germline genetic alterations to confirm the presence of new types of hereditary polyposis syndrome.
The pathogenicity of somatic variants of cancer including CRC is assessed by examining general population data, functional data, predictive data, cancer hotspots, and computational evidence (13). Therefore, numerous patients with CRC and healthy controls have been registered to establish reliable evidence. However, the registered CRC patients' own environmental factors and gut microbial compositions considerably differ. The potential role of epigenetic alterations has been reported in links between obesity, gut microbiota, and colorectal cancer. For colorectal cancer progression, high-fat diet-induced obesity leads to epigenetic remodeling of the acetylation landscape based on the gut microbiota, promotes changes in DNA methylation, and enhances production of deoxycholic acid, a secondary bile acid that is produced solely by Gram-positive gut bacteria and known to cause DNA damage through reactive oxygen species production (14). Therefore, pure genetic/epigenetic factors for colorectal tumorigenesis should be detected under the same environmental and microbiological conditions if possible. One way of accomplishing that is to compare genetic/epigenetic profiles among the normal mucosa, pre-cancerous lesions, and cancer lesions obtained from patients with identical backgrounds at the same time. In the present study, we analyzed the normal colonic mucosa, LST-NG lesions, and sigmoid cancer lesion of the same patient obtained immediately following colorectal resection.
In the present study, genetic analysis using customized multigene panels revealed only one APC frameshift variant in one LST-NG lesion, while the remaining three LST-NG lesions had no pathogenic variants. Metz et al reported that more than 90% of the LST lesions examined exhibited an APC mutation, but it did not exhibit the mutation frequency of an LST-NG lesion (15). Sugimoto et al detected loss of heterozygosity (LOH) at the APC locus in 60% of the LST-NG lesions they examined, whereas only 28% LST-G lesions harbored the mutation (16). In contrast, precise analysis by Sugai et al showed that null APC variants (i.e., nonsense and frameshift type pathogenic variants) were numerous in LST-G lesions compared with LST-NG lesions (17). These previous reports suggest that somatic pathogenic APC variants play a role in the occurrence of LST-NG lesions but are not the main contributors to tumorigenesis. In the present study, we detected somatic pathogenic alterations in TP53 and FBXW7, both of which variants were only present in the cancer lesion. Previous studies have demonstrated that the synergistic contributions of wild type FBXW7 and TP53 proteins contribute to the suppression of gastrointestinal cancer (18,19), and most FBXW7 mutations in cancers, including CRC, exhibit a TP53 mutation (20)(21)(22). Therefore, we speculated that our study patient had a cancer lesion that simultaneously lost the two tumor-suppressors that usually cooperate in the inhibition of tumorigenesis. In addition, Sakai et al have suggested that the TP53 mutation is more closely involved at an earlier stage in LST-NG lesions than in LST-G lesions during cancer development (11). In the same manner, the TP53 mutation might occur in an earlier phase of the patient's cancer lesions than the phase in which LST-NG lesions appear, and the mutation may continuously influence sigmoid tumor progression to the advanced level.
We further performed epigenome-wide analysis to determine whether there were any pathogenic epigenetic alterations that cause tumorigenesis after the LST-NG phase. Of the 766 DMSs identified, 756 were hypermethylated only in the cancer lesion, and the methylation levels in the LST-NG lesions were as low as in the normal colonic mucosa (Group a). This result was expected since Sakai et al were able to cluster LSTs into two epigenotypes in 108 LST samples (51 LST-G and 57 LST-NG lesions), i.e., intermediate-and low methylation-groups. The authors found that the intermediate methylation epigenotype was associated with LST-G lesion morphology, while the low methylation LSTs mostly reflected LST-NG lesion morphology (10,11). When we assessed the remaining 10 DMSs, all of which were categorized in the same cluster group (Group b), we noticed that the methylation levels of all 10 DMSs were as high as those in the cancer lesions. Moreover, all 10 DMSs were located in the CpG island region, indicating that the genes where the 10 DMS were located play roles in tumorigenesis by silencing the pre-cancerous phase of the LST-NG lesions. We further determined whether the CpG island regions, including the 10 DMSs, were methylated to the same extent in the LST-NG lesions as in the cancer lesion and found that ZNF625, LONRF2, SDC2, and WDR17 may have been methylated at the promoter region in both the LST-NG lesions and the cancer lesion. Among those four genes, SDC2 has been investigated most extensively regarding methylation in colorectal neoplasms. SDC2 has the chromosomal locus 8q22.1 and encodes syndecan-2 protein. The syndecan-2 protein participates in cell proliferation, cell migration, and cell-matrix interactions via its extracellular matrix proteins receptor (23)(24)(25)(26)(27), and altered syndecan-2 expression has been detected in several different tumor types (28)(29)(30). As reported, overexpressed SDC2 has tumor activity in CRC (31)(32)(33), and it apparently exerts its oncogenic character when activated. Oh et al first reported the hypermethylation of SDC2 in colorectal adenoma as well as in CRC, indicating its contribution to tumorigenesis (34). Their results have been corroborated by other groups, and various useful evaluation methods using stool samples, blood, and urine have been demonstrated (35)(36)(37)(38)(39)(40). In contrast to many previous reports concerning SDC2 in colorectal adenomas and CRC, little is known about alterations in ZNF625, LONRF2, and WDR17 concerning CRC tumorigenesis. ZNF625 has the chromosomal locus 19p13.2 and encodes zinc finger protein 625, which is predicted to enable DNA-binding transcription factor activity, but has not yet been completely analyzed. Lin et al reported that among 228 hypermethylated promoter-associated CpG islands, ZNF625 is one of the most frequently hypermethylated genes in colorectal cancer (41). LONRF2 has the chromosomal locus 2q11.2 and encodes LON peptidase N-terminal domain and ring finger 2. The gene is conserved in various species including chimpanzees, mice, dogs, chickens, zebrafish, and frogs. Hua et al reported LONRF2 hypermethylation in the rectal adenocarcinomas from 171 patients (42). WDR17 has the chromosomal locus 4q34.2, encodes WD repeat-containing protein 17, and is abundantly expressed in the retina and testes. It has been suggested as a candidate gene for retinal disease (43). In anal cancer, WDR17 is hypermethylated, regardless of HIV infection status (44), but to date there has been no report on WDR17 methylation in CRC. Considering the findings of this study along with those of previous studies, we suggest that SDC2 hypermethylation contributes to colorectal tumorigenesis at the adenoma stage, and is not limited to LST-NG lesions. Although little is known about the methylation of ZNF625, LONRF2, and WDR17 concerning CRC tumorigenesis, it is possible that methylation of the CpG island promoters at ZNF625, LONRF2, and WDR17 plays a unique key role in the tumorigenesis of LST-NG lesions.
However, the present study has some limitations: i) the genetic/epigenetic analysis was performed using a different number of samples in each group, that is, only one cancer lesion, four LST-NG, and one normal mucosa; ii) only one patient was analyzed.
In conclusion, we successfully demonstrated the acquired genomic/epigenomic status of pre-cancerous and cancerous phases under identical germline and environmental conditions by analyzing a patient with multiple LST-NG lesions and sigmoid colon cancer. We detected four genes methylated at the CpG island promoters during the LST-NG lesion phase. Although rare, patients with both pre-cancer and cancer lesions should be further investigated to elucidate the contribution made by pure somatic genomic/epigenomic alterations to tumorigenesis.
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Ischemic stroke of unclear aetiology: a case-by-case analysis and call for a multi-professional predictive, preventive and personalised approach
Due to the reactive medical approach applied to disease management, stroke has reached an epidemic scale worldwide. In 2019, the global stroke prevalence was 101.5 million people, wherefrom 77.2 million (about 76%) suffered from ischemic stroke; 20.7 and 8.4 million suffered from intracerebral and subarachnoid haemorrhage, respectively. Globally in the year 2019 — 3.3, 2.9 and 0.4 million individuals died of ischemic stroke, intracerebral and subarachnoid haemorrhage, respectively. During the last three decades, the absolute number of cases increased substantially. The current prevalence of stroke is 110 million patients worldwide with more than 60% below the age of 70 years. Prognoses by the World Stroke Organisation are pessimistic: globally, it is predicted that 1 in 4 adults over the age of 25 will suffer stroke in their lifetime. Although age is the best known contributing factor, over 16% of all strokes occur in teenagers and young adults aged 15–49 years and the incidence trend in this population is increasing. The corresponding socio-economic burden of stroke, which is the leading cause of disability, is enormous. Global costs of stroke are estimated at 721 billion US dollars, which is 0.66% of the global GDP. Clinically manifested strokes are only the “tip of the iceberg”: it is estimated that the total number of stroke patients is about 14 times greater than the currently applied reactive medical approach is capable to identify and manage. Specifically, lacunar stroke (LS), which is characteristic for silent brain infarction, represents up to 30% of all ischemic strokes. Silent LS, which is diagnosed mainly by routine health check-up and autopsy in individuals without stroke history, has a reported prevalence of silent brain infarction up to 55% in the investigated populations. To this end, silent brain infarction is an independent predictor of ischemic stroke. Further, small vessel disease and silent lacunar brain infarction are considered strong contributors to cognitive impairments, dementia, depression and suicide, amongst others in the general population. In sub-populations such as diabetes mellitus type 2, proliferative diabetic retinopathy is an independent predictor of ischemic stroke. According to various statistical sources, cryptogenic strokes account for 15 to 40% of the entire stroke incidence. The question to consider here is, whether a cryptogenic stroke is fully referable to unidentifiable aetiology or rather to underestimated risks. Considering the latter, translational research might be of great clinical utility to realise innovative predictive and preventive approaches, potentially benefiting high risk individuals and society at large. In this position paper, the consortium has combined multi-professional expertise to provide clear statements towards the paradigm change from reactive to predictive, preventive and personalised medicine in stroke management, the crucial elements of which are: Consolidation of multi-disciplinary expertise including family medicine, predictive and in-depth diagnostics followed by the targeted primary and secondary (e.g. treated cancer) prevention of silent brain infarction Application of the health risk assessment focused on sub-optimal health conditions to effectively prevent health-to-disease transition Application of AI in medicine, machine learning and treatment algorithms tailored to robust biomarker patterns Application of innovative screening programmes which adequately consider the needs of young populations
Clinically manifested strokes is only the "tip of the iceberg"
In 2003, Leary and Saver published evidence-based estimates of the annual incidence of first silent stroke in the USA, which indicated that for the year 1998 there were about 11 million first-ever silent MRI brain infarcts (SBI, called also "covered brain infarction") or haemorrhages versus 770,000 symptomatic clinically manifested stroke cases [4]. These data indicate that clinically manifested strokes contribute a noticeable but minor share of the entire issue of brain infarction, which is estimated to be about 14 times greater in the population than the currently applied reactive medical approach, per definition [5], and is capable to identify and manage. Specifically, lacunar stroke (LS), being also characteristic for SBI, represents up to 30% of all ischemic strokes. Silent LS is frequently diagnosed during routine health check-ups as well as during autopsy in otherwise healthy individuals without stroke history. Depending on the focus of the study and patient stratification criteria, the reported prevalence of SBI/silent LS is 6 to 55% in the investigated populations [6][7][8][9][10][11][12][13][14]. Here, it is worth noting that SBI is more prevalent in females whereas clinically manifested stroke is reported to be more prevalent in males [6,[15][16][17][18][19]. There are obvious sex differences in the symptomatic interpretation of Cerebrovascular Diseases (CVD) and cerebrovascular infarcts, which has been traditionally considered more prevalent in the population and, therefore, described more precisely. Consequently, female-specific symptoms might be simply overlooked or interpreted wrongly resulting in poorer outcomes [20]. Further, SBI-related prevalence
Global epidemics of stroke: status quo and prognoses in figures
Due to the reactive medical approach applied to disease management, stroke has reached epidemic scale worldwide. In 2019, the global stroke prevalence was 101.5 million people, of which 77.2 million (about 76%) suffered from ischemic stroke; 20.7 and 8.4 million suffered from intracerebral and subarachnoid haemorrhage, respectively [1]. Globally in the year 2019 -3.3, 2.9 and 0.4 million individuals died of ischemic stroke, intracerebral and subarachnoid haemorrhage, respectively.
During the last three decades, the number of cases has increased substantially. The World Stroke Organization (WSO) reported an increase in stroke incidence of 70.0%, death from stroke of 43.0%, stroke prevalence of 102.0%, and disability-adjusted life-years lost of 143.0% [2]. The current prevalence of stroke is 110 million patients worldwide with more than 60% below the age of 70 years with the highest rates of ischemic stroke recorded for the highincome countries in North America, the Middle East and Southeast Asia, amongst others [3]. Prognoses presented by the WSO are pessimistic: globally, 1 in 4 adults over the age of 25 is predicted to suffer a stroke in their lifetime [3]. Although age is the best known contributing risk factor, over 16% of all strokes occur in teenagers and young adults aged 15-49 years with increased incidence trend. The socioeconomic burden of stroke is enormous since stroke is the leading cause of disability. Current global costs of stroke are estimated at 721 billion US dollars which is 0.66% of the global GDP [2]. data are lacking for young populations [6] significantly hindering targeted primary prevention and effective protection of young populations.
Silent brain infarction as an indicator of associated systemic disorders -the prominent examples and pathomechanisms behind the health-to-disease transition
"…silent strokes aren't so silent" said Dr. Gorelik who every day examines people with abnormal brain scans indicating a silent stroke -see "Silent strokes' found accidentally need treatment" by American Heart Association News [21] to prevent severe pathologies linked to subtle changes indicated by SBI.
SBI as an independent marker for ischemic stroke risks
Existing SBI strongly predicts a next silent infarction [22] and indicates the highest risk of ischemic stroke in affected individuals. Amongst the selected patient cohorts, the prevalence of SBI in the ischemic stroke patients was 57% [23].
Cognitive impairment and dementia
The onset of cognitive impairment and dementia are leading consequences of stroke. In a cohort of Japanese Alzheimer's disease patients, every 3 rd patient was co-diagnosed with SBI [24]. Similar observations have been made from autopsies of individuals with a history of dementia [25][26][27]. Small vessel disease and lacunar brain infarctions are considered strong contributors to cognitive impairments and dementia in the general population [28]. In particular, SBI localised in the thalamus negatively affects cognitive tasks such as short-term memory performance [29][30][31], whereas non-thalamic SBI correlates with a decline in psychomotor speed tasks [23].
Affective disorders and suicide
Significant data indicate that SBIs below 3 mm are associated with symptoms of depression; in fact, SBI can be diagnosed in up to 50% of elderly people with major depression [23,[32][33][34]. Bipolar disorder type II with a suicide attempt, secondary to a lacunar state has been reported [35].
Migraine
Significant associations were found between migraine attacks and silent brain infarction [36,37]. Females are 3 times more likely to be affected by migraine; high oestrogen levels increase risks of migraine with aura, thromboembolism, secondary ischemia-induced aura and ischemic stroke [38].
Normal-tension glaucoma
Silent cerebral infarction (SCI) has been proposed to be an independent risk factor for progression of visual field loss in patients with normal-tension glaucoma (NTG) [39]. The correlation between SCI and NTG is considered indicative for small vessel diseases, a major contributor to the NTG pathology, microvascular dysregulation and endothelial dysfunction characteristic for the NTG patient cohort and NTGspecific type of nerve degeneration resulting in progressive visual field loss. Vascular geometry may affect perfusion efficiency and the functional link between SCI, small vessel disease and normal-tension glaucoma [40].
Pathomechanisms behind the health-to-disease transitiona vicious circle of the stroke development
A better understanding of the mechanisms underlying the health-to-disease transition in stroke is key point for costeffective health risk assessment, predictive diagnostics, patient stratification and targeted prevention which might be offered to individuals at risk. Stroke development shares several risk factors, e.g., affected metabolic pathways and molecular targets with other stress-related diseases such as cancers and diabetes mellitus type 2 as detailed below.
Systemic effects of the reciprocal cancer-stroke promotion: what comes first?
Cancer-associated ischemic stroke is well-described in the literature: As reported recently, cancer pathomechanisms are functionally linked to endothelial dysfunction, coagulation abnormalities, activated pro-inflammation, and platelets adhesion, which collectively increase the risk of thromboembolism and ischemic stroke [41]. Additionally, vascular toxicity and secondary dysfunction are adverse effects of current anti-cancer therapeutics. It is also to be noted that stroke survivors are diagnosed with cancer at almost double the rate of the general population [42]. Though, a study dedicated to the cancer incidence in young adults suffering from ischemic stroke demonstrated median time from pre-stroke cancer to stroke of about 4.9 (1.0-9.5) years, whereas from stroke to post-stroke cancer by 6.7 (2.7-10.9) years [43]. Thus, the question "What comes first -cancer or stroke?" has not yet been clarified. However, per evidence, both stress-related diseases share several risk factors, affected metabolic pathways and molecular targets, namely: -Oxidative stress -Disturbed microcirculation -Endothelial dysfunction (ET-1) -Compromised mitochondrial health -Pro-inflammation -Connective tissue impairments -Extensive tissue remodelling, amongst other, collectively leading to the vicious cycle in the development of the systemic hypoxia-reperfusion, ischemic lesions, activated MMPs, blood brain barrier (BBB) breakdown, brain infarction and metastases [44][45][46][47][48].
However, by far not every cancer patient develops stroke and not all patients with a history of stroke are at high risk of cancer. Indeed, patients with brain malignancies and cancers spreading metastases to the brain such as triple-negative breast cancer [49] are at high risk of stroke as well as cancer patients with secondary vascular dysfunction who underwent vasculo-toxic therapies causing thromboembolic type of stroke [41]. On the other hand, subtle systemic hypoxicischemic effects causing low-grade inflammation have been proposed to be involved in the development of particularly aggressive metastatic cancers [48] the pathomechanisms of which are considered to be similar to those of non-healing wounds [50]. Consequently, for this type of cancer, SBI diagnostics might be particularly useful to indicate associated systemic effects -the concept to be further considered by the follow-up studies. Figure 1 summarises facts and hypotheses involved in the reciprocal cancer-stroke promotion.
Diabetic retinopathy is indicative for the stroke risk
Due to pronounced vascular ageing, individuals affected by diabetes mellitus (DM) have twice the risk of stroke compared to non-diabetics [61]. Microvascular dysfunction is characteristic for DM. Hyperglycaemia, obesity, insulin resistance and hypertension have been described as the main drivers of DM-related microvascular dysfunction. Cerebral microvascular dysfunction is present already in pre-diabetic stages suggesting that cerebral microvascular disease is an early step in the overall cascade of DM pathophysiology Fig. 1 Schematic presentation of the health-to-disease transition in the stroke development and reciprocal cancer-stroke disease promotion; blood-brain barrier, BBB; and metalloproteinases, MMPs. Suboptimal health conditions representing reversible damage are relevant for the cost-effective primary healthcare (blue frame) and are based on the individualised health risk assessment and targeted primary prevention. At this stage, subtle changes may include imbalanced stress, enhanced endothelin-1 blood plasma levels indicating pronounced vasoconstriction of peripheral vessels, increased stiffness of peripheral vessels co-diagnosed with connective tissues deficits/disease as demonstrated in pregnant women with the Flammer syndrome [45][46][47]51], enhanced homocysteine levels in blood plasma potentially leading to small vessel disease and associated SBI [52], blood pressure fluctuations with remarkable nocturnal lows [6] and low-grade inflammation [53,54], all relevant to the manifestation of hypoxia-reperfusion and systemic ischemic lesions. These changes may result in irreversible damage leading to SBI, retinal microvascular changes, systemic inflammation, mitochondrial impairments, pre-cancerous lesions and pre-metastatic niches -altogether leading to the reciprocal cancer-stroke promotion in a "vicious cycle". To this end, thromboembolic stroke is frequently observed in (treated) patients with cancer diagnoses. In turn, brain metastases are characteristic, e.g., for patients diagnosed with the triple-negative breast cancer and the Flammer syndrome phenotype demonstrating extensive vasoconstriction and systemic hypoxic-ischemic lesions including SBI [48,55]. Extensive tissue remodelling plays a key role in both, neurodegeneration (stroke) and metastatic cancer performed by the core of metalloproteinases, which are excellent indicators and pathology-associated biomarkers [49,[56][57][58][59][60] predisposing to lacunar and haemorrhagic stroke, cognitive dysfunction, and depression [62]. For pre/diabetes care, it is crucial, to stratify patients at high versus low risk of stroke based on highly specific biomarker patterns, including the presence of diabetic retinopathy (DR), which has been associated with an increased risk of life-threatening systemic vascular complications, including stroke, coronary heart disease, and heart failure [63]. Due to the particularly high sensitivity of retinal cells to oxidative stress, retinal health provides valuable insights into patients' risk of future vascular disease and death. Early DR manifestation is associated with an increased risk of stroke in DM. This correlation is particularly robust for DM type 2, suggesting that DR is an important biomarker for the prediction of stroke in this patient cohort [64]. Specifically, proliferative DR has been proposed as the biomarker for accelerated promotion of CVD complication in DM [65]. In fact, increased severity of DR carries a heightened risk for cerebrovascular accidents, myocardial infarction, congestive heart failure and severity of the corresponding disease outcomes in patients with type 2 DM [66].
Cryptogenic strokes: unidentifiable aetiology or underestimated risks?
According to different statistical sources, cryptogenic strokes (CSs) account for 15 to 40% of the stroke incidence [67]. A generally accepted definition for CSs currently does not exist, although several classification systems have been provided, namely the Trial of Org 10,172 in Acute Stroke (TOAST) that defines CSS as a stroke of undetermined aetiology which is not caused by large artery atherosclerosis, cardioembolism, and small vessel occlusion. The Causative Classification of Stroke System (CCS) is a computerised algorithm determining causative and phenotypic stroke subtypes and detecting uncommon and undetermined causes including incomplete evaluation and cryptogenic stroke, and ASCOD (Atherosclerosis, Small Vessel Disease, Cardiac Causes, Other, and Dissection), which grades the likelihood that the disease was causally related to the stroke: from 0 = absence of the disease and 1 = potentially causal up to 9 = insufficient workup to rule out the disease [67]. Despite the difference between individual classification systems, the main concept is similar, namely, cryptogenic stroke is an ischemic stroke with no identifiable aetiology based on a diagnosis of exclusion.
Contextually, the question does arise, whether cryptogenic stroke is fully referable to unidentifiable aetiology or rather to underestimated risks, which have been demonstrated by recent studies but still not taken into account when considering the causes of CS, e.g., in the computerised algorithms for the automatic disease recognition, causality and phenotyping, pre-stages, corresponding biomarker patterns, etc. Considering the latter, translational research might be of great clinical utility to realise innovative predictive and preventive approaches, potentially benefiting individuals at high risk and society at large. This concept may be illustrated with the stroke cases analysed below.
Ischemic stroke case-by-case
Case 1: Young ischemic stroke associated with cancer A 49-year-old woman (BMI = 16.22 kg/m2) was referred to the hospital due to a sudden onset of left-sided hemiplegia. The patient has generalisation of endometrial adenocarcinoma (pT2pN1 (3/45) M0, G2, R0 treated from 2018 with radiotherapy and chemotherapy, FIGO IIIC2, paclitaxel/ carboplatina). Brain metastases were irradiated with Leksell gamma knife in 2019. The patient underwent outpatient oncological treatment. Multimodal CT of the brain revealed MCA/ACA occlusion (T-occlusion) right-sided. The patient was treated with intravenous thrombolysis (Actilyse) and mechanical thrombectomy. Re-canalisation TICI 2c was achieved and neurological findings improved to moderate left-sided hemiparesis. Later, there was a progression of the oncological disease and the patient died 10 months later due to the generalisation of endometrial adenocarcinoma in Hospic, mRS 6.
The cause of stroke: MCA/ACA occlusion (T-occlusion) is most likely a pro-coagulant condition caused by cancerendometrial adenocarcinoma (see Fig. 1).
Case 2: Young ischemic stroke associated with the pronounced Flammer syndrome phenotype and severe course after SARS-CoV-2 infection
A 37-year-old woman (BMI = 23.94 kg/m2) was referred to the hospital due to the onset of vision problem and clumsiness of the left limbs. At admission, left-sided homonymous hemianopsia and left-sided hemiataxia were detected. Multimodal CT revealed an acute ischaemic lesion in the left cerebellar hemisphere (penumbra without core), CT chest/lung confirmed viral pneumonia compatible with SARS-CoV-2 infection confirmed by the corresponding PCR test. Further, the patient demonstrated the Flammer syndrome phenotype with pronounced vascular maladaptation, migraine with accompanying symptoms, low BMI at young age, suboptimal sleep patterns, a tendency to low blood pressure and specific psychosocial behavioural patterns such as a pronounced meticulous personality, amongst other signs and symptoms. This is of special interest as the recently emerging field of affective immunology addresses the multidirectional-communication between emotion regulation, personality traits and immune functioning, mainly focusing on the association between pro-inflammatory signalling and free radical production by the immune system and personality traits. Upcoming findings will enable a more detailed evaluation of these processes and their interplay for their contribution and importance in the context of prediction and prevention also in ischemic stroke.
The patient was treated with intravenous thrombolysis (Actilyse) with excellent result. Other examinations: normal transthoracic and transesophageal echocardiography and ECG Holter, negative thrombotic panel, negative panel of autoimmune diseases, negative test for Fabry disease. Diffusion-weighted MRI scans 1 month after stroke onset showed no infarction. Full recovery of neurological deficit, mRS 0.
The cause of stroke: ischaemic lesion in the left cerebral hemisphere is most likely a pro-coagulant condition caused by a combination of the strongly pronounced Flammer syndrome phenotype and severe course of the SARS-CoV-2 infection.
Case 3: Young ischemic stroke associated with the pronounced Flammer Syndrome phenotype and stress overload
A 45-year-old woman (BMI = 24.8 kg/m2) was referred to hospital due to a sudden onset of stand and gait ataxia and hypestesia on the left side of her face. Multimodal CT of the brain at admission was normal. MRI revealed an ischaemic lesion (6 × 5 × 4,5 mm) on the left side of the medulla oblongata. Recanalization therapy was not used due to exceeded time window, the patient was treated with acetylsalicylic acid and rosuvastatin, crystaloids and rehabilitation. In the past medical history, her premature birth (birth weight 1350 g) was recorded. Further, the patient demonstrated the Flammer syndrome phenotype with migraine, vascular maladaptation, thermal dysregulation, extremely low BMI at young age, Sicca syndrome and dehydration, suboptimal sleep patterns, dysmenorrhea, a tendency to low blood pressure and specific psychosocial behavioural patterns such as a pronounced meticulous personality, amongst other signs and symptoms. The patient is a smoker. Further, she reported exposure to enormous stress several months before stroke onset. Results of genetic testing showed Leiden heterozygosity and negative Fabry disease test. There were normal transthoracic and transesophageal echocardiography and ECG Holter (7 days). The patient was discharged with residual hemiataxia and instability, disability pension, mRS 3.
The cause of stroke: ischaemia in the medulla oblongata is most likely a pro-coagulant condition caused by a combination of the pronounced Flammer syndrome phenotype and stress overload which, however, has to be objectively measured.
Predictive phenotyping and targeted primary prevention of ischemic stroke are feasible: a detailed case presentation
At the end of 2021, a 59-year-old woman underwent commercially available and internationally validated predictive diagnostic test based on mitochondrial health quality measurements (check-point and consultation by responsible general practitioner specialised in family medicine). Reason for the test was diagnosis of causes of sub-optimal health condition the patient worried about. In the patient's medical history, the following information was considered highly relevant: -Ischemic stroke in the family history (thrombotic vein occlusion of mother at the age of 77 years and lacunar stroke of father at the age of 88 years); -Pronounced Flammer syndrome phenotype of the patient including low body mass index, low blood pressure, migraine with aura, tinnitus, strong vasospastic reactions under stress conditions accompanied with a significantly increased endothelin-1 level (3.2 pg/ml) in blood serum, retinal ischemic lesions diagnosed early in life (35 years of age; check-point and consultations by specialised ophthalmological clinic), and connective tissue impairments, amongst others; -As a carrier of the Flammer syndrome phenotype, the patient is a highly stress-sensitive person that is further aggravated by her meticulous personality and permanent work/stress overload caused by a highly ambitious international academic career of the patient.
Indeed, the predictive health quality test performed, demonstrated characteristic non-compensated stress overload patterns which, in the specific case of this patient reflect ischemicreperfusion episodes associated with vasospastic reactions of peripheral vessels and increased endothelin-1 levels [68]. The following recommendations were provided to the patient: 1. Treatment with commercially available scavenges as bioactive protection against acute stress such as emotional and psychological stress situations [68-70] 2. Evaluation of single stressors to apply individualised therapeutic approaches [71,72] 3. Brain MRI was recommended to check for SBI as the most reliable biomarker for ischemic stroke prediction.
Follow-up report.
-The patient is taking orally L-cysteine (500 mg, Gall Pharma) as prophylaxis against expected stress situations. -The patient is taking ginkgo biloba (120 mg) and magnesium as daily nutritional supplement to improve microcir-culation and to protect small vessels against inappropriate vasoconstriction. -Brain MRI (check-point and consultations by the specialised diagnostic radiology unit) performed according to the recommendations has confirmed the predicted SBI (Fig. 2). However, given successful mitigation measures, no recent ischemic lesions have been detected. The current health condition of the patient is considered stabilised.
Conclusions in the framework of 3P medicine
Due to the reactive medical approach applied to the disease management, stroke reached an epidemic scale worldwide, and current WSO prognoses are pessimistic: globally, 1 in 4 adults over the age of 25 years is predicted to get diseased on stroke in their lifetime. Moreover, the incidence trends to increase particularly for young stroke patients below 50 years of age. The corresponding socio-economic burden is enormous. Further, clinically manifested strokes are only the "tip of the iceberg": apparently, the total size is about 14 times greater in the population than the currently applied reactive medical approach, per definition is capable to identify and manage. Specifically, the lacunar stroke builds up to 30% of all ischemic strokes being also characteristic for silent brain infarction. Silent LS is diagnosed mainly by routine health check-up and autopsy in individuals without stroke history: the reported prevalence of silent brain infarction is 6 to 55% in the investigated populations. To this end, silent brain infarction is an independent predictor of ischemic stroke. There are significant sex and age differences in symptomatic interpretation of the silent brain infarction: female sex and youth-specific symptoms are frequently overlooked or wrongly interpreted that significantly hinders targeted primary prevention and effective protection of females and young populations. Further, small-vessel disease and silent lacunar brain infarctions are considered strong contributors to cognitive impairments, dementia, depression and suicide, amongst others in the general population. In sub-populations such as diabetes mellitus type 2, specifically proliferative DR is an independent predictor of the ischemic stroke. According to different statistical sources, cryptogenic strokes account for up to 40% of the entire stroke incidence. The question does arise whether cryptogenic stroke is fully referable to unidentifiable aetiology or rather to underestimated risks. Considering the latter, translational research might be of great clinical utility to realise innovative predictive and preventive approaches, potentially benefiting at high-risk individuals and society at large.
In this position paper, the consortium involved has combined multi-professional expertise to provide clear statements towards the paradigm change from reactive to predictive, preventive and personalised medicine in stroke management, the crucial elements of which are the following: The examination was performed on the 3Tesla-MRI with weight-adjusted intravenous contrast medium administration (CMA, gadolinium). Sequences obtained: FLAIR, axial scan; DWI, ADC map, axial scan; SWI axial scan; axial FLAIR 3D scan; Time-of-Flight MR angiography; axial T2tse scan; 3D T1-weighted sequences after CMA with coronary and axial reconstructions. Results and conclusions: Although the cerebral vasculature is well-structured and intact without any detectable pathological changes (D), small vessels clearly show signs of micro-angiopathy; several lacunar microinfarction zones, white matter hyper-intensities and micro-haemorrhages (A, B, C) are evident; no any recent ischemic lesions have been recorded
A B D C
-Consolidation of multi-disciplinary expertise including family medicine, predictive and in-depth diagnostics followed by the targeted primary and secondary (e.g. treated cancer) prevention of the silent brain infarction -Application of the health risk assessment focused on suboptimal health conditions to effectively prevent healthto-disease transition -Application of AI in medicine, machine learning and treatment algorithms tailored to robust biomarker patterns -Application of innovative screening programmes considering the needs of young populations
Declarations
Ethics approval Ethics approval FN/1252/01 dated on July 2 nd 2020 has been provided by the Ethical Commission of University Hospital Plzen and Faculty of Medicine in Plzen, Charles University, Prague, Czech Republic.
Conflict of interest
The authors declare no conflict of interest. OG is the Editor-in-Chief of the journal, but had no involvement in, influence over, or access to the details of the peer review process of this work.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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v2
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2022-11-19T16:31:17.552Z
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2022-11-17T00:00:00.000Z
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253654069
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s2ag/train
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Comparison of the state of brain metabolism and psycho emotional disorders in patients with postmastectomy syndrome
BACKGROUND: The most common consequence of radical treatment of breast cancer is postmastectomy syndrome a complex of changes in the lymphocirculatory system, central and peripheral nervous system, skeletal and muscular apparatus, that significantly reduce the quality of life and working capacity of women. In recent years, special attention has been paid to the study of psycho emotional disorders in this group of patients. A promising method for preclinical diagnosis of anxiety and depressive disorders in patients with postmastectomy syndrome may be positron emission computed tomography with fluorine-18 labeled glucose 2(18F)-fluoro-2-deoxy-D-glucose, which makes it possible to deduce typical patterns of changes in glucose metabolism in cerebral structures in various depressive and anxiety states.
AIM: The purpose of this study is to study the relationship between brain metabolism and psycho emotional status in patients with postmastectomy syndrome.
MATERIALS AND METHODS: In our work, the sample consisted of 28 patients who underwent radical treatment for breast cancer, who underwent an assessment of the psycho-emotional state using the State-Trait Anxiety Inventory and Zung scales for self-assessment of depression. Positron emission tomography was also performed with 18-fluorodeoxyglucose.
RESULTS: The study revealed that 71% of patients showed an increased level of anxiety, 64% showed signs of depression. Positron emission tomography data revealed the following areas of hypometabolism in patients with anxiety-depressive disorders: parietal cortex, inferior parietal lobule, precuneus, superior temporal gyrus, prefrontal cortex, posterior cingulate cortex.
CONCLUSION: Thus, typical zones of changes in glucose metabolism in patients with psycho emotional disorders have been identified, which makes it possible to improve the accuracy of diagnosing these conditions, as well as to develop the most effective ways to prevent and treat them.
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v2
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2022-11-19T16:41:52.013Z
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2022-11-17T00:00:00.000Z
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253657379
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s2ag/train
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SYNTHESIS AND CHARACTERIZATION OF SOME NOVEL HETERONUCLEAR BIS-THIAZOLIDINONE DERIVATIVES AND EVALUATION OF ITS ANTIMICROBIAL AND IN VITRO CYTOTOXIC PROPERTY
A series of novel bis-thiazolidinone derivatives 3(a-j) have been synthesized by the cyclization of thiosemicarbazones 2(a-j) with chloroacetic acid and sodium acetate. The integrated heterocyclic compounds were featured by chemical and spectroscopic methods such as IR, 1H NMR and 13C NMR. All the synthesized compounds have been screened for their antimicrobial activity against Gram-positive and Gram-negative bacteria such as Staphylococcus Aureus, Bacillus licheniformis, Klebsiella pneumoniae, Esherichia coli and antifungal activity against Aspergillus niger and in vitro cytotoxic activity against human cancer cell line (HeLa cell) and Vero cell line, using MTT assays but showed no activity.
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v2
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2022-11-19T16:42:31.802Z
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2022-11-17T00:00:00.000Z
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253661484
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s2ag/train
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An enigmatic tale of the missed and migrated tail
We, report a case of missing and migrated intrauterine device presenting as vague abdominal pain. The patient had symptoms of vaginal discharge and menstrual disturbances as polymenorrhagia and leukorrhea. She was evaluated for a suspected malignancy of the ovaries. During evaluation on pelvic CT scan, the missing and migrated Intrauterine Contraceptive Device (IUCD) was located in the small bowel and tumor markers were normal. We treated her with antibiotics for presumptive diagnosis of pelvic inflammatory disease and then posted her for the evaluation of the missing IUCD and the tubo-ovarian masses by endoscopy. The missed IUCD was seen embedded in the terminal ileum with intraperitoneal adhesions and the tubo-ovarian masses. The IUCD was removed endoscopically enterotomy of ileum which was sutured. The patient recovered well and was discharged on the 6th post-operative day.
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v2
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2022-11-20T14:10:38.409Z
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2022-11-17T00:00:00.000Z
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253671914
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s2ag/train
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A cell circuit approach to dissect fibroblast-macrophage interactions in the tumor microenvironment
The tumor microenvironment (TME) is composed of various nonmalignant cell types that interact with each other and with cancer cells, impacting all aspects of cancer biology. The TME is complex and heterogeneous, and thus simplifying systems and concepts are needed. Here we provide a tractable experimental system and powerful mathematical circuit concepts to identify the main molecular interactions that govern the composition of the TME. We focus on two major components of the TME - cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs), define their interactions and verify our predictions in mouse and human breast cancer. We measure the population dynamics starting from many initial conditions of co-cultures of macrophages and organ-derived fibroblasts from mammary, lung, and fat, and explore the effects of cancer-conditioned medium on the circuits. We define the circuits and their inferred parameters from the data using a mathematical approach, and quantitatively compare the cell circuits in each condition. We find that while the homeostatic steady-states are similar between the organs, the cancer-conditioned medium profoundly changes the circuit. Fibroblasts in all contexts depend on autocrine secretion of growth factors whereas macrophages are more dependent on external cues, including paracrine growth factors secreted from fibroblasts and cancer cells. Transcriptional profiling reveals the molecular underpinnings of the cell circuit interactions and the primacy of the fibroblast autocrine loop. The same fibroblast growth factors are shared by the co-cultures and mouse and human breast cancer. The cell circuit approach thus provides a quantitative account of cell interactions in the cancer microenvironment.
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v2
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2022-11-20T14:10:43.799Z
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2022-11-17T00:00:00.000Z
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253671867
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s2ag/train
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Deciphering the maturation of tertiary lymphoid structures in cancer and inflammatory diseases of the digestive tract using imaging mass cytometry: from high-level data to a simple architectural and functional grading
Objective Persistent inflammation can promote the development of tertiary lymphoid structures (TLS) within tissues resembling the secondary lymphoid organs (SLO) as lymph nodes (LN). The composition of the TLS across different organs and diseases could be of pathophysiological and medical interest. In this work, we compared TLS to SLO and between cancer and inflammatory diseases of the digestive tract. Design Colorectal and gastric tissues with different inflammatory diseases and cancers from the department of pathology of CHU Brest were analyzed based on 39 markers using imaging mass cytometry (IMC). Unsupervised and supervised clustering analyses of IMC images were used to compare SLO and TLS. Results Unsupervised analyses tended to group TLS per patient but not per disease. Supervised analyses of IMC images revealed that LN had a more organized structure than TLS and non-encapsulated SLO Peyer’s patches. TLS followed a maturation spectrum with close correlations between germinal cell (GC) markers’ evolution. The correlations between organizational and functional markers made relevant the previously proposed TLS division into three stages: lymphoid-aggregates (LA) (CD20+CD21-CD23-) had neither organization nor GC functionality, non-GC TLS (CD20+CD21+CD23-) were organized but lacked GC’s functionality and GC-like TLS (CD20+CD21+CD23+) had GC’s organization and functionality. This architectural and functional maturation grading of TLS pointed to differences across diseases. Conclusion TLS architectural and functional maturation grading is accessible with few markers allowing future diagnostic, prognostic, and predictive studies on the value of TLS grading, quantification and location within pathological tissues in cancers and inflammatory diseases. KEY MESSAGES - What is already known on this topic: Tertiary lymphoid structures (TLS) arise in organs under various pathological conditions and can be of prognostic significance. - What this study adds: This study deciphers the composition of TLS in digestive cancers and inflammatory diseases using massively multiplexed (39 markers) imaging mass cytometry (IMC). Beyond the term TLS, this study points to the heterogeneity of these structures in terms of composition and maturation but also the relevance of a simple architectural and functional three-stage grading of TLS. - How this study might affect research, practice, or policy: This preliminary study paves the way for future studies evaluating the diagnostic, prognostic and theranostic values of TLS maturation grading, quantification and location within tissues as novel biomarkers in inflammatory diseases and cancers.
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v2
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2022-11-20T16:14:21.279Z
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2022-11-17T00:00:00.000Z
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253703727
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s2orc/train
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Postoperative Impact of Pontocerebellar Angle Surgery on the Quality of Life in Patients with Vestibular Schwannoma
Background: Vestibular Schwannomas are benign tumors arising from the VIII CN. Surgical treatment is indicated in case of tumors larger than 2.5 cm in the cerebellopontine angle or in the case of cranial nerve dysfunction. The aim of the present study was to evaluate the QoL by means of the PANQOL questionnaire in a group of surgically treated patients mainly affected by large and giant VS Methods: All patients underwent preoperative and postoperative otoneurological evaluation and gadolinium enhanced MRI and they completed, independently, the PANQOL questionnaire at last follow up. Results: 70% of patients presented with large Koos III or IV VS Each domain of PANQOL showed a strong correlation with the total PANQOL score. In relation to the postoperative facial nerve function, patients with poorer function showed significantly lower score in the facial dysfunction and pain, patients with postoperative balance problems showed a significantly lower PANQOL score for domains of balance and pain. Conclusions: This study showed that postoperative QoL of patients was acceptable even if there were some domains that were more affected, such as hearing and balance domains; therefore, the lowest scores suggest the need for vestibular rehabilitation programs and strategies that improve postoperative hearing.
Introduction
Vestibular schwannomas (VS) are benign, slow-growing tumors arising from the VIII CN and constitute 8% of all intracranial neoplasms and 90% of cerebellopontine angle (CPA) lesions [1].
Due to the slow growth of these tumors, treatment options include surgical excision, stereotactic radiosurgery (SRS), and conservative management/active surveillance. Individual patient management depends on various factors including age, medical comorbidities, size and location of the tumor, and hearing status [2].
Surgical treatment is generally indicated in the case of tumors larger than 2.5 cm in the cerebello-pontine angle with the primary aims of complete tumor removal and the preservation of facial function and the patient's quality of life (QoL) [3].
In Italy, the preferred surgical approaches for VS removal are the translabyrinthine (TL) and retrosigmoid (RS) [3]. TL is a presigmoid transmastoid approach that allows the exposure of the internal auditory canal (IAC) and cerebellopontine angle (CPA) after the removal of the posterior labyrinth and the presigmoid bone. It is therefore the best option when hearing preservation is not an issue and, compared to the RS approach, allows the identification of the facial nerve both at the root entry zone of the brainstem and at the fundus of the IAC. The RS approach is mainly an intradural approach that allows a large view of the CPA; however, cerebellar retraction is needed, and the fundus of the IAC is difficult to expose especially in the case of hearing preservation with the risks of subtotal removal [4].
The evaluation of QoL in VS patients has become increasingly important in recent years. In a systematic review, Barker-Collo et al. [2] have reported that surgical treatment does not improve the QoL in patients affected by small-to-medium size tumors; therefore, initial observation has been proposed as the first therapeutic option in these patients [1]. In the case of large and giant VS, surgical treatment represents the only therapeutic option, especially in the case of cranial nerve dysfunction [3] and therefore the evaluation of the surgical results as well as the post-operative QoL must be considered at the time of surgical planning. The Penn Acoustic Neuroma Quality of Life Questionnaire (PANQOL) is a disease-specific tool proposed by Shaffer et al. [5] that measures the QoL of VS patients, evaluating the effect of the tumor and of the treatment in six specific domains: balance, energy, hearing, anxiety, face, general health and pain. Lucidi et al. [6] have recently adapted the questionnaire in Italian and have evaluated the QoL of VS patients treated with three surgical techniques. The aim of the present study was to evaluate the QoL by means of the PANQOL questionnaire in a group of surgically treated patients mainly affected by large and giant VS; in addition, the internal consistency and reliability of the Italian PANQOL questionnaire and factors that may predict patients' QoL were evaluated.
Participants
Between April 2018 and January 2022, 31 patients affected by VS underwent microsurgical tumor removal and represent the study group. Patients affected by skull base pathologies other than VS, patients with neurofibromatosis type 2, those who had received multiple active treatments, or those who had undergone previous microsurgical tumor removal were excluded.
A retrospective chart review was conducted for the included patients containing preoperative (sex, age, hearing impairment, tumor side and size), intra-operative (surgical approach, grade of resection, time of surgery) and post-operative characteristics and symptoms (facial paresis, balance problems, postoperative complications).
Procedures
All patients underwent pre-operative and post-operative evaluation consisting of clinical history, complete otoneurological evaluation of the cranial nerves, vestibular bed side examination (spontaneous nystagmus evaluation, Romberg test, Unterberger test, Head Shaking Test and clinical Head Impulse Test), tonal and speech audiometry and gadolinium enhanced MRI.
Tumor size was classified according to Koos et al. [7] in four stages. Facial function was classified according to the House-Brackmann scale (HB) [8], while pre-operative hearing was classified according to the classification system of the Committee on Hearing and Equilibrium of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) 1995 [9]. Tumor removal was classified in terms of the percentage of tumor removed by resection as: gross total resection (no macroscopic residual of tumor resection 100%); near total resection (the residual consists of only a small, thin capsular peel, <25 mm 2 , <2 mm thick); subtotal resection (a substantial portion of tumor remains >25 mm 2 , >2 mm thick removal) as proposed by Bloch et al. [10]. Complications were classified as intraoperative or post-operative. Average follow-up was 8 months (range 6-18 months).
The Italian version of the PANQOL questionnaire was used [6]. PANQOL comprises 26 multiple-choice questions that focus on the following areas: balance (six items), energy (six items), hearing (four items), anxiety (four items), face (three items), general health (two items) and pain (one item). Patients were asked to rate each item from 1 (strongly disagree) to 5 (strongly agree). A total instrument score was calculated as the unweighted average of the domain scores and reported on a scale from 0 to 100 (worst to best QoL). The questionnaires were completed by the patients independently in our clinics at last follow-up.
Statistical Analyses
Descriptive statistics of the PANQOL questionnaire were performed. Reliability was measured with one measure tool Cronbach's Alpha. A domain correlation matrix with Spearman coefficient was created and a subgroup analysis was performed with measures such as the Wilcoxon rank-sum test and Kruskal-Wallis test. Nonparametric tests were utilized considering the lack of the normal distribution of the domain scale. A Bonferroni correction was performed in the case of multiple comparisons. Data were considered statistically significant with a p value (α) < 0.05. All statistical analyses were performed using the software SPSS statistics (IBM-1 New Orchard Road, Armonk, NY, USA) version 28.0.1.1.
Patients Characteristics
Thirty-one patients undergoing surgery for VS between April 2018 and January 2022 were included in this study. Fifteen were female and 16 were male with a median age of 54.2 years (range, 16-79 year). Sixteen VS were located on the left side, while 15 were found on the right side. Seventy percent of patients presented with large Koos III or IV VS (Table 1). Twenty-six patients underwent TL approach and five patients the RS approach; resection was gross total in 23 patients and near total in eight ( Table 2). All patients presented with a normal pre-operative facial nerve function, while post-operative facial function evaluated at last follow-up is reported in Table 3. Of the patients, 61.3% presented a facial function HB grade 1 or 2; 22.6% grade 3 and 16.1% worse than grade 3. Table 3. Post-operative facial function.
House-Brackmann Scale
Value % At last follow-up, all patients presented with vestibular areflexia in the operated side, and 12 (38.7%) reported balance problems. Otoneurological examinations showed cerebellar ataxia in four cases (all Koos III-IV), central nystagmus in two cases (all Koos III-IV) and postural instability in the remaining six cases (three cases Koos I-II and three cases Koos III-IV).
Mortality was 0% and no intra-operative complications occurred. Nine patients developed post-operative complications. In two cases, additional surgery (a lumbo-peritoneal and a ventriculo-peritoneal shunt) was needed because of post-operative hydrocephalus (both Koos IV). In one case, a CSF leak was managed with temporary lumbar drainage, while all other complications (pulmonary embolism and cerebellar ischemia) were successfully managed with medical treatment ( Table 2).
PANQOL Results
Internal consistency was measured by Cronbach's alpha and was high for all domains (balance: 0.761; hearing: 0.778; anxiety: 0.74; facial dysfunction: 0.81; energy: 0.748; pain: 0.818; general health: 0.795). Domain scores were calculated on a scale between 0 and 100 as previously described (0 1 /4 worst to 100 1 /4 best QOL), and a total instrument score was calculated as the equal average of all domain scores (10): x = (actual raw value − lowest possible raw value)/possible range of raw value × 100 (1) Table 4 reports the average scores (with SD) and the range (min and max) for each domain and for the total score. Average scores were greater than 50 for balance, anxiety, facial dysfunction, energy and pain, while they were lower than 50 for hearing and general health. A non-parametric correlation test (Spearman correlation matrix) on each domain of PANQOL questionnaire was performed. All domains showed a strong correlation with the total PANQOL score and several showed a robust correlation with others (Table 5). A single sample analysis was performed in order to investigate factors influencing post-operative PANQOL score. The following factors were evaluated: pre-operative tumor size, pre-operative hearing level, post-operative facial dysfunction, post-operative balance, and post-operative complications. The type of surgical approach was not evaluated because of the small number of patients undergoing RS removal.
In relation to the pre-operative size, median PANQOL score was lower in patients with Koos III and IV VS (52.9) compared to Koos I and II (59); however, the difference was not significant. The "general health" and "facial dysfunction" domain scores were also not significantly lower in Koos III and IV. In relation to the post-operative facial nerve function, patients with poorer function showed significantly lower scores in the "facial dysfunction" (p = 0.032) and "pain" (p = 0.014) domains, while the total scores and all other domains were not significantly different. Twelve patients with post-operative balance problems showed a significantly lower PANQOL score for the domains of "balance" (p = 0.019) and "pain" (p = 0.033), while statistical significance was reached for the "facial dysfunction" domain (p = 0.049) without Bonferroni correction for multiple comparisons (Table 6). Pre-operative hearing level as well as the presence of post-operative complications did not correlate with post-operative QoL.
Discussion
The evaluation of the QoL in patients undergoing different types of treatment has become increasingly important in the otological and otoneurological literature.
In recent years, disease-specific questionnaires have been proposed in order to improve the measurement of specific diseases or their treatments on the patient's QoL. The process of translation and validation of these questionnaires in different languages allowed the comparison of results and the evaluation of the impact of specific symptoms on QoL in different populations.
For example, the validation of the Chronic Otitis Media Questionnaire-12 (COMQ-12) in different languages including Italian has allowed the evaluation of the effect of chronic otitis media on patients' QoL in different populations [11].
The PANQOL is a disease-specific tool that has been increasingly used over the years to evaluate the QoL of VS patients and has been translated and validated in several languages including Italian [6]. In the present study, the Italian version of the PANQOL questionnaire presented high Cronbach's alpha, as has also been reported by other authors in different languages [5,[12][13][14][15], confirming its good reliability as a specific tool for analyzing the quality of life of VS patients.
In the present series, VS patients presented with Koos III and IV tumors in 71% of cases, representing a typical population of a multidisciplinary otolaryngologic and neurosurgical clinic. In fact, while smaller tumors have different therapeutic options, larger VS need surgical treatment and therefore the evaluation of the post-operative QoL has become increasingly important in patient counselling.
In comparison with a "normal population," as well as with VS patients that are conservatively managed, the QoL of surgically treated patients has been reported to be poorer [1,2,16,17]. Selection bias has been reported since patients that undergo a watch-and-wait protocol usually present with smaller tumors and better hearing compared to those undergoing surgery. In cases of larger VS, even with the use of a not-disease-specific questionnaire such as the SF-36, Turel et al. [18] have reported that patients affected by large and giant VS may improve their QoL after surgery. The results of the present study showed that post-operative QoL of patients was acceptable even if there are some domains that were more affected such as the general health, hearing and balance domains.
While hearing and balance also represent the most affected domains in other series of surgically treated patients [12,13,16], general health was reported to be significantly impaired only by Pruijn et al. [16]. Such non-homogeneous results have been attributed to the fact that only two questions explore the general health domain in the PANQOL questionnaire and lead to a poor internal consistency of this scale [12]. The removal of the VS together with the drilling of the labyrinth in the case of the TL approach induce complete unilateral vestibular ablation and hearing loss. A single side deafness has been associated with poor sound localization and speech discrimination especially in noisy environments [19]; in addition, the hearing level of the contralateral ear represents a crucial factor in speech discrimination [20]. In the present series, mean age at surgery was 54.1 years, suggesting that more than half of the patients may possibly present with some degree of age-related hearing loss [21,22]. Even if hearing can be rehabilitated with CROS systems and bone anchored hearing aids, these aids restore a pseudo-binaural hearing that does not improve speech discrimination [23] nor sound-localization [24]. Cochlear implantation is the only device that is able to restore binaural hearing, but is not feasible in most VS surgeries due to the cochlear nerve damage induced by the tumor [25]. The poor results obtained in the balance domain are instead associated with the complete ablation of the vestibular function after TL approach and VS removal; even if a central compensation is expected after surgery, not all patients obtain a good balance and several prognostic factors have been reported [26], among them the size of the tumor and vestibular rehabilitation. In the present series, most of the patients presented with large VS and none underwent vestibular rehabilitation, suggesting the need for post-operative rehabilitation programs.
In patients operated on for small tumors, delayed compensation occurred, while in patients operated on for larger tumors, both delayed compensation and central vertigo occurred. As reported by other authors [27], early vestibular rehabilitation should be implemented in all patients and particularly in the elderly and those affected by large VS The facial dysfunction domain was associated with the least impact on QoL. In the present series as reported by other authors [1,12,16], there was a high correlation between post-operative facial nerve function according to the HB classification and the self-reported facial dysfunction PANQOL domain. Although the majority of the patients presented with large tumors, 61.3% presented with grade 1 or 2 facial function and only 6% of patients presented with grade 6. Near total removal was performed in eight cases in order to preserve the facial nerve anatomy and function and has been proposed as the standard of care when the facial nerve anatomy is at risk, especially in the case of large VS [28].
Anxiety, energy and pain domains, together with facial dysfunction, presented the highest scores and therefore in the present series do not represent factors that impair the QoL. The low incidence of reported headache was associated with the number of TL approach that, compared to RS, has been shown to be less associated with post-operative headache [29].
In the present series, no pre-operative factor was associated with post-operative QoL decrease; in particular, size did not influence the post-operative QoL in any domain. Similar results were reported by other authors [12,30,31], supporting a conservative management in smaller tumors and suggesting active treatment for larger tumors [13]. Finally, as also reported by Glaas et al. [12], patients with post-operative balance problems had lower scores in the balance domain as well as pain and facial dysfunction, corroborating the need for post-operative vestibular rehabilitation programs.
Although the present study presents many limitations, such as the limited sample and the absence of a control group, it evaluates the QoL of a group of Italian patients affected by large VS and surgically treated mainly by the TL approach. The low scores obtained in the hearing and balance domains suggest the need for specific rehabilitation programs and for strategies that improve post-operative hearing. Specific tools aimed at the evaluation of the hearing dysfunction as well as therapeutic strategies aimed at the restoration of the binaural hearing should be implemented.
Conclusions
The evaluation of the QoL in VS patients is important in the otological and neurotological literature. This study showed that the post-operative QoL of patients was acceptable even if there were some domains that were more affected, such as the hearing and balance domains. The lowest scores suggest the need for rehabilitation programs and strategies that improve post-operative hearing. Institutional Review Board Statement: This study was approved by the University of Bari institutional research board (Approval code #7191) and it was conducted in accordance with the Principles of Ethics for Medical Research Involving Human Subjects set in the Declaration of Helsinki and its subsequent amendments.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The statistical analysis plan, study protocol, and informed consent of the included patients are present in the Supplementary File; identified data and raw data are available upon motivated request to the corresponding author ([email protected]) and may be reused to reproduce research, to make public assets available to the public, to leverage investments in research, and to advance research and innovation.
Conflicts of Interest:
The authors declare no conflict of interest.
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v2
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2022-11-17T06:18:03.756Z
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2022-11-23T00:00:00.000Z
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253551807
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s2ag/train
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[Clinical characteristics of digestive system cancers metastatic to the heart].
Objective: To investigate the clinical features of patients with cardiac metastases from digestive system tumors. Methods: This retrospective study collected and analyzed the medical records of patients with cardiac metastases from digestive system tumors who received treatments in the Cancer Hospital, Chinese Academy of Medical Sciences between January 1999 and January 2021. Kaplan-Meier method was used for survival analysis. Results: A total of 19 patients were identified. The primary tumors were esophageal squamous cell carcinoma (n=7), gastric or gastroesophageal junction adenocarcinoma (n=6), hepatobiliary cancers (n=3) and colorectal cancers (n=3). 16 patients had pericardial metastases, 2 patients had right atrium metastases, and 1 patient had left ventricle metastasis. The most common symptom was dyspnea, which was present in 8 cases. 7 patients received locoregional treatment, while 11 patients underwent systemic therapies. The median overall survival from diagnosis of primary cancer was 31.4 months, and the median overall survival time from diagnosis of cardiac metastasis was 4.7 months. Conclusion: Cardiac metastasis from digestive system tumors is associated with low incidence and a poor prognosis. Systemic treatment remains the cornerstone of management, while novel anti-tumor drugs may improve therapeutic efficacy.
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v2
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2022-11-18T15:30:04.555Z
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2022-11-18T00:00:00.000Z
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253598082
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s2ag/train
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Neuro-ophthalmic complications of immune checkpoint inhibitor therapy: Current status and future directions
Since 2011, use of immune checkpoint inhibitors (ICI) in cancer immunotherapy dramatically expanded, both alone and in combination with either a different cancer treatment or with two different ICIs. With this increase in use have come a myriad of adverse effects from enhanced immune activation, including ophthalmic and neurologic immune related adverse events (irAE). Neuro-ophthalmic immune related adverse events (NOirAE) associated with use of ICIs are increasingly recognized and their severity may actually limit use of potentially life-saving immunotherapy. NOirAEs comprise a wide variety of presentations involving both the central and peripheral nervous system. They cause afferent or efferent visual dysfunction, including among them optic neuropathy and edema, orbital inflammatory disease, and ocular myasthenia. While treatment for irAEs typically involves immunosuppression with corticosteroids, there is no expert consensus regarding best practices for treatment of NOirAEs and whether to stop ICI immunotherapy for the cancer or not. This state-of-the-art review explores the pathophysiologic basis for NOirAEs, provides a framework for categorizing them within neuro-ophthalmology, and discusses what is needed to close the current knowledge gaps in diagnosis and management of an increasing population of cancer patients requiring neuro-ophthalmic care.
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v2
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2022-11-18T20:02:04.471Z
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2022-11-18T00:00:00.000Z
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253623247
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s2ag/train
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Projected Cancer Burden, Challenges, and Barriers to Cancer Prevention and Control Activities in the State of Telangana
Background and Aim: The Telangana cancer care program is a proactive, comprehensive initiative encompassing infrastructure development, human resource skilling and ensuring financial protection to those below poverty line. The broad aim of this exercise was to identify modalities to augment the Telangana State Cancer control Plan to implement a sustainable comprehensive cancer care model for Telangana. Methods: We conducted in-depth interviews of stakeholders (17 patients and 25 providers) to identify barriers and challenges to access existing cancer care system; calculated the estimated magnitude of cancer and commensurate workload (in terms of visits to tertiary cancer care system for chemotherapy, radiotherapy, and surgery and human and equipment requirement) for the next 15 years (from 2021 to 2036). Using the anecdotal evidence and information from stakeholders interviews, we developed patient-journey funnels for cancer patients, which helped us to appreciate at what levels of care leakages occur. Results: We estimated a 28% increase in the number of new cancer cases per year and the resultant workload: number of visits, chemotherapy, radiotherapy, surgeries, specialized human resources and equipment, from 2021 to 2036. Stakeholders mentioned delayed access to healthcare system as the main reason for the poor prognosis of patients. The common reasons cited for delayed access were: poor cancer-literacy including prevailing myths and misconception, financial barriers, and rural residence. Patient journey funnel for cancer care revealed major leakage from screened positive to diagnosis confirmation step. Patient leakage varies from ~70% to 90% from screened positive till treatment completion. Conclusion: Govt. of Telangana has initiated several measures to strengthen the healthcare system and to promote the uptake of cancer care services to manage the rising burden of cancer and resultant increasing workload. However, there is ample scope for further improvement (such as improved healthcare access, reduced patient leakage, commensurate human skills and infrastructure development etc.) to deliver comprehensive cancer care services in the state.
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v2
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2022-11-19T06:16:20.121Z
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2022-11-18T00:00:00.000Z
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253627078
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s2ag/train
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Probiotics enhances anti-tumor immune response induced by gemcitabine plus cisplatin chemotherapy for urothelial cancer.
Chemotherapy drugs, such as gemcitabine and cisplatin (GC), are frequently administered to patients with advanced urothelial carcinoma, however, the influence of the gut microbiota on their action is unclear. Thus, we investigated the effects of GC on the gut microbiome and determined whether oral supplementation with a probiotic mixture of Lactobacillus casei Shirota and Bifidobacterium breve enhanced the anti-tumor immune response. After subcutaneous inoculation with MBT2 murine bladder cancer cells, syngenic C3H mice were randomly allocated into eight groups. Gut microbiome cluster pattern was altered in both the GC and oral probiotic groups (P = 0.025). Both tumor-bearing conditions (no treatment) and GC chemotherapy influenced Pseudoclostridium, Robinsoniella, Merdimonas, and Phocea in the gut. Furthermore, comparison of the GC-treated and GC+probiotics groups revealed an association of four methyltransferase family enzymes and two short-change fatty acid-related enzymes with oral probiotics use. A significant difference in tumor volume was observed between the GC and GC+probiotic groups at week 2 of treatment. Additionally, decreased recruitment of cancer-associated fibroblasts and regulatory T cells, and activation of CD8+ T cells and dendritic cells were observed in the tumor microenvironment. Our findings reveal the positive effects of a probiotic mixture of Lactobacillus and Bifidobacterium in enhancing anti-tumor effects through the gut-tumor immune response axis. Future clinical trials are needed to evaluate the full benefits of this novel supplement with oral probiotics in patients with advanced urothelial carcinoma.
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v2
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2022-11-19T06:16:20.189Z
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2022-11-18T00:00:00.000Z
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253626944
|
s2ag/train
|
Protein-binding approaches for improving bioaccessibility and bioavailability of anthocyanins.
Color is an important characteristic of food. Over the last 15 years, more attention has been paid to natural colorants because of the rising demand for clean-label food products. Anthocyanins, which are a group of phytochemicals responsible for the purple, blue or red hues of many plants, offer a market advantage. In addition, anthocyanin-rich foods are associated with protection against cardiovascular disease, thrombosis, diabetes, cancer, microbial-based disorders, neurological disorders, and vision ailments. However, the real health value of anthocyanins, whether as a natural colorant or a functional ingredient, is dependent on the ultimate bioaccessibility and bioavailability in the human body. Many animal and human clinical studies revealed that, after intake of anthocyanin-rich foods or anthocyanin extracts, only trace amounts (< 1% of ingested content) of anthocyanins or their predicted metabolites were detected in plasma after a standard blood draw, which was indicative of low bioavailability of anthocyanins. Protein binding to anthocyanins is a strategy that has recently been reported to enhance the ultimate bioactivity, bioaccessibility, and bioavailability of anthocyanins as compared to anthocyanins delivered without a protein carrier. Therefore, in this review, we address anthocyanin properties in food processing and digestion, anthocyanin-protein complexes used in food matrices, and changes in the bioaccessibility and bioavailability of anthocyanins when bound into anthocyanin-protein complexes in foods. Finally, we summarize the challenges and prospects of this delivery system for anthocyanin pigments.
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v2
|
2022-11-19T06:16:20.237Z
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2022-11-18T00:00:00.000Z
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253626144
|
s2ag/train
|
Fatal Retroperitoneal Bleeding in Neurofibromatosis Type 1: A Clinically Occult Complication.
ABSTRACT
Neurofibromatosis type 1 (NF1) is a common, autosomal dominant neurocutaneous syndrome. The most frequent clinical manifestations include multiple neurofibromas, café-au-lait spots, dystrophic scoliosis, benign and malignant peripheral nerve sheath tumors, and paragangliomas. Neurofibromatosis type 1 vasculopathy is a less well-recognized constellation of vascular pathologies that can cause significant medical complications in patients with NF1. A rare manifestation of this process is neurofibroma infiltration of vasculature with resultant bleeding. The case presented herein illustrates a rare example of a massive fatal hemorrhage due to disruption of a large paraspinal artery in the setting of a diffuse, infiltrative neurofibroma. This case highlights the potential of benign neurofibromas to infiltrate major blood vessels, leading to extensive bleeding and death.
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v2
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2022-11-19T06:16:20.271Z
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2022-11-18T00:00:00.000Z
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253627607
|
s2ag/train
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Synthesis of Homo- and Heteromultivalent Fucosylated and Sialylated Oligosaccharide Conjugates via Preactivated N-Methyloxyamine Precision Macromolecules and Their Binding to Polyomavirus Capsid Proteins.
Glycoconjugates are a versatile class of bioactive molecules that have found application as vaccines and antivirals and in cancer therapy. Their synthesis typically involves elaborate functionalization and use of protecting groups on the carbohydrate component in order to ensure efficient and selective conjugation. Alternatively, non-functionalized, non-protected carbohydrates isolated from biological sources or derived through biotechnological methods can be directly conjugated via N-methyloxyamine groups. In this study, we introduce such N-methyloxyamine groups into a variety of multivalent scaffolds─from small to oligomeric to polymeric scaffolds─making use of solid-phase polymer synthesis to assemble monodisperse sequence-defined macromolecules. These scaffolds are then successfully functionalized with different types of human milk oligosaccharides deriving a library of homo- and heteromultivalent glycoconjugates. Glycomacromolecules presenting oligosaccharide side chains with either α2,3- or α2,6-linked terminal sialic acid are used in a binding study with two types of polyomavirus capsid proteins showing that the multivalent presentation through the N-methyloxyamine-derived scaffolds increases the number of contacts with the protein. Overall, a straightforward route to derive glycoconjugates from complex oligosaccharides with high variability yet control in the multivalent scaffold is presented, and applicability of the derived structures is demonstrated.
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v2
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2022-11-19T15:04:43.538Z
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2022-11-18T00:00:00.000Z
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253631353
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s2orc/train
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A misplacement of a ureteral stent into the abdominal aorta: a case report of a rare retrograde ureteral stenting complication
Background Cervical cancer is often associated with malignant ureteral obstruction and consequent hydronephrosis. Hydronephrosis caused in this way can be resolved by placing ureteral stents or performing a percutaneous nephrostomy. Complications that may occur during the retrograde ureteral stent placement are usually mild, but serious complications such as stent migration into the cardiovascular system are also possible. Here we present an unusual case where a ureteral stent entered the abdominal aorta during the cystoscopic ureteral stenting, which was resolved by a cystoscopic stent removal kept in check by endovascular catheters. Case presentations The 48-year-old female patient was treated in the regional secondary healthcare facility due to bilateral hydronephrosis caused by cervical cancer. The patient had bilateral percutaneous nephrostomies and ureteral stents. Due to the calcification of the left ureteral stent, an urethrorenoscopy with lithotripsy of the calculus in the left ureter was performed in the regional secondary healthcare facility, and the ureteral stent was cystoscopically replaced. The control radiography of the urinary tract showed a misplacement of the left ureteral stent, and a computed tomography showed that the stent was located in the abdominal aorta. The patient was referred to the University Clinical Center of Serbia, where a ureteral stent was cystoscopically removed from the abdominal aorta under the control of endovascular catheters. The patient was in good general condition at all times, with no signs of bleeding, and she was discharged from the hospital on the fourth postoperative day. Conclusions The migration of a ureteral stent into the abdominal aorta and the cardiovascular system in general is a rare type of ureteral stenting complication whose treatment requires a multidisciplinary approach. In order to prevent such complications, it is necessary to strictly adhere to the indications for the ureteral stent placement in the case of malignant ureteral obstruction. Also, this procedure should be performed according to the current guidelines and controlled by an X-ray or ultrasound.
Background
Cervical cancer can frequently cause a malignant ureteral obstruction in any stage of the disease and can then lead to hydronephrosis [1]. In such cases, hydronephrosis can be treated by a ureteral stent placement or by percutaneous nephrostomy (PCN). Ureteral stent placement is considered appropriate for the initial treatment of hydronephrosis; however, in numerous cases this treatment can be either inapplicable or ineffective, in which Open Access *Correspondence: [email protected] case, the placement of PCN is required [1][2][3]. In certain selected cases, the choice of treatment could depend on the patient's or urologist's preferences. Ureteral stenting, regardless of whether it is performed via the antegrade or retrograde method, could cause numerous complications, such as hematuria, pain, infection, calcification or fragmentation [4]. Complications are usually mild, but could at times be severe and difficult to treat, such as stent migration into the cardiovascular system or even the pleural cavity [5][6][7][8]. In such cases, the treatment of these complications is complex, and requires a multidisciplinary approach.
We present a rare case where a ureteral stent entered the abdominal aorta during the cystoscopic ureteral stenting, which was resolved by a cystoscopic stent removal kept in check by endovascular catheters without the need for embolization.
Case presentation
A 48-year-old female patient was treated for 6 years by a urologist in a regional secondary healthcare facility for bilateral hydronephrosis, caused by cervical cancer treated with radical radiotherapy and chemotherapy. The treatment of bilateral hydronephrosis was performed with the bilateral placement of ureteral stents and PCNs which were regularly and periodically replaced. Due to an unsuccessful cystoscopic replacement of the ureteral stent on the left side 1 month prior to that, and due to calcification around the stent in the left ureter, the urologist in charge indicated that ureteroscopy with lithotripsy of the calculus in the left ureter should be performed. The patient underwent a procedure of the left side ureteroscopy with ultrasonic lithotripsy of the 6 mm ureteral calculus, positioned approximately 6 cm from orifice, under general anesthesia, in the designated healthcare facility. Due to technical reasons, the already existing ureteral stent on the left side was removed cystoscopically, and another ureteral stent was inserted into the left ureter.
Following the procedure, a three-way urinary catheter was placed into the bladder, and a slightly red urine was formed. The patient coped quite well, she was in good general postoperative condition, without pain, hemodynamically stable, with normal heart rate, afebrile. In the first 10 minutes following the procedure, there was a mild macroscopic haematuria, after which her urine became completely macroscopically clear.
On the same day, radiography of the urinary tract was performed, when it was seen that the ureteral stent on the left side was dislocated, and that it was in summation with the spine (Fig. 1). An urgent computerized tomography of the abdomen and pelvis with contrast was performed, which showed that the ureteral stent went through the left ureter in the length of 65 mm from the orifice, piercing through the left internal iliac artery at approximately 35 mm from the bifurcation of the left common iliac artery, continuing through the abdominal aorta where its tip was at the level of the 11th thoracic vertebrae (Figs. 2 and 3). Neither the contrast extravasation nor the presence of a hematoma was observed. The patient was referred to the Clinic of Urology of the University Clinical Center of Serbia that same day for further multidisciplinary treatment.
Following the admission to the Clinic of Urology, and due to high risk from potential complications, the patient was initially admitted to the intensive care unit. At the admission, the patient was conscious, communicative, without pain, afebrile (36,5 °C), with normal vital signs (blood pressure 110/80 mmHg, heart rate 70-80/min). The physical examination revealed the abdomen to be in line with the chest cavity, without defacement, insensitive to pain during palpation, bilateral PCN were present with clear urine. Laboratory investigations at the admission indicated the following: red blood cells 2,32 × 10 12 /L, hemoglobin 101 g/L, hematocrit 0,321 L/L, white blood cells 9,9 × 10 9 /L, platelets 151 × 10 9 /L.
The following day, the patient was transferred to the Clinic for vascular and endovascular surgery, so that the cystoscopic extraction of the ureteral stent could be performed. After the adequate preparation of the surgical area and under the local anesthesia and fluoroscopic monitoring, 5F Cobra catheter was inserted through the right femoral artery into the patient's left internal iliac artery, at the place where the ureteral stent penetrated the aortic wall. A control Vertebral catheter was inserted through the left femoral artery into the left common iliac artery. Aortography was performed, and the position of the ureteral stent indicated that it penetrated the aortic wall, and that its proximal end was in the aorta at the level of the thoracoabdominal junction (Fig. 4). A rigid cystoscope was inserted into the bladder. Cystoscopic pincers were used to grab the ureteral stent in the left orifice, the stent was then pulled out and then extracted from the aorta, common iliac artery and bladder under the fluoroscopic control. Following the procedure, angiography indicated that there was no extravasation of contrast from the aorta and the left common and internal iliac artery (Fig. 5). Therefore, there was no need for embolization. The patient successfully underwent the procedure, and was then transferred back to the Clinic of Urology that same day. During hospitalization, the patient stated she felt well, she was haemodynamically stable, without visible signs of bleeding, and without reduced levels of hemoglobin. The patient was discharged from the Clinic of Urology on the fourth postoperative day with stable vital signs.
Discussion and conclusions
Hydronephrosis is frequently developed in patients suffering from cervical cancer, and is commonly associated with more advanced disease stages [1]. It occurs as a consequence of a tumor or lymph nodes growth, inflammation or fibrosis in pelvis, which results in pain occurrence, infection, or renal malfunction due to obstruction. Furthermore, it is considered to be an indicator of a bad prognosis in such patients [9].
There are several hydronephrosis treatment models for patients with cervical cancer and the placement of ureteral stents is considered the primary choice for the initial treatment of ureter obstruction [1,2]. The treatment of hydronephrosis can be performed with the insertion of PCNs in cases when the placement or replacement of ureteral stents is not possible or considered inefficient, such as in the case of advanced hydronephrosis, renal malfunction, infection or pain occurrence [3]. The studies conducted so far have shown that in 16-58% of patients with malignant ureteral obstruction, the placement of ureteral stents is unsuccessful [3,10]. In their study from 2004, Ku et al. have shown that hydronephrosis treatment outcomes, where ureteral stents or PCNs were used, were similar [11]. Results from a more recent study by Netsch et al. have shown that there is no difference in the survival rate and complication occurrence among patients with malignant ureteral obstruction who were treated by a ureteral stent placement and a PCN placement [12].
When deciding on the kind of hydronephrosis treatment, significant factors are the patient's and urologist's preferences [13,14], which in our case was the decisive factor, as both sides were for the PCN removal.
Ureteral stents have been used since 1967 in the treatment of ureteral obstruction caused by calculosis, stenosis, trauma or tumor compression, regardless of whether they are used as a temporary or permanent solution [15]. The placement of ureteral stents can be antegrade or retrograde. In this case, a ureteral catheter is placed using a retrograde approach with the aim of treating hydronephrosis caused by cervical cancer. One of the complications connected with the ureteral stent placement is calcification which can occur around the stent [16], which is exactly what happened in this case, and which demanded endolithotripsy of the calculus and a stent replacement. Furthermore, complications may include a stent misplacement and migration [16]. In the case of our patient, during the cystoscopic placement, the stent migrated from the left ureter to the left iliac artery and to the abdominal aorta. During the retrograde stent placement, an X-ray monitoring is advised, but in our case, as in the majority of such cases, an X-ray was not possible, therefore the control radiography was done after the procedure had been performed.
The removal of ureteral stents from the circulatory or vascular system requires a multidisciplinary approach, and today it can be performed with an open, laparoscopic or endovascular approach [5][6][7]. In the case of our patient, the ureteral stent migrated into the abdominal aorta thus penetrating the ureter wall as well as the wall of the iliac artery which were damaged and fragile because of radiotherapy, frequent stent replacements, calculosis and ureterorenoscopy. Additionally, the urologist did not strictly follow the procedure of the ureteral stent placement that should be followed up by an X-ray, and the choice of the ureteral stenting in this case of malignant obstruction is questionable.
Until now, several cases of ureteral stent migration into the vein blood vessels, right ventricle and atrium or pulmonary arteries have been described [5][6][7]17], but we have found only one case of stent migration into the abdominal aorta [18]. In that case, the stent migrated from the ureter which was damaged during laparoscopic hysterectomy. The stent was cystoscopically removed with the placement of endovascular catheters and X-ray monitoring. During the procedure, transcatheter superselective embolization of the inferior gluteal artery was performed, at the place where the ureteral stent entered the bloodstream. In our case, after the removal of the ureteral stent, there were no signs of contrast extravasation from the blood vessels, and thus there was no need for embolization. In order to prevent ureteral stent migrations, it should first be considered if there are any indications for the placement of the ureteral stent or PCN. When a urologist decides to perform a ureteral stent placement, and when the circumstances and conditions allow it, the procedure should be performed according to the existing guidelines. It is advised that the procedure be monitored by ultrasound or an X-ray. After the procedure, it is necessary to monitor the patient's symptoms, as the symptoms and other signs can indicate that there may be complications. Furthermore, it is necessary to perform a radiographic evaluation of the stent position following the intervention. In the case of a stent misplacement or migration, the patient should be immediately treated, and a stent migration into the vascular or circulatory system requires a multidisciplinary approach.
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v2
|
2022-11-19T15:04:54.536Z
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2022-11-18T00:00:00.000Z
|
253631404
|
s2orc/train
|
The experiences and perceptions of female breast cancer patients regarding weight management during and after treatment for oestrogen-receptor positive disease: a qualitative study
Background Weight gain is commonly observed during and after breast cancer treatment and is associated with poorer survival outcomes, notably in women with oestrogen-receptor positive disease. The aim of this qualitative study was to investigate the experiences and perceptions of oestrogen-receptor positive (ER +) female breast cancer patients (BCPs) regarding weight management behaviours during and after treatment. Secondly, to gain insight into the experiences of healthcare professionals (HCPs) regarding the provision of weight management advice to patients undergoing treatment. Methods Four focus groups involving 16 BCPs having a median (range) age of 51 (35–70 y) and three focus groups involving 21 HCPs aged 46 (29–62) were held at a university campus, local cancer support centre or clinical site. Data were analysed using Framework analysis. Results Four overarching themes (and 10 subthemes) were identified: (1) Treatment; (2) Support for lifestyle behaviour change; (3) Information availability for BCPs; (4) Knowledge of current evidence amongst HCPs. The physical and psychological consequences of treatment influenced motivation for weight management amongst BCPs. Social support for health promoting behaviours was viewed as important but was conflicting, requiring context-specific considerations. BCPs said they would have welcomed access to credible information (guided by HCPs) about the potential detrimental health effects of excess body weight and weight gain, together with advice on weight management via healthy eating and physical activity. HCPs felt that they had insufficient knowledge of public health dietary and physical activity recommendations or evidence-based interventions to confidently offer such advice. HCPs expressed concern that raising weight management issues would exacerbate distress or invoke feelings of guilt amongst BCPs, and cited time pressures on patient consultations as additional barriers to providing weight management support. Conclusion The study yielded novel insights into factors influencing weight management behaviours amongst overweight ER + BCPs. The results suggest that evidence-based information and support, which addresses key physical and psychological challenges to physical activity and dietary behaviours, offers the best route to sustainable weight management in this population. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-10238-7.
Background
Weight gain is commonly observed during and after breast cancer treatment due to chemotherapy and endocrine therapies, induced menopause, changes in metabolism and food intake and decreased physical activity [1,2]. Systematic review evidence shows that women who are overweight and obese at diagnosis, and those who gain weight, have poorer breast cancer survival outcomes than women of a healthy weight, irrespective of menopausal status [3]. Excess body weight after breast cancer also increases the risk of type 2 diabetes mellitus and cardiovascular disease [4,5].
The adverse impact of excess body weight on survival outcomes is clearly shown for women with oestrogenreceptor positive (ER +) breast cancer [2,6], which accounts for 70% of all breast cancer cases [7]. Higher body fat increases the risk of ER + recurrence because of increased aromatase activity and circulating levels of oestrogens and androgens [8]. This is compounded by other risk factors, including abnormal insulin and adipokine metabolism, impaired anti-tumour immunity and chronic low-grade systemic inflammation [2,9]. Although associations between intentional weight loss and survival outcomes after primary breast cancer treatment are not yet well established [10,11], the significant body of observational evidence pointing to the adverse impact of weight gain provides a strong rationale for the development of accessible and adoptable weight management interventions.
Previous qualitative studies have identified a range of important barriers to adopting and adhering to health promoting behaviours amongst breast cancer patients (BCPs). These include treatment-related physical symptoms which impede physical functioning (e.g., lymphoedema which restricts upper-limb range of motion), fatigue, pain, lack of confidence, body image concerns, fears about health behaviour change due to feelings of vulnerability, co-morbidities and conflicting priorities (e.g., work commitments, family caring duties, etc.) and low motivation [12][13][14][15]. In contrast, important facilitators have been identified as desire to lose weight [14], access to supervised exercise and dietary education, feeling a sense of control, peer support and having an opportunity to regain a sense of normality [16]. Women commonly experience deficits in the availability of clear and simple information on lifestyle, citing insufficient support from healthcare professionals (HCPs) [15]. Group-based interventions can provide an opportunity for peer-to-peer support as a means of addressing barriers to health-promoting behaviours and building the skills and confidence needed for dietary and physical activity behaviour change. Successful group-based weight-loss interventions in breast cancer patients have used a variety of delivery formats, including face-to-face workshops for 8-15 women alongside remote support methods such as telephone, emails, text-messaging and printed mail-outs [17][18][19][20][21].
In the UK, support for health behaviour change after primary treatment for breast cancer is limited to that provided by prominent cancer charities. For example, Breast Cancer Now offer an on-line course and book/ printed materials ("Moving Forward") to help women adjust to life after breast cancer treatment. This takes place over half a day for three or four weeks and aims to provide information, support and professional guidance on how to cope with and adjust to life after breast cancer treatment [22]. Macmillan Cancer Support offer the Recovery Package after completion of primary treatment comprising a Holistic Needs Assessment, treatment and cancer care reviews with a HCP and an education/support event such as a Health and Wellbeing Clinic [23]. There remains a gap however, in longer-term provision of tailored (bespoke) lifestyle support, specifically designed to address the barriers to effective weight loss that many women experience after primary treatment for breast cancer. This means that offering a route to accessible and adoptable weight management support would address an important unmet need for women and their treating clinicians at what is frequently an opportune 'teachable moment' for patients [24].
Understanding and addressing the challenges women face adopting and adhering to healthy lifestyle behaviours during and after breast cancer treatment from the perspective of both BCPs and HCPs is an important first step in designing effective and sustainable weight loss interventions for this population. Current guidelines for cancer survivors recommend achieving/maintaining a healthy body weight, engaging in regular physical activity and eating a healthy dietary pattern that is high in vegetables, fruits and whole grains and low in calorific foods and beverages, processed meats and alcohol [25][26][27]. However, recent survey evidence showed that while a high proportion of breast cancer patients reported at least one positive nutrition or physical activity behaviour after diagnosis or treatment, several treatment-related barriers (fatigue, stress, changes in appetite/taste disturbances, pain and discomfort) impeded the adoption of these health behaviours [28]. Furthermore, a recently published American College of Clinical Oncology Guideline paper emphasised the need for more research into diet and weight management strategies for people undergoing cancer treatments [29]. Thus, the aim of this qualitative study was to investigate the experiences and perceptions of overweight ER + BCPs regarding weight management (healthy dietary behaviours and physical activity) during and after primary treatment and ongoing hormone therapy. Secondly, to gain insight into the experiences of HCPs regarding the provision of weight management advice to BCPs undergoing treatment for ER + disease.
Participant recruitment
A purposive sample of female breast cancer patients attending routine follow-up clinical visits were invited to participate in the study by members of their clinical team or a research nurse. Women were eligible to participate in the study if they were over 18 years of age and were more than eight weeks since completion of chemotherapy (providing time to reflect on their experience of adjuvant treatment) and less than 36 months since completion of primary treatment for ER + breast cancer (for accurate recall of experiences), with a BMI ≥ 25 kg/m 2 . Women being prescribed hormone therapies were eligible. HCPs from five UK National Health Service (NHS) Trusts were recruited by the lead investigators. Emails containing details of the study were sent to HCPs involved in the care of breast cancer patients and they were invited to respond to the research team for further information.
Procedures and data collection
Our underlying philosophy was constructivist [30], recognising the individual nature of experience and the impact of BCPs' wider life experiences on their perspectives of weight management behaviours. Four focus groups involving 16 BCPs and three focus groups involving 21 HCPs were held at a university campus, a local cancer support centre or at a clinical site between December 2018 and January 2019. Each session lasted between 70 and 89 min (mean = 81 min). Initially, two HCP focus groups took place, however, due to emerging qualitative data highlighting the importance of appropriate and safe lymphoedema management, an additional focus group was arranged with lymphoedema practitioners. The focus groups were led by an experienced female qualitative researcher (either SW, a physiotherapist or KP, a physical activity public health expert, both were qualified in exercise prescription for cancer patients), with assistance from at least one other member of the research team (HC, SW, KP or JS), and all sessions were audiorecorded. Topic guides were developed on the basis of study objectives, previous literature and knowledge of important issues for BCPs amongst members of the research team gleaned from previous research and clinical practice. Topic guides explored barriers and facilitators to weight management (healthy eating and physical activity) from the perspectives of BCPs and HCPs during and after ER + breast cancer treatment (Table 1). Participant characteristics are presented in Table 2. All participants provided written, informed consent prior to data collection and the study was approved by the Northwest Preston NHS Research Ethics Committee (18/NW/0400). The research was conducted and reported in accordance with qualitative research reporting guidelines [31].
Data analysis
Audio-recordings were transcribed anonymously, verbatim. Two researchers independently analysed the data using framework analysis [32], comprising: (i) familiarisation with the data; (ii) identification of a thematic framework (informed by a combination of a priori topics and emergent issues); (iii) indexing (i.e. applying framework codes to the data); (iv) charting (i.e. summarising indexed data); and (v) mapping and interpreting the data (assessing meaning, differences and similarities etc.) [33]. Transcripts were initially analysed and charted in relation to the a priori topics that guided focus group discussions, with the BCP and HCP focus groups coded into this framework separately. Commonalities and differences between the views and experiences of BCPs and HCPs were used to create overarching themes and subthemes, before the data from the two participant groups were coded in relation to one another. NVivo (version 11; QSR International, Melbourne, Australia) was used to organise the data. At each stage of the process the guiding framework was modified in relation to the emergent content within the data. The researchers liaised at several points to discuss data saturation, which was confirmed during thematic analysis when no new codes, categories or themes emerged from the data. The coding framework represented all relevant data, and there was a high level of agreement between analysers. The emergent themes were discussed and the final themes refined. This approach to data analysis was somewhat deductive, framing the analysis within the a priori topic guide, yet the data were borne out of original transcripts from interviews and focus groups [34].
Results
Four overarching themes (and 10 subthemes) were identified: (1) Treatment; (2) Support for lifestyle behaviour change; (3) Information availability for BCPs; (4) Knowledge of current evidence amongst HCPs. Example quotes from BCPs and HCPs are used to illustrate each theme (and subtheme), with the latter also including a gender identifier in parentheses (F or M). Lymphoedema practitioners (all female) are denoted by the letter L. A more complete list of quotes to illustrate each theme (and subtheme) is presented in the Supplementary Table.
Theme 1: treatment
The physical and psychological impacts of treatment emerged as an important theme negatively influencing motivation for weight management behaviours amongst BCPs. BCPs and HCPs considered the medications prescribed during treatment, the psychological impact of a cancer diagnosis and altered dietary patterns due to taste disturbances, altered food choices, comfort eating and the perceived convenience of, or craving for, less healthy foods (e.g., sugary foods) to be important factors impeding motivation for healthy lifestyle behaviours.
Side-effects of treatment
Several side-effects of breast cancer treatment have the potential to influence weight gain. BCPs talked about how their side-effects impacted their activity levels and diet. HCPs said they make patients aware of the side-effects of hormone therapies during patient consultations and these messages are reinforced via printed literature. Some HCPs provided very general advice on how to counteract common side-effects, such as loss of bone mineral density.
HCP(M)16: And I think for those of us who are initiating certainly aromatase inhibitors, we will of necessity warn them of the risk of osteoporosis... and as part of that would be to say the most useful thing you can do to avoid it is to be as active as possible. So, we would say that.
Pre-treatment expectations
BCPs were under the impression that they would lose weight during their treatment and were surprised that they gained weight (all reported gaining weight during treatment).
BCP4: I had a misconception that I was going to lose weight on chemotherapy, and it was exactly the opposite.
Prioritisation of treatment
The adoption of healthy lifestyle behaviours for weight management was regarded as low priority during treatment because of the physical and emotional strain of the breast cancer diagnosis and disruption to normal routines imposed by the treatment.
BCP2: I put on two stone during treatment… part of that was the steroids, but part of that was the oh shit I'm going to die so I might as well eat cake, because why wouldn't you do that? HCP(F)1: Their normal pattern goes out of the window, and they're just trying to get through the treatments, and diet isn't their priority, nor exercise.
Theme 2: support for lifestyle behaviour change
Support for lifestyle behaviour change from HCPs along the breast cancer treatment pathway was perceived to be minimal amongst BCPs. For some BCPs, motivation to engage with weight management behaviours (particularly physical activity) was strongly influenced by the support of significant others, including their peer-group (other BCPs). However, for some BCPs, friends and family tended to be over-protective, and others wanted to 'move on' from their cancer experience which made them reluctant to seek support from their peer-group.
Support within the clinical pathway
There was a perceived lack of support from HCPs, and contrary to promoting healthy lifestyle behaviours, there were some instances of HCPs encouraging BCPs to do the opposite.
HCP(F)2: [Towards the end of treatment]… their head is moving into a different place… I would say there's definitely a shift in terms of the questions that they're asking, and what they are open to receiving in terms of information.
Another major barrier to providing lifestyle support was the time constraints imposed on clinical appointments.
HCP(F)9: I think to be honest it's time constraints. I mean with the best will in the world it would be lovely to sit down with everybody for half an hour or so and chat to them, but you don't have that. You don't have time to do that if you're doing everything else.
Suggestions for overcoming the barrier imposed by time constraints, included enabling all HCPs along the care pathway to apply consistent messages via a "brief intervention" approach and/or sign-posting/referring patients onto community services, but with some reservations concerning the latter as such services can often be short-lived.
Support from family and friends
BCPs who reported having family and friends that provided verbal encouragement (e.g., be more active, eat healthily, etc.), were more likely to adopt healthy lifestyle behaviours during and after primary treatment. Other BCPs recounted experiences in which family and friends had discouraged them from engaging in healthy lifestyle behaviours, feeling that they had been through too much to be concerned with healthy eating and or the need for regular exercise and/or that it might be detrimental, and this negatively affected their motivation for health behaviour change.
Peer-support
Several BCPs expressed the importance of peer-support in motivating them to adopt healthy lifestyle behaviours and in their motivation for attending support sessions with similar others who they could share experiences with.
BCP16: Everybody in the room then is feeling what you're feeling and everybody will come up with a question and be comfortable to expand on that, because you're all in a similar position and you can all relate to it positively, can't you?
For some BCPs, it was more important to move on from their cancer experience and they did not want the constant reminder of what they had been through by attending support groups or exercise classes with other patients.
Theme 3: information availability for BCPs
BCPs felt poorly supported, in terms of the information they received about the potential impact of healthy lifestyle behaviours on treatment outcome and their general health and wellbeing as they progressed through the treatment pathway. This meant that if they had the motivation to adopt healthy lifestyle behaviours, they were unsure about what to do for the best and the onus was often on them to seek relevant information from other sources.
Patient receptiveness to information
BCPs were acutely aware of the lack of support for lifestyle behaviour change during the breast cancer treatment pathway. Some BCPs said they would have been very receptive to information (guided by HCPs) about the broader health impacts of breast cancer treatment, as well as advice on weight management via healthy eating and physical activity. The need for tailored advice and information to meet the specific needs of patients was also highlighted.
The need for credible information
BCPs felt that credible information sources were essential due to the huge volume of conflicting information available on the internet that can get very confusing and may have a dubious evidence-base. In addition, many 'myths' or outdated information continue to circulate, such as this example from a BCP: BCP9: Every time you take a hit of sugar you suppress your immune system. So potentially you're… not giving [your body] a fighting chance really. You're suppressing that natural immunity all the time.
Some BCPs said they were given information from HCPs in the form of leaflets but were left to interpret that information themselves and often resorted to looking up information on the internet. Some of the advice received from HCPs was perceived as vague: BCP15: I was told just eat a healthy diet and, yeah, not to really change anything particularly. So, it almost felt a bit vague, I felt like they weren't really sure when I asked them the questions.
Theme 4: knowledge of current evidence amongst HCPs
Many HCPs said they had insufficient knowledge of the most recent scientific dietary and physical activity evidence and public health guidance to confidently discuss such matters with BCPs and would only be able to provide general advice if asked.
Knowledge-gap
Many HCPs discussed how they were unfamiliar with current evidence in support of the benefits of healthy eating and regular exercise for BCPs and this was clearly evident amongst BCPs.
BCP11: I want to try and do a healthy diet and way of living, and keep this cancer in remission. What's the best thing that I can do? And that's where I don't seem to be getting a lot of information back as to what should I actually be eating?
Furthermore, BCPs lacked confidence in the ability of exercise and fitness professionals to provide breast cancer-specific advice. The lack of evidence-based guidance underpinned a sense of nervousness and generated fears about engaging or re-engaging with exercise programmes amongst BCPs.
BCP2: This is why, I mean this is one of the reasons why I haven't gone back fully to exercising and doing what I want to do, because I'm so paranoid, I have a sleeve, I'm so paranoid about what it's going to do to my arm.
In contrast, the lymphoedema practitioners said that they use their evidence-based knowledge to empower women to be able to self-manage their lymphoedema through exercises that have been shown to be effective. They had a much better grasp of research evidence supporting the important role that exercise and weight management play in lymphoedema management. They were also more proactive in promoting this evidence to their patients, feeling it was fundamental to their role and essential for patient care. This group of HCPs were part of a specialist lymphoedema clinic (private company) commissioned to deliver the local NHS lymphoedema service for cancer and other patients. The lead nurse is an advocate of exercise and weight management and empowers her team to this end via training opportunities.
Experiential knowledge gained
BCPs and HCPs recounted how they had gained some level of experiential knowledge about the health benefits of weight loss and regular exercise on important health outcomes following a breast cancer diagnosis. This was a source of positive motivation for some HCPs to provide lifestyle advice and for some BCPs to engage in more healthy, active lifestyles.
BCP4: I was confused for a while after treatment. I'd put on so much weight, and I was having all these joint pains and couldn't bend over to put my shoes on without not being able to breathe... But then once I got back down below a certain weight, those things went away.
Discussion
This qualitative study explored the experiences and perceptions of ER + BCPs and HCPs regarding weight management behaviours during and after treatment and the provision of weight management advice as part of the care pathway. During treatment, women recounted how concerns about weight management were overshadowed by the physical and emotional strain of their diagnosis and disruption to normal routines caused by hospital appointments. However, as they emerged from their primary treatment, physical changes such as increased body weight, change in body shape, shoulder mobility issues and a lack of knowledge and/or confidence to engage in healthy lifestyle behaviours (particularly physical activity and structured exercise) were important barriers to health behaviour change, consistent with previous evidence [12][13][14][15].
Studies suggest that a cancer diagnosis can act as a "teachable moment", prompting women to adopt healthier lifestyle behaviours after a breast cancer diagnosis and/or treatment [24,35]. However, conflicting evidence exists and the possibility of study selection bias (study samples being biased towards cancer survivors who have changed their behaviours) also needs to be taken into account [24]. Although recent evidence of modest improvements in dietary and physical activity behaviours within three years of a breast cancer diagnosis provides support for the "teachable moment" [36], improvements may not be maintained over the longer-term [37]. The low priority participants gave to weight management behaviours during treatment resonates with qualitative data from other studies [38,39] but contrasting evidence suggests that long-standing, deep-routed concerns about excess body weight can overshadow the emotional stress of a breast cancer diagnosis in some women [40]. This demonstrates the complex interaction between the emotional consequences of a breast cancer diagnosis and existing weight management concerns. Furthermore, it emphasizes the need for weight management advice to be timely and cognizant of the balance between emotional distress and existing body weight sensitivities if the "teachable moment" is to be capitalized on by HCPs [12]. Evidence from previous studies suggests that the optimal time to implement weight management support is after primary treatment for the majority of BCPs [41,42]. This is consistent with the views expressed by HCPs in the present study and the feeling amongst BCPs that they "drop off the edge of the earth" after primary adjuvant treatment and are left to their own devices to improve their lifestyles.
Our results also show that the physical and emotional barriers impeding the adoption of weight management behaviours in BCPs can be compounded by a lack of awareness, regarding the likelihood of weight gain during breast cancer treatment. Weight management in cancer patients has been dominated by concerns about unintentional weight loss secondary to treatment or progressive disease [11] and is likely to have influenced pre-treatment weigh loss expectations amongst BCPs. This could be an important barrier to the adoption of healthy lifestyle behaviours by precluding the need to seek weight management advice. In addition, women felt ill-informed and poorly supported and expressed their nervousness and fears about engaging in physical activity or structured exercise. This was due to a lack of evidence-based knowledge and guidance regarding the types of exercise it is safe to engage in, without exacerbating common sideeffects, such as fatigue and lymphoedema, as reported previously [43]. There were also concerns that nutrition and exercise professionals may have insufficient experience of breast cancer and are therefore unable to provide the support that BCPs need to overcome the physical and emotional challenges experienced during recovery from primary treatment. Consistent with previous evidence [12,43], BCPs also spoke about the conflicting information that is available through social media channels. This lack of support, in conjunction with personal sensitivities related to weight gain and other bodily changes (e.g., change in body shape, impaired shoulder mobility, etc.), underscored their lack of confidence for health behaviour change. Consequently, the need for credible, evidencebased advice and support from competent HCPs as a means of rebuilding confidence and motivation for physical activity and healthy eating, emerged as a key theme in the analysis, consistent with previous research [12,44].
These findings highlight a training gap for HCPs, which is needed to build the required competences for the provision of evidence-based (and bespoke) weight management support to addresses the health behaviour change challenges faced by BCPs. This training and associated support programmes for BCPs should aim to develop evidence-based knowledge, while drawing on key tenets of health behaviour change theory. For example, according to the Health Belief Model, individuals are likely to adopt health behaviours when they perceive their susceptibility to an illness and its seriousness and believe that the benefits of behaviour change outweigh the perceived barriers [45]. Thus, tailored (and timely) educational support for improving knowledge of the adverse effects of weight gain and benefits to be gained from healthy dietary choices and physical activity could tip the balance in favour of behaviour change [46]. Strategies aimed at developing perceived competence for weight management behaviours, while being mindful of flexible options to help meet individual needs and preferences (facilitating autonomy), is also consistent with intrinsically motivated behaviour change according to Self-Determination Theory [47]. Furthermore, establishing a platform for frequent peer-to-peer contact and support would help to fulfil the need for relatedness (sense of belonging to a social group) in self-determined behaviour change [47]. Having the opportunity to share experiences with "similar others" is consistent with previous research, in which empathy received from women perceived to be "in the same boat" and "same as you" helped BCPs to move on from feeling isolated to feeling accepted, while also providing subtle motivational peer-pressure for health behaviour change [14,16]. However, it is also important to note that some BCPs wanted to move on from their cancer experience and felt that this would not be served by attending weight management support groups with other patients.
The perceived role of family and friends in supporting health behaviour change was more complex, with recollections of conflicting advice and support given. While evidence suggests that support from family and friends can be a powerful motivational factor for health behaviour change in cancer patients [48], its absence and/or active discouragement from significant others (linked with "over-protection") has been identified as a barrier to participation in physically active lifestyles in cancer patients [38,46]. The motivation for health behaviour change amongst BCPs in the present study was influenced either in a positive or negative way, depending on the encouragement or discouragement received from family and friends during and after primary treatment. This shows that social support for health behaviour change is a complex issue for BCPs, requiring context-specific considerations. Furthermore, these findings suggest that support programmes which include family and friends in endeavours to change the health behaviours of BCPs could be beneficial [39].
Many HCPs (with the exception of the lymphoedema practitioners) said they had insufficient knowledge of upto-date scientific dietary and physical activity evidence to confidently discuss such matters with BCPs and would only be able to provide general advice if asked. This perceived lack of evidence-based knowledge amongst HCPs is well-reported and highlights a potential training gap [49,50]. Furthermore, because of HCP concerns about exposing patient sensitivities during weight management conversations [49,51], interventions aimed at redressing this training gap should consider how such advice could be sensitively provided. Time constraints imposed on clinical appointments were identified as another barrier to such provision with NHS cancer care pathways, in accordance with previous findings [49], which limits delivery options to brief consultant-led conversations within the care pathway. Evidence suggests there is potential to increase the number of brief intervention consultations with appropriate training [52], consistent with the Making Every Contact Count (MECC) initiative [53], but this approach is likely to have minimal impact [54,55]. Alternatively, having the opportunity to signpost/refer women onto credible and motivational weight management information and support after primary treatment was viewed positively by HCPs, as in other studies [38,42], and could help to overcome the challenge of time constraints during consultations. For these reasons, a bespoke programme of evidence-based weight management support that dovetails with the NHS breast cancer care pathway could be the optimal solution for promoting health behaviour change and developing the skills and confidence women need for effective and sustainable weight loss.
Strengths and limitations
Participants attending the HCP focus groups came from diverse backgrounds and most professions involved in breast cancer care, including specialist nurses, oncologists and lymphoedema practitioners, enabling a broad range of relevant views. Likewise, the wide age-range of overweight BCPs involved in the focus groups (35-70 years) and varied weight management experiences allowed for a diverse representation of views. Emerging data from the BCP and HCP focus groups regarding questions and concerns around diet, physical activity and lymphoedema, dictated that further specialist input from lymphoedema practitioners was required. An additional focus group with the latter ensured that data saturation had been reached regarding all relevant topics raised by BCPs and HCPs. A potential limitation of this research was the exclusive use of focus groups as a means of understanding the perceptions of BCPs and HCPs regarding weight management support. Focus groups might be less effective for gleaning detailed information than individual interviews but capitalise on group dynamics to stimulate discussion. In the focus group sessions, new topics that may have not been explored otherwise were frequently opened or expanded upon following comments from other members of the group. However, it may have been difficult for some participants to fully express their views in the focus group setting and a mixed qualitative approach might have yielded more indepth data.
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v2
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2022-11-19T15:05:06.938Z
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2022-11-18T00:00:00.000Z
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253630984
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s2orc/train
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The effect of preoperative biliary drainage on postoperative complications of pancreaticoduodenectomy: a triple center retrospective study
Background Biliary obstruction which is a major complication of pancreas and periampullary tumors could result in cholangitis, coagulopathies, gastrointestinal symptoms, and impaired wound healing. Pancreaticoduodenectomy (PD) is still the standard approach for pancreas resection and imposes high risk of morbidity and mortality to patients. To reduce the high risk of PD and address the biliary obstruction, the use of preoperative biliary stenting was increased. However, available literature doubts its efficiency. Methods A total of 147 patients who underwent PD between September 2012, and February 2022, at three medical centers were identified. Patients were grouped based on biliary stent placement. Non-jaundiced patients with and without preoperative biliary drainage (PBD) were compared. Results The incidence of overall complications (34.2% versus 45.8%) and mortality (17.8% versus 24.3%) did not differ in the PBD group compared to the no PBD group. There was no difference in complications and mortality in non-jaundiced patients with and without PBD. Patients with drainage duration of > 30 days experienced more overall complications compared to patients with less than 30 days drainage duration (12 (50.0%) and three (15.8%) patients, respectively, p-value = 0.019). Conclusions PBD does not significantly increase the post-operative burden on patients who undergo PD. However, we cannot overlook the financial burden that PBD places on the patient and the healthcare system, as well as the difficulties related to endoscopic retrograde cholangiopancreatography (ERCP). Therefore, biliary stenting should not be routinely practiced in the absence of a valid indication, such as severe jaundice, pruritus, cholangitis, delayed surgery for neoadjuvant treatment, or referral to a tertiary facility.
Introduction
One of pancreatic and periampullary tumors' main complications is biliary obstruction [1], which could result in cholangitis, coagulopathies, and gastrointestinal symptoms. Cholestasis precipitates bacterial growth within the bile. Bacteria enter circulation after elevated biliary pressure have damaged the hepatic cell and bile microduct Open Access *Correspondence: [email protected] barrier. Ensuing reduction in hepatic blood circulation impairs liver metabolic and synthetic function, which in turn results in a chain of events including oxidative stress, decreased plasma albumin, impaired coagulation cascade, and immune system disturbances. Due to the bile salts deficiency, gut normal flora grows excessively, and subsequent intestinal mucosal barrier disruption causes bacterial translocation, and increased endotoxin concentration which ultimately impairs wound healing [2].
Pancreaticoduodenectomy (PD) which is the standard procedure for resecting pancreatic and periampullary tumors, is associated with high mortality and morbidity [3,4]. According to the above-mentioned reasons, hyperbilirubinemia was hypothesized to be a determining factor in PD outcome. Thus, it was believed that addressing biliary obstruction with preoperative biliary drainage (PBD) would reduce the post-operative complications [5].
In the initial studies, performing PBD yielded promising results. PBD can be accomplished by biliary stenting using endoscopic retrograde cholangiopancreatography (ERCP) or by placement of a percutaneous transhepatic catheter (PTC) [6]. However, given the preoperative complications of PBD, the benefits of this method have been doubted. Throughout the last decade numerous studies have published to address the diversity of outcomes and to reach an agreement. Limited studies have stated favorable effects of PBD on postop outcome in selected patients [7][8][9], while others report equivalent or even adverse effects of PBD on postoperative complications [10][11][12]. Latest guidelines suggest selective use of PBD in the following circumstances: cholangitis, neoadjuvant therapy, delayed surgery, and bilirubin level of ≥ 15 mg/ dL [13].
Recent studies are coming to an agreement that performing PBD neither improves nor harms the outcome of PD. However, many studies are single-centered, and there is limited evidence from developing countries where patients have difficulty accessing a pancreatic surgeon. Furthermore, decisive variables, such as duration between onset of symptoms and operation, and drainage duration, have not been fully addressed. We have conducted this study to report the result of PBD in an Iranian population.
The participants were identified by extensive review of medical records.
Inclusion criteria were patients undergoing PD with periampullary neoplasm or other benign pathologies. Exclusion criteria were combination of PD with other surgeries and placement of PTC prior to surgery. One patient underwent simultaneous PD and liver transplant, and 23 patients with PTC placement were excluded from the study.
To reduce the effect of biliary obstruction, we further divided the study population into two groups based on biliary obstruction and then assessed variables within the obstructed patients. Biliary obstruction was defined as a subjective report of jaundice prior to admission or a total bilirubin level of > 2 mg/dL. Then patients were grouped based on their PBD situation. Patients who received preoperative biliary stenting were referred to as the PBD group.
Jaundice management
Most of the patients underwent ERCP and stent placement as part of a gastroenterology assessment before referral to a pancreatic surgeon. Stent size and type (metallic or plastic) were decided by the gastroenterologist. In the event of unsuccessful biliary stenting, PTC was employed. Stent exchange was performed either due to stent disfunction or cholangitis.
Operation
During the study period, PDs were performed by 12 surgeons who used similar techniques. Prophylactic intravenous antibiotics were administered prior to surgery. Based on the surgeon's opinion either a classic or a pylorus-preserving Whipple was performed. Following resection, pancreaticojejunostomy, choledochojejunostomy, and gastrojejunostomy were performed sequentially. At the end of the procedure surgical drains were placed. Patients were admitted to the intensive care unit (ICU) for at least 24 h following surgery and were given prophylactic antibiotics.
Variables
Data of patients' demographic, symptoms, past medical history, and the American Society of Anesthesiologists (ASA) score were extracted from medical records. Preoperative assessments included preoperative laboratory values within a week prior to surgery, and pathology evaluation. Furthermore, operation duration, estimated blood loss, and postoperative complications were collected.
A weight loss of > 10% in six months was documented. Drainage duration was computed from the date of initial placement until the surgery date. Length of hospital stay was considered the hospitalization days after the surgery.
Surgery duration was calculated from the start of anesthesia until the dressing of the surgical wound. Postoperative complications within 30 days after surgery or during hospitalization were recorded. Primary surgical complications were defined as the occurrence of severe postoperative complications (Clavien-Dindo ≥ 3) [15]. Secondary surgical Complications included delayed gastric emptying (DGE), postoperative hemorrhage, postoperative pancreatic fistula (POPF), intraabdominal abscess, and wound infection. Presence of grade C DGE [16], severe postoperative hemorrhage [17], and grade C POPF [18] according to the International Study Group of Pancreatic Society (ISGPS) criteria, were extracted from the medical records. Overall complications were defined as the sum of all complications. Mortality was defined as deaths within 90 days after surgery.
Ethics
Research Medical Ethics Committee of Shahid Beheshti university of medical sciences reviewed and approved this study (approval number: IR.SBMU.MSP.REC.1398.1008). This study was conducted in accordance with the Declaration of Helsinki and institutional ethics guidelines. For the use of clinical data, written informed consent was obtained from the patients.
Statistical analysis
Hospitalization was reported as median and interquartile range (IQR). Other continuous variables were presented as mean ± standard deviation (SD). Categorical variables were shown as count (percentage). Kolmogorov-Smirnov test was applied to evaluate continuous variables for normal distribution. Comparisons of parametric and nonparametric continuous variables were executed using Student t-test and Mann-Whitney U test, respectively. Categorical variables were analyzed using Chi-square or Fisher's exact test. Also, the effect of different risk factors on overall morbidity and mortality was evaluated using univariate and multivariate analyses. Risk factors with a p-value of less than 0.1 in the univariate analysis, were used for multivariate regression modeling. Data were analyzed with SPSS version 26 (IBM, Armonk, NY, USA). A p-value of < 0.05 was considered statistically significant.
Results
Overall, 147 patients were included in the study with mean age of 57.4 ± 12.3 (range 20-87, median = 58.0) years. Ninety-six (65.3%) patients were male. The most prevalent symptoms were jaundice, abdominal pain, and weight loss, respectively. Eight patients undergoing PBD experienced fever, and two of them developed cholangitis prior surgery. Hypertension and diabetes were most prevalent comorbidities. Detailed description of patients characteristics is shown in Table1.
All patients
Concerning patient demographics, the only significant differences between PBD and no PBD groups were prevalence of jaundice (p-value < 0.001) and fever (p-value = 0.03) before surgery. Also, no PBD group had significantly higher levels of WBC (p-value = 0.02), bilirubin (p-value < 0.001), and alkaline phosphatase (p-value < 0.001) compared to PBD group.
Patients with biliary obstruction
After excluding non-obstructed patients, there was no significant difference in symptoms and medical history between PBD and no PBD groups. the mean duration of symptoms was considerably longer in PBD group (136 ± 149 versus 67 ± 50). Patients undergoing PBD had lower total bilirubin and alkaline phosphatase levels (p-value < 0.001) before surgery (Table2).
Postoperative outcome
Overall postoperative morbidity and mortality was 40.7% and 21.1%, respectively. Primary surgical complications rate was 15.0% and wound infection was the most common secondary surgical complication with 14.8% incidence. Other common complications were hemorrhage, intraabdominal abscess, POPF, and DGE, respectively.
There was no difference in post-operative morbidity and mortality between PBD and no PBD group (Table 3).
Postoperative outcome in patients with biliary obstruction
Post-operative morbidity and mortality were similar between PBD and no PBD group in patients with biliary obstruction (Table 4).
PBD subgroup analysis
In the PBD group, bilirubin level of > 10 mg/dl was associated with higher secondary surgical complications (75.0% versus 21.0%, p-value = 0.041). In the no PBD group, mortality rate was higher in patients with a bilirubin level of greater than 15 mg/dl (40.0% versus 11.1%, p-value = 0.018). Drainage duration was not associated with increase in primary surgical complications, wound infection, hemorrhage, intraabdominal abscess, POPF, DGE, mortality, or hospitalization (p-value > 0.05 m). However, patients with drainage duration of > 30 days significantly experienced more overall complications compared to patients with less than 30 days drainage duration (12 (50.0%) versus three (15.8%) patients, respectively, p-value = 0.019).
Patients with plastic and metallic stent did not differ in any complications, mortality, or hospitalization (p-value > 0.05). Patients with stent exchange were comparable to patient without exchange regarding complications, mortality, and hospitalization (p-value > 0.05).
Univariate and multivariate analyses in all patients
Univariate and multivariate analyses were conducted to detect independent predictors of outcomes in all patients (Table 5). In univariate analysis, total bilirubin, estimated blood loss and surgery duration affected overall morbidity; however, these variables did not significantly increase or decrease the probability of overall morbidity. In addition, no predictors were identifiable in the multivariate analysis of overall morbidity. In univariate and multivariate analyses of mortality, no risk factors were identified.
Univariate and multivariate analyses in patients with biliary obstruction
Univariate and multivariate analyses were carried out to identify independent predictors of outcomes in obstructed patients (Table 6). Although stent placement, nausea and vomiting, total bilirubin, estimated blood loss and surgery duration were selected for multivariate analysis of overall morbidity, these risk factors did not significantly change the possibility of overall morbidity. In univariate and multivariate analyses of morbidity, no risk factor significantly impacted the outcomes.
Discussion
Obstructive, painless jaundice is still the most typical scenario of periampullary malignancies. Before being referred to a pancreatic surgeon, most patients had already undergone biliary stenting during an upper endoscopy as part of a malignancy workup. Attempts to alleviate blockage with regular preoperative biliary drainage (PBD) have failed to show an advantage in patient outcomes, although previous research had previously revealed that impaired hepatic function and nutritional state are induced by cholestasis.
In this retrospective study, the incidence of postoperative complications of PD was compared between patients receiving endoscopic retrograde biliary drainage (ERBD) and patients without PBD. Regarding postoperative complications, there was no association between stent placement and incidence of wound infection, hemorrhage, intraabdominal abscess, POPF, and DGE. Primary, secondary, and overall surgical complications, as well as mortality, were not significantly different between ERBD and no PBD groups. Despite a growing body of evidence showing no advantage for routine PBD, in practice, PBD continues to be widely performed. Several studies have even shown the inferiority of PBD compared to surgery first approach. Fang et al. in a meta-analysis study, showed that the relative risk of overall complications is higher in the ERBD group compared to people without ERBD (rate ratio 1.66; 95% confidence interval (CI):1.28-2.16; p-value = 0.001) [19]. Similarly, PBD using stent placement was associated with an increased risk of overall complications compared with immediate surgery in a meta-analysis conducted by Scheufele et al. (odds ratio 1.40; CI: 1.14-1.72; p-value = 0.002) [20]. However, in our study, the incidence of overall complications was not significantly different in the ERBD group compared to the no PBD group (34.2% versus 45.8%).
Interestingly, in more updated studies, complications tend to be milder and limited to wound infection. [22]. Additionally, some studies report similar results regarding wound infection [20,[23][24][25]. Nevertheless, in our study, there was no difference in the incidence of wound infection between groups. Regarding postoperative hemorrhage, intraabdominal abscess, POPF, and DGE, our results were similar to a retrospective study conducted at Massachusetts General Hospital, showing no considerable difference between patients who received ERBD and no PBD patients [23]. Although some studies have reported an increase in postoperative DGE [24,26], we did not observe any significant difference between ERBD and no PBD groups.
We did not found any difference in postoperative hospitalization between PBD and no PBD groups (median of 12 and 12 days, respectively), which is comparable to the results of Webra et al. [22]. However, one study has reported a shorter length of hospital stay in patients receiving biliary stating [21], and conversely, a randomized trial by van der Gaag et al. demonstrated significantly longer hospitalization in the PBD group [25].
Although operation duration was associated with an increased risk of developing wound infection, intraabdominal abscess, DGE, secondary surgical complications, and overall complications, stent placement did not significantly affect surgery duration.
Based on our study, severe hyperbilirubinemia (bilirubin > 15 mg/dl), regardless of stent placement, was associated with higher overall complications. Furthermore, our study highlights the risk of secondary surgical complications in patients with bilirubin level of > 10 mg/dl in the PBD group due to stent failure. Consistently, in previous studies a bilirubin level of greater than 7.5-15 mg/ dl is reported to be associated with higher rates of post operative complications [1,[27][28][29]. Interestingly a recent meta-analysis revealed that in bilirubin level of > 15 mg/ dl, surgery outcomes do not differ in the PBD and no PBD groups [30].
Based on our observation, patients with drainage duration of more than 30 days experienced more overall complications. However, a retrospective study on 304 patients by Scheufele et al. showed that patients with drainage duration of > 4 weeks and < 4 weeks did not differ in survival [31].
Our study did not identify any difference in surgery outcomes between metallic or plastic stent use. This is comparable with the results of a recent meta-analysis by Watanabe et al., which demonstrated no significant difference in stent-related (risk ratio 0.74; CI: 0.32-1.71) and postoperative complications (risk ratio 0.73; CI: 0.45-1.17) [32]. However, the results of a prospective study demonstrated that PBD-related complication rates were higher in patients receiving plastic stents compared to the metallic stent (46% versus 24%) (relative risk of plastic stent use 1.9, CI: 1.1-3.2; p-value = 0.011) [33]. Moreover, in a network meta-analysis, Lee et al. revealed that metallic stents have fewer stent-related complications than plastic stents. Whereas the postoperative outcomes were comparable in both groups (odds ratio 0.99; CI:0.65-1.49; p-value > 0.05) [34].
The PBD group had a higher prevalence of ampullary and pancreatic tumors, whereas the no PBD group had a higher prevalence of neuroendocrine tumors. The anatomical origins of neuroendocrine tumors and the fact that they are less prone to result in obstructive jaundice might be the reasons for this variation.
The current study had some limitations, including its retrospective nature, limited capacity to detect mild outcomes, lack of access to the patients' medical records prior to stent placement, and inability to diagnose POPF at its initial stage.
Conclusions
In conclusion, PBD does not significantly increase the post-operative burden on patients who undergo PD. However, we cannot overlook the financial burden that PBD places on the patient and the healthcare system, as well as the difficulties related to ERCP. Therefore, biliary stenting should not be routinely practiced in the absence of a valid indication, such as severe jaundice, pruritus, cholangitis, delayed surgery for neoadjuvant treatment, or referral to a tertiary facility.
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2022-11-19T15:25:23.665Z
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2022-11-18T00:00:00.000Z
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253632101
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s2orc/train
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Efficacy and safety of metronomic chemotherapy in maintenance therapy for metastatic colorectal cancer: A systematic review of randomized controlled trials
Background: The current studies on metronomic chemotherapy in mCRC are all aimed at patients after multi-line therapy failure, and only a few studies have focused on maintenance treatment after successful first-line therapy. Methods: The PubMed, Embase, Cochrane Library, Wanfang, CNKI, and VIP were searched, and the relevant data was extracted, including media progression-free survival (mPFS), media overall survival (mOS), and grade 3/4 adverse events (AEs). Results: We included 4 randomized controlled trials (RCTs), 2 RCTs showed that metronomic maintenance chemotherapy could significantly improve mPFS compared to observation group; another RCT showed that metronomic maintenance chemotherapy group did not have low mPFS than the bevacizumab maintenance treatment (MT). The final RCT showed that dual-agent metronomic chemotherapy combined with bevacizumab MT did not improve mPFS compared with bevacizumab MT. The 3 RCTs showed that the metronomic maintenance therapy could not effectively improve mOS in mCRC compared to observation group or bevacizumab MT, while another RCT reported that the mOS in metronomic maintenance chemotherapy group was similar to bevacizumab MT. AEs was mostly mild and manageable. Grade ≥ 3 AEs are mostly nonhematological toxicity, and no deaths related to AEs were reported. Conclusion: This systematic review indicates that metronomic chemotherapy for mCRC MT can improve mPFS in some patients and is relatively safe. However, improvements in OS in most RCTs are arguable. Therefore, we need further studies to verify its long-term efficacy.
Introduction
According to the Global Cancer Statistics Report (2020), there were 935,173 deaths from colorectal cancer worldwide, accounting for 9.4% of all cancer deaths, and the mortality rate ranking second in the world. [1] From 2000 to 2018, due to a change in diet and lifestyle, the incidence of colorectal cancer among adults (aged 50 and over) rose from 20% to 61%. [2] As the early symptoms of colorectal cancer are not obvious, most patients are diagnosed in the advanced stage (they lose the chance of surgical intervention), and the 5-year survival rate is only 14%. [2] Currently, the treatment of metastatic colorectal cancer (mCRC) is mainly through cytotoxic drugs (such as oxaliplatin, capecitabine, and irinotecan) or combined targeted therapy with monoclonal antibodies (such as bevacizumab and cetuximab). The induction therapy prolongs life, and a small number of patients with microsatellite instability-high(MSI-H) can benefit from immune checkpoint inhibitors. Induction Medicine chemotherapy kills tumor cells when the maximum tolerated dose of the drugs is used (a high-intensity antitumor therapy), but the real challenge is to find ways to consolidate this curative effect. Should one choose to continue induction therapy with the original regimen, maintenance treatment (MT) with standard doses of some drugs, or simply conduct regular observations? If the original regimen is continued, the adverse drug reactions caused by a long-term treatment, such as cumulative use of oxaliplatin > 1000 mg/m 2 , will increase the risk of peripheral neurotoxicity in patients. [3] In 2020, Sonbol and coworkers performed a network META-analysis of randomized controlled trials (RCT) of diverse MT strategies in patients with mCRC. They showed that only after 16 weeks of first-line induction of FPOX or FPIRI, subsequent treatment strategies could be applied. Compared with the standard-dose fluorouracil ± bevacizumab MT group and the observation group, patients cannot be maintained from the original induction regimen until disease progression. Also, the comparison of the effectiveness between the standard dose fluorouracil ± bevacizumab MT group and the observation group showed that although the former can significantly improve progression-free survival, OS was not statistically difference. [4] In this regard, it is necessary to explore a new MT strategy for these patients. At present, other MT strategies for mCRC are still being explored, such as the use of EGFR inhibitors, but no optimal MT for mCRC is reported. [5] To achieve the purpose of prolonging the life of patients, MT also needs to take into account the potential adverse reactions caused by drugs and their impact on the quality of life of the patients. Can metronomic chemotherapy (MC) be used for the MT of mCRC? As early as 2000, some studies proposed MC for the treatment of advanced malignant tumors, and it has been found effective in tumors of different origins, including breast cancer, lung cancer, and ovarian cancer. [6][7][8][9] MC can achieve the purpose of anti-tumor by using continuous administration of drugs at low doses. This can also reduce the incidence and severity of adverse drug reactions and prevent drug resistance. Therefore, MC may bring new hope to patients who need MT to provide anti-tumor efficacy even after the first-line therapy is over. However, the current studies on MC in mCRC are all aimed at patients after multi-line therapy failure, and only a few studies have focused on MT after successful first-line therapy. Therefore, the purpose of this study is to assess the efficacy and safety of MC in mCRC patients and to provide appropriate medication references for patients whose disease is in remission or under stable conditions after first-line induction therapy and in need of MT.
Study selection
The databases of scientific literature, including PubMed, Embase, Cochrane library, China Wanfang, China CNKI, and China VIP, were searched for relevant articles published as of February 28, 2022. The free words and subject headings search method was used. The following keywords were used for the search: "Colorectal Neoplasms" or "Colorectal Tumors" or "Colorectal Carcinoma" and "maintenance treatment" or "metronomic."
Inclusion criteria
The inclusion criteria were as follows: RCTs of MC for mCRC; Eastern Cooperative Oncology Group performance status (PS) ≤ 2 points; received at least 16 to 24 weeks of first-line induction chemotherapy, and the efficacy assessment was response or stable disease; The treatment group received single-drug or dual-drug MC ± targeted drug MT, while the control group received observation or targeted drug MT; adequate hematologic, hepatic and renal function.
Exclusion criteria
The exclusion criteria were as follows: Multiple lines of chemotherapy; severe toxicity caused by induction chemotherapy; cardiovascular disease poorly controlled by medication; a history of neurological or psychiatric disorders.
Data extraction
Our teams formulated the search method. Two reviewers checked the articles separately, shortlisted relevant literature, and extracted information. Arguments were solved after discussion with our team. The first author, the year of publication, country, sample size, media progression-free survival (mPFS), media overall survival (mOS), and grade 3/4 adverse events (AEs) were extracted.
Quality evaluation
Two reviewers independently performed the quality evaluation of the scientific articles included in this study. Modified Jadad score was a tool to assess the quality of RCT. Then, the risk of bias for each article was assessed by RevMan software (version 5.4). Disagreements on the quality evaluation process was solved after discussion with our team till a consensus was reached.
Pooled data analysis
A qualitative synthesis of the eligible studies was conducted in the form of a table showing the research characteristics, clinical characteristics, and reported efficacy and safety values. Metaanalysis was not performed because data on relevant outcomes were insufficient for quantitative synthesis and the tabulated results indicated high methodological heterogeneity between the studies.
Eligible studies
The literature screening process was conducted as recommended by the PRISMA statement. 877 articles were retrieved from the databases search, including 139 from PubMed, 771 from Cochrane Library, 6 from Embase, 14 from CNKI, 31 from China Wanfang, and 16 from China VIP. We excluded 780 articles because of the following reasons: duplicate papers, reviews, irrelevant topics, retrospective studies, and studies reporting in vitro test results. After a full-text review, 8 articles were selected, and 4 were excluded. Among the selected articles, 1 article could not be extracted, 1 lacked outcome indicators, 1 was a single-arm trial, and 1 was non-MC. The remaining 4 papers were eligible. [10][11][12][13] Flow chart is shown in Fig. 1.
Quality evaluation
The quality of the RCTs were assessed by modified Jadad score. The scoring system includes 4 itemsrandom sequence, allocation concealment, blinding, withdrawal, and failure in follow-up. The score value of 1-3 was considered low-quality literature, and 4-7 were of high quality. However, allocation concealment and blinding were not used in some of these studies. The results showed that 2 of the articles are 5 points, and the other 2 are 3 points. And the results of articles bias risk showed that there were 2 articles with low risk of bias, and 2 articles with unclear risk of bias, as shown in Fig. 2.
Characteristics of the included studies
The basic characteristics are shown in Table 1. In total, 836 patients were included in the 4 RCTs, [10][11][12][13] 1 each from China, Italy, Switzerland, and New Zealand. All 836 patients were split into 2 groups. The treatment group received single-agent or double-agent MC ± bevacizumab (2 studies used capecitabine; one study used capecitabine + bevacizumab, and another study involved capecitabine + cyclophosphamide + bevacizumab). The control group was either on placebo or treated with only bevacizumab as MT. Thus, 413 patients were treated with MC, and 423 were either observed or given bevacizumab monotherapy MT. [10] Hagman et al conducted the first RCT with capecitabine MC maintenance versus bevacizumab maintenance in 67 patients with stable disease after 18 weeks of XELOX/FOLFOX/FOLFIRI/XELIRI induction chemotherapy; follow-up was done till 34.5 months. The statistics displayed that mPFS and mOS of capecitabine MC group were not inferior to those of the bevacizumab MT, with mPFS of 3.7 and 3.9 months and mOS of 28 and 26.4 months, respectively. However, the mPFS and mOS results between the 2 groups were not statistically analyzed. [11] Simkens et al divided 557 patients into treatment and observation groups. Both groups received 6 cycles of induction chemotherapy (capecitabine + oxaliplatin + bevacizumab), and then only the treatment group received 2). Although the mOS of MC group was better than observation group, there was no significant statistical difference. [12] In another phase Ⅲ RCT, Cremolini et aladministered capecitabine + cyclophosphamide MC + bevacizumab maintenance or bevacizumab monotherapy MT to 165 patients after 8 cycles of induction therapy with FOLFOXIRI + bevacizumab. A follow-up after 47.8 months showed mPFS of 10.3 months and 9.4 months (HR 0.94 70%C:I 0.82-1.09, P = .680) and mOS of 22.5 months and 28 months (HR 1.16 95%CI: 0.99-1.37, P = .336) in capecitabine + cyclophosphamide MC + bevacizumab maintenance and observation groups. However, the data was statistically insignificant. The results suggest that double-agent MC + bevacizumab MT did not significantly improve PFS or OS in mCRC. [13] 3. 5
. The safety of metronomic maintenance chemotherapy in mcrc
All the 4 eligible studies reported adverse reactions of grade ≥ 3with an incidence rate of 36.36% (304/836), which were then classified as hematological toxicity and non-hematological toxicity. The incidence of hematological toxicity was 3.29% (10/304), mainly due to neutropenia. The incidence of non-hematological toxicity was 96.71% (294/304) which manifested as hand-foot syndrome (HFS), mucosal inflammation, and diarrhea. The mPFS of patients with BRAF (V600E) mutation (treated with capecitabine MC + bevacizumab and observation group) were 9.5 months and 2 months (HR 0.19 95%CI: 0.08-0.44, P < .0001), mOS was 15.8 months and 13.6 months (HR 0.32 95%CI: 0.14-0.73, P = .007), respectively. [12,14] 3.6..2. Based on the location of the primary tumor. Two of the 4 eligible RCT studies had performed subgroup analysis. Cremolini et al used the location of the primary tumor (left colon or right colon) as a grouping factor, and the subgroup showed that the primary tumor location had no significant effect on patient mOS. It was 25.4 months (95%CI: 13.7-43.1) for tumors originating in left hemicolon and 23 months (95%CI: 12.5-45.3) for the right hemicolon (HR 0.90 95%CI: 0.66-1.24, P = .522). [13] Goey et al performed a post hoc analysis of the CAIRO3 trial conducted by Simkens et al This analysis also used primary tumor location as a grouping factor to explore its impact on patient survival, and the results showed that patients with right colon could benefit from capecitabine MC. However, in patients with primary tumors at left hemicolon, capecitabine MC treatment had improved mPFS but not the OS. [12,14]
Discussion
We included 4 articles, all of which were RCTs, and their conclusions varied on whether metronomic MT of mCRC patients could improve their mPFS and mOS. In terms of mPFS, 2 RCTs studies showed that metronomic maintenance chemotherapy could significantly improve mPFS in patients compared to the observation group; another RCT showed that the metronomic maintenance chemotherapy group did not have low mPFS than the bevacizumab MT group. The final RCT study on mCRC patients, treated with induction chemotherapy (FOLFOXIRI), showed that dual-agent MC combined with bevacizumab MT did not improve mPFS in patients compared with bevacizumab MT. In terms of mOS: the 3 RCTs studies showed that the metronomic maintenance therapy could not effectively improve mOS in mCRC patients compared to the the observation group or bevacizumab MT, while another RCT reported that the mOS in the metronomic maintenance chemotherapy group was similar to the bevacizumab maintenance group. In all the 4 RCTs included here, AEs in patients was mostly mild and manageable. Grade ≥ 3 AEs are mostly non-hematological toxicity, and no deaths related to AEs were reported. The drugs currently used for MC of mCRC mainly include fluorouracils (FU), camptothecins, and cyclophosphamide. [15][16][17] Three of the 4 included RCT studies included capecitabine in the MT, and the fourth RCT was on capecitabine + cyclophosphamide dual-drug MC. Capecitabine is a prodrug of 5-FU, which is initially converted into 5ʹ-deoxyfluridine by carboxylesterase and cytidine deaminase in vivo, then transformed into 5-deoxy fluoruridine by cytidine phosphorylase, and finally converted into active 5-FU. This design can greatly reduce the expression of fluorouracils in the gut and bone marrow, thereby reducing adverse drug reactions. [18] Studies have shown that mCRC patients who require salvage therapy can benefit from capecitabine MC. One study [19] included 68 patients with mCRC who were unable to receive standard chemotherapy due to adverse drug reactions or failure of chemotherapy at one or more metastatic sites. A single-arm study, with a 6.5-month follow-up, of low-dose capecitabine (1500 mg daily) in patients showed that capecitabine MC had moderate activity and was well-tolerated in mCRC who had received multiple lines of chemotherapy or were frail. In recent years, studies have shown that cyclophosphamide MC can inhibit the growth of tumor blood vessels not only to achieve the anti-tumor efficacy but to enhance the immune response as well. [20][21][22] To verify the effectiveness of cyclophosphamide MC in enhancing the immune response generated by MVA-5T4 vaccination, Scurr et al divided 52 patients, with stable and inoperable mCRC after induction chemotherapy, into 4 groupscyclophosphamide MC group (50 mg/bid, d1-7, d15-21), MVA-5T4 treatment group, cyclophosphamide MC + MVA-5T4 group, and the observation group. The results showed that cyclophosphamide MC can reduce Foxp3 + Tregs (T regulatory cells) and prolong PFS. Also, the patients did not experience any grade ≥ 3 AEs. Although low-dose cyclophosphamide did not increase the immune activity of the MVA-5T4 vaccine, it induced a beneficial immune response, prolonged survival, and showed better tumor efficacy. [17] Current MT for mCRC patients is mostly based on standard-dose chemotherapeutics, which is different from our studies based on MC. Luo et al studied the efficacy and safety of capecitabine monotherapy MT in mCRC patients who received 18 to 24 weeks of XELOX regimen after induction chemotherapy. The study randomly assigned patients to the capecitabine standard-dose maintenance group (capecitabine 1000 mg/m 2 , d1 to 14, twice a day, then stopped for one week for every 3 weeks of drugs; continued this cycle) and the observation group. After 29 months of follow-up, although the mPFS of the capecitabine group was significantly improved compared to the observation group, the improvement in OS was insignificant. In terms of safety, compared with the observation group, the incidence of grade 3/4 AEs in the capecitabine group was 41.9%, which was significantly higher than that in the observation group (22.4%). Among all the AEs, the most common were neutropenia in 12.5% of patients (17/136), HFS in 5.9% (8/136), and mucositis in 5.9% (8/136). Throughout the trial, 8.8% (12/136) of patients in the capecitabine group had dose reductions due to HFS (50%) and diarrhea (25%). Thus, mCRC patients had tolerable adverse reactions, and capecitabine standard-dose maintenance may be considered an appropriate choice after induction chemotherapy with XELOX or FOLFOX. [23] In the 4 included RCTs, 2 RCTs used capecitabine 500 mg/bid metronomic maintenance therapy, 1 used capecitabine 500 mg/tid + cyclophosphamide 50 mg/d double-agent metronomic maintenance therapy, and the last one used capecitabine 625 mg/bid metronomic maintenance therapy continuously. The incidence of grade 3/4 AEs in the capecitabine 500 mg/bid group was less than 35%. Geng and coworkers showed that the incidence of HFS ≥ grade 3 was 8% (2/25). [10] In the study by Hagman not reported. [11] Capecitabine 500 mg/tid + cyclophosphamide 50 mg/qd double-agent group had a rate of ≥ grade 3 AEs in 16.9%, of which the incidence of the HFS was 9.1% (7/77). [13] Capecitabine 625 mg/bid group had a higher incidence of grade 3 AEs of about 60% (167/278), but no grade 4 AEs occurred. It may be due to the slightly higher incidence of AEs due to the combinatorial effect of bevacizumab. The common grade 3 AEs in the capecitabine 625 mg/bid group were hypertension in 24% (68/278), the HFS in 23% (64/278), and peripheral neuropathy in 10% (27/278). [12] A total of 27 patients in the capecitabine 625 mg/bid group stopped treatment due to drug-related AEs.
Although the incidence of the HFS was higher than that in the observation group, it did not affect the quality of life of patients. It can be seen that with respect to the incidence of ≥ grade 3 AEs, capecitabine 500 mg/tid + cyclophosphamide 50 mg/ qd double-agent group has a low incidence of grade ≥ 3 AEs, capecitabine 500 mg/bid group had a slightly higher incidence, but the highest was observed in capecitabine 625 mg/bid group. Generally, the incidence of grade 3/4 AEs in metronomic maintenance therapy was lower than that in capecitabine standard dose MT, and the incidence of grade 3/4 AEs in HFS was lower.
There are few reports of drug discontinuation due to AEs in metronomic maintenance therapy. Economically, the cost of MT for patients with standard-dose chemotherapy is usually higher than that of MC. As early as 2005, Bocci et al conducted a pharmaceutical economics evaluation of cyclophosphamide/methotrexate MC in patients with metastatic breast cancer (under palliative care) and showed that it was significantly different from 11 single-agent or combination chemotherapy (e.g., vinorelbine, docetaxel, gemcitabine, paclitaxel, and docetaxel + carboplatin). The low-dose cyclophosphamide/methotrexate has been evaluated as a cost-effective/ cost-saving option for metastatic breast cancer patients under palliative care. [24] There is no economic analysis of capecitabine MC, capecitabine standard doses, and other maintenance regimens, but hopefully, this will be addressed in future studies.
Studies have found that the prognosis of mCRC is related to the location of the primary tumor and the gene mutations in KRAS, RAS, and MSS. The prognosis when the tumor originates in the left colon is better than that of the right colon. [25] Then the origin of the primary tumor and the status of KRAS, RAS, and MSS may also have a certain impact on the benefit of metronomic maintenance therapy. In this systematic review, 3 RCTs have conducted genetic assessments of patients and associated it with the effectiveness of MC. Among them, 1 study only considered KRAS mutations in mCRC patients. The results indicated that in patients with KRAS mutations, the mPFS and mOS of capecitabine MC were similar to bevacizumab MT. Two RCTs have investigated whether the origin of the primary tumor can benefit from metronomic maintenance therapy, but the included studies had small sample sizes to conclude about the benefits of metronomic maintenance therapy being associated with the primary tumor location. However, the induction chemotherapy regimens used in mCRC patients in the included studies were different (included XELOX, FOLFOX + bevacizumab, CAPEOX + bevacizumab, XELOX/FOLFOX/XELIRI/ FOLFIRI + bevacizumab). Hence, the question arises of whether different induction chemotherapy regimens and induction chemotherapy, with or without bevacizumab, affect the efficacy and safety of metronomic maintenance therapy? Also, how to determine the dose and schedule of MC after first-line induction chemotherapy? These questions will probably be answered in future research.
There are some limitations to this systematic review. Firstly, there are inconsistencies in the experimental group and the observation group in the eligible RCT studies, which may have influenced the conclusion and application of the study. Therefore, the results of this study should be comprehensively considered in combination with the actual situation of patients when applied in clinical practice. Secondly, the number of studies included here is very small (only 4 RCTs are included), which may affect the reliability of the results and the extrapolation of the conclusions. Thirdly, the included RCTs were not blinded, and only 2 used randomized control for allocation concealment; the remaining 2 were not subject to allocation concealment, which may affect the credibility of the study, resulting in a lower quality of the included studies. Finally, 2 RCTs in the included studies were sponsored by pharmaceutical companies, and in one of the studies, the sponsor participated in the trial design. Although the sponsor did not participate in the specific implementation of the specific trial, it may have a certain impact on the trial results. This systematic review suggests that further clinical research should pay attention to adopting sufficient randomization methods, allocation scheme concealment, and blinding methods in research design and methods to reduce various biases such as selectivity, implementation, measurement, and attrition. Research results should provide detailed and fully transparent research information for readers to judge the authenticity of the research results.
Conclusion
This systematic review indicates that MC for mCRC MT can improve mPFS in some patients and is relatively safe. Most of the adverse reactions were mild and manageable, and no AE-associated deaths were reported. However, improvements in OS in most RCTs are arguable. Therefore, we need further studies to verify its long-term efficacy.
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2022-11-20T06:15:57.538Z
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2022-11-18T00:00:00.000Z
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253671097
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s2ag/train
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Nontoxic Natural Polymeric Particle Vehicles Derived from Hyaluronic Acid and Mannitol as Mitomycin C Carriers for Bladder Cancer Treatment.
Hyaluronic acid/mannitol (HA/MN)-based particles were designed as mitomycin c (MMC) delivery vehicles through the crosslinking of 1:0, 3:1, 1:3, and 0:1 mole ratios of HA/MN to investigate their potential use in bladder cancer therapy. The HA/MN-MMC particles prepared by the microemulsion crosslinking method were of 0.5-10 μm size with a zeta potential value of -36.7 mV. The MMC carrier potential of the HA/MN-MMC particles was investigated by changing HA/MN ratios in the particle structure. The MMC loading capacity of neat HA particles was 5.3 ± 1.1 mg/g, whereas HA/MN (1:3) particles could be loaded with about three times more drug, for example, 18.4 ± 0.8 mg/g. The kinetic of MMC drug delivery from the HA/MN-MMC particles were tested in vitro in bladder cancer conditions for example, pH 4.5, 6, and 7.4. The HA-MMC particles released approximately 70% of the loaded drug in 300 h, while 43% of the loaded drug was released from the HA/MN-MMC particles within 600 h under physiological conditions, pH 7.4, 37 °C. The cytotoxicity of HA-based particles on healthy L929 fibroblast cells and HTB-9 human bladder cancer cells was investigated in vitro via MTT tests. Bare MMC inhibited about 90% of L929 fibroblast cells even at 100 μg/mL, but the cell viabilities in the presence of HA-MMC and HA/MN-MMC particles were 85 ± 5 and 109 ± 7% at 1000 μg/mL, respectively. The HA/MN-MMC (1:3) particles at 1000 μg/mL were found capable of destroying half of HTB-9 human bladder cancer cells within 24 h. Interestingly, the same particles at 50 μg/mL destroyed almost all the cancer cells with 8 ± 5% cell viability in 72 h of incubation time. The designed HA/MN-MMC (1:3) particles were found to afford a chemotherapeutic effect on the tumor cancers while reducing the toxicity of MMC against L929 fibroblast cells.
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v2
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2022-11-20T16:33:08.138Z
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2022-11-18T00:00:00.000Z
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253693389
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s2ag/train
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EVALUATION OF THE USEFULNESS OF SHEAR WAVE ELASTOGRAPHY IN THE DIAGNOSTIC PROCESS FOR NONCANCEROUS SOFT TISSUE MASSES
Current study was carried out to assess efficacy of SWE as an adjunct to sonography in investigating superficial benign lesions. The retrospective study was conducted in Radiology department of Nishtar Medical Hospital from July 2021 to July 2022. The study included 45 patients with 49 lesions. Doppler and grayscale ultrasound and SWE was performed. Conventional ultrasonic features like depth (mm), size (mm), margin (ill defined or well defined), vascularity (present or absent) and echogenicity (isoechoic, anechoic, hyperechoic or hypoechoic) were investigated. Median shear modulus and ultrasonic features were documented. Mean shear modulus for epidermoid, ganglion and lipomatous cyst was 23.6kPa, 5.9kPa and 9.1kPa respectively. Difference between lipomatous tumors, ganglion cysts and epidermoid cysts were significant (P=0.018). Median shear modulus of epidermoid cyst was higher than ganglion cysts and lipomatous tumors. Shear wave elastography is valuable modality for diagnosis of superficial benign soft tissue masses through direct quantitative analysis.
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2022-11-20T16:34:21.862Z
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2022-11-18T00:00:00.000Z
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253703780
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s2ag/train
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Palliative cancer patients pain reduction methods during opioid epidemic era
Introduction: Opioids are the most commonly used medication in palliative cancer pain treatment due to their proven effectiveness. However, modern anti-pain treatment concentrates not only on analgesics, but simultaneously on the detection of conditions affecting and intensifying pain sensation. Many studies have shown potential of other, non-opioidal palliative cancer pain treatment with additional positive effect on patient’s general quality of life.
Aim of the study: The purpose of our review is to introduce the issue of the use of opioids and draw attention to other non-opioidal pain reduction methods as well as to indicate directions for further potential researches.
Methods and materials: We have reviewed the literature available in the PubMed, Google Scholar, Science Direct database using the keywords: „cancer patients and opioids”; „pain and cancer”; „chronic pain”; „palliative cancer”. We excluded abstracts, comments, and non–English language articles.
Results:
The methods outlined in this review will not affect pain reduction to the same extent as opioids, but they offer a chance to reduce it to a level that allows patients to maintain a normal life. In the light of opioid epidemic era literature shows new approaches to treating pain such as analgesics, including antidepressants, anticonvulsants, Vitamins, cannabis and nonpharmacological methods and showing their potential for wider use in palliative cancer patients treatment.
Conclusion: Besides opioids, there are many factors that affect pain reduction, however, their analgesic potential require additional studies on larger groups of patients.
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v2
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2022-11-20T16:40:14.981Z
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2022-11-18T00:00:00.000Z
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253703707
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s2ag/train
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Validation of HPV triage in cytology-based cervical cancer screening for ASC-US cases using Japanese data.
OBJECTIVE
In Japan, cervical cancer screening consists of a cytology examination performed once every 2 years. We verified whether the risk of cervical intraepithelial neoplasia (CIN) 3 disease or higher (CIN3+) was equivalent to that of cytology negative cases (negative for intraepithelial lesion or malignancy [NILM]) for patients with a cytological diagnosis of "atypical squamous cells of undetermined significance (ASC-US)" who tested negative for human papillomavirus (HPV).
METHODS
Data from a total of 22,925 cases who had undergone cervical cancer screening at least twice or who had completed follow-up examinations after cervical screening at a single facility between April 2013 and April 2018 were analyzed. The cumulative incidence of CIN3+ was calculated for each category of initial cytology finding and HPV result (NILM, > ASC-US, ASC-US/HPV (unknown), ASC-US/HPV+, and ASC-US/HPV-). The statistical analysis was conducted using the Cox proportional hazards model.
RESULTS
The hazard ratio for the cumulative incidence of CIN3+ in 2 years relative to that for NILM cases was 2.7 (95% confidence interval=1.0-7.8) for > ASC-US cases, 0.5 (0.1-1.7) for ASC-US/HPV (unknown), 0.8 (0.3-2.4) for ASC-US/HPV+ cases, and 0.3 (0.1-1.0) for ASC-US/HPV- cases.
CONCLUSION
Because the cumulative incidence of CIN3+ at 2 years for the ASC-US/HPV- cases was sufficiently low, compared with that of the NILM cases, we considered it reasonable and safe to perform HPV triage for ASC-US cases and to allow HPV-negative cases to return for their next screening in 2 years, which is the same follow-up schedule as that for NILM cases.
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v2
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2022-11-23T16:08:58.089Z
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2022-11-18T00:00:00.000Z
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253783959
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s2ag/train
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Quality of life in glioblastoma after the introduction of temozolomide: a systematic review
Introduction: Gliomas are primary tumors of the central nervous system with an aggressive pattern of progression with a poor prognosis in terms of survival and quality of life. The current standard treatment consists of surgery with maximum excision associated with radiotherapy and chemotherapy, based mostly on the use of temozolomide. Since its introduction, the quality of life of patients undergoing this therapy has not been widely targeted and evaluated. Objective: To verify the quality of life of patients with glioblastoma after the introduction of temozolomide in the therapeutic protocols. Methods: A systematic literature review guided by the PICO and PRISMA protocol was conducted; PubMed, Medline and Lilacs databases were consulted. Results: Initially, 77 studies were found, after selection criteria, 35 articles were analyzed. No statistically significant change was found in overall quality of life in studies that analyzed temozolomide therapy versus different control therapies. Conclusion: The association of temozolomide with surgery and radiotherapy proved to be neutral, with no significant negative or positive impacts on the quality of life of patients with glioblastoma.
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v2
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2022-11-19T15:05:49.566Z
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2022-11-19T00:00:00.000Z
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253631764
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s2orc/train
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Development and validation of a risk prediction model for overall survival in patients with nasopharyngeal carcinoma: a prospective cohort study in China
Objective Nasopharyngeal carcinoma (NPC) is prevailing in Southern China, characterized by distinct geographical distribution. Aimed to predict the overall survival (OS) of patients with nasopharyngeal carcinoma, this study developed and validated nomograms considering demographic variables, hematological biomarkers, and oncogenic pathogens in China. Methods The clinicopathological and follow-up data of the nasopharyngeal carcinoma patients obtained from a prospective longitudinal cohort study in the Chongqing University Cancer Hospital between Jan 1, 2017 and Dec 31, 2019 (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm{n}=1635$$\end{document}n=1635). Cox regression model was used to tested the significance of all available variables as prognostic factors of OS. And independent prognostic factors were identified based on multivariable analysis to model nomogram. Concordance index (C-index), area under the receiver operating characteristic (AUC), calibration curve, and decision curve analysis (DCA) were measured to assess the model performance of nomogram. Results Data was randomly divided into a training cohort (1227 observers, about 70% of data) and a validation group (408 observers, about 30% of data). At multivariable analysis, the following were independent predictors of OS in NPC patients and entered into the nomogram: age (hazard ratio [HR]: 1.03), stage (stage IV vs. stage I–II, HR: 4.54), radiotherapy (Yes vs. No, HR: 0.43), EBV (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge 1000$$\end{document}≥1000 vs.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$<1000$$\end{document}<1000, HR: 1.92), LAR (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$>6.15$$\end{document}>6.15 vs.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\le 6.15$$\end{document}≤6.15, HR: 2.05), NLR (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$>4.84$$\end{document}>4.84 vs. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\le 4.84$$\end{document}≤4.84 HR: 1.54), and PLR (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$>206.33$$\end{document}>206.33 vs.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\le 206.33$$\end{document}≤206.33, HR: 1.79). The C-indexes for training cohort at 1-, 3- and 5-year were 0.73, 0.83, 0.80, respectively, in the validation cohort, the C-indexes were 0.74 (95% CI 0.63–0.86), 0.80 (95% CI 0.73–0.87), and 0.77 (95% CI 0.67–0.86), respectively. The calibration curve demonstrated that favorable agreement between the predictions of the nomograms and the actual observations in the training and validation cohorts. In addition, the decision curve analysis proved that the nomogram model had the highest overall net benefit. Conclusion A new prognostic model to predict OS of patients with NPC was developed. This can offer clinicians treatment making and patient counseling. Furthermore, the nomogram was deployed into a website server for use.
Introduction
Nasopharyngeal carcinoma (NPC) is an epithelial carcinoma originating from the nasopharyngeal mucosal tissue, with the characteristic of distinct geographical distribution of occurrence [1,2]. NPC occurred highly in East and Southeast Asia [1], and it particularly prevalent in Guangdong and Guangxi, the regions of southern China [3]. In 2019 the number of NPC deaths in China reached 28,659, accounting for 40% of NPC deaths worldwide [4]. China accounts for a significant proportion of mortality of NPC over the world, especially in southern China [5,6].
Epstein-Barr virus (EBV) is one of the most common causative agents, and it can be detected in all types of NPC [7]. Radiotherapy is the primary treatment choice for NPC treatment due to the radiosensitive characteristic of NPC tumor [8]. And precise staging is crucial for reducing mortality in patients with NPC. However, heterogeneities of clinical outcomes of the NPC patients with the same clinical stage and degree of EBV were reported in considerable recent research. Those findings indicated that it is not enough to refine the prediction of outcomes for NPC patients only considering single factors. Recently, a major current focus in the area of prognostic of NPC is to find more risk factors to get a more accurate predictive model. Numerous studies have demonstrated that hematological biomarkers were associated with survival outcomes of NPC patients, such as lymphocyte-albumin ratio (LAR), neutrophil-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) [9][10][11]. However, the literature related to the survival outcomes of the cohort among NPC patients that take various factors into account in model design are limited. Therefore, in this study, our objective was to develop a clinically useful prognostic model in which demographic variables, hematological biomarkers, and oncogenic pathogens were considered to predict overall survival among NPC patients in the region.
Data source
The data set used in the retrospective cohort study was obtained collecting 1635 patients with NPC from the Chongqing University Cancer Hospital tumor database between Jan 1, 2017 and Dec 12, 2019. The inclusion criteria were as follows: (1) age ≥ 18 years; (2) histologically confirmed primary NPC; (3) the main treatment occurred in our hospital; (4) completed baseline clinical information and follow-up information; (5) completed the entire course of the treatment of radiotherapy, chemotherapy and targeted therapy. The exclusion criteria for this study were as follows: no follow-up records and a history of cancer treatments. The present study was performed according to the guidelines of the Declaration of Helsinki and was approved by the ethics committee of the Chongqing University Cancer Hospital. Written informed consent was obtained from all subjects.
Variables
IN this study, we employed demographics, including age, sex (female and male), ethnicity (Han and others), marriage (married and others), and occupation (worker/clerk, self-employed/unemployed, professional and technical personnel, and others). The clinical characteristics were selected, including clinical stage which was classified according to the American Joint Committee on Cancer Staging Manual (8th edition), pathological (non-keratinized differentiation, non-keratinized undifferentiated, and keratinized squamous cell carcinoma and others), and transfer information. We also abstracted therapeutic methods information like radiotherapy, chemical-therapy and targeted-therapy. Finally, we retrieved laboratory variables, which consisted of EBV, LAR, NLR, and PLR and selected the cutoff point using X-tile. Continuous variables in laboratory data were transformed into categorical variables based on cutoff values: EBV ( < 1000 , ≥ 1000 ), LAR ( ≤ 0.13 , > 0.13 ), NLR ( ≤ 4.84,> 4.84 ), PLR ( ≤ 206.33 , > 206.33).
The endpoint of interest in this study was the overall survival (OS) of NPC patients, calculated from the date of first diagnosed as NPC to the time of death or the last follow-up was set as the end.
Construction of nomogram
Patients were randomly divided into train group (1227 observers, about 70% of data) and a validation group (408 observers, about 30% of data). The nomogram model was developed using training cohort. The univariate Cox regression analysis was performed to verify the prognostic significance of each covariate as factor of OS. And entered the variables with p-value < 0.05 to the multivariate Cox regression model to analyze the association between each variable and OS to find independent risk factors. The nomogram was created based on the risk Conclusion: A new prognostic model to predict OS of patients with NPC was developed. This can offer clinicians treatment making and patient counseling. Furthermore, the nomogram was deployed into a website server for use.
Keywords: Nasopharyngeal carcinoma, Nomogram, Overall survival, Prognosis score calculated by the final Cox regression model that was constructed by stepwise process.
Model performance and validation
Concordance index (C-index) and area under the receiver operating characteristic (AUC), calibration curve, and decision curve analysis (DCA) were used to assess the model performance of nomogram. C-index was used to estimate the accuracy of the model calculating the difference between predicted value and actual one. The calibration curve was evaluated using a plot to estimate the performance of accordance of the prediction and e reality. DCA calculates a clinical "net benefit" for one or more prediction models in comparison to default strategies of treating all or no patients.
Statistical analysis
The data analysis was performed using SPSS software
Characteristics of the training and validation cohorts
Patients with nasopharyngeal carcinoma enrolled in follow-up visit were randomly split between training (n = 1227, 70%) and validation cohorts (n = 408, 30%), from the Chongqing University Cancer Hospital tumor database platform.
Independent prognostic factors in the training cohort
In the training cohort (n = 1227), the independent prognostic factors were performed using Cox proportional hazards models and modeled results were reported in Table 2. The following variables were significant as predictors of OS on univariable analysis: age, occupation (only professional and technical personnel), stage, radio therapy, chemical therapy, EBV, LAR, NLR, and PLR (all p < 0.05 ). Reddy etc. found that keratinization may be a vulnerable aid in predicting response to therapy for NPC [12]. And Luo etc. demonstrated that differentiation is close to EBV, which indicates that it was a link between EBV and NPC [13]. Based on clinical consensus and the previous research, we kept the pathological in the model.
Developing the prognostic nomogram model
Independent predictors on multivariable analysis were selected for the development of nomogram model to predict 1-, 3-and 5-year OS in nasopharyngeal carcinoma patients (Fig. 1). Each variable was converted to a point score based on corresponding Cox estimated regression coefficients and the sum of the values was positioned to the total point table to obtain the probability of OS. Fig. 2. In addition, the calibration curve at 1-, 3-, 5-year survival of the model performed well, showing good agreement between the predictions of the nomograms and the actual observations in the training and validation cohorts (Fig. 3.). Moreover, the decision curve analysis was used to test the predictive ability of the nomograms. The DCA results of the four models showed that, except for a small range of predicted probability threshold between 75 and 90%, the nomogram model displayed a positive net benefit in the train set (Fig. 4.).
Risk-stratifying ability of the nomogram
Based on the predictive risk scores calculated by the nomogram model, the study subcategorized the training and validation cohort into low-risk group (the prognostic risk score was less than the threshold) and high-risk group (the prognostic risk score was greater than the . And the Kaplan-Meier survival curves for OS presented significant differences between the two groups in the training and validation cohort ( p < 0.0001 ) (Fig. 5).
Webserver development for the nomogram
We developed an easily accessible webserver for the nomogram model of NPC (https:// nomog ramwe bserv erofn pc. shiny apps. io/ DynNo mapp/). The survival plot and probability of the patient can be displayed by selecting the corresponding indexes and survival time on the left side of the webserver board (Fig. 6). For example, the probability of one patient with the following characteristics at 1-year is 0.82: 65-year-old, stage 3, with non-keratinizing differentiation, no radiotherapy, no chemical-therapy, EBV ≥ 1000, LAR > 6.15, NLR ≤ 4.84, PLR ≤ 206.33, and the probability of the patient with same characteristics at 3-year and 5-year was 0.55, 0.46, respectively.
Discussion
In the present study, we used the follow-up database from the Chongqing University Cancer Hospital to establish a novel nomogram prognostic model of NPC and complete internal verification, by incorporating demographics, hematological biomarkers, and oncogenic pathogens. And a user-friendly online calculator was developed to help clinicians in treatment decision making.
Several previous studies have been published using the nomogram to predict the OS of NPC patients. In 2018, Wu and colleagues evaluated a nomogram for predicting long-term OS for patients wish NPC using demographic variables and TNM stage [14]. And Huang etc. developed a prognostic nomogram to reveal the relationship between EBV and NPC in 2021 [15]. Although the western region is not a high incidence area of nasopharyngeal carcinoma, the survival prediction of its patients should not be ignored. Inflammatory markers have been widely used in various cancers, but rarely in NPC, thus we established a prognostic model considered several systemic inflammation parameters and EBV.
Several of our findings are worth highlighting. First, age, stage, radio therapy, EBV, LAR, NLR and PLR were recognized as independent prognostic parameters based on the univariate and multivariate cox regression analysis, and the conclusion was in general agreement with previous reports [9,[16][17][18]. EBV infection is the most common causal agent [19] and a useful prognostic factor of NPC [20], and has been used to assess the disease progression and population screening [21]. LAR is a novel independent prognostic risk factor [9] and have a strong survival predictive power for OS in NPC [18]. Li etc. concluded that NLR could be an attractive indicator for evaluating the 5-year OS in NPC patients with stage III [22]. High PLR was associated with poor OS in NPC patients [23]. Notably, chemical therapy was suggested that it did
(A)
ROCs for overall survival training cohort (B) ROCs for overall survival validation cohort not reach statistical significance to be a prognostic factor in our study, and it was unlike some previous results [24]. Considering the chemotherapy sensitizing the tumor to the toxic effects of the radiotherapy [25] and the choice to chemotherapy depending on clinical risk, for example, the results obtained in the study is reasonable. Radiotherapy is the primary curative treatment of NPC, and combing chemotherapy with radiotherapy is a rational option in the treatment of locoregionally advanced NPC [26]. Therefore, to avoid missing important factors and based on the clinical features, we conduct model incorporating chemotherapy. In addition, it should be noted that through the univariate model, the correlation between pathological and NPC was of no significance, which was contradictory to other researches. In our study, the calibration curve pointed optimal accordance between predicted survival probability and actual value, which indicated good repeatability and reliability of the model. training and validation cohorts). In addition, the DCA curves illustrated a better performance of survival predictions of nomogram than the models with stage, EBV, and stage + EBV. In conclusion, the results were suggested that our nomogram was a reliable and precise prognostic tool to predict OS in NPC patients. Our study is not devoid of limitations. First, there may exist a potential source for selection bias based on the serious inclusion and exclusion criteria. Second, our samples were collected from a single center from a nonendemic region in China and lack of external verification. It is necessary for our study, aimed at exploring the performance of combination of OS and disease-free survival, to design a multicenter randomized controlled study in the next step.
Conclusion
Patients with NPC have heterogeneous survival outcomes, which can be predicted using our novel prognostic model. And it can support help in clinicians deciding treatment and patient counseling. Furthermore, the nomogram was deployed into a website server for use.
Author contributions SM and HL conceived and designed the study. Analysis and interpretation of statistics were performed by XL, WZ. SM wrote initial drafts of the paper. XL and HL handled the data collection and statistical analysis. GW, AS, YW and YW made revisions to the article. All the authors read and approved the final manuscript.
Funding Support for this work was provided by the Chongqing Performance Incentive and Guidance Project for Scientific Research Institutions (cstc2020jxjl130016), Chongqing Key Disease Prevention and Control Technology Project (2019ZX002).
Data availability
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation, to any qualified researcher.
Declarations
Ethics approval and consent to participate We followed the ethical principles for medical research involving human subjects as laid down in the Declaration of Helsinki. The studies involving human participants were reviewed and approved by The Ethics Committee of Chongqing University Cancer Hospital.
Competing interests
There are no potential conflicts of interest among the authors.
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Organelle-targeted therapies: a comprehensive review on system design for enabling precision oncology
Cancer is a major threat to human health. Among various treatment methods, precision therapy has received significant attention since the inception, due to its ability to efficiently inhibit tumor growth, while curtailing common shortcomings from conventional cancer treatment, leading towards enhanced survival rates. Particularly, organelle-targeted strategies enable precise accumulation of therapeutic agents in organelles, locally triggering organelle-mediated cell death signals which can greatly reduce the therapeutic threshold dosage and minimize side-effects. In this review, we comprehensively discuss history and recent advances in targeted therapies on organelles, specifically including nucleus, mitochondria, lysosomes and endoplasmic reticulum, while focusing on organelle structures, organelle-mediated cell death signal pathways, and design guidelines of organelle-targeted nanomedicines based on intervention mechanisms. Furthermore, a perspective on future research and clinical opportunities and potential challenges in precision oncology is presented. Through demonstrating recent developments in organelle-targeted therapies, we believe this article can further stimulate broader interests in multidisciplinary research and technology development for enabling advanced organelle-targeted nanomedicines and their corresponding clinic translations.
INTRODUCTION
Cancer-related mortality remains to be an ongoing monumental global crisis, with an estimated 19.3 million new cases and 10 million cancer deaths worldwide (9.9 million excluding nonmelanoma skin cancer) in 2020, according to the International Agency for Research on Cancer. [1][2][3] Developing highly efficient and precise cancer theranostics is an extremely important research area for public health and society development. Notably, nanomaterials have garnered substantial attention in cancer theranostics due to their superior performance in pharmacokinetics/pharmacodynamics (PK/PD), side-effect reduction and ease of formulation with multifunctionalities. [4][5][6] However, approximately only 0.7% of administrated nanomedicines could reach their final (sub)cellular targets due to physiological and pathological barriers, and these nanomedicines often exhibit severely compromised efficacy. 7,8 Furthermore, the clinical application of nanomedicines is often hindered by risks of low bio-availability, amplified dose, and rejection effects. 9 Precision medicine, or personalized medicine, offers the potential for best-practice interventions in cancer treatment with subcellular organelles representing ideal targets. [10][11][12] In the era of precision medicine, targeting molecular-based pathogenesis becomes an established paradigm of cancer therapeutic agent development. 13,14 Organelle-targeted therapies focused on the highly-sensitive and precise attack on specific organelles have received substantially growing research interest (Fig. 1). 15 These strategies can accurately regulate the transport processes of therapeutic agents from the plasma membrane to action targets, boosting drug efficiency while maintaining necessary concentrations to induce apoptosis. 16 Therefore, organelletargeted strategies hold great potential to overcome physiological and pathological barriers, greatly decrease therapeutic threshold dosage, minimize side effects and, ultimately, boost treatment outcomes.
Subcellular-targeting strategies are a very promising cancer modality, while the structures and death-induced modes of organelles still remain elusive. Additionally, transport of nanomaterials to organelle targets can be restricted by cell crowding and complex cell environments, such as cytoskeletal structures. 17 Therefore, a subtler design of organelle-targeted nanoplatforms based on unique organelle characteristics is desired to meet demands of varying cancer treatment modalities, allowing the ability to achieve maximized treatment efficacy. 18 Subcellular organelles, such as the nucleus, mitochondria, lysosome and endoplasmic reticulum (ER), can maintain a balance between cell proliferation and death, while modulating cell metabolism functions. 19 This article will primarily focus on discussing these essential therapeutic targets for advanced cancer treatments. We will introduce target structures, intracellular organelle functions, organelle-mediated cell death and intervention mechanisms, and design guidelines for advanced organelle-targeted cancer nanosystems. We also aim to provide a comprehensive review on the efficacy of cancer therapy methods based on subcellular organellespecific oncology, which is vital in revolutionizing treatments and enabling the curing of cancer.
KEY MILESTONES IN DEVELOPING ORGANELLE-TARGETED THERAPIES
Organelle-targeted therapies, which seek to enhance the efficacy of therapeutic agents, have been studied for nearly 70 years.
These strategies are built on the basis of organelle spatial structure analysis, organelle-mediated death signaling pathway investigation, and therapeutic agent development. Herein, this section focuses on discussing critical studies in the development of organelle-targeted therapies in cancer treatment and outline these hallmark events in Fig. 2.
Nucleus-targeted therapy The nuclear pore plays an essential role in bi-directional nucleocytoplasmic transport, first described in 1950. 20 In 1993 and 2004, the first cryo-electron microscopy map and tomography of nuclear pore complexes were obtained, respectively. These important milestones were instrumental in determining the nucleocytoplasmic transportation mechanism for macromolecules. 21,22 With continuous, extensive research efforts focusing on elucidating nuclear structure, transport of macromolecules between the cytoplasm and nucleus was progressively recognized and then widely accepted. In 1984, Kalderon et al. 23 first proposed nuclear localization signals based on the simian virus 40 (SV40), which assists macromolecules across the nuclear envelope (NE). Notably, the earliest research of nuclear-targeted photodynamic therapy (PDT) was in 1997 by Akhlynina. 24 Since then, much effort has been made to understand transport mechanisms, which drives nucleus-targeted therapy development. [24][25][26][27] Concurrently, there has been growing attention to signal transduction pathways in response to DNA damage, which facilitated more comprehensive investigations into nuclear-targeted strategies. [28][29][30] Mitochondrial-targeted therapy Mitochondria are essential organelles that generate most of energy supply for cells, control metabolic pathways, and regulate cell death. Early critical research in determining mitochondrial structure was conducted from 1953 to 1956. [31][32][33] Since then, mitochondrial-targeted molecules have facilitated antioxidant mitochondrial accumulation based on their unique structure. [34][35][36][37] Additionally, great efforts have been delivered to understand cell death caused by mitochondrial. Research focused on mitochondria-mediated death signaling pathways have received significant attention since when the "Warburg Effect" was first proposed in 1956. 38 Multiple lines of evidences have implicated mitochondrial dysfunction, such as apoptosis. 39,40 Mitophagy and ferroptosis were first proposed in 2005 and 2019, respectively. 41,42 In general, personalized therapeutic strategies toward mitochondria is an important research area in cancer treatment.
Lysosomal-targeted therapy Since the first discovery of lysosomes in 1955 by Christian de Duve, research investigations into lysosome structure-function relationships has led to significant progress in obtaining a deep understanding. 43 In 1963, following the groundbreaking study of morphological processes of lysosomes under electron microscopy, Christian de Duve coined the term "autophagy." 44 In 1984, Glaumann et al. 45 revealed that iron overload can result in increased lysosomal fragility, 45 inspiring various studies on lysosome-mediated cell death. In 2018, cell death triggered by lysosomes was officially termed lysosome-dependent cell death (LDCD) by Nomenclature Committee on Cell Death. 46 However, a study published in 1989 found that the heat shock protein (hsp70) family can regulate intracellular protein degradation in lysosomes. 47 In 2004, Gyrd-Hansen et al. 48 found that hsp70 promoted cancer cell viability by stabilizing the lysosomal membrane. The safeguarding function of hsp70 predicts poor therapy efficacy in lysosome-targeted therapies; thus, hsp70 is a potential target to enhance LDCD sensitivity and induce cell death.
ER-targeted therapy In 1953, the first high-resolution images of the ER were successfully obtained. 49 Since then, many research studies have enabled a deep understanding about ER functions, such as the relationship between the ER and cell death; unfolded protein response (UPR) under ER stress occupies a pivotal position in cell death. In the late 1990s, IRE1, PERK, and ATF6 were reported as ER Fig. 1 Organelle-targeted therapy boosts cancer treatment outcomes by allowing for maximum accumulation of therapeutic agents in targets, triggering specific cell death pathways stress sensors and UPR activators to safeguard ER functions or trigger ER-mediated cell death signaling pathways. [50][51][52][53] With improved understandings of the UPR mechanism, many proteasome inhibitors were developed to trigger cancer cell death. For example, bortezomib was approved by FDA for cancer treatment in 2003. 54 Moreover, understandings about the relationship between the ER and mitochondria in Ca 2+ response was first obtained in 1993 55 and then enhanced in 2010, 56 which provided important fundamental insights to provide new therapeutic avenues for ER-targeted therapeutic agent development. Organelle-targeted therapies: a comprehensive review on system design for. . . Yang et al. Organelle-targeted therapeutic agents are experiencing an explosive growth in research and technology development as a very promising treatment strategy, with the ability to precisely attack specific molecular targets. It is anticipated that organelletargeted strategies will continue to attract growing interests and very likely become a pillar of precision medicine in future cancer treatment.
NUCLEUS-TARGETED STRATEGIES FOR PRECISION ATTACK LIFE BLUEPRINT
Structural components of nucleus The nucleus has been considered as the fundamental, functional building block of the cell in regards to activity regulation, including proliferation, apoptosis, and metabolism. 57 However, the structure of the nucleus remains elusive and has been a subject of lively debate. The nucleus, in many cases, was believed to have a nucleoskeleton, while people also proposed that it had no more than a transient complex and a membrane-bound bag of genetic materials. 58 With exciting development in cryo-electron microscopy, X-ray crystallography, and computer-aided proteomic techniques, significant advances in understanding the nuclear structure and nucleocytoplasmic transport pathways have been achieved in recent years. 59,60 These findings suggest that the nucleus has its own distinct substructure that acts as a dynamic organelle, rather than a rigid framework. 60 The nucleus, in eukaryotic cells, is enclosed by a double membrane separating the nucleoplasm and cytoplasm, termed as nuclear envelope (NE). 58 As a compartment border, the NE ensures versatile communication, secured macromolecule exchange between nucleus and cytoplasm, and genetic material safeguarding. Moreover, the NE is highly adaptable and dynamic, which can be reflected by its disassembly and reformation during mitosis, composition fluctuations during differentiation and deformation, and transient rupture or repairment under mechanical pressure. 61 The double membrane is designated as the inner nuclear membrane (INM) and the outer nuclear membrane (ONM), which are essential components in transporting millions of molecules per second in bi-directional nucleocytoplasmic trafficking. 62 Nucleocytoplasmic exchange processes are regulated by nuclear pore complexes (NPCs), which are approximately 30-50 nm in diameter and 50-80 nm in length, whose central channels perforate the NE as a bridge. 63,64 The NPC is a highly modular symmetrical scaffold, composed of an eightfold symmetrical ring and spoke assembly, cytoplasmic fibers, and a filamentous nuclear basket. 65,66 Furthermore, more than 500 copies of 30 different nucleoporins (nups) proteins are conserved with biochemical stability, arranged within the building blocks of NPCs. 67 A specific phenylalanine-glycine nucleoporin (FG Nups) harbors intrinsically disordered FG-rich domains, which occupied the central channel of NPCs to achieve selective transport within milliseconds. 68,69 Nucleocytoplasmic transport exhibited a robust profile under the assist of FG Nups, however, the physical properties of cargoes exert a non-negligible impact on the efficiency in nucleocytoplasmic trafficking. 70 Relatively small cargoes can permeate freely through NPCs, while large macromolecules are impeded. These large cargoes can only achieve rapid transport through NPCs with the aid of karyopherin and FG Nups interaction.
Bi-directional nucleocytoplasmic trafficking with high efficiency and selectivity in complex and crowded conditions is enabled by the highly-organized sub-units of the nucleus, coordinating with one another. 71 Elaborate structures of the NPC allow macromolecules to perform precise and efficient shuttling between the nucleus and cytoplasm for contributing to cellular homeostasis. The malfunction of nucleocytoplasmic trafficking can lead to protein mis-localization and directly affect gene expression, signal transduction, and diseases. 72 Therefore, understanding the organizing framework of the nucleus, including the substructure inside cell nuclei as well as how the nucleocytoplasmic transport is coordinated and regulated, is the essential basis for nucleartargeted transportation.
From DNA damage to cell death The nucleus is the most prominent organelle of the eukaryotic cell, which serves as the container of the majority of cellular genetic information and coordinates gene expression. 73,74 Genome integrity is paramount to life as irrevocable damage to nuclear DNA (nDNA) can adversely impact cellular function, viability, and growth. However, thousands of DNA lesions are constantly attacked per day. 75 To stay alive, the ability of an individual cell to act appropriately is important, especially when their genome is threatened by an intrinsic or extrinsic insult. 76 If damage is too severe or the attempted repair is ineffective, the DNA damage response (DDR) can trigger a proapoptotic signaling cascade and initiates an apoptotic response. 77,78 Notably, DDR defects are a pervasive hallmark of cancer cells, causing detrimental mutation accumulation.
Double-strand breaks (DSBs), which is the most severe type of DNA damage, occur when the phosphodiester backbone is disrupted on both strands. 76 Most DNA-damaging agents can cause DSBs to trigger apoptosis. 79 DSBs are considered to be a crucial initiator of the apoptosis signaling pathway. 80 Ataxia telangiectasia mutated kinase (ATM), Rad3-related, and ataxia telangiectasia-related (ATR) kinases, which initiate DNA damage checkpoints and DDR, are activated upon DSBs. Subsequently, additional checkpoint kinases, Chk1 and Chk2, are phosphorylated by ATM and ATR kinases and act downstream to activate p53. P53 then transactivates pro-apoptotic genes through transcription, such as Bax, Fas, and Puma, and induces apoptosis. Furthermore, DNA damage-mediated cell death is not solely regulated through genome regulation as complex enzymatic reactions also play an important role. 74 Cells intricately respond to DSBs by evoking related signaling pathways that may ultimately trigger DNA repair or initiate cell death-related pathways to eliminate the damage. 80 Uncovering the relationship between DNA damage and cell death can allow a deep understanding about the pathogenesis of cancer as well as the development of more effective therapies.
DNA intervention strategies Irreversible damage to DNA is a key driving force of cell death perturbation. Common DNA intervention mechanisms either arrest transcription, lesion DNA repairing processes, or block DNA replication. 81 Therefore, it is apparent that DNA intervention is a crucial mechanism in apoptosis, exerted by many toxins in cancer treatment (Table 1). A strategy of great interest and potential is to enhance cancer cell apoptosis efficiency of toxins by amplifying DNA damage.
Chemotherapeutic agents. DNA-damaging chemotherapeutic agents, such as cisplatin and doxorubicin (DOX), are widely used in chemotherapy, where they can interfere with DNA replication and transcription. Cisplatin cytotoxicity, for example, is caused by the formation of interstrand, or intrastrand, adducts with DNA, which destroys DNA function and induces irreparable DNA lesions. 82,83 Another typical DNA toxin, DOX can intercalate into stranded DNA to form DNA adducts for increasing torsional stress. Moreover, DOX inhibits enzyme topoisomerase II, thus preventing DNA replication and inducing DNA breaks. 84 In addition, several molecule inhibitors, such as Elimusertib, Prexasertib, and PHI-101, have been developed to disrupt DDR signaling pathways, thus promoting cancer cell death. As shown in Table 2, many nucleartargeted therapeutic agents are currently under clinical trials.
The efficiency of chemotherapeutic drugs acting on DNA depends on the pharmacological effective drug concentration at Organelle-targeted therapies: a comprehensive review on system design for. . . Yang et al. the nuclear site. However, it is pertinent to point out that onlỹ 1% of cisplatin and~0.4% of Dox could pass through intracellular barriers and reach the nucleus in a pharmacological concentration. 85,86 Notably, successful transport to the nucleus utilizing a nuclear-targeted strategy would significantly boost therapeutic outcomes as the damage in the nucleus is destructive to cells. As an example, Fan et al. 87 constructed biomimetic nanocarriers (GTDC@M-R) based on erythrocyte membraneencapsulated graphene oxide quantum dots (GOQDs) for DOX and CS-6 delivery. TAT and RGD peptides were attached to the surface of GOQDs and erythrocyte membranes to achieve dualtargeting of the nucleus and triple-negative breast cancer (TNBC) cell membranes. In this study, Gamabufotalin (CS-6) markedly reduced aggressiveness in TNBC via down-regulation of vascular endothelial growth factor (VEGF) expression and inhibited angiogenesis. As a result, GTDC@M-R regulated the signaling pathway of apoptotic (BAX/Bcl-2 and p53) and metastasis (COX-2 and VEGF), which effectively suppressed tumor growth, invasion, and metastasis.
Thermal ablation. High temperature can inhibit DNA replication, [88][89][90] due to thermal-mediated enzyme denaturation related to DNA replication (such as DNA polymerase α and β responsible for DNA replication and repair). Additionally, the aberrant condensation of nuclear matrix proteins induced by hightemperature leads to blockage of DNA replication. 90 Collectively, the local high temperature locally at the nucleus, leads to defects in normal DNA functions and is responsible for thermal-mediated death of cells.
Nuclear-targeted thermal ablation nanoplatforms only require low-power density to achieve high-efficiency therapeutic treatment, which may be a practical approach for optimal cancer treatment. 91,92 To date, gold nanomaterials such as nanorods, nano-stars, nanocages, and nano-shells have been employed as nuclear-targeted thermal ablation nanoplatforms due to their remarkable surface plasmon resonance (SPR) effect, highefficiency in light-to-heat conversion, and excellent photothermal stability, which can collectively enhance the therapeutic efficacy. 93 Pan et al. 94 synthesized a nuclear-targeted therapeutic system (GNRs-NLS) which caused DNA damage and DNA repair process failed at low NIR intensity (0.2 W/cm 2 ), resulting in apoptosis without excessive inflammation.
Phototoxicity. It is pertinent to note that excessive reactive oxygen species (ROS) can serially damage DNA upon lipophilic photosensitizer accumulation in the nucleus under laser irradiation, leading to single-strand breaks and inactivated DNA repair enzyme. 95,96 Moreover, photodynamic therapy (PDT) induces destabilization of [Ca 2+ ] and ROS-induced nuclear-pore expansion which directly damage the nucleus and lead to apoptosis. 97,98 Nuclear-targeted photosensitizer chlorin (Ce6) was first developed by Akhlynina in 1997, showing that nuclear-targeted PDT enhanced therapeutic effects with EC 50 can decrease by almost 2000-fold. 24 Accurate bombardment has increased viability in PDT, with the nucleus as the damage-sensitive site, which demonstrates that nuclear-targeting as an effective strategy for enhancing PDT in cancer treatment.
nDNA expression interference. Gene therapy is a promising strategy for enabling a permanent cure in cancer research. Extraneous genes (double-strand DNA (dsDNA), single-strand DNA (ssDNA), plasmid DNA, antisense oligonucleotide, and small interfering RNA) are developed to interrupt, eliminate, or correct genetic defects and anomalies to alter endogenous gene expression. [99][100][101] As of November 2017, gene therapy clinical studies were reviewed to encompass 2,597 trials within 38 countries. 102 However, a lack of full understanding about its safety and efficiency within this rapid-developing technological area Organelle-targeted therapies: a comprehensive review on system design for. . . Yang et al. currently hinders their practical implementation. Therefore, developing innovative, safe and robust approaches to achieve accurate therapeutic agent nuclear accumulation to circumvent existing challenges is of great significance in cancer treatment.
The future of nuclear-targeted therapies design Nuclear translocation continues to be a complicated spatiotemporal challenge. Entry into the nucleus is considered to be regulated by the transport kinetics of NPCs. 103,104 Deciphering the nuclear import machinery will further facilitate the construction of nuclear-targeted nanosystems.
Passive diffusion. Passive diffusion is the equilibration of relatively small cargoes, macromolecules with up to 40 kDa in molecular weight, between the cytoplasm and the nucleoplasm, which is a result of Brownian motion and without specific interaction with the NPC domain (Fig. 3). 105 The specific size and shape of cargoes are found to be the permeant determinants of passive diffusion rates. 69 The passive diffusion behavior of cargoes through the NPC highly depends on the size threshold. Paine et al. 106 proposed that the patent radius of NE pores is approximately 45 Å, which can restrict molecular movement in nucleocytoplasmic transport. This observation revealed the sieving properties of NE. Since then, substantial research has been performed on size limitation in passive diffusion and it was found that passive diffusion is relatively fast for small cargoes with molecular weight in the range tof 20-40 kDa. 107 Passive transport rates became more restricted and inefficient beyond such thresholds.
Recent in vitro studies evaluated the effect of size-dependency on permeability and intranuclear accumulation of tiopronin-covered Au nanoparticles (Au-TIOP NPs) with diameters of 2, 6, 10, and 16 nm in MCF-7 cancer cells. 108 After 24 h, the larger Au-TIOP NPs were primarily localized in the cytoplasm. However, Au-TIOP NPs with diameters less than 10 nm could efficiently enter the nuclear. Additionally, folic acid (FA) modified carbon quantum dots (CDs) with diameters smaller than 9 nm exhibited excellent nuclear translocation efficiency in oral cancer cells. 109 In general, cargo sizes smaller than 9 nm enable efficient and unrestricted permeation to the NE, whereas larger cargoes exhibit limited transport.
Cargo morphologies can modulate the rate and efficiency of barriers crossing during nuclear import. 110 Gaus et al., 27 were inspired by the shape of pathogens, which developed nanoparticles with varying shapes (including vesicles, micelles, rods, and worms) and identical surface chemistry. Their results demonstrated that rod-and worm-shaped nanoparticles with a high-aspect-ratio tended to have higher nuclear accumulation. Therefore, highaspect-ratio nanoparticles seem to be more promising for nuclearspecific accumulation, enabling a significant increase in concentration within the nucleus compared to spherical nanoparticles. The well-defined size and geometry of nanomaterials are key design parameters that need to be considered for designing nucleartargeted therapeutic nanoplatforms.
Active targeting. Passive diffusion depends on several critical properties of nanomaterials as it is driven by a concentration gradient. 69,105 Highly dynamic and disordered proteins inside of each NPC, such as FG Nups, can impede the nuclear entry of macromolecules larger than 9 nm in diameter (or molecular weight higher than 40 kDa). 111 Moreover, nuclear accumulation through passive diffusion ceases as the concentration between nucleoplasm and cytoplasm reaches equilibrium. 112 The efficiency of passive diffusion is frequently limited by several factors, as such it is not the preferred choice for nucleus targeting. Fortunately, the nuclear localization signal (NLS) facilitates transport and accumulation of macromolecules in the nucleus. 113 It has been reported that NLS-containing cargos are actively transported into the nucleus through NPCs within 30 min. 114 The NLS was first reported based on the simian virus 40 (SV40) large T-antigen by Kalderon et al. in 1984, and has attracted significant attention since then. 23 The NLS sequence has been classified into classical NLS (cNLS) and non-classical NLS (ncNLS). 115,116 The most well-characterized NLS is the cNLS, which contains a single stretch of a basic amino acid sequence of 4-8 amino acids, primarily including positively charged lysine (K) and arginine (R) residues, with the essential functional sequence of cNLS being K-(K/R)-X-(K/R). 117 For example, the sequence of simian virus 40 large T antigen (SV40T) is PKKKRKV. For comparison, ncNLSs do not require these characteristics, such as the prolinetyrosine nuclear localization signal (PY-NLS). 118 The import pathway of NLS cargoes is shown in Fig. 3. This nucleocytoplasmic transport is orchestrated by nuclear transport receptors, referred to as karyopherins, where "importins" and "exportins" regulate the import and export of signal-specific cargoes. Within the cytoplasm, importin α (Impα) recognizes and binds to NLS cargoes and, subsequently, forms a heterodimer complex with importin β (Impβ), which can be abbreviated as NLS-cargo•Impα • Impβ. Subsequently, Impβ explicitly interacts with the FG Nups to form the NLS-cargo•Impα • Impβ, which is localized at the nucleus. Ran guanosine triphosphate (RanGTP) then dissociates the complex by inducing spatial conformation changes of Impβ, resulting in NLS-cargo and Impα releasing into the nucleus. Finally, through the assist of RanGTP and Cse1, Impα is transported to the cytoplasm where it lies in wait for the next round of cargo transform. 119 This process is also applicable to the non-classical nucleocytoplasmic transport pathway, where Impβ directly binds and transports ncNLS-cargos without involving impα. 120 NLS is necessary for translocating large nanoparticles into the nucleus. In 1988 it was found that NLS-coated gold nanoparticles with a diameter of 26 nm were successfully transported across the NPC and achieved nuclear localization. 25 These results led to the understandings that the threshold size of NLS-cargoes through the NPC was 26 nm. Up until 2001, the feasible diameter of NLS-cargoes complexes was speculated to increase by about 8 nm, due to classical nucleocytoplasmic transport principle development. 107 In 2002, Kann et al. 121 re-defined the threshold of cargo-receptor-gold complexes; NLS cargoes as large as 39 nm in diameter were able to across the NPC without disassembly process occurred. Moreover, it is currently under investigation that if chitosan nanoparticles with diameters varying between 25-150 nm, 122 or polymeric nanoparticles with diameters~234 nm, 123 can achieve nuclear accumulation under the regulation of NLS.
It is still confounding that the NPC allows nanocarriers to traverse, whose immense sizes far exceed the maximum pore diameter of the NPC. The intranuclear accumulation of large nanoplatforms is separated from the participation of Impα/β, NLS, and RanGTP, due to the selective barrier of NPC. 124 Interestingly, the interaction between NPCs and impα·impβ·NLS-cargos can result in NPC barrier reduction, nuclear pores, and NLS-cargo deformation. 124 These results can be used to explain how large nanocarriers bypass the NPC barrier and enter the nucleus.
The significance of NLS in the nucleocytoplasmic transport of macromolecules is clear. Strategies involving NLS incorporation also significantly impact nuclear transport efficiency of ultra-small nanoparticles. 125,126 Nevertheless, the electrostatic interactions between NLS and nanoplatforms may disrupt the stability of NLS, resulting in the failure of active nuclear-targeted transport. 127 Therefore, randomized utilization NLS may not enhance the efficiency of nuclear translocation. Understanding the elaborate nucleocytoplasmic transport trafficking pathway in specific cells is vital for enabling the nuclear entry.
Nuclear envelope permeability enhancement. The NE, a complex double-membrane system, separates the nucleus and the cytoplasm while safeguarding nuclear compartmentalization. 128,129 It was generally accepted that NE transient rupture only occurs during mitosis. 130,131 However, recent studies revealed that reactive oxygen species (ROS) and mechanical forces allow the NE to dismantle in a spatiotemporally controlled manner (Fig. 3). Enhancing NE permeability by controlling nuclear compartmentalization may facilitate the nuclear entry of large nanomaterials whose dimensions exceed the NPC restrictions.
ROS are highly destructive chemicals that predominantly lead to lipid peroxidation in the membrane. 132,133 Excessive accumulation of ROS damages phospholipids directly by affecting the fluidity and permeability of lipid bilayers, and ultimately compromises membrane integrity. More importantly, ROS may attack bio-membranes and subsequently induce various types of cell death, such as apoptosis, autophagy and ferroptosus. 134,135 Wu et al. 136 utilized light irradiation to stimulate ROS generation and facilitate nanoplatform nuclear entry. The nanoplatforms were fabricated with polyamine-containing polyhedral oligomeric silsesquioxane (POSS), polyethylene glycol (PEG), and rose bengal (RB), denoted as PPR NPs. Under mild-light irradiation, PPR NPs generate single oxygen species ( 1 O 2 ), which escapes from the endolysosomal compartment, and further accumulates near the nucleus to increase the permeability of NE. PPR NPs successfully deliver payloads that scarcely cross the NPC into the nuclei, and therefore functional payloads cause irreversible damage to cancer cells.
Nuclear envelope rupture, or the NER effect, is another mechanism that increases NE permeability and promotes large macromolecule passive migration. However, transient NER with incomplete sealing of the NE may yield exposure of DNA to the cytoplasm, which then leads to DNA damage. The NER effect can be controlled by vapor nanobubble-mediated photoporation of Au NPs. 137 Upon laser activation, the temperature of perinuclear Au NPs rapidly increase and short-lived vapor nanobubbles (VBN) accumulate around the perinucleus. Following VBN collapse, high-pressure shock waves occur which lead to mechanical force impairments of NE, and the large nanoplatforms accumulate inside the nucleus due to the incomplete NE. Therefore, nuclear photoporation in a spatiotemporally controlled manner provides a powerful tool to achieve specific nuclear targeting therapeutics in oncology. Unlike the NLSmediated nuclear translocation strategy, enhanced permeability of NE allows larger sized nanoparticles to enter the nucleus and be highly efficacious.
Nuclear pore expansion. The nucleocytoplasmic transport efficiency of large nanomaterials could be determined by inducing nuclear pore expansion, for example, under the effect of Design guidelines of nuclear-targeted nanosystems. Small cargoes cross the nuclear envelope through passive diffusion, without interaction with NPC. Active targeting is necessary for large cargoes. NLS-cargoes interact with nuclear transport receptors to achieve large cargoes accumulation in the nucleus. Nuclear envelope permeability enhancement allows ROS and mechanical forces to enhance the permeability of the nuclear envelope, and thus facilitate large cargo nuclear transportation. Nuclear pore expansion is a direct manner that dexamethasone regulates to expand the size of NPC. INM inner nuclear membrane, ONM outer nuclear membrane, NPC nuclear pore complexes, NLS nuclear localization signal, Impα importin α, Impβ importin β, RanGTP Ran guanosine triphosphate dexamethasone (Dex), which is a commonly applied synthetic glucocorticoid (Fig. 3). 138,139 A plethora of studies have been performed to elucidate Dex-mediated behaviors of nucleocytoplasmic transport. Shahin et al. 26 observed the possible effects of Dex on Xenopus laevis oocytes during nucleocytoplasmic transport, which were visualized by atomic force microscope (AFM). It was found that the apparent diameter of NPCs was remarkably enlarged up to almost 60 nm within 90 s after injecting Dex. Specifically, Dex induced dilation and conformational changes in NPCs within the ONM due to the triggering of an intracellular signal cascade in the nucleus.
Dilation behavior of NPCs, mediated by Dex, is of vital significance for nuclear translocation of nanoplatforms. Similar results were found in the study by Kastrup et al. 140 where NPCs of Xenopus laevis oocytes dilated to 110 nm within minutes of Dex treatment, followed by increased expansion in the NPC with diameters up to~140 nm after 5-11 min. Furthermore, pores up to 300 nm in diameter were also observed. Dex is highly specific and selective to glucocorticoid receptors (GR), expressed in almost every nucleus. 26 Consequently, Dex has been employed to achieve cancer-cell-specific nuclear-targeted therapeutic agent delivery. In one of the studies performed by Ye et al., 141 Dex was used to modify WS2 nanocomposites to achieve precision with ROS-and thermal-sensitive subcellular organelles, causing irreversible damage to cancer cells. It is now recognized that Dex could be employed to enhance the nuclear pore expansion, assisting nuclear-targeted strategies by regulating nuclear entry behaviors and promoting nuclear translocation of macromolecules.
MITO-BOMB: MITOCHONDRIAL-TARGETED STRATEGIES
A brief overview of mitochondria In the 1950s, mitochondria were first postulated to be linked to cellular bioenergetics after the Krebs cycle discovery. 142 Following in-depth investigations into the cell biology of mitochondria, it was found that they can serve as the fundamental centers of cell death, controlling a plethora of signaling cascades. 143 Mitochondria often participate in and orchestrate complex cellular processes, from controlling cell division and differentiation, to regulating cell growth and death. 144,145 Their versatile functionalities are associated with mitochondrial architecture and biochemical activity.
Mitochondria are defined as dynamic organelles with complex intramitochondrial compartments, including the outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), and the mitochondrial matrix (MM), as shown in Fig. 4a. 60,146 Each intramitochondrial compartment provides unique biochemical reaction environments to maintain homeostasis and regulate metabolism.
The OMM, as the interface between the mitochondria and cytoplasm, coordinates the process of small molecule permeation and mediates the transduction of mitochondrial signals. 147 In addition, the OMM serves as the membrane contact site to exchange constituents between the mitochondria and other organelles, including the lysosome and ER. Specific host proteins in OMM, such as translocases, can mediate mitochondrial precursor protein transport. The IMM, another mitochondrial membrane, exhibits a heavily folded structure which can be further divided into the inner boundary membrane (IBM) and mitochondrial cristae. 148 IBM that runs in parallel to the OMM harbors high amounts of channel transporters which shuttle ions and mitochondrial respiration complexes. 149 The IMM invaginates and forms the cristae that provides optimal surface areas for mitochondrial respiration. The cristae is a critical site for the oxidative phosphorylation pathway (OXPHOS), as it hosts various respiratory chain complexes as well as F 1 F o -ATP synthase. 150,151 Moreover, cytochrome c, the caspase activator during apoptosis, can be localized at the intracristal compartment. Thus, the IMM not only participates in mitochondrial respiration and mitochondrial energy conversion, but also impacts the apoptosis.
The OMM and IMM are separated by the IMS, which acts as a critical buffer between the cytoplasm and the MM. The IMS is essential for mitochondrial metabolism and free radical scavenging, especially for maintaining cellular homeostasis. 152 The MM is involved in metabolic reactions by regulating tricarboxylic acid, fatty acid oxidation, and amino acid metabolism. 153 Moreover, MM contains mitochondria genetic material, mitochondrial DNA (mtDNA), which encodes mitochondrial proteins for ATP production. Mutations of mtDNA often cause mitochondrial dysfunction, ultimately resulting in a devastating array of mitochondrial diseases. 154 Obtaining a comprehensive understanding about the relationship between mitochondrial structure, function, and biochemical activity will promote the development of therapeutic modulation based on mitochondrial dynamics.
Consequences of mitochondrial dysfunction
As a double-edged sword, mitochondria not only generate the majority of cell chemical energy, but they are also critical modulators of programmed cell death (Fig. 4b). Dozens of death signaling pathways are localized in the mitochondria which exert lethal functions in pathological conditions. 155 Mitochondria, as essential regulators, control the activation of the intrinsic apoptosis pathway. The mitochondrial outer membrane permeabilization (MOMP) represents a critical event during intrinsic apoptosis. 156 Several factors have been identified that can contribute to mitochondrial permeability transition pore (mPTP) opening, such as signal transducers protein P53, AKT kinase activating protein BH3, pro-apoptotic factors (Bax, Bak, Bid and Bad) or anti-apoptotic factors (Bcl-2, Bcl-XL, Mcl-1). 157,158 MOMP directly leads to the release of apoptotic factors (cytochrome c, Smac/Diablo, Omi/HtrA2) from the mitochondria and into the cytoplasm. Further multimeric apoptosome is recruited when cytochrome c binds to APAF1, and activates procaspase 9. 144,155 Subsequently, executioner caspase 3 and 7 are activated, initiating a caspase cascade for cancer cells apoptosis. 159 Of note, MOMP and cytochrome c release are feature points of intrinsic apoptosis.
Mitochondria also play a crucial role in non-apoptotic cell death, particularly in mitophagy and necroptosis. Mitophagy refers to the process of the degradation or elimination of dysfunctioning, impaired or depolarized mitochondria, for maintaining homeostasis of the intracellular environment and normal cellular function. 160 Increased evidences have indicated that mitophagy suppresses metastatic growth in the early stage of cancer and promotes advanced cancer survival. 161,162 As an emerging target, mitophagy is available for invasive cancer treatment. 163 Necrosis is always considered as an accidental, uncontrolled, and highly inflammatory form of cell death. 144,164 However, some studies pointed out that its occurrence may be regulated. In some circumstances, necrosis is closely related to mitochondrial dysfunction, such as reactive oxygen species (ROS) overgeneration and ATP depletion, also termed necroptosis. 165 As a non-apoptosis, pro-inflammatory, and caspaseindependent cell death modality, ferroptosis is regulated by the lethal accumulation of iron-dependent lipid peroxides. 166 Under normal conditions, most iron is sequestered into iron-binding proteins and controlled by glutathione peroxidase 4 (GPX4) and glutathione during utilization. 167 Iron is required in vital processes, such as respiration and DNA synthesis, and acts as a co-factor in the Fenton reaction to generate highly reactive hydroxyl radicals. 41 Nevertheless, excessive iron loading leads to oxidative damage through the Fenton reaction, killing the cell by attacking lipid bilayers of membranes. 134 Recently, studies have shown the relationships between mitochondria and ferroptosis. Gao et al. 41 found that MMP hyperpolarization is related to cysteine-deprivation-induced (CDI) ferroptosis. In addition, Fang et al. 168 observed that mitochondria-targeted antioxidants (Mito-TEMPO) enable the suppression of DOX-induced ferroptosis-induced heart damage. These findings support the effect of the mitochondria on ferroptosis. While great progress has been made in this area, studies of mechanisms and relationships between ferroptosis and mitochondria are still in their infancy; much remains to be investigated. Unraveling the relationship between mitochondria and cell death will inform the design of Fig. 4 Personalized therapeutic strategies toward mitochondria. a The structure and function of mitochondria are displayed, with emphasis on the TCA cycle and β-oxidation. In contrast, cancer cells rely on the "Warburg effect" to achieve energy supply. b Mitochondrial dysfunctions to trigger cell death include metabolism disruption, redox state imbalance, and perturbation of mtDNA. Once mitochondrial damage and mPTP opening occur, cell death may occur by mitophagy, apoptosis, necroptosis, or ferroptosis. IMM inner mitochondrial membrane, OMM outer mitochondrial membrane, MM mitochondrial matrix, PDK dehydrogenase kinases, GAPDH glyceraldehyde 3-phosphate dehydrogenase, ROS reactive oxygen species, GSH glutathione, Cyt C cytochrome C treatment solutions for improved cancer therapeutic effects. More importantly, further development of therapeutic agents which target mitochondrial-mediated cell death pathways will be expected to cure difficult-to-treat tumors.
Intervention of mitochondria to control cancer cell fate In 1956, Otto Warburg first proposed that the mitochondrial respiration defect has crucial involvement in cancer pathophysiology. 38 Multiple hallmarks of cancer have been associated with mitochondrial dysfunctions, such as unlimited proliferation, anabolism enhancement, and apoptosis pathway impairment. 169,170 Mitochondrial DNA (mtDNA) mutations have been reported in various cancers. 170 The reprogrammed metabolism negatively affects mitochondrial metabolism for facilitating adaption of cancer cells to tumorigenic microenvironment. 171 Therefore, mitochondria represent a promising target for eradicating cancer cells (Table 1). In general, mitochondrial membrane potential (MMP) loss, MPTP opening, and MOMP trigger proapoptotic protein released from IMS, which promotes apoptosmone formation and caspase cascade reaction activation, resulting in cell apoptosis. 172 Herein, we will introduce a series of intervention mechanisms that cause mitochondrial structure and function abnormalities (Fig. 4b).
Metabolism disruption. As vital organelles for energy generation, mitochondria can convert glucose, fatty acids, and amino acids to adenosine triphosphate (ATP), which rely on interwoven complex biochemical processes, including oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and β-oxidation. 173 Distinct from normal cells, cancer cells rely on aerobic glycolysis as the predominant energy source, known as the Warburg effect. [174][175][176] The intermediate metabolites during aerobic glycolysis, nucleotides, lipids, and amino acids, satisfy the energy demand of cancer cells for rapid growth and proliferation. 177,178 Additionally, tumor migration, invasion, and metastasis are more prone to develop due to glycolysis, creating a tumor microenvironment with acidification and hypoxic. 179,180 Metabolism remodeling directly drives anti-apoptosis occurrence of the most aggressive malignant tumors. Therefore, metabolism interference can help promote cancer cell apoptotic.
As demonstrated by several studies, multiple isoforms of pyruvate dehydrogenase kinases (PDKs) are universally overexpressed in cancer cells, resulting in pyruvate dehydrogenase complex (PDC) inactivation and OXPHOS compromise. [181][182][183] PDKs are thus defined as the essential target for inhibiting glycolysis from rearranging metabolic pathways and, subsequently, the cell death. 184 Kolb et al. 185 constructed a mitochondrial-targeting system (Mito-DCA) to inhibit glycolysis by impeding PDK1 function. The orphan drug dichloroacetate (DCA) and lipophilic triphenylphosphonium cation (TPP) were selected as mitochondrial kinase inhibitors and mitochondrial-targeting factors. Mito-DCA can enhance therapeutic efficacy by reversing the glycolytic phenotype of cancer cells. Another type of glycolytic inhibitor, 3-bromopyruvate (3-BP), can block the function of hexokinase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which are involved in the glycolytic process, and ultimately induces apoptosis of cancer cells. 186,187 Liposome nanoparticles have been developed for site-specific, local delivery of 3-BP, minimizing side effects such as hepatotoxicity as well as being appliable to additional aerobic glycolysis-targeting drugs. 188 Strong evidences have indicated that glycolysis might serve as the essential target to enhance therapeutic effects. As of now, there are 46 anti-cancer drugs with glycolysis targets, including 3-BP and DCA, that have entered clinical development or clinic translation ( Table 2). 189 Therefore, regulating glycolysis-related pathways would help develop glycolysis inhibitors to achieve suppression of tumors, which will usher in a new dawn in the age of cancer treatment.
Redox state imbalance. The majority of ROS by-products are generated in mitochondrial respiration. 190,191 During this process, approximately 2% of oxygen is converted to ROS precursors, such as superoxide anion radical. Nevertheless, if not detoxified, intracellular ROS may cause disturbances in mitochondrial functions (when over a critical threshold), such as MPTP, MOMP, and mtDNA damage. [192][193][194][195] Under these circumstances, an imbalance of intracellular ROS results in irreversible cell apoptosis. Moreover, ROS, as the signaling molecules, can initiate the signaling path of proliferation and promote the formation of blood vessels, which are essential for developing distant metastases of malignant cells. 196 Therefore, many aberrant proliferative cancer cells are characterized by elevated levels of ROS relative to the antioxidant level of a system, termed oxidative stress. High levels of oxidative stress render cancer cells more vulnerable to the effects of exogenous substances, which can cause an imbalance in redox homeostasis. 197 The redox state of mitochondria is a tempting target for the efficient treatment of cancer because mitochondria are susceptible to damage from oxygen radicals. 198,199 Currently, several photosensitizers as well as therapeutic agents have entered clinical trials, such as β-lapachone (ARQ 501), menadione (2-methyl-1,4-naphthoquinone), and motexafin gadolinium, which participate in redox cycles in the respiratory chain to trigger excess generation of ROS for cancer treatment (Table 2). However, the excess endogenous antioxidant GSH in cancer cells scavenges ROS, making it very difficult to accumulate up to toxic levels. 170,196 A mitochondrial oxidative stress amplifier was designed by Liang et al. 200 Specifically, mitoCAT-g, supported by carbon dots loaded with atomically gold atoms (CAT-g) and conjugated with mitochondrial-targeted agent TPP and ROS generation agent cinnamaldehyde (CA) was investigated for their cancer treatment capabilities. Intracellular GSH was depleted due to covalent Au-S bonding generated between atomic gold and GSH. Therefore, ROS-mediated damage may occur once CA generated abundant amounts of ROS. MitoCAT-g drives the alteration of mitochondrial membrane potential (MMP) by modulating oxidative stress, leading to mitochondrial dysfunction, and ultimately resulting in cell apoptosis.
Perturbation of mtDNA. Mitochondrial DNA, or mtDNA, consists of circular double-stranded DNA with a length of 16.6 Kb, 201 which is indispensable during the biochemical process of energy production and metabolism, primarily responsible for encoding polypeptides of the respiratory chain. The encoding of 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs) associated with mitochondrial proteins is inseparable from the participation of mtRNA. 195,202,203 Indeed, mtDNA is more susceptible to oxidative damage than nuclear DNA (nDNA) due to the lack of histone protection and inefficient DNA repair capacity; thus, mtDNA has an extremely high mutation frequency. 204,205 Mutations and deletions of mtDNA lead to mitochondrial dysfunction and can affect the electron transfer of the respiratory chain and the efficiency of ATP production, resulting in the dysregulation of cell proliferation and differentiation and enhancing the risk of carcinogenesis. 206,207 Therefore, repairing and/or degrading mutated mtDNA are crucial for improving patient prognosis and therapeutic outcomes.
Small interference RNA (siRNA), or exogenous therapeutic DNA, have been used to regulate mtDNA expression for tumor suppression. 208,209 However, these free therapeutic genes could not achieve endosome/lysosome escape and mitochondrial localization. Weissig et al. 210 designed a mitochondrial-targeted DQAsome vehicle to target delivery plasmid DNA (pDNA), where pDNA-mitochondrial leader sequence (MLS) peptide was loaded into mitochondriotropic cationic "bola-lipid"-based vesicles to form DQAplexes-DNA complexes (DQAplexes). DQAplexes could escape from endosomes and further selectively release pDNA to the site of mitochondria, achieving the goal of therapeutic transgenes to express into mitochondria.
Smart design of mitochondria-targeted nanosystems As mitochondria function is closely associated with cell death, mitochondria-targeted therapeutic agents represent a promising approach to eradicate cancer cells. [211][212][213][214] However, unlike nuclei, mitochondria are highly impermeable organelles, where the transport and permeation of therapeutic agents are challenging due to the double-membrane architectures. 191,215 The IMM has a complex structure composed of more than three times the proteins/lipids compared to cell membranes. 216 Additionally, oxidative phosphorylation that occurs in IMM can cause large MMP, usually between −160 mV and −180mV. 217 The complex IMM structure with high negative membrane potential and hydrophobicity render it difficult for macromolecules and bioagents to transport through the IMM for reaching the MM. 218 There is a strong need for developing mitochondrialtargeted nanoplatforms which can satisfy the key requirements of cancer treatment.
Delocalized lipophilic cation. The large, negative membrane potential and high lipid content of mitochondria collectively favor selective transportation and mitochondrial accumulation of lipophilic cations. Commonly used delocalized lipophilic cations (DLCs) include triphenylphosphonium (TPP), aedualinium (DQA), berberine (BBR), rhodamine, and cyanine dyes. 154,219,220 Among these molecules, TPP acts as the representative DLC, which is commonly used in mitochondrial-targeted nanosystem construction. 221 According to the Nernst equation, TPP enables passage rapidly through the mitochondrial membrane and achieves almost 1000-fold accumulation in the mitochondria, driven by the MMP (at −180mV) and hydrophobic effect. 222 Thus, TPP can play an instrumental role for mitochondrial-targeted therapeutic vehicle construction in malignant cancer treatments. 214,220,223 DLC (as mitochondrial-targeted agents) has been widely employed in constructing various biomolecule probes and therapeutic agents. 224 At high concentrations, it stimulates or even induces cytotoxicity against mitochondria which results in cell death. Underlying toxicity from DLCs primarily involves inhibiting F0F1-ATPase, limiting the activity of a respiratory enzyme, interference with mtDNA, and/or inducing mitochondrial membrane depolarization. These phenomena can cause mitochondria dysfunction and decreasing ATP generation. 218,223 Additionally, the use of DLC is limited by the polarity of cargoes. DLC acts as well-investigated carriers of lipophilic or small polar molecules, yet exhibiting unsatisfactory efficiency in large polar molecule mitochondrial transportation. 144 Peptide. An emerging strategy to target mitochondria is using peptide-based nano-systems, in which the sequence or the structural motif of the peptide could be rationally designed depending on the needs, compared with the DLC system. Inspired by cell-penetrating peptides (CPP), Horton et al. 225 first designed a mitochondrial penetrating peptide (MPP) and confirmed its promotion of cell internalization and intramitochondrial localization. Among them, methylated lysine (K), arginine (R), phenylalanine (F), and cyclohexylalanine (Fx) were selected as the MPP units in order to respond to the unique lipophilic characters and negative potential of mitochondrial membranes. Localization analysis in HeLa cells demonstrated that MPP exhibited excellent mitochondrial localization and facilitated mitochondrial membrane fusion, further corroborated by additional studies with similar results. 226 MPP exhibits excellent mitochondrial-targeted ability and protects mitochondrial anoxia from damage, as well as provide great potential in mitochondrial-targeted nanoplatforms design. MPP, with expected pharmacokinetic profiles, is currently undergoing active development focused on mitochondrial-related diseases.
In addition to MPP, Szeto-Schiller (SS) peptides, XJB peptides, and ATAP peptides are also used for mitochondrial-targeted nanoplatform construction. SS peptides were initially developed as antioxidants for reducing ROS generation and inhibiting mitochondrial permeability transition. 227 Later, SS peptides were observed to cross the IMM based on the electrostatic interactions to achieve mitochondrial accumulation. SS-31 (D-Arg-Dmt-Lys-Phe-NH 2) is a SS peptide utilized for ischemic brain injuries by scavenging the toxic ROS, reaching phase II trials. XJB-5-131 peptide (Leu-D-Phe-Pro-Val-Orn) is a derivative of gramicidin S. 37 Unlike other peptides, XJB-5-131 peptide can enter the IMM, rather than relying on MMP to achieve mitochondrial localization. 144,228 Mitochondrialtargeting peptides are an intriguing platform for allowing structure design and biopharmaceutical function by manipulating the subsequence of a peptide.
Mitochondrial targeting sequence. In mitochondria, 98% of proteins are encoded from the nuclear genome and synthesized in the cytoplasm, which are then translocated to different compartments of mitochondria. 229 Notably, highly-efficient migration of the precursor proteins to mitochondria depends on an N-terminal or C-terminal mitochondrial targeting sequence (MTS). 230 MTS primarily includes the N-terminal sequences and tail-anchored sequence composed of a positively charged and hydrophobic stretch of 20-40 amino acid residues, so MTS possesses a hydrophobic surface containing positive charges. 144,231,232 Evidences have been presented that MTS could be recognized by the mitochondrial import protein and further inserted into the OMM and IMM, or undergo interactions with the mitochondrial protein import complex, which ultimately achieve translocation across the mitochondrial membranes. 36 Moreover, it is worth noting that MTS exhibits broad applicability in transporting various polar molecules. Therefore, it is important to select site-specific mitochondrial-targeted MTS for target-specific therapies, according to the heterogeneity of the disease. While MTS can exhibit excellent biocompatibility, MTS-cargo transportation is limited by the MTS transport channel size in the IMM and OMM to a certain extent. 233 Cardiac cells, for example, allow NP transport through the OMM only when sizes are below 3 nm, while the IMM restricts NPs with sizes greater than 2 nm. 233,234 Therefore, MTS faces stringent cargo size limitations. As such, developing versatile nano-systems with varying shapes/sizes, can provide a promising alternative solution to meet the need of specific mitochondrial compartment localization. Fortunately, increases in DLC-mediated MMP and peptide-mediated membrane fusion promote macromolecular translocation in mitochondria. It is essential to select the most appropriate correlation of mitochondrial-targeting agents, according to the cargoes unique physio-chemical properties and the reaction site-specific targets (IMM, OMM, and IMS), which can maximize the treatment efficacy.
LYSOSOMES-TARGETED STRATEGIES-TWISTING CELL SUICIDE SWITCH
Structure of lysosome The lysosome, known as the "suicide bags" of the cell, were first described by Christian de Duve in 1955. 43 This simplified understanding of the organelle has deeply evolved since, and now it is perceived as a crucial component in degrading and recycling cellular waste (Fig. 5a). 235 Broadly speaking, lysosomes are spherical or ellipsoidal, which is no more than 1 μm in size with primarily perinuclear distribution. The shape, size, and quantity of these features vary largely depending on the cellular state and cell type. 236,237 Lysosomes are single membrane-enclosed vesicles composed of a 7-10 nm phospholipid bilayer, containing a unique Fig. 5 The personalized therapeutic strategy toward lysosomes. a Lysosomes play a vital role in exocytosis, endocytosis, autophagy, and cell death. b LMP induction, as a typical approach, can be triggered by ROS, toxin reagents, radiation, and magnetic fields, eventually leading to caspase-dependent cell death. Proton pump inhibition is another strategy that enables overcoming MDR. Furthermore, HSP70 inhibition and iron release increase sensitivity to lysosomal-dependent cell death (LDCD). LMP lysosomal membrane permeabilization, ROS reactive oxygen species, Cyt C cytochrome c, PPI proton pump inhibitors, HSP 70 heat shock protein 70 acidic lumen with a pH of 4.5-5.0. 238,239 The acidic lumen is an integrated system maintained by proton pump V-ATPases, ion channels, and membrane transport proteins, to collectively provide an optimal environment for the degradation of hydrolytic enzymes. 240,241 Up to now, almost 60 hydrolytic enzymes have been found in lysosomes, including sulfatases, proteases, phospholipases, and phosphatases. They can participate in autophagy and process the digestion and recycling of macromolecules, organelles, and exogenous substances to remobilize nutrients and maintain cellular homeostasis. 242,243 Furthermore, lysosomes are inseparable from various essential processes including plasma membrane repair, mitogenic signaling, energy metabolism, immune responses. [244][245][246] Lysosomal function defects impose a heavy burden, with approximately 50 monogenic diseases associated with lysosomal dysfunction, such as lysosomal storage disorders (LSDs) caused by mutations of lysosomal proteins. 247 Thus, lysosomes are of fundamental physiological importance in cell life activities and are anticipated to be an emerging target for multiple diseases.
Lysosome responding to cell death Lysosomes, the vital command-and-control organelle for cellular metabolism and signaling, is associated with cell survival and death, including apoptosis, necrosis, and autophagy. 238,248,249 It has been reported that lysosomes can stimulate cancer cell invasion, angiogenesis, and drug resistance, correlated with poor prognosis. Even though lysosomes increase the tumorigenic potential of cancer, they are more fragile, with higher instability and sensitivity to the death of cancer cells. 250 In some particular situations, lysosome-mediated cell death programs (initiated with hydrolytic enzyme release) are termed lysosomal-dependent cell death (LDCD). 251 The two-sided effects primarily depend on the location of lysosomal enzyme release, which is related to the process of lysosomal membrane permeabilization (LMP) and exocytosis (Fig. 5b). 252 Intracellular released cysteine cathepsins result in cancer cell diminishment, whereas they are prooncogenic if extracellulary released as they then promote angiogenesis and migration of cancer cells.
Compared with a normal cell, lysosomes of cancer cells exhibit a stark difference in volume, number, and distribution, which are strongly associated with carcinogenesis. 253,254 On average, cancer cells express lysosomes near the plasma membrane about three times as much compared to normal cells. Additionally, the increased expression of lysosomal hydrolases is a widespread phenomenon in the majority of cancer cells, related to the poor prognosis of tumors. Previous studies noted that the expression of cathepsins is upregulated in cancer cells. The extracellular mis-localization of lysosomal cathepsins stimulates tumor angiogenesis, thus promoting tumor growth, invasion, and metastasis. 255,256 Moreover, sphingosine kinase SK23-25 is overexpressed in tumor cells, while acid sphingomyelinase is downregulated, causing the disordered sphingolipid metabolism to affect lysosomal function and membrane structure and increasing lysosomal biogenesis. [256][257][258][259][260] Abnormal lysosomes increase the tumorigenicity potential, whereas lysosomes with thinner membranes and enlargement volumes can be de-stabilized in cancer cells, increasing cell death sensitivity. 242 One critical process that is closely linked to the LDCD is LMP. 250 LDCD is triggered by the leakage of hydrolytic enzymes into the cytoplasm, predominantly hydrolases, leading to a series of responses that are associated with cell death, such as chromatin condensation, DNA fragmentation, phosphatidylserine exposure, plasma membrane blebbing, and aberrant degradation of cellular components. 261 The releasing extent of cathepsin into the cytoplasm determines cell death mechanisms, such as apoptosis and/or necroptosis. 246 Executioner caspases are activated by the moderate release of cathepsin, transmitting a complex signaling cascade that eventually results in LDCD. In contrast, a massive release of lysosomal cathepsins can lead to cell necrosis due to the damage to the lysosomal membrane. Additionally, lysosomal calcium release plays an essential function in this process. Thus, lysosomal membrane integrity is critical for maintaining cellular homeostasis and regulating cellular physiological functions. 262 Moreover, during apoptosis, lysosomes could interact with mitochondria. 242,263 After oxidative stress, low concentrations of hydrogen peroxide drive LMP before inducing mitochondrial dysfunction. Mitochondrial dysfunction causes overproduction of ROS and impairs lipid metabolism, eventually triggering LMP. Ultimately, lysosomes play integral roles in initiating and executing cell death.
Future targeting to lysosome for intervention Lysosomes are crucial organelles that participate in extensively crucial cellular processes. 250 Intervention targets of the biochemical pathways mediated by lysosomes have been demonstrated as innovative therapeutic strategies that can induce programmed cell death (Table 1 and Fig. 5b).
LMP induction. LMP has been demonstrated to be an effective strategy to trigger LDCD, 263 where massive lysosomal leakage can cause cytoplasmic acidification and uncontrolled degradation of cellular components leading to potential cell death. Indeed, lysosomes in cancer cells are more vulnerable to LMP due to oncogenes downregulating lysosomal membrane protection proteins, which are highly glycosylated glycoproteins. 264 Additionally, hydrolysis of sphingomyelin, where lysosomal membranes are rich in, sensitizes cancer cells to LMP. 258,265 Cancer cells with enlargeable lysosome size and number are thus more vulnerable to LMP-mediated apoptosis.
Among various external and internal stimuli, intracellular second messengers (ROS and sphingosine), lysosomal toxin reagents, and radiation primarily contribute to lysosome instability and disrupt the lysosomal integrity, which can cause poreformation and LMP initiation. 245,266 Additionally, LMP induction by magneto-mechanical effect of particles (TMMEP) is an emerging research area. The magnetic vibrations of these nanoparticles, induced by a mechanical force, leads to cancer cell destruction. 267 Cheng et al. 268 synthesized highly-magnetized, zinc-doped iron oxide nanoparticles to mechanically destroy cancer cells at low frequency by rotating magnetic fields (15 Hz and 40 mT). Lysosomal membrane integrity is disrupted by the magnetically anisotropic aggregates, leading to LMP-induced cell death. Moreover, iron oxide nanoparticles are also widely used to initiate lysosomal permeabilization at pulsed magnetic fields. 269 Harnessing LMP emerges as a primary strategy for constructing the lysosomal-targeted therapeutic agents. Given the diverse strategies available for inducing LMP, a method that efficiently destroys lysosomes is promising for eliminating damaged cells. As such, a key objective of LDCD will be a better understanding of the LMP mechanism and LMP-inducing agent action.
Proton pump inhibition. The vacuolar H + -ATPase (V-ATPase), an evolutionarily multi-subunit complex, acts as proton pumps responsible for regulating the acidic environment of the intracellular, acidic lumen of lysosomes, and extracellular space. 270 The acidic environment of the lysosome is primarily maintained by V-ATPase pumping protons into the lysosomal lumen. However, abnormalities in the V-ATPase proton pump promotes intracellular alkalinization and extracellular acidification processes, which are commonly observed in invasive tumors. 271 More importantly, the V-ATPase proton pump also significantly impacts the multidrug resistance (MDR). 272,273 In particular, weakly basic anticancer drugs (such as anthracyclines) are prone to protonation in acidic environments. The drug entering the cytosol is hindered by accumulation in lysosomes following protonation, thus leading to drug resistance. 274,275 MDR cancer cells usually exhibit V-ATPase activity enhancement, which treatment can be further complicated. 276 Therefore, regulating V-ATPase activity may enhance the chemosensitivity of MDR cancer cells toward chemotherapeutic drugs.
In recent years, much attention has been focused on targeting tumor acidity and improving the microenvironment to inhibit cancer cell metastasis and reverse MDR. [277][278][279] Unlike conventional cytotoxic anticancer drugs, proton pump inhibitors (PPI) target tumor microenvironments to achieve efficient tumor killing. 277 Of which, pantoprazole, omeprazole, and lansoprazole have been confirmed to exhibit efficient anti-cancer activity by suppressing cell viability and metastasis, facilitating cell apoptosis (Table 2). Moreover, PPI participates in a complex biological process that modulates cancer progression through proteinprotein interactions and various signaling pathways. 277 However, long-term PPI usage can lead to serious side-effects which may affect nutrient absorption and lead to complications, enhancing the incidence of cancer through heterogeneous tumors. 280 Consequently, further investigations about the action mechanism of PPI is necessary to determine a more precise action mode with lesion targets; proper caution is imperative, regarding adverse effects when treating cancer cells with PPI.
HSP70 inhibition. Several small molecules, such as heat shock protein 70 (HSP70), have been identified as lysosome membrane stabilizers, which can prevent LMP. 254 The overexpression of HSP70 in cancer cells improves the resistance of membrane instability enhancing cell survival. 281 HSP70 binding to LMPinducing factors (such as p53) limits the membrane rupture or dysfunction. 282,283 Moreover, HSP70 interacts with bismonoacylglycerol-phosphate (BMP), forming Hsp70-BMP to improve sphingolipid hydrolysis and eventually promoting the stability of lysosomal membranes. 281,284 The suppression of HSP70 function is therefore an emerging target for cancer therapy. 285,286 Applying HSP70 inhibitors (2-phenylethynesulfonamide, PES) or inhibiting the related regulators of HSP70 expression (such as heat shock factor 1, HSF1) to down-regulate the expression of HSP70 is a well-recognized entity for enhancing the sensitivity of LMP in cancer cells. 287,288 Iron release. Iron is the most abundant transition metal in the human body, playing a vital role in the human health. 289,290 Specifically, iron participates in many biological processes, such as electron transport, enzymatic reactions, oxygen transport, and DNA synthesis. 291 Previous studies have noted iron concentration discrepancies between normal and cancer cells. 292 Lysosomes accumulate a significant portion of iron with redox activity due to the degradation of iron-containing metalloproteins. 293 Once excessive iron in the labile iron pool is released to the cytoplasm, it can act as a pro-oxidant factor contributing to excess ROS generation based on the iron-catalyzed Fenton reaction. Subsequently, a range of biological responses can occur, such as DNA damage and organelle rupture, also termed ferroptosis cell death. [294][295][296] Therefore, developing efficient methods for inducing ferroptosis cell death is important for lysosomal targeting cancer treatment.
Lysosome-targeted nanosystems design Lysosome-targeted treatment strategies significantly contribute to the development enhanced cancer therapy, as the accumulation of therapeutic nanoplatforms within the lysosome are more accessible than in other organelles. 297,298 Studies have confirmed that exogenous cargo modified by a specific ligand or by optimizing with specific physicochemical properties could be internalized by cells upon receptor-mediated endocytosis, and eventually accumulated in lysosomes.
The intercellular internalization pathways of cell surface components and extracellular macromolecules primarily involve clathrin-dependent endocytosis (such as receptor-mediated endocytosis) and clathrin-independent endocytosis (phagocytosis, micropinocytosis, and caveolin-mediated endocytosis). 299 One of the most well-characterized forms of endocytosis is the receptor-mediated endocytosis, also referred as RME, which is responsible for cellular internalization between specific ligands and cell surface receptors. [300][301][302] During endocytosis, the plasma membrane invaginates to form luminal vesicles that are then fused with endosomes to enter the endolysosomal membrane system. 303 The extracellular materials eventually arrive in specific lysosomal locations under the endocytosis pathway. Therefore, after modifications with specific receptors, the therapeutic agents can enter lysosomes from the extracellular environment by interacting with a high-affinity ligand on the surface of cancer cells. 304,305 As a result, active-targeting receptor-mediated endocytosis may be a promising strategy to achieve accumulation in lysosomes.
The physicochemical properties of nanoplatforms (such as size, charge, and flexibility) significantly impact lysosomal retention. Human HT29 colon cancer and SKB3 breast cancer cells which express chimeric receptors were utilized as a model to investigate the endocytosis efficiency of size-and rigiditydependent nanoparticles. 306 The internalization rate of larger and more rigid nanoparticles was found to be much slower than that of smaller nanoparticles. In general, cationic nanoparticles can penetrate the cell membrane barrier more efficiently than anionic nanoparticles due to the positively charged surfaces favor electrostatically interactions with the negative charges of cell membranes. [307][308][309] Furthermore, cationic nanoparticles induced membrane depolarization, resulting in membrane permeabilization that ultimately contributes to cell death. 310,311 A type of mixed-charge nanoparticle was constructed through reasonable regulation of positively and negatively charged ligand ratios by Borkowska et al., 312 termed [+/−]NPs, which could selectively target lysosomes with improved cell internalization efficiency accompanied with negligible cytotoxicity to normal cells. The [+/-]NPs induced lysosomal swelling and disrupted lysosomal integrity, ultimately triggering the death of cancer cells.
Additionally, lysosome-targeted fragment modification is another strategy that has been applied to achieve nanoplatform accumulation within lysosomes. Alkylated piperidine fragments are trapped within lysosomes as they protonate in an acidic environment, which can then be used as targeting factors. 313 Daum et al. 313 designed a novel prodrug based on lysosometargeting ROS amplifiers. Specifically, N-alkylaminoferrocene was modified with an alkylated piperidine fragment to achieve lysosome targeting. The prodrug was activated by high ROS concentration in lysosomes, eventually disrupting the cell cycle by attacking lysosomes and disrupting ROS balance. N,N-dimethylpropane-1,3-diamine could also be used for lysosome-targeting with fluorescent chemosensor (Lyso-HS) modification. The tertiary amine of Lyso-HS can be protonated under the lysosomal microenvironment, and thus Lyso-HS remains in the lysosome and allows for H 2 S detection. 314
ER-TARGETED STRATEGIES-A PERTURBATION SITE OF PROTEIN HOMEOSTASIS
Structure of ER The ER is one of the largest and most complicated intracellular organelles, spanning from the outer NE up to the boundary of the cell membrane. 315,316 Depending on the dynamic membranous network of tubules, lamellae, and vesicles, the ER communicates with various cellular organelles, including the mitochondria, Golgi apparatus, and cell membrane, and facilitates protein and lipid transport between various compartments. 317,318 This important organelle is the central hub for protein folding and processing, lipid and sterol biosynthesis, and intracellular calcium storage and buffering.
The ER lumen contains a protein quality monitorization system that modulates the correct folding and complex formation of expressed proteins, 319,320 where only correctly folded polypeptides are delivered to their destination following release from ER. Almost 30% of nascent proteins are folded in the ER lumen with the assist of a series of molecular chaperones. 321 Unfolded or misfolded proteins can trigger unfolded protein response (UPR) signaling pathways to transport them out of ER and to subsequent degradation by the proteasome. 322 If unfolded or misfolded proteins are not promptly removed, perturbations of ER homeostasis can lead to severe ER stress. 323 A series of diseases, such as diabetes mellitus, Alzheimer's disease, many cardiovascular conditions, and inflammation-related diseases, have been found to be linked to overactive ER stress. [324][325][326] More recently, mounting evidence suggests that UPR plays a critical role in the survival and maintenance of cancer cells. 327 More importantly, as a Ca 2+ storage compartment, the ER regulates the equilibration of intracellular Ca 2+ homeostasis. 328 In general, resting cytosolic Ca 2+ concentration is between 50-100 nM, which is significantly lower than the 100-800 μM in the ER. 329 Indeed, high Ca 2+ concentration in the ER is a requisite for the functioning of ER chaperones, 330 which is also essential for maintaining an oxidizing environment in ER lumen to promote disulfide bone formation during protein processing.
Unfolded protein response: friend or foe? Many studies indicated that the ER plays a pivotal role in initiating apoptosis. As discussed above, ER stress occurs when protein misfolds during biosynthesis. In response to ER stress, UPR is activated to address the unfolded or misfolded protein threat and re-establish normal ER function. 322,331 In the ER membrane, three transmembrane proteins (PERK, IRE1α, and ATF6) have been recognized to ER stress and promote pro-survival pathways. However, if prolonged ER stress or UPR recovery fails, the apoptotic signaling pathway will be activated to remove damaged cells (Fig. 6). 332 Proapoptotic protein C/EBP homologous protein (CHOP/ GADD153) regulates ER stress-induced apoptosis and promotes cell death. 333,334 When ER stress persists, PERK phosphorylates eIF2α and subsequently activates and upregulates the expression of transcription factor 4 (ATF4), which directly triggers CHOP/ GADD153 mediated ER-stress-induced apoptosis. 335 Moreover, after activating cleavage, the ATF6 (p50ATF60) cleavage product upregulates the expression of pro-apoptosis protein, such as CHOP, and consequently induces apoptosis. 336 Additionally, IRE1α regulates another ER stress-induced cell death pathway, where it recruits the adapter molecule TNF receptor-associated factor 2 (TRAF2) and subsequently activates apoptosis signal-regulating kinase 1 (ASK1) and c-jun N-terminal kinase (JNK), eventually leading to cell death. 337 Likewise, Ca 2+ in the ER plays an integral role in the ER stressmediated cell apoptosis. While Ca 2+ flux and leakage from the ER occur, significant amounts of Ca 2+ can enter and accumulate in the MM along the ER-mitochondria contact sites, collapsing the mitochondrial function. 338 Mitochondrial Ca 2+ overloading is intimately associated with cell death, where a high concentration of Ca 2+ can trigger mPTP opening and release mitochondrial pro-apoptosis factors to initiate apoptosis. 339 These examples indicate the ER is crucial in deciding cell survival and death. Fig. 6 Unfolded protein response (UPR) is a valuable target in cell death. Protein misfolding or unfolded can occur as a disturbance in ER homeostasis, leading to ER stress. Chemotherapeutic agents, ROS, proteasome inhibitors, and HSP 90 inhibitors as ER stress inducers perturb ER homeostasis differently. If ER stress is not resolved in a timely fashion, unfolded or misfolded proteins accumulate in ER, and UPR triggers cell death via ATF6, PERK and IRE1α mediated signaling pathways. Importantly, fluctuations in ER and mitochondrial Ca 2+ homeostasis can initiate mitochondrial-mediated cell death. UPR unfolded protein response, ROS reactive oxygen species, HSP 90 heat shock protein 90, mPTP mitochondrial permeability transition pore, CHOP C/EBP homologous protein, ATF6 p50ATF60, ATF4 transcription factor 4, TRAF2 TNF receptor-associated factor 2, ASK1 apoptosis signal-regulating kinase 1, JNK c-jun N-terminal kinase Go in for the kill: how to trigger unfolded protein response? In comparison to normal cells, cancer cells reprogram their intrinsic metabolism patterns to adapt unfavorable environments for survival and then relentlessly proliferate. 340 A wide variety of studies have indicated that ER stress and UPR activity are directly correlated with tumor invasion, metastasis, and chemo-resistance to different types of cancer. 341-343 ER stress and UPR overactivation are common phenotypes in most cancer cells, 344 where UPR overactivation enables the management of protein translation to protect cells from ER stress damage, and thus, increase cancer viability under unfavorable environments. 345 Many studies also suggest that changes in UPR component expression, such as GRP78/BIP, UPR trans-activators XBP1, and ATF6, have been detected in numerous types of human cancer. 346 Therefore, increasing ER stress could be a potential strategy for therapeutic intervention.
Excessive ER stress causes pro-apoptosis signaling pathway activation, eventually causing cell death. Different ER stress inducers, such as chemotherapeutic (curcumin and celecoxib) and ROS, can target the ER and subsequently induce ER-stress apoptosis (Fig. 6). 347,348 In addition, Ca 2+ imbalance and intracellular hypoxia environments accelerate ER stress and ER dysfunction, followed by cell apoptosis. 349 The clinical significance of UPR as a vital target has been increasingly recognized in cancer therapy. 334 As proteasome inhibitors, Bortezomib, Nelfinavir, and Atazanavir, have been employed against prostate, lung, breast, and colon cancer in clinical trials (Table 2). 350,351 They are involved in UPR activities, leading to misfolding protein accumulation in the ER and generating enhanced ER stress. Alternatively, HSP90, as molecular chaperones, can participate in the folding process of substrate proteins during UPR. 352 They are frequently mutated or overexpressed in tumors to protect from ER stress damage. 353 HSP90 inhibitors disrupt HSP90 client protein folding, such as oncogenic proteins, and eventually lead to cell death. 354 It is worth mentioning that UPR is overactivated in cancer cells, which is not always the case in normal cells. The difference of cancer cells compared with normal cells on UPR can be exploited to reduce toxic side effects during cancer treatment.
Strategies to achieve ER accumulation Given that it serves several essential roles in apoptosis, targeted delivery of therapeutic agents into the ER is of significant importance for cancer therapy. However, therapeutic agents which can selectively navigate into ER is a daunting task due to the ER's complex structure, containing a vast 3D interconnected network of different thicknesses. 355 The ER-targeting strategy is of great clinical importance as it provides a key target for anticancer drug development and cancer treatment advancement.
Small molecules. ER-targeting small molecules specifically bind to the surface of the ER, accumulate, and disrupt ER function. 356 Sulfonamide ligands have been extensively developed for small molecule drugs and drug delivery vehicle modification due to its low toxicity, high efficiency, and high selectivity. 357 They specifically recognize and bind to sulfonylurea receptors with high affinity, which are potassium-selective ion channel proteins highly expressed on ER membranes.
Glibenclamide, a sulfonamide urea derivative, can assist commercial fluorescent probes, such as ER-Tracker Red and ER-Tracker Green, into the ER to achieve membrane visualization. Additionally, the dansyl and toluenesulfonyl groups in N-(2aminoethyl)-5-(dimethylamino) naphthalene-1-sulfonamide can serve as typical sulfonyl ligands that endow therapeutic agents with ER-targeting ability. 358,359 Basu et al. 360 engineered 17AAG-ER-NPs with an ER targeting group (toluenesulfonyl) and HSP90 inhibitor (17AAG, ER stress inductor) to trigger ER stress-mediated cell death. This nanoplatform prompted remarkable anticancer efficacy at sub-micromolar concentration, providing a promising alternate for cancer treatment. Chen et al. 361 synthesized polymeric, reduction-sensitive NPs, which were loaded with an ER-targeting photosensitizer containing toluene sulfonamide, to induce ER stress by local ROS generation and subsequent immunogenic cell death (ICD) activation.
ER-targeting peptides. ER is the primary site of protein biosynthesis, and their localization signal peptides with homing properties can assist ER molecular chaperones in delivering their duty. 362 The KDEL peptide was first used to enhance protein accumulation in the ER in 1987, and then extensively used as an ER-retention sequence for ER recognition and localization. 363 KDEL with the C-terminal sequence Lys-Asp-Glu-Leu motif could recognize and bind specifically to KDEL receptors (KDELR) to promote ER accumulation via a coat protein I (COPI)-mediated retrograde pathway. 364,365 Wang et al. 366 showed the evidence that KDEL facilitates ER transportation through monitoring trafficking pathways of KDEL-Au NPs. Interestingly, the KDEL peptide-mediated ER translocation pathway evades lysosomes to prevent degradation and protect cargos. These featured characteristics of the KDEL peptide make it e an attractive tool for ER retention of therapeutic agents in treating cell malignancies.
Plasma membrane
The plasma membrane primarily consists of a phospholipid bilayer structure in which various proteins and lipid species are inhomogeneously incorporated. 367 Developing the role of the plasma membrane in cancer treatment has received significant research interests. The uncontrolled growth of cancer cells relies on plasma membrane reprogramming to satisfy rigorous requirements of biosynthesis and bioenergetics due to their rapid division. 368 Aberrant upregulation of several lipogenic enzyme expressions, such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), has been observed in many cancers, directly resulting in fatty acid synthesis and cholesterol metabolism alteration in cancer cells. 369,370 Furthermore, an increase in sphingolipids and cholesterol content promotes abnormalities of plasma membrane permeability, contributing to reduced drug influx, P-gp efflux, and increased intracellular vesicle-mediated drug entrapment, which can trigger multidrug resistance (MDR) in drug-resistant cancer cells. 371,372 Therefore, therapeutic strategies for plasma membranes may offer a practically feasible approach to improving treatment efficacy and enhancing the sensitivity of current anti-cancer therapies.
To enable plasma membrane-targeted personalized strategies, photodynamic therapy (PDT) has been extensively studied. Severe ROS damage to the plasma membrane may directly inhibit cell proliferation and migration, inducing apoptosis by destroying cellular integrity and activating the immune system. 373 However, plasma membrane retention suffers from cellular uptake and endocytosis. 374 Recently, efforts have been focused on optimizing therapeutic agent structures to prolong retention times in the plasma membrane. A pH (low) insertion peptide (pHLIP) allows for insertion into the plasma membrane spontaneously after selftransformation. A pH-driven, membrane-anchoring photosensitizer (pHMAPS), with pHLIP and protoporphyrin IX, has been designed to achieve membrane localized PDT. 375 It was observed that pHMAPS can cause significant damage to cancer cells and significantly inhibited tumor growth, due to cytotoxic ROS generation and accumulation near the plasma membrane. Moreover, cracked cancer cell membrane (CCCM), 376 lipophilic palmitic acid (PA), 377 membrane fusogenic liposomes (MFLs), 378 and cholesterol 379 have also been applied to promote cell membrane anchoring via insertion or fusion approaches.
Notably, plasma membrane-anchoring therapies can protect therapeutic agents with significant efficacy, by safe-guarding from lysosomal degradation and retention. These strategies demonstrate another influential, promising target for precision cancer medicine. Despite their remarkable potential, these studies are still in the initial stages and more investigations are needed to improve/confirm their viability through further system optimizations, such as prolonging membrane retention time.
Peroxisome
The origin and nature of peroxisomes have been actively debated since 1950s. 380 It is now accepted that peroxisomes are semiautonomous organelles which are involved in lipid metabolism regulation, fatty acids auxiliary processing, plasmalogen synthesis, and ROS modulation. 381 Recent studies indicate that peroxisome plays a significant role in regulating cancer initiation and progression. 382 Peroxisomes have been observed to support cancer cell energy supply by providing a lipid substrate. 383 Alteration of enzymatic activities and protein levels related to lipid processing in peroxisomes has been identified in numerous cancer types, such as prostate, breast, liver, and ovarian cancer. 384,385 Elevated peroxisomal fatty acids and ether phospholipids in peroxisomes help cancer cells survive various stresses, and contribute to tumor progression in an oxygen-depleted tumor environment. [386][387][388] More importantly, evidence suggests that peroxisomes can cooperate with mitochondria by connecting vesicular pathways or fission machinery. 389,390 A variety of enzymes that regulate ROS production and clearance reside both in peroxisomes and mitochondria, suggesting a possible link of metabolic cross-talk between peroxisome and ROS homeostasis. 391,392 The loss or overproduction of Mpv17, which encodes peroxisomal proteins, directly leads to intracellular ROS production reduction or enhancement. 393 In addition, peroxisomes play an important role in resisting ROS-mediated apoptosis and influencing cancer cell growth. Increases in peroxisome amounts were observed in vorinostat-resistance lymphoma cells against ROS damage, and further peroxisomal proteins PEX3, PEX11B, and PMP70 were also upregulated. 394 These results indicated the role of peroxisomal participation in ROS metabolism, suggesting peroxisome metabolism may be a potential therapeutic target against cancer progression and circumvent drug resistance.
Peroxisome metabolism could be a desired target for future cancer therapeutic agents. Considering the interplay between the mitochondria and peroxisome, peroxisome disruption may rewrite metabolism pathways and have profound therapeutic effects. However, a few peroxisome activity modulators have been developed without preclinical and clinical trials. Peroxisome metabolism intervention therapies with therapeutic nanosystems are still underexplored. As such, further investigations of the peroxisome-mediated molecular pathway and the development of specific therapeutic agents are required, which may drive additional impactful cancer therapy treatments.
Golgi apparatus
The Golgi apparatus exists as a series of flattened membranebound sacks (cisternae), organized in a perinuclear lace-like reticulum in a cis-to-trans fashion. 395,396 Cellular homeostasis is highly reliant on the proper functioning of the Golgi apparatus in protein sorting and trafficking. 397 It has been shown that Golgi glycosylation abnormalities are closely related to the occurrence and metastasis of cancers. 398 It is thus conceivable that the fragile Golgi apparatus provides an opportunity for specific cancerdirected therapeutic approaches.
Various Golgi-disturbing agents have been developed, such as brefeldin A (BFA), monensin, nocodazole, and retinoic acid (RA), which can directly attack protein trafficking pathways mediated by the Golgi apparatus or induce ion imbalances, and subsequently, disturb the Golgi apparatus and induce apoptosis. [399][400][401] Ma et al. 402 constructed chondroitin-modified lipid nanoparticles (CSNs) to deliver RA and DOX. DOX+RA-CSNs efficiently accumulated in the Golgi apparatus to damage their structures and inhibit extracellular matrix (ECM) protein production, resulting in liver cancer cell apoptosis. Aside from chondroitin sulfate (CS), a series of novel targeting ligands can anchor in the Golgi apparatus, such as cysteine derivatives (protein kinase D and galactosyltransferase), phenylsulfonamide derivatives, and aminoquinolies. [403][404][405][406] To date, few Golgi therapeutic agents are applicable for cancer curing. Future research directions may include revealing molecular interference targets of the Golgi, exploring the mechanisms of Golgidisturbing agents, and new targeting tags to drive the course of cancer therapy.
CONCLUSION AND OUTLOOK
With the rapid and intensive research and technology development across biology, medicine, and materials science, precision subcellular-targeted nanoplatforms have become an important research topic for cancer treatment across the globe. This review presents three major organelles, including the nucleus, mitochondria, lysosomes and ER, while summarizing the unique characteristics and various functions of each organelle to unveil the hallmarks and potential for these therapeutic targets. Furthermore, underlying guidelines of organelle-targeted nanoplatform constructions are discussed according to specific characteristics of each organelle. Advancing the understandings of the interplay between organelle characteristics and functions with nanoplatform construction guidelines will be essential for enabling improved organelle-targeted therapeutic agents for future oncology development.
The organelle-targeted strategy holds tremendous potential in next-generation cancer therapies, which has gradually become the primary approach for personalized cancer treatment. While the controlled delivery at the organelle level has been achieved, their adaption to current medical practice have yet to be fully exploited. Here, we provide a brief perspective on couple ongoing challenges with several potential solutions: (1) incomplete understandings of molecular pathogenesis of tumor heterogeneity lead to noticeable variation in treatment effects in different individuals with identical treatments. More in-depth studies and enhanced comprehension of aberrant cellular signaling pathways and molecular regulators on cancer are urgently needed, with particular focuses on chemotherapy and gene therapy. Investigations of the underlying intrinsic molecular regulatory mechanisms and development of novel molecular targets are essential for enabling organelle-targeted treatment for majority cancers; (2) efficacy and safety following organelletargeted cancer treatment requires additional study. Blood clearance and retention times for targets of nanomaterials along with physiological monitoring of individuals are still lacking. Therefore, long-term tracking of safety and treatment efficacy, as well as establishing a primate experimental model are indispensable procedures, which are necessary to validate their benefits for translations to clinical applications.
Overall, this article presents a broad and comprehensive review on the topic of potential organelle-target characteristics and the underlying system design guidelines for therapeutic agent construction. We highlight the importance of organelletargeted therapeutic strategies for precision medicine in cancer therapeutics, which will be very important for the development of emerging organelle-targeted nanomaterials and their associated future implementation. Looking forward, we believe this formidable technology holds great potential to revolutionize cancer therapy at the interface of biology, nanomaterials, and medicine.
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v2
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2022-11-19T15:06:07.630Z
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2022-11-19T00:00:00.000Z
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253631639
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s2orc/train
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The diminution and modulation role of water-soluble gallic acid-carboxymethyl chitosan conjugates against the induced nephrotoxicity with cisplatin
The toxicity of cisplatin (CDDP) toward the renal tubules and its severe effects on the proximal tubules limits its further use in cancer therapy. The current study was undertaken to evaluate the protective effects of gallic acid-grafted O-carboxymethyl chitosan (GA@CMCS) against nephrotoxicity induced by CDDP in rats. Renal injury was assessed in the GA@CMCS/CDDP-treated rats using kidney injury molecule-1 (KIM-1). Moreover, the levels of reduced glutathione (GSH), malondialdehyde (MDA), and nitric oxide (NO) were measured. The comet assay was performed to measure the DNA damage. The renoprotective activity of GA@CMCS was supported by histo- and immuno-pathological studies of the kidney. GA@CMCS significantly normalized the increases in kidney homogenate of KIM-1, MDA, and NO-induced by CDDP and significantly increased GSH as compared with the CDDP group. GA@CMCS also significantly protects rat kidneys from CDDP-induced histo- and immuno-pathological changes. Both biochemical findings and histo- and immuno-pathological evidence showed the renoprotective potential of GA@CMCS against CDDP-induced oxidative stress, inflammation, and renal dysfunction in rats. In conclusion, GA@CMCS has been shown to mitigate the nephrotoxicity impact of CDDP in cancer therapy.
Instrumentation, Elemental analyses for C, H, N and S were performed with a Perkin-Elmer 263 elemental analyzer. FT-IR spectra were recorded on a BRUKER Tensor-37 FT-IR spectrophotometer in the range 400-4000 cm -1 as KBr discs or in the 4000-550 cm -1 region with 2 cm -1 resolution with an ATR (attenuated total reflection) unit (Platinum ATR-QL, Diamond). For signal intensities the following abbreviations were used: br (broad), sh (sharp), w (weak), m (medium), s (strong), vs (very strong). NMR-spectra were obtained with a Bruker Avance DRX200 (200 MHz for 1 H) or Bruker Avance DRX500 (500 MHz for 13 C) spectrometer with calibration to the residual proton solvent signal in D 2 O ( 1 H NMR: 4.79 ppm) against TMS with δ = 0.00 ppm.
Multiplicities of the signals were specified s (singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). The particle shape of new materials was examined using transmission electron microscope (TEM). The images were taken by a JEM-2011F microscope (JEOL, Japan) operated at 200 kV. The morphology of the formed micro and nano-composites was investigated using Scanning electron microscopy (SEM, Hitachi S-7400, Hitachi, Japan) supported with energy dispersive -X-ray (EDX) to determine the elemental analysis of the formed products..
Synthesis and characterization of CS and LMWCS
Synthesis of chitosan (CS), The crabs were obtained from Bay of Suez coast. The terminals and operculum of crabs were removed and the shells (~1500 g) are scraped free of loose tissue and washed individually in lightly saline water, then separated from cephalothoraxes, salted (5 kg of NaCl per 250 g of shells), washed thoroughly in distilled water and placed in Ziploc bags and refrigerated overnight. The crab's exoskeletons were crushed into smaller pieces using a meat tenderizer and dried in the sun (25-30 °C) for 3 days, and finally oven-dried for a week at 65°C until constant weight. After that, the dried shells were grinded, sieved, and the fraction below 80 µm was used hereafter. The extraction method proposed here involved three chemical treatment steps, with each step followed by rinsing with distilled water until a neutral pH was reached. In the first step (demineralization process), 100 g of shrimp shells powder was immersed in 1000 mL of 0.5 M HCl at ambient temperature (25 °C) under constant stirring for 24 h. After washing with distilled water, the second step of the procedure was the deproteination stage in which the residue was immersed in 1000 mL of 1 M NaOH under vigorous stirring at 60 °C for 24 h. Then the proteins were removed by filtration. Distilled water was used to wash the residue to neutral. Then, the residue was subjected to the above procedure two more times. The chitin obtained still had a slight pink colour. Further decolourisation was achieved by soaking chitin in 250 mL of 1% KMnO 4 for 1 h. followed by 250 mL of 1% oxalic acid for 2 h. The amount of 250 mL of 95% ethanol and 200 mL of absolute ethanol were sequentially used to remove ethanol-soluble substances from the obtained crude chitin and to dehydrate the chitin. Finally the chitin was dried in air at 50 °C overnight. Yield 86.34 g (86.34% based on 100 g of crab shell powder). The final step is the deacetylation process in which the purified chitin was deacetylated to form chitosan by treating 10 g of chitin with 100 mL 2 M NaOH under stirring at 60 °C for 72 h. After filtration, the residue was washed three times with hot deionized water at 60 °C. The crude chitosan (7.9 g) was obtained by drying in an air oven at 50 °C overnight. The obtained crude chitosan was purified by dissolution in 1% (v/v) aqueous acetic acid until a homogenous solution is obtained, filtered through 22 µm Whatman filter paper to remove insoluble impurities, then precipitated by titration with 1 M NaOH until pH value of 8.5, and finally washed several times with distilled water. Yield 6. At the end of the addition, the suspension was refluxed for 1 h. It was poured into stirred water (4 L) preheated to 80 °C. The precipitate was decanted, washed five times with water until neutral pH, and separated by filtration. The resulting polymer was purified by dialysis against water for 3 days and isolated by lyophilization
Preparation of low molecular weight chitosan (LMWCS), Chitosan solution (2%) was prepared in 1%
CH 3 COOH by stirring for 24 h in room temperature. A solution of H 2 O 2 30% (4.4%) was then used to achieve chemical degradation of chitosan in 30 for 1.5 h. Adjusting the pH of the solution to approximately 7 was carried out by using 1 M NaOH solution. As the pH increased, part of chitosan was precipitated. Thereafter, the solution was centrifuged at 6000 rpm for 30 minutes to separate sediments. The upper phase of the centrifuged solution included water-soluble chitosan (WSCS), which was soluble in neutral pH, and the lower phase consisted of low molecular weight chitosan (LMWCS). Then, WSCS was vacuum filtered with the aid of appropriate filter paper, while the wet LMWC was submitted to ultrasonic irradiation with an amplitude of 100 Hz for 20 minutes in order to break the chains further. The final product was dried under vacuum at 40 °C for 48 h and chracterized based upon FTIR, 1 H NMR and molecular weight (MW) determination by viscometric measurements using an Ubbelohde Capillary Viscometer (0.5 mm). Average molecular weights were calculated from [η] = k.M α equation, where η intrinsic viscosity, k = 1.81 x 10 3 (cm 3 /g) and α = 0.93 determined in 0.25M CH 3 COONa and 0.25M CH 3 COOH solution at 25 °C.
Structural characterizations of LMWCS
Molecular weight, microanalytical data, degree of acetylation (DA)The molecular weights of CS and LMWC samples can be calculated based upon their intrinsic viscosities (η) according to the Mark-Houwink-Sakurada (MHS) (502.76 and 21.83 mL/g, respectively) and were found to be 713.6 and 24.5 kDa, respectively.
As shown, the molecular weight of LMWC system was decreased by ~97% compared to the native CS. As the LMWCS was obtained by partial deacetylation of chitin followed by partial degradation of CS, so the proposed empirical formula for LMWCS is (β-D-Glc-NHAc) DA (β-D-Glc-NH 2 ) 100-DA . Elemental analysis (EA) provides a powerful tool for calculation the degree of acetylation (DA) calculated according to the following equation. Fig. S1.
Determination of degree of carboxymethylation (DC)
The DC of CMCS derivatives was determined by dissolving CMCS derivative (0.3 g) in 0.1 mol L −1 HCl (30 mL) and titrating with 0.1 mol l −1 aqueous NaOH. The DC value was calculated as follows [S1]: where V NaOH and C NaOH were the volume and molarity of aqueous NaOH, respectively, m CMCS was the mass of CMCS (g), and 161 and 58 are the molecular weights of the glucosamine (chitosan skeleton unit) and the carboxymethyl group, respectively.
Determination of grafting degree of GA@CMCS
The measurement of GA content in GA@CMCS was carried out according to the Folin-Ciocalteu method with slight modification. 1 Briefly, the freeze-dried GA@CMCS powder was dissolved in deionized water at 1 mg/mL. Then, 0.5 mL of each sample was mixed with 1 mL of Folin-Ciocalteu reagent (10 times dilution) and reacted for 5 min in the dark, followed by the addition of 2 mL 15% Na 2 CO 3 and deionized water till 10 mL.
The mixture was shaken and maintained under room temperature for 2 h and the absorbance of the reaction mixture was read at 750 nm using UV-vis spectrophotometer (Evolution 201, Thermo Fisher Scientific Inc., Waltham, MA, USA). GA was used as a standard and the grafting degrees of GA@CMCS were expressed as mg of GA equivalents per g of GA-CS (or weight percent). Figure S1. Mechanism of action of CDDP including (i) cellular uptake, (ii) aquation/activation, (iii) Platination of DNA, and (iv) cellular processing leading to apoptosis
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v2
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2022-11-20T06:15:58.591Z
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2022-11-19T00:00:00.000Z
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253670843
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s2ag/train
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Surgical salvage of human papillomavirus-positive oropharyngeal cancer: Secondary analysis of a randomized controlled trial.
BACKGROUND
Survival outcomes are generally better for human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) than other forms of head and neck cancer. However, less is known about oncologic outcomes, late adverse events, and gastrostomy tube dependence associated with salvage surgery after the failure of definitive chemoradiation in patients with HPV+ OPSCC.
METHODS
A secondary analysis of the Radiation Therapy Oncology Group 1016 randomized trial, which compared radiotherapy plus cetuximab to radiotherapy plus cisplatin in patients with HPV+ OPSCC, was performed. The oncologic and adverse event outcomes for patients who underwent salvage surgery were examined.
RESULTS
Among the 805 patients who were assigned to treatment and were eligible for analysis, 198 developed treatment failure. Salvage surgery was required for 61 patients (7.6%), with 33 patients undergoing salvage surgery after locoregional failure (LRF) and 28 patients undergoing salvage neck dissection within the 20 weeks after treatment. Patients with LRF who underwent salvage surgery experienced improved overall survival in comparison with patients with LRF who did not undergo surgery (45% vs. 17% at 5 years after treatment; hazard ratio, 0.41; 95% confidence interval [CI], 0.23-0.74). Surgical salvage after LRF was associated with similar frequencies of late grade 3/4 dysphagia in comparison with LRF without surgery (24% [95% CI, 13%-41%] vs. 20% [95% CI, 12%-32%]; p = .64) and with similar gastrostomy tube dependence at 2 years (29% [95% CI, 15%-49%] vs. 13% [95% CI, 5%-28%]; p = .12).
CONCLUSIONS
Salvage surgery in patients with HPV+ OPSCC is associated with favorable survival and adverse event outcomes.
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v2
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2022-11-21T16:17:30.738Z
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2022-11-19T00:00:00.000Z
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253727679
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s2ag/train
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Intrinsic resistance and efficacy of immunotherapy in microsatellite instability-high colorectal cancer: A systematic review and meta-analysis.
Some patients with microsatellite instability-high colorectal cancer (MSI-H CRC) have shown a poor response to immunotherapy in clinical trials. We investigated the intrinsic resistance to and efficacy of immunotherapy in patients with MSI-H CRC. The PubMed and Web of Science databases were searched using keywords such as "colorectal cancer," "immunotherapy," and "clinical experiment." Random-effects models were used to generate the combined complete response, partial response, stable disease, progressive disease, objective response rate (ORR), disease control rate (DCR), and incidence of adverse events. We then performed a subgroup analysis based on the ORR and incidence of intrinsic resistance. The meta-analysis included seven clinical trials. The incidences of complete response, partial response, stable disease, and progressive disease summarized by the random-effects model were 8%, 37%, 26%, and 25%, respectively. The ORR and DCR were 45% and 71%, respectively. The ORRs of programmed cell death protein 1 inhibitor (anti-PD-1), programmed death ligand 1 inhibitor (anti-PD-L1), and anti-PD-1 combined with cytotoxic T lymphocyte-associated antigen 4 inhibitor (anti-CTLA-4) immunotherapy were 38%, 54%, and 57%, respectively. The ORR of immune checkpoint inhibitors for first- and third-line therapy was 56% and 32%, respectively. Dual-drug immunotherapy significantly reduced the incidence of intrinsic resistance to immunotherapy (12% vs 31%). The incidences of intrinsic resistance to first-line therapy and second-line and later therapy were 29% and 26%, respectively. Approximately 25% of patients with MSI-H CRC had intrinsic resistance to immunotherapy. Anti-PD-1 combined with anti-CTLA-4 significantly increased the ORR, thereby reducing the incidence of intrinsic resistance. Moving immunotherapy into earlier lines of therapy, although not reducing the incidence of intrinsic resistance, can improve the ORR in patients with MSI-H CRC.
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v2
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2022-11-22T16:08:47.989Z
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2022-11-19T00:00:00.000Z
|
253753636
|
s2ag/train
|
A Rare Axillary Cutaneous Squamous Cell Carcinoma: A Case Report and Literature Review
Background: Non-melanoma skin cancer is the most frequent tumor in Brazil and the world. One of its forms, squamous cell carcinoma (SCC) predominantly affects the old white population in areas of high exposure to the sun. Most SCCs are indolent, evolving with a cure rate higher than 90% within five years. Rarely, metastasis occurs mainly in regional lymph nodes, but it can also happen in the lungs, liver, brain, skin, and bones.
There are currently many treatment options; based on the stratification of the neoplasm as high or low risk, an appropriate approach is defined.
Case presentation: This report presents the case of a patient with high-risk squamous cell carcinoma affecting an area not exposed to solar radiation and without any other previous triggering factor, which is quite uncommon for this type of tumor. The rarity of the case stems from the lack of scientific reports on the occurrence of SCC in the axillary region, without a history of local chronic inflammatory lesions. The Portuguese, English, and Spanish languages were used to search the database of the main scientific platforms Pubmed, Cochrane Library, Scielo, and Lilacs, with no results similar to the case reported.
Conclusion: Despite the fact that the axillary area is not sun-exposed, squamous cell skin cancer manifested as an extensive lesion that required a complex surgical resection with flap repair. Such findings highlight the importance of a thorough physical exam and work-up to diagnose lesions in their early forms which require simple resection procedures and avoid late diagnoses resulting in complex procedures. Such an approach reduces the risk of various complications like wound infection or dehiscence, flap ischemia, or necrosis, among others.
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v2
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2022-11-21T06:16:49.016Z
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2022-11-20T00:00:00.000Z
|
253707680
|
s2ag/train
|
Hematological and neurological expressed 1 (HN1) activates c-Myc signaling by inhibiting ubiquitin-mediated proteasomal degradation of c-Myc in hepatocellular carcinoma.
Hepatocellular carcinoma (HCC) has a poor prognosis due to the usually advanced stage at diagnosis. Sustained activation of the MYC oncogene is implicated in the development of HCC; however, the molecular mechanisms of MYC deregulation in HCC are poorly understood. Here, real-time PCR and western blotting were used to measure the expression of hematological and neurological expressed 1 (HN1) in HCC cells. Expression of HN1 and MYC in clinical specimens was analyzed using immunohistochemistry. The role of HN1 in HCC proliferation, migration, and invasion was explored in vitro and in vivo. MYC expression was measured using real-time PCR and western blotting. MYC transcriptional activity was assessed using a luciferase reporter system. Expression of MYC target genes was quantified using real-time PCR. Protein interaction between MYC and HN1 was assessed using co-immunoprecipitation and western blotting. We identified HN1 as a novel regulatory factor of the glycogen synthase kinase (GSK) 3β-MYC axis. HN1 expression is elevated in liver tumor tissues and cells, and significantly correlates with poor survival in HCC patients. Upregulation of HN1 promotes, and silencing of HN1 represses, the proliferation and metastasis of liver cancer cells in vitro and in vivo. Moreover, our results demonstrate that HN1 sustains stabilization and persistent activity of MYC via interaction with GSK3β in HCC. Importantly, the tumor-promoting effects of HN1 on HCC cells were attenuated by suppressing MYC. In conclusion, constitutive activation of MYC by HN1 promotes the progression of HCC; therefore, HN1 might be a novel therapeutic target for HCC.
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v2
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2022-11-21T14:44:37.805Z
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2022-11-20T00:00:00.000Z
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253709981
|
s2orc/train
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Preoperative chemotherapy combined with para-aortic lymph node dissection has clinical value in the treatment of gastric cancer with para-aortic lymph node metastases
Background Lymph node metastases often occur in advanced gastric cancer, with some patients presenting with metastases in the para-aortic lymph nodes. There are persistent Controversies about the benefit of para-aortic lymph node dissection (PAND). Our purpose is to probe whether PAND following preoperative chemotherapy had any clinical significance in individuals with PALNs in gastric cancer. Material and methods To retrospectively analyze the clinical data of 86 gastric cancer patients (40 in the D2 + PAND group and 46 in the D2 group) who attended the abdominal surgery department of Zhejiang Cancer Hospital between September 1, 2008, and July 30, 2018. Results In the D2 + PAND group (40 cases), the average number of lymph nodes cleared per case was 4.3 in group 16 (16a2, 16b1), and the postoperative pathology confirmed lymph node positivity in 16 cases, with a metastasis rate of 40%. The median overall survival times were 63 and 34 months for the patients in the D2 + PAND group and D2 group, respectively. The 3-year overall survival (OS) compared to the D2 group (D2 + PAND 69.1% vs. D2 50%, P = 0.012) and a statistically significant difference in 3-year disease-free survival (DFS) (D2 + PAND 69.6% vs. D2 38.3%, P = 0.007). Lymph node dissection extent and recurrence of para-aortic lymph nodes were independent prognostic variables for the patients. The recurrence rate was reduced in the D2 + PAND group compared to the D2 group (D2 + PAND 7.5% vs. D2 26.1%, p = 0.023). Conclusions For patients with gastric cancer whose imaging suggests metastasis in the para-aortic lymph nodes, preoperative chemotherapy combined with PAND is an effective and safe treatment that may benefit patient survival.
Introduction
According to the World Health Organization's 2020 data report, China has approximately 480,000 new instances of gastric cancer (GC) and ranks third in the world in terms of new malignant tumors, with 370,000 deaths from malignant tumors, ranking third in the world [1]. Advanced gastric cancer is the most common type of stomach cancer in China, accounting for approximately 80% of the cases.
According to some studies, the metastatic rate of advanced gastric cancer para-aortic lymph nodes (PALNs) can range from 18 to 40% [2][3][4][5]. The efficacy of para-aortic lymph node dissection (PAND) for PALN metastases has been controversial. The 3rd version of the Japanese Guidelines for the Treatment of Gastric Cancer defines PALN as M1 based on the Japanese JCOG9501 trial, which denied the therapeutic efficacy of prophylactic D2 + PAND [6,7]. Based on the outcomes of the JCOG0001 and JCOG0405 investigations, Japanese researchers now consider that preoperative chemotherapy followed by D2 + PAND in patients with circumscribed para-aortic lymph node metastases from gastric cancer can benefit patients with a 5-year overall survival (OS) of 53% [8,9]. The follow-up endpoint of a Chinese phase II clinical study of D2 lymph node dissection alone after chemotherapy for gastric cancer with PALN metastases was not fulfilled, but the median survival time has now reached 29.8 months [10]. However, other studies have concluded that the use of neoadjuvant chemotherapy before minimally invasive radical gastrectomy with D2 lymph node dissection does not increase postoperative complications [11]. Patients with ypN0 have a good prognosis because the lymph nodes are indeed negative before neoadjuvant chemotherapy or because the pathology achieves a complete response after treatment [12]. Neoadjuvant chemotherapy appears to be associated with a higher rate of postoperative complications compared to surgery alone [13]. Currently, the clinical significance of preventative D2 + PAND surgery has been ruled out, but the clinical significance of curative D2 + PAND surgery is unknown.
We retrospectively analyzed the clinical and pathological data of patients with gastric cancer who were found to have metastatic lymph nodes in the parietal abdominal aorta 16a2 and 16b1 regions at the initial CT diagnosis and who underwent radical gastric cancer surgery after preoperative chemotherapy. The goal of this research was to probe whether parietal aortic lymph node dissection following preoperative chemotherapy had any clinical significance in individuals with metastatic parietal aortic lymph nodes in gastric cancer.
Selection criteria and patients
We retrospectively analyzed the clinical and pathological data of consecutive patients with histologically confirmed GC/Esophagogastric junction cancer (EGJC) and who received surgical treatment at the Department of Abdominal Oncology Surgery at Zhejiang Cancer Hospital between September 1, 2008, and July 30, 2018. The inclusion criteria were as follows: (1) The pathological examination from biopsy confirmed gastric adenocarcinoma. (2) A preoperative CT revealed enlarged lymph nodes in the 16a2 and 16b1 areas adjacent to the abdominal aorta. (3) The patient had normal function of major organs. (4) The patient received at least 3 cycles or more of first-line chemotherapy before surgery (chemotherapy regimen was not limited). (5) Radical gastric cancer surgery was performed (R0 excision). The criteria for exclusion were as follows: (1) previous gastric surgery; (2) associated with other malignant tumors; (3) other distant metastases; (4) received radiotherapy; (5) unable to tolerate chemotherapy and surgery; and (6) incomplete data. Finally, 86 patients were included in this study.
According to the extent of lymph node dissection after surgery, the patients were classified into 40 patients in the D2 + PAND group (observation group) and 46 patients in the D2 group (control group). Before surgery, all patients had a gastroscopy to confirm that they had stomach cancer, and CT and B-mode ultrasound were used to rule out distant metastases such as metastases to the supraclavicular lymph nodes, liver, and peritoneum. There were 61 men and 25 women among the patients, with a median age of 59 (28-74) years. The patients' clinical data, such as tumor location, surgical method, lymph node dissection extent, anastomotic method, and postoperative complications, were also collected.
This study was approved by the ethics committee of the Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital) (No. IRB-2020-300). Informed consent from the patients was waived by the ethics committee of the Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital) (No. IRB-2020-300) because of the retrospective nature of this study, and conformed to the tenets of the Declaration of Helsinki (as revised in 2013).
Treatment method
All patients received 3 or more cycles of preoperative chemotherapy, including SOX (oxaliplatin + S-1) in 44, DOS (docetaxel + oxaliplatin + S-1) in 4, PS (paclitaxel + S-1) in 14, EOX (epirubicin + oxaliplatin + capecitabine) in 2, ECX (epirubicin + cisplatin + capecitabine) in 9, XELOX (oxaliplatin + capecitabine) in 9, FOLFOX (oxaliplatin + fluorouracil + calcium folinate) in 1, and DCF (docetaxel + cisplatin + fluorouracil) in 3. After evaluation of the efficacy of the preoperative chemotherapy, radical surgery for D2 gastric cancer was considered when the lymph node size after chemotherapy was suggested by CT and when the metastasis next to the abdominal aorta disappeared or shrunk to < 1.0 cm. When the metastatic lymph nodes adjacent to the abdominal aorta were > 1.0 cm, radical surgery for D2 + PAND gastric cancer was considered.
Intraoperative lymph node dissection of the para-aortic lymph nodes: the transverse colonic hepatic flexure was freed inward and downward, Kochers' maneuver was made, the left hemicocele was freed and turned up to the right, the posterior part of the pancreas was freed up to the left side of the abdominal aorta, and the 16a2, 16b1 lymph nodes were cleared from left to right, starting from the superior border of the abdominal aortic trunk up to the superior border of the inferior mesenteric artery. The fine lymphatic vessels were meticulously ligated or sutured (Fig. 1A, B).
Assessment and follow-up
Following surgery, all patients were followed up every 3 months for the first year, every 6 months for the next 1 to 3 years, and every 12 months after 3 years. Outpatient and telephone follow-ups were used, and each outpatient review included an assessment. For the evaluation: a thorough medical history, physical examination, serum tumor markers, and radiographic exams (enhanced CT or PET-CT) were obtained. Assessment of PALNs for recurrence (enlarged lymph nodes ≥ 1 cm in diameter present in the para-aortic abdomen) by enhanced CT or pet-ct findings. The patients were followed for 3 to 142 months, with a median follow-up period of 34 (20, 51.25) months. The final follow-up date was November 10, 2021.
Statistical methods
GraphPad Prism 8.0 software was used for statistical analysis. The rank sum test was used to compare measurement data. The chi-square test was used to compare count data, which were expressed as the number of cases (n) and the rate (%). The Kaplan-Meier method was used to plot survival curves, and the log-rank approach was employed to test significance. For univariate and multifactorial prognostic analyses, Cox models were applied. The differences were judged to be statistically significant at P < 0.05.
Comparison of clinicopathological characteristics between the patients in the D2 + PAND group and D2 group
Between September 1, 2008, and July 30, 2018, 86 patients were eligible for enrollment. In the D2 + PAND group, there were 40 patients, while in the D2 group, there were 46 patients. Sex, age, enlarged lymph nodes in the first 16a2 and 16b1 areas, chemotherapy regimen, tumor location, Borrmann type, grade of differentiation, and level of neurological invasion were not significantly different between the two groups ( Table 1).
Comparison of the surgical modalities and complications between the D2 + PAND group and the D2 group
In terms of the surgical resection range, reconstruction, intraoperative hemorrhage, and postoperative hospital length of stay, there was no statistically significant difference between the D2 + PAND group and the D2 group. The D2 + PAND group had a median time to surgery of 198 (189, 205.5) min, while the D2 group had a median time to surgery of 167 (156, 178) min. There was a statistically significant difference in the median time to surgery between the two groups (Z = 7.138, P = 0.001) ( Table 2).
The D2 + PAND group had one case of intestinal blockage, two cases of celiac leakage, one case of abdominal infection, and one case of anastomotic leakage. In the D2 group, there was one instance of duodenal stump leakage, one case of celiac leakage, one case of abdominal infection, two cases of anastomotic leakage, and two cases of pulmonary infection; the frequency of postoperative complications was not statistically significant (χ 2 = 0.132, P = 0.717). There were 3 patients who had postoperative recurrence of abdominal para-aortic lymph nodes in the D2 + PAND group, 12 patients in the D2 group, and this was a statistically significant difference (χ 2 = 5.133, P = 0.023) ( Table 3).
Lymph node metastasis in 16 groups
In the D2 + PAND group (40 patients), 172 lymph nodes were eliminated in 16 groups (16a2, 16b1), with an average of 4.3 lymph nodes cleared per case. Postoperative pathology revealed 63 positive lymph nodes. The lymph node-positive group had 16 instances, while the lymph node-negative group had 24 cases. There was a 40% metastatic rate.
Prognostic analysis of the D2 + PAND group versus D2 group
The patients in the D2 + PAND group had a statistically significant difference in their 3-year OS compared to the D2 group (D2 + PAND 69.1% vs. D2 50%, P = 0.012) (Fig. 2) and a statistically significant difference in 3-year disease-free survival (DFS) (D2 + PAND 69.6% vs. D2 38.3%, P = 0.007) (Fig. 3). The degree of lymph node dissection, nerve invasion, and recurrence of para-aortic lymph nodes in the abdomen were all linked to the patient overall survival in the univariate Cox (Table 4).
Discussion
In the JCOG9501 trial, the Japanese Society of Clinical Oncology (SCSG/JCOG) found no survival benefit from prophylactic D2 + PAND compared to D2 lymph node dissection alone in patients with advanced gastric cancer [7]. In this study, 523 patients with advanced gastric cancer were recruited to examine the prognostic impact of D2 + PAND surgery vs. D2 surgery alone. According to the findings here, there was no significant difference in 5-year recurrence-free survival or 5-year overall survival. The D2 + PAND group, on the other hand, had a higher rate of postoperative complications. As a result, the use of preventive D2 + PAND in the treatment of advanced gastric cancer has been denied. After excluding the patients with a leathery stomach or who had more than 15 positive lymph nodes, Tokunaga et al. [14] found that the 5-year OS after D2 + PAND clearance reached 28.6% in patients with positive para-abdominal aortic lymph nodes. The 5-year OS after D2 + PAND was just 17% in a study by the Italian Gastric Cancer Research Group (GIRCG) [15]. In all of the investigations, D2 + PAND did not improve the survival in individuals with gastric cancer and who had metastases to the para-aortic lymph nodes. Chemotherapy followed by D2 + PAND improves the prognosis in individuals with focal para-aortic lymph node metastases from gastric cancer. JCOG conducted three phase II clinical trials (JCOG0001, JCOG0405, and JCOG1002). to determine whether preoperative chemotherapy combined with D2 + PAND is effective and safe in patients with advanced gastric cancer. In the JCOG0001 experiment [9], the patients were given two or three cycles of irinotecan and cisplatin, followed by D2 + PAND gastrectomy. The 3-year survival rate was 27%. Following that, 53 gastric cancer patients were enrolled in the JCOG0405 trial [8], which had similar enrollment criteria to the JCOG0001 trial. Preoperative chemotherapy with S-1 and cisplatin was given to all of the patients. By using the same examination for all patients, a gastrectomy with D2 + PAND was performed, and the R0 resection rate was 82%. Their findings revealed a 64.7% RR, a 58.8% 3-year survival rate, and a low frequency of major adverse events with no treatment-related fatalities. In comparison to the JCOG0405 study, preoperative DCS (doxorubicin, cisplatin, and S-1) in the JCOG1002 study failed to produce sufficient efficiency in patients with extensive lymph node metastases [16]. Based on the results of these phase II trials, S-1 in combination with cisplatin was considered more effective than irinotecan in combination with cisplatin. The 5th edition of the Japanese guidelines for gastric cancer also suggests a combination of preoperative chemotherapy combined with D2 + PAND surgery for patients with lymph node metastasis in the NO. 16a2/b1 group alone [17].
Preoperative chemotherapy (capecitabine and oxaliplatin) followed by a combined D2 gastrectomy had adequate R0 resection rates, according to a study by Chinese researchers [10]. Another real-world study found that D2 gastrectomy alone is safe and effective in patients with gastric cancer who have metastatic abdominal paraaortic lymph nodes and respond well to preoperative chemotherapy [18]. Nonetheless, for patients with gastric cancer with para-aortic lymph node metastases, no relevant controlled studies have directly revealed the advantages and disadvantages of preoperative chemotherapy followed by D2 + PAND surgery over D2-only surgery. As a result, we undertook this study to explore whether preoperative chemotherapy combined with paraaortic lymph node dissection had any therapeutic value in the treatment of patients with gastric cancer and paraaortic lymph node metastases.
In this study, there were no statistically significant differences between the D2 + PAND group and the D2 group in terms of sex, age, preoperative chemotherapy regimen, tumor location, tumor differentiation, or other characteristics, and the two groups were comparable. In comparison to conventional D2 radical gastric cancer surgery, D2 + PAND radical gastric cancer surgery was more difficult and required more operative time, but there were no significant differences in the intraoperative bleeding, postoperative complication rate, or postoperative length of hospital stay between the groups. Seckin et al. [19] found that D2 + PAND surgery, when compared to D2 surgery, did not enhance the length of postoperative hospital stay or the rate of postoperative complications. Other research has found that a routine D2 + PAND surgery can be conducted safely and with no increase in the postoperative mortality [20]. We believe that D2 + PAND surgery is safe and practical and that it can be conducted in specialist centers and has a low surgical risk. However, D2 + PAND radical gastric cancer surgery takes longer to complete than D2 radical gastric cancer surgery. The intricate retroperitoneal anatomy that is involved with the removal of the para-aortic lymph nodes, the restricted intraoperative view, and the potential for blood vessel damage may all play a role in its difficulty. D2 + PAND should only be performed in a cancer center with skilled surgeons, as there are some dangers associated with the formation of complications, such as celiac fistula, in some rare cases. Further prognostic analysis revealed that combined D2 + PAND radical surgery for gastric cancer after preoperative chemotherapy improved overall survival and disease-free survival compared to D2 radical surgery for gastric cancer. Also, the extent of lymph node dissection was an independent factor affecting overall survival. The overall survival rates of patients with gastric cancer with para-aortic lymph node metastasis after neoadjuvant chemotherapy combined with surgery were 80% and 48% at 1 and 3 years, respectively, and the disease-free survival rates were 72% and 38% at 1 and 3 years, according to the findings of a domestic study. This study found that neoadjuvant chemotherapy followed by surgery can dramatically improve these patients' prognoses. Based on these findings, we infer that preoperative chemotherapy followed by D2 + PAND surgery may result in a survival benefit for patients with gastric cancer and metastatic abdominal para-aortic lymph nodes. The overall survival rates of patients with gastric cancer with paraaortic lymph node metastasis after neoadjuvant chemotherapy combined with surgery were 80% and 48% at 1 and 3 years, respectively, and the disease-free survival rates were 72% and 38% at 1 and 3 years, respectively, according to the findings of a Chinese study. This study found that neoadjuvant chemotherapy followed by surgery can dramatically improve these patients' prognoses [21]. Based on the results of this study, we also infer that preoperative chemotherapy followed by D2 + PAND surgery may result in a survival benefit for patients with gastric cancer and metastatic abdominal para-aortic lymph nodes.
In our research, individuals with gastric cancer who were evaluated or paraaortic lymph node metastases based on CT at the initial visit had a postoperative pathologically confirmed metastatic rate of 40%. Furthermore, we discovered that the non-PAND group had a greater rate of PALN recurrence than the PAND group, and multifactorial analysis revealed that para-aortic lymph node recurrence was an independent risk factor for prognosis. Some studies have indicated a considerable reduction in the rate of recurrence in the retroperitoneal area in D2 + PAND patients [22]. They considered that D2 + PAND surgery for metastatic lymph nodes in the para-aortic area would be helpful. According to the abovementioned findings, eliminating the para-aortic lymph nodes in the abdomen may minimize the risk of retroperitoneal recurrence. Furthermore, paraaortic lymphatic recurrence in the abdominal aorta was found to be an independent prognostic factor in our study, which may also be the reason for the better OS in the D2 + PAND group than in the D2 group in our research.
As this study is a retrospective analysis, there are certain shortcomings: (1) the sample size of this study is small; (2) this study is a single-centre retrospective study and further prospective, multi-centre clinical studies are needed to confirm it. More prospective studies are needed to evaluate the optimal indication for D2 + PAND following preoperative chemotherapy in patients with gastric cancer as well as para-aortic lymph node metastases.
Conclusion
In conclusion, we believe that the rate of PALN metastasis is higher in gastric cancer patients who have first-visit imaging that suggests paraaortic lymph node metastasis. Although D2 + PAND is a difficult procedure, preoperative chemotherapy combined with para-aortic lymph node dissection can improve overall survival, disease-free survival. And reduce the risk of retroperitoneal lymph *Statistically significant (p < 0.05)
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v2
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2022-11-22T06:17:22.697Z
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2022-11-20T00:00:00.000Z
|
253733556
|
s2ag/train
|
Safety of anti-SARS-CoV-2 messenger RNA vaccine in lung cancer patients undergoing anticancer chemotherapy: A multicenter, prospective, observational, patient-reported outcome study.
BACKGROUND
COVID-19 incidence is high in patients with cancer. The fatality rate was high for the Delta variant, necessitating infection prevention by vaccination. This study evaluated the safety of a SARS-CoV-2 vaccine in patients with advanced lung cancer receiving anticancer therapy.
METHODS
We prospectively enrolled patients receiving anticancer drugs for advanced lung cancer and planning SARS-CoV-2 vaccination. Early side effects within 7 days of vaccination were evaluated using patient-reported outcome (PRO) surveys. Chi-square test and multivariate logistic regression analyses were used.
RESULTS
Post-vaccination PROs were collected from 406 patients (252 were males). The mean age was 72 years. Treatment at the time of initial vaccination included chemotherapy, immune checkpoint inhibitors (ICI), a combination of chemotherapy and ICI, targeted therapy including tyrosine kinase inhibitors, and others in 115, 93, 45, 147, and six cases, respectively. The vaccines administered were BNT162b2 and mRNA273 in 361 and three cases, respectively and unknown in 42 cases. A total of 16.1% of patients developed fever (38°C) after the second mRNA vaccination (95% confidence interval: 12.6%-20.1%). This rate is comparable to data previously reported in 120 patients and slightly higher than that of healthy participants of the BNT162b2 study. Patients receiving treatment with cytotoxic anticancer agents were more likely to have high fever. Multivariate analysis showed no correlation between fever frequency and patient background. No serious initial adverse events due to vaccination were observed.
CONCLUSIONS
Anti-SARS-CoV-2 mRNA vaccination is safe; however, post-vaccination fever is more common in patients undergoing lung cancer treatment than in healthy individuals.
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v2
|
2022-11-21T14:10:24.650Z
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2022-11-21T00:00:00.000Z
|
253709591
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s2orc/train
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Distinct clinical pattern of colorectal cancer patients with POLE mutations: A retrospective study on real-world data
Objective: Studies have demonstrated an association between somatic POLE exonuclease domain mutations (EDMs) and the prognosis of colorectal cancer (CRC). However, the prognostic value of POLE non-EDMs remains unclear. This retrospective study aimed to explore the possible relationships between POLE mutation subtypes and CRC prognosis. Methods: The 272 CRC patients from the First Affiliated Hospital of Zhengzhou University (ZZ cohort) and 499 CRC patients from The Cancer Genome Atlas database (TCGA cohort) were retrospectively collected. The cases were divided into subgroups based on POLE mutation sites and microsatellite instability (MSI) status. The continuous variables were compared among three subgroups with Kruskal-Wallis tests. Pairwise comparisons between three groups were performed by Bonferroni correction method, and adjusted p < 0.05 was considered statistically significant. The categorical variables were compared with Chi-square test and Fisher’s exact test. The Kaplan—Meier curves and Cox regression models were conducted to evaluate prognostic values of POLE mutations. Results: In the ZZ cohort, POLE EDMs (2.6%) were significantly associated with younger age (p = 0.018) and localized in the left colon (p = 0.001). POLE non-EDMs were significantly associated with MSI-high status (p < 0.001) and localization in the right colon (p = 0.001). In the TCGA cohort, the tumor mutation burden (TMB) of both POLE EDM tumors (p < 0.001) and POLE non-EDM tumors (p < 0.001) was significantly higher than that of POLE wild-type (WT) tumors. A similar trend was observed in the ZZ cohort, although there were no significant differences. In the ZZ cohort, the POLE EDM group had higher progression-free survival (PFS) (p = 0.002) and overall survival (OS) (p = 0.042) than the POLE non-EDM group and POLE WT group. We also report one CRC patient harboring a germline POLE mutation who received camrelizumab and exhibited long-term stable disease. Conclusion: Both POLE-EDMs and POLE non-EDMs were associated with significantly increased TMB in CRC and may be biomarkers for CRC treatment and prognosis. Current evidence does not support an effect of POLE non-EDMs on PFS and OS. A significant association between POLE EDMs and improved PFS and OS may exist, but future studies with larger sample sizes are needed. Entire coding region of the POLE gene should be screened.
Introduction
The global incidence and mortality of colorectal cancer (CRC) rank first among gastrointestinal cancers (Sung et al., 2021). The poor prognosis of CRC is mainly due to its insidious onset, as approximately 25% of patients have metastasized CRC at the time of diagnosis, resulting in limited treatment options (Andre et al., 2015;Bryan et al., 2018). CRC is a highly heterogeneous cancer that develops mainly by affecting the expression and behavior of genes related to cell growth and differentiation (Fearon, 2011). In recent years, increasing studies have indicated that mutations in the DNA polymerase gene POLE mutation may be important for guiding CRC management, and are a potential biomarker for treatment and prognosis (Huhns et al., 2020).
The nuclear DNA replication-repair-associated polymerases Pol α, Polδ and Polε all belong to the polymerase B family (Doublie and Zahn, 2014). During replication, the main function of POLε is to lengthen the leading strand. The catalytic subunit of POLε has 5′ to 3′ DNA polymerase activity and 3′ to 5′ exonuclease activity and is capable of the timely removal of erroneous bases generated during replication to ensure the fidelity of DNA replication. This catalytic subunit is encoded by POLE (Henninger and Pursell, 2014). In 2012, The Cancer Genome Atlas (TCGA) exome sequencing project conducted a complete genome analysis of 224 CRC cases and showed that POLE mutation is closely related to an ultra-hypermutated phenotype (TMB >100 mut/Mb) (Cancer Genome Atlas, 2012). Subsequently, several studies have shown that CRC patients carrying POLE mutations often have TMB and infiltration of immune cells in tumors (Forgó et al., 2020;Picard et al., 2020). The aggregation of epitopes in tumors makes them more susceptible to immune checkpoint inhibitors (ICIs). To date, microsatellite instability-high (MSI-H)/deficient mismatch repair (dMMR) is the only widely recognized specific biomarker related to the positive effect of ICIs in CRC treatment (Andre et al., 2020). However, only 5% of CRC patients have MSI-H/dMMR (Battaglin et al., 2018). POLE mutations have the potential to serve as a specific biomarker to screen for candidates who may benefit from ICIs. In addition, similar to MSI in nonmetastatic CRC, POLE mutations also imply lower recurrence and metastasis rates. For stage II CRC patients whose need for adjuvant therapy is still controversial, POLE mutations indicate a better prognosis and may be important evidence for guiding treatment decisions.
In the predictions of treatment and prognosis of CRC, somatic POLE mutations have been reported to be a promising candidate biomarker. However, most studies have focused on POLE exonuclease domain mutations (EDMs) or individual mutation points. The significance of POLE non-EDMs in CRC remains unclear. Thus, this retrospective study investigated the clinical characteristics and prognostic value of POLE mutation subtypes in a real-world dataset. A similar analysis was carried out in a TCGA dataset, and the results of the two cohorts are compared and discussed.
Patients
The Chinese cohort included 272 CRC patients treated at The First Affiliated Hospital of Zhengzhou University (ZZ cohort) between January 2016 and December 2020. The latest follow-up date was 1 March 2021. All patients were pathologically diagnosed with primary CRC by tissue biopsy and underwent NGS. Ethics committee approval was obtained from the institutional research ethics board -KY-1040
DNA sequencing
The genomic profiling was conducted by a hybridization capture-based NGS assay using a commercial panel consisting of 520 cancer-associated genes (OncoScreen Plus, Burning Rock Biotech), spanning 1.64 Mb of the human genome (Wang et al., 2022). Tissue DNA was fragmented using Covaris M220 (Covaris, MA, United States) followed by end repair, adapter ligation and purification of fragments with sizes between 200 and 400 base pairs. Fragment size and quality were assessed with high-sensitivity DNA kit using Bioanalyzer 2100 (Agilent Technologies, CA, United States). Subsequently, the Indexed samples were sequenced on the NovaSeq 6000 platform (Illumina, Inc., CA, United States) with 150-base pair read lengths.
Sequence data were analyzed using the Burning Rock analysis system. Concisely, raw reads were aligned to the reference human genome (hg19) using Burrows-Wheeler Aligner (version 0.7.10). Variant calling was implemented using VarScan (version 2.4.3) with the following filtering steps to retain high-confidence variants: loci with depths ≥100, at least eight supporting reads for single nucleotide variations (SNVs), at least two and five supporting reads for Indel variants. Single nucleotide polymorphisms (SNPs) were all removed.
TMB and MSI calculation
TMB was defined as the number of non-synonymous variants per megabase of genome examined, and was estimated using the OncoScreen Plus panel (OncoScreen plus, Burning Rock, Guangzhou, China) with a total size of 1.003 Mb of coding regions. Hotspot variants, copy number variations, structural variants, and germline SNPs are not counted.
MSI status of tumor and plasma samples was determined using a read-count-distribution-based approach that utilizes a given set of repeat lengths of coverage as the prime characteristic of each microsatellite locus. A locus is classified as unstable if more than 30% of the total number of microsatellite markers in the sample is below this threshold.
Statistical analysis
Overall survival (OS) was defined as the time from histological diagnosis of CRC to death. Progression-free survival (PFS) was defined as the time from first-line therapy to the first tumor progression or recurrence. The end date was defined as the date of the last follow-up visit if there was no cancer recurrence or death. Continuous variables are described as the mean and standard deviation or the median and the interquartile range. Categorical variables are described with frequencies and percentages. The continuous variables (age at diagnosis, TMB) were compared among POLE EDM, POLE non-EDM and POLE WT groups with Kruskal-Wallis tests. Pairwise comparisons between three groups were performed using the Bonferroni correction method, and an adjusted p value < 0.05 was considered statistically significant. The same method was used to compare TMB levels among POLE non-EDM (MSI-L/MSS), POLE WT (MSI-H) and POLE WT (MSI-L/MSS) groups. The categorical variables were compared among POLE EDM, POLE non-EDM and POLE WT groups with Chisquare test and Fisher's exact test. Survival function curves were generated using the Kaplan-Meier method (Ying et al., 2021). Survival differences among groups were evaluated by the log-rank test. Univariate and multivariate Cox regression models were employed to evaluate the prognostic value of POLE mutations for OS and PFS (Burke et al., 2017). All statistical analyses were performed with SPSS version 23.0 software (IBM, Chicago, IL). A two-tailed p value < 0.05 was considered statistically significant.
Molecular characteristics of POLE mutations
The protein distribution of POLE mutations is shown in Figure 1. In the ZZ cohort, the somatic POLE mutation rate was 7.7% (21 out of 272), including 2.6% (7 out of 272) of POLE EDMs and 5.1% (14 out of 272) of POLE non-EDMs. Five of the seven POLE EDMs were known pathogenic mutations (V411 L in 1 case, P286R in 4 cases). A mutation of unknown significance (E396 fs) was detected in 2 cases ( Figure 1A). The location and genetic characteristics of each POLE mutation are shown in Table 1. Compared with POLE WT tumors, POLE non-EDM tumors were mainly MSI-H (p < 0.001). Most POLE EDM tumors were MSI-L/MSS; however, the difference was not significant.
Frontiers in Genetics frontiersin.org 03 In the TCGA cohort, compared with POLE WT tumors, both POLE EDM tumors (median TMB = 115.3 mut/Mb, p < 0.001) and POLE non-EDM tumors (median TMB = 64.2 mut/ Mb, p < 0.001) exhibited a significantly increased TMB. A similar trend was observed in the ZZ cohort; however, in the pairwise comparisons, the Bonferroni corrected p values indicated no significant difference between each pair of groups (p > 0.05).
Given that POLE non-EDM tumors are mostly MSI-H (ZZ cohort p < 0.001; TCGA cohort p < 0.001), this study further
Prognostic value of POLE mutations
All patients were divided into 3 subgroups: the POLE EDMs, POLE non-EDMs and POLE WT groups. In the ZZ cohort, the 272 CRC patients were followed for a median of 16.8 months. Since no patients in the POLE EDM group had progressed by the last follow-up, the median PFS was not reached. Based on the stratified log-rank test, the PFS rate of the POLE EDM group was significantly higher than that of the POLE non-EDM (median = 22.0 months, χ 2 = 5.407, p = 0.020) and POLE WT groups (median = 14.6 months, χ 2 = 8.830, p = 0.003) (Figure 2A). The OS of the POLE EDM group and POLE non-EDM group were not reached. There was no significant difference in OS among these three groups (p = 0.056) ( Figure 2B (MSI-L/MSS) group (median = 38.2 months) on OS ( Figure 4B). With the univariate and multivariate Cox regression models, POLE EDM and POLE non-EDM were both prognostic protective factors (HR<1) without statistical significance levels ( Table 3, Supplementary Table S1). Distant metastasis and advanced clinical stage (stage III-IV) were independent risk Frontiers in Genetics frontiersin.org 07 factors for shortened PFS while age ≥60 and poor differentiation (G3) were independent risk factors for shortened OS.
In the TCGA cohort, the median follow-up periods of 499 CRC patients was 22.0 months. The similar analyses were also performed among these three subgroups in the TCGA cohort; however, there were no significant differences on PFS and OS among the groups. With the univariate and multivariate Cox regression models of the TCGA cohort, POLE EDM and POLE non-EDM were both prognostic risk factors (HR>1) without statistical significance levels (Table 3, Supplementary Table S2). Lymph node metastasis and pT4 were independent risk factors for shortened PFS. Distant metastasis, pT4 and age >60 years are independent risk factors for shortened OS.
A patient with an inherited germline POLE mutation treated with camrelizumab
Polymerase proof-reading associated polyposis and Lynchlike syndrome are inherited cancer susceptibility syndromes associated with germline POLE mutations. (Elsayed et al., 2015;Vande Perre et al., 2019). Such patients often progressively develop CRC or extraintestinal tumors (Bellido et al., 2016). Identifying germline POLE mutations may help to understand the pathogenesis of CRC, reduce the morbidity and mortality, and guide treatment. The germline POLE mutations identified in CRC patients published from 2017 to 2020 are summarized in Table 4. There were two relatively rare cases in which p. V411L was previously described as a somatic hotspot alteration and p. V474I was located outside the ED. This study also reports a rare case.
A male patient with abdominal pain, abdominal distention, and difficulty defecating was referred to our center in December 2019.
Medical imaging examination and tissue biopsy suggested bowel obstruction and rectal adenocarcinoma with multiple lymph node metastases. He received first-line treatment with an oxaliplatin plus capecitabine regimen. He developed adrenal metastasis 3 months later and was treated with bevacizumab. However, rectal occupation progressed soon after this addition, so the above regimen was stopped. Treatment with "FOLFIRI + bevacizumab" began on 16 April 2020; however, the effect was poor. The tumor continued to progress, and the patient presented with liver metastasis 2 months later. NGS results of a 41-gene panel suggested the presence of a POLE mutation (exon 45, S2084 fs), KRAS mutation (G12S), TP53 (R2084 fs) and MSS. The administration of anlotinib and camrelizumab began on 8 June 2020 and was continued until the last follow-up (6 December 2021), with no progression observed (PFS >18 months) (Figures 5, 6). The POLE mutation was an inherited germline mutation located outside the ED; this variant has not been previously identified in a large population database. According to the ACMG 2015 guidelines, this variant was evaluated as a hereditary variant with possible pathogenicity. The patient had MSS but received sustained longterm benefit from immunotherapy. It is believed that POLE mutation may be used to predict the response to ICIs.
Discussion
This study included mutations inside and outside of the POLE exonuclease domain. We aimed to explore the molecular pathological features and prognostic value of different POLE mutation subtypes. As previously reported, in the Asian population, POLE EDMs are mainly found in the left colon and relatively young CRC patients (Hino et al., 2019;Hu et al., 2021), whereas POLE non-EDMs are more common in the right colon.
Somatic POLE mutations were evenly located throughout the POLE gene with no apparent tendency to cluster as shown in Figure 1. In the ZZ cohort, the frequency of POLE EDMs was 2.6%, while the frequency of POLE non-EDMs was 5.1%; in the TCGA cohort, the frequencies were 1.8% and 4.8%, respectively. This finding is consistent with the previously reported frequency of POLE mutations in CRC (Campbell et al., 2017). Although the frequency of POLE mutations is low, its unique high immunogenicity has attracted widespread attention.
Tumors harboring POLE EDMs often manifest with a high TMB, which is associated with an enhanced intertumoral immune response and better outcome (Llosa et al., 2015). This discovery was first reported in the TCGA whole-exome sequencing project in 2012 and is a critical first step for moving treatment of toward precision therapy. In addition, some tumors harboring only POLE non-EDMs also exhibited elevated mutation burdens, such as C810 and E978. This study showed that both POLE EDMs and POLE non-EDMs were associated with significantly increased TMB (Table 2). Since the POLE EDM Frontiers in Genetics frontiersin.org 08 Frontiers in Genetics frontiersin.org 09 tumors in this study were mostly MSI-L/MSS, and the POLE non-EDM tumors were mostly MSI-H, we analyzed the data again after excluding the interference of MSI status and still reached the same conclusion. POLE EDM tumors tended to have ultra-hypermutated phenotypes (TMB>100 mut/Mb), and POLE non-EDM tumors tended to have hypermutated phenotypes (TMB>10 mut/Mb). Although consistent with previous reports that POLE EDMs are predominantly MSS, this study identified 3 cases of CRC harboring both POLE E396fs and MSI-H (ZZ cohort, 2 cases; TCGA cohort, 1 case) (Stenzinger et al., 2014;Kawai et al., 2021). All 3 cases were stage II CRC with prolonged PFS. The significance of this mutation merits further study. This study indicated that mutation location is not a determining factor for the predictive value of POLE mutations. Thus, it is necessary to thoroughly assess POLE mutations throughout the coding region.
In tumors with MSI/dMMR or POLE mutations, the production of new antigens is caused by a large accumulation of nonsynonymous substitution and/or frameshift mutations. Major histocompatibility complexs can present these new antigens to the immune system, thereby enhancing the immune system's attack on tumor cells. In recent years, several patients with both POLE EDMs and MSS have been reported to obtain clinical benefit from ICI treatment (Guerra et al., 2017;Keenan et al., 2021). A study of a cohort of 295 patients with stage II CRC indicated that POLE mutant tumors have significantly elevated mutation levels (Domingo et al., 2016). These patients have a better prognosis and may not require adjuvant treatment. Studies have indicated that the predicted amount of new antigens in MSI/dMMR tumors is 10-50 times those in MSS tumors, and in POLE mutant tumors produce 15 times the amount of new antigens compared to that of MSI/dMMR tumors (Shinbrot et al., 2014;Howitt et al., 2015). Therefore, the prognosis and treatment response of CRC patients with POLE mutations may be improved and enhanced.
MSI and POLE mutations have similar effects on tumors. To exclude the influence of MSI status and thus determine the prognostic value of POLE mutation itself, this study conducted 3 subgroup analyses according to POLE mutation and MSI status. Additionally, the prognostic value of MSI status and POLE mutation was compared.
First, this study divided all patients into three groups: the POLE EDM, POLE non-EDM and POLE WT groups. In the ZZ cohort, we found that POLE EDM tumors were less prone to recurrence or progression than POLE WT tumors (Figure 2A). POLE non-EDM tumors did not show a PFS advantage. Moreover, no difference in OS was observed among the groups ( Figure 2B). Subsequently, we divided the patients into POLE EDM, POLE WT (MSI-H) and POLE WT (MSI-L/MSS) subgroups. In the ZZ cohort, POLE EDM and POLE WT (MSI-H) tumors had better OS and PFS outcomes than POLE WT (MSI-L/MSS) tumors (Figure 3). POLE EDMs and MSI-H status had similar roles in improving the prognosis of CRC. Finally, we divided the patients into POLE non-EDM (MSI-L/MSS), POLE WT (MSI-H) and POLE WT (MSI-L/MSS) subgroups. POLE non-EDM tumors did not show improvement or deterioration of PFS or OS in the ZZ cohort (Figure 4). Based on the above subgroup analyses, POLE EDMs and MSI-H statue improve clinical outcomes to a similar degree. Currently, POLE non-EDMs do not demonstrate this beneficial effect.
In the univariate and multivariate Cox regression models, POLE EDMs and POLE non-EDMs were both protective factors for PFS and OS prolongation (HR<1) in the ZZ cohort but did not reach statistical significance levels (Table 3). We considered that the accuracy and validity of the Cox regression model was reduced due to the high proportion of censored data for most patients who did not reach the clinical outcome of PFS or OS.
In this study, the above 3 subgroup analyses were also performed in the TCGA cohort; however, POLE mutations did not show an effect on the PFS or OS outcomes. Paradoxically, POLE mutation may be a risk factor for reduce PFS and OS in the TCGA cohort (HR > 1). It should be noted that the cases in the TCGA cohort were diagnosed from 1998 to 2013. However, the clinical application of ICIs has only gradually been realized in the past 5 years. POLE mutations and MSI-H statue are both factors closely related to the effect of immunotherapy. Therefore, the above contradictory results are likely related to the application of ICIs. In addition, it is worth noting that only 12 (2.4%) cases in the TCGA dataset were Asian, and the differences between ethnic groups cannot be ignored.
Somatic POLE mutations have the potential to guide personalized treatment, thereby improving clinical outcomes. The discovery of germline POLE mutations is highly important for reducing the incidence of CRC. Esteban et al. reported a germline POLE mutation (V474I) located outside the ED (Esteban-Jurado et al., 2017). This study also identified a potentially pathogenic germline POLE non-EDM (S2084 fs). This metastatic rectal cancer patient progressed rapidly after early treatment but obtained continued benefits after receiving camrelizumab and anlotinib (PFS >18 months). Interestingly, after two cycles of application of this regimen, MRI scans showed that the metastasis in the right lower lobe of the liver first increased and then gradually decreased and remained stable after continuous administration ( Figure 6). We suggested the efficiency of ICI treatment should not be evaluated too soon after application due to the temporary increase in reactivity.
This study excluded the effect of MSI status on tumors and extended the scope of the study to the entire region of the POLE gene. We fully analyzed the clinico-molecular pathological features of POLE EDM tumors and POLE non-EDM tumors and the prognostic impact of POLE mutation subtypes from different aspects. In addition, this study compared the difference between the effects of POLE mutation and MSI status on CRC. Our study also had a few limitations. First, patients with POLE mutations had a high survival rate and PFS rate, and the insufficient follow-up time resulted in Frontiers in Genetics frontiersin.org insufficient statistical power for some subgroups. We need to continue to closely follow-up with these patients. Second, the total number of POLE mutation was small, and additional studies are required to verify the applicability of the findings in this study. Third, racial differences in the clinical characteristics and prognosis of CRC patients with POLE mutations should be explored further in future studies.
In conclusion, both POLE EDMs and POLE non-EDMs were associated with significantly increased TMB in CRC, which is an important biomarker for CRC treatment and prognosis. It is also necessary to study the entire region of the POLE gene. POLE EDMs may be significantly associated with prolonged PFS and OS; however, the evidence is currently insufficient. Future studies need larger sample sizes to provide more data. The current data do not support the impact of POLE non-EDMs on CRC prognosis. Future studies need to eliminate the interference caused by ethnicity and treatment to analyze the specific role of POLE genes more accurately.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Ethics statement
The studies involving human participants were reviewed and approved by the First Affiliated Hospital of Zhengzhou University. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.
Author contributions
MJ conceived and designed this paper. Study implementation and feasibility analysis for JH and JS. YJ analyzed and explained the results. SJ modified the later versions. HZ and SJ revised the pictures and the manuscript.
Funding
This study was conducted with support from the Health Commission of Henan Province (Nos. SBGJ202102136 and SBGJ202102137). The work of YJ was supported by Zhengzhou University (Grant No. 32212456
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Epigenetic-related gene mutations serve as potential biomarkers for immune checkpoint inhibitors in microsatellite-stable colorectal cancer
Background Combination therapy with immune checkpoint inhibitors (ICIs) may benefit approximately 10-20% of microsatellite-stable colorectal cancer (MSS-CRC) patients. However, there is a lack of optimal biomarkers. This study aims to understand the predictive value of epigenetic-related gene mutations in ICIs therapy in MSS-CRC patients. Methods We analyzed DNA sequences and gene expression profiles from The Cancer Genome Atlas (TCGA) to examine their immunological features. The Harbin Medical University Cancer Hospital (HMUCH) clinical cohort of MSS-CRC patients was used to validate the efficacy of ICIs in patients with epigenetic-related gene mutations (Epigenetic_Mut). Results In TCGA, 18.35% of MSS-CRC patients (78/425) had epigenetic-related gene mutations. The Epigenetic_Mut group had a higher tumor mutation burden (TMB) and frameshift mutation (FS_mut) rates. In all MSS-CRC samples, Epigenetic_Mut was elevated in the immune subtype (CMS1) and had a strong correlation with immunological features. Epigenetic_Mut was also associated with favorable clinical outcomes in MSS-CRC patients receiving anti-PD-1-based therapy from the HMUCH cohort. Using immunohistochemistry and flow cytometry, we demonstrated that Epigenetic_Mut samples were associated with increased anti-tumor immune cells both in tumor tissues and peripheral blood. Conclusion MSS-CRC patients with epigenetic regulation impairment exhibit an immunologically active environment and may be more susceptible to treatment strategies based on ICIs.
Current clinical and investigational studies of screening MSS-CRC patients who would benefit from ICIs treatment are limited. PD-L1 expression is a classic biomarker, but the Keynote-028 study demonstrated that PD-L1 + MSS-CRC patients could not benefit from ICI therapy (8). POLD1/POLE mutations are predictive but occur in only 1% of MSS-CRC patients (9). Biomarkers such as tumor mutation burden (TMB), tumor-infiltrating lymphocytes (TIL), neo-antigen load (NAL), and immune-regulatory gene expression profiling (iGEP) may allow the selection of clinical patients for ICIs. However, the lack of uniform detection methods and validated cutoffs limit the use of these methods (5,(10)(11)(12). Several emerging biomarkers, such as gut microbiota and T-cell-receptor (TCR) sequencing, have also shown predictive value, although they are not yet clinically applicable (7,13). DNA damage response (DDR) gene mutations may induce a hypermutational phenotype (14), and recent studies have shown that patients with MSS-CRC and mutations in the DDR system have better immune responses and outcomes following ICI therapy (15,16). However, the pathogenicity of different DDR gene mutations in MSS-CRC remains unclear, and their incidence is significantly lower than in endometrial, ovarian, or biliary tract cancers (17).
Epigenomic alterations can affect tumor immunogenicity and anti-tumor responses by regulating genome stability and chromatin accessibility (18). Additionally, several epigenetic-related gene mutations have been shown to exhibit predictive functions in ICI therapy for multiple types of tumors. ARID1A, an AT-rich interactive domain-containing protein 1A, is a component of the switching defective/sucrose non-fermenting (SWI/SNF) complex that plays a role in chromatin remodeling (19), and increasing evidence suggests that ARID1A alterations are correlated with better outcomes after ICI therapy for bladder cancer, nonsmall-cell lung cancer (NSCLC), and gastric cancer (20, 21). ARID1A mutation is defined as an immunologically active subgroup in MSS-CRC patients with abundant intratumoral T-cell infiltration (22). Lysine methyltransferase 2 (KMT2) family members facilitate transcription and gene accessibility by methylating lysine 4 on histone H3 (H3k4) (23), and KMT2 family mutations have also been linked to a favorable response to ICIs in multiple cancers (24). Furthermore, as identified using clustered regularly interspaced short palindromic repeats (CRISPR), KMT2D mutant tumors exhibit an increased mutation burden, IFN-g-stimulated antigen presentation, and a higher sensitivity to ICIs. Moreover, disruption of DNA methylation signatures has been identified as a marker of anti-PD-1 therapy efficacy in NSCLC (25), and TET1, a DNA demethylase, enhances the immunotherapeutic effect (26). Although this evidence points to the role of epigenetic regulation in anti-tumor immune responses, there is no clinical data on the association between comprehensive epigeneticrelated gene mutations (mutations in genes that are involved in epigenetic modifications) and the clinical benefit of ICIs in MSS-CRC.
Given the proposed role of epigenetic regulation impairment in predicting the response to ICIs, we hypothesize that epigenetic-related gene mutations in MSS-CRC cause hypermutation and improve the expression of immune response gene sets. As a result, we conducted this study to clarify the value of epigenetic-related gene mutations as an indicator of immunotherapy efficacy in patients with MSS-CRC. For this purpose, we analyzed whole-exome sequencing (WES) data from TCGA to study TMB, frameshift mutation (FS-mutation), and immune characteristics of Epigenetic_Mut and Epigenetic_Wt groups of MSS-CRC samples. Additionally, in a Chinese clinical MSS-CRC cohort of 89 patients who received PD-1-based treatment, we found that Epigenetic_Mut was associated with favorable clinical outcomes. Here, we report the relationships between epigenetic-related gene mutations and TMB, FS-mutation, immunomodulatory mRNA expression signature, and ICI therapy efficacy in patients with MSS-CRC.
Epigenetic-related gene status definition
Epigenetic-related gene status (Epigenetic_Wt or Epigenetic_Mut) was defined based on the presence of a lossof-function (LOF) variant in 68 genes that have been proposed as core genes of epigenetic regulation (18). Supplementary Table 1 presents a detailed description. Nonsense, frameshift, and splice site changes within consensus regions and start lost/ gained variants were considered to be LOF variants. Missense and in-frame variants were excluded from the analysis.
DNA extraction and sequencing
For the TCGA cohort, gene mutation data were acquired using the GDC Data Portal. We assessed the mutational status of epigenetic-related genes in CRC using exome-sequencing data from HMUCH. For analysis, DNA was extracted using a DNA Kit (Applied Biosystems, Foster City, CA, USA), from whole blood samples or formalin-fixed paraffin-embedded (FFPE) tissues of each patient. The lymphocytes from the whole blood samples were isolated by centrifugation at 1,600 × g for 10 min in red cell lysis buffer (Tiangen, RT122, Beijing, China) at 25°C, and DNA was extracted using a genomic DNA kit (Tiangen, DP304, Beijing, China). We sheared the DNA into fragments of 150-200 bp using an ultrasonicator and used a KAPA Kit (KAPA Biosystems, Wilmington, MA, USA) to prepare DNA fragment libraries for the Illumina platform (Illumina HiSeq X-Ten, Illumina, USA). Probe hybridization capture technology and Illumina highthroughput sequencing were used to detect the exonic regions and some intronic regions of 825 tumor-related genes (Genetron Health Co., Ltd. Beijing, China) (Supplementary Table 2).
Analysis of MSI status, TMB, and FSmutation in the TCGA and HMUCH cohorts MSI status for the TCGA cohort was determined using the MSI sensor (version 0.5). In brief, for MSI sensor scores < 3.5, samples were considered to be MSS; otherwise, they were considered MSI (27). Published studies using the TCGA cohort provided FS-mutation and TMB data (28)(29)(30), and MSI status for the HMUCH cohort was determined using a 3730 sequencer (Life Technologies, Carlsbad, CA, USA). For this purpose, whole blood samples or prepared FFPE tissue were diluted to 2 ng/mL or 20 ng/mL, respectively, followed by the addition of 2.8 mL of ddH 2 O, 4 mL of 2.5× Buffer A, 2 mL of 5× MSI Primer Mix, and 0.2 mL of Taq DNA Polymerase I. PCR amplification was carried out as follows: pre-denaturation at 95°C for 5 min; followed by 30 cycles at 94°C for 30 s, 60°C for 1 min, and 70°C for 1 min; and then a final extension at 60°C for 30 min. Finally, the temperature was reduced to 15°C, and the samples were centrifuged at 3,000 × g for 1 min. NR-21 and BAT-26 were labeled with blue fluorescent dye, BAT-25 with green dye, and NR-24 and MONO-27 with yellow dye. Finally, tumors were classified as MSI-H if two or more markers showed instability; otherwise they were classified as MSS.
Analysis of the consensus molecular subtypes (CMSs) in the TCGA cohort
Consensus molecular subtypes (CMSs) are classification systems for CRC and include immune (CMS1), canonical (CMS2), metabolic (CMS3), and mesenchymal (CMS4) subtypes. These subtypes were identified through a large-scale analytical study and have unique molecular and metabolic characteristics (31).
Immune-related signature analysis
Our study compared the RNA expression of patients with Epigenetic_Mut and Epigenetic_Wt using gene signatures for the IFN-g pathway and other immunological responses (Supplementary Table 3) (12,32). We obtained TCGA transcriptome profiles from the GDC data portal, and used transcripts per kilobase million (TPM) normalization to normalize gene expression. The geometric mean of gene expression levels in the log 2 (TPM + 1) format was used to evaluate immune signatures.
Clinical outcomes
The objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were the main clinical outcomes of interest. The Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 was used for the assessment of ORR and divided into complete response (CR) and partial response (PR). DCR was defined as CR, PR, or stable disease (SD) lasting more than six months. PFS was evaluated from when immunological therapy was initiated until progression or death, and patients who did not progress were examined at the last scan. OS was evaluated from the start of ICI therapy until patient death or the end of the trial, and the patients with whom we lost contact were classified based on the date of last contact.
Immunohistochemistry (IHC)
Primary tumor paraffin sections of 4 mm were processed for immunochemistry to evaluate CD8 + and FOXP3 + lymphocytes according to the following protocol: roast, deparaffination, and rehydration before performing heat-mediated antigen retrieval with EDTA buffer (pH 9.0), inactivation of endogenous peroxidase activity with 3% H 2 O 2 , incubation with antibody against CD8 (ab101500, 1:500; Abcam, Cambridge, UK) or against FOXP3 (ab200334, 1:500; Abcam) at 4°C overnight, exposure to a DAB IHC Detection Kit after incubation with biotinylated secondary antibodies, and counterstaining with Mayer's hematoxylin solution. An open-source platform for biological-image analysis (Fiji/ImageJ) was used to estimate the densities of CD8 + and FOXP3 + lymphocytes.
Statistical analysis
Fisher's exact test was used to analyze the relationship between epigenetic-related gene mutations and the ORR or DCR, and the Kaplan-Meier method and log-rank test were employed to examine the PFS and OS probabilities of the Epigenetic_Mut and Epigenetic_Wt CRC groups. Based on the Mann-Whitney U-test, TMB, FS-mutation, tumor-infiltrating lymphocytes, expression of immune-related genes, and immune signatures were compared between the Epigenetic_Mut and Epigenetic_Wt CRC groups. Statistical analysis was conducted using two-sided tests with a nominal significance level of 0.05 using R version 3.5.2.
Results
The mutational landscape of epigeneticrelated genes of MSS-CRC in the TCGA cohort A total of 68 epigenetic-related genes involved in 13 different pathways were included in the current research, including genes involved in modifying DNA, histones, and protein complexes that reshape chromatin structure (Supplementary Table 1). In the TCGA cohort, MSI-H and MSS-CRCs had epigenetic-related gene mutation frequencies of 66.67% (50/75) and 18.35% (78/ 425), respectively. The three most frequently mutated pathways in the MSS-CRC cases from TCGA were SWI_SNF, Histone_methylase, and CHD ( Figure 1A), and the epigeneticrelated genes ARID1A, KMT2C, and RSF1 had the highest mutation rates in the TCGA cohort ( Figure 1B).
Epigenetic-related gene mutations are linked with the TMB, FS-mutation, and molecular subtype of CRC High levels of TMB and FS-mutations (FS_mut) reflect a high degree of genomic instability and potential immunogenicity of a tumor, and both of these are therefore potential biomarkers of immune checkpoint inhibitor responsiveness. Hence, we examined the relationships between TMB, FS_mut, and epigenetic-related gene mutation status. In TCGA cohort, epigenetic-related gene mutations were associated with an increased incidence of TMB in MSS-CRC (median mutation rate of 4.76/mb vs. 4.99/mb in Wt and Mut cases, respectively; p = 7.4e-05; Figure 2A). A higher rate of FS_mut was also linked with epigenetic-related gene mutations in MSS-CRC (median frameshift mutation rate of 1.39/mb vs. 1.79/mb in Wt and Mut cases, respectively, p = 3.5e-06; Figure 2B). Molecular subtypes of CRC (CMS) are currently a highly recognized classification method for CRC that can accurately guide patient treatment and prognosis. CMS1, also known as the immune subtype, has better immune activity and high reactivity to ICIs. Here, we analyzed the distribution of Epigenetic_Mut samples based on molecular subtype in all CRC and MSS-CRC cases ( Figures 2C, D). Among the CMS1-CRC cases, 74.12% were Epigenetic_Mut samples (74.12%, 63/85), but in MSS CMS1-CRC cases, this rate was 40% (12/30). Both for all samples and MSS CRC specifically, Epigenetic_Mut samples were enriched in the CMS1 (immune subtype) group.
Epigenetic_Mut is related to increased immune activity in MSS CRC
To identify the tumor immune microenvironment, we compared Epigenetic_Mut and Epigenetic_Wt for immune signatures, tumor-infiltrating lymphocytes, and expression of immune checkpoints and key genes. We demonstrated that epigenetic-related gene mutations increased the expression of immune response genes, including those involved in the IFN-g pathway, antigen presentation, and cytotoxic T-cell function ( Figure 3A). In addition, the expression of NK cell-related genes was increased in the Epigenetic_Mut group. Other immune cells also showed an upward trend, but no statistical difference was observed due to the limited cohort size ( Figure 3B). Finally, we compared the expression of immune checkpoints and key genes between the two groups. In line with the immune response pathway, several immune checkpoints and key genes were upregulated in the Epigenetic_Mut group. In particular, the expression of LAG3 and HAVCR2 was significantly elevated, and elevated levels of TNFRSF4, PDCD1, and IL4l1 were very nearly statistically significant ( Figure 3C).
Epigenetic_Mut predicts favorable clinical outcomes following ICI therapy
Next, to validate the function of epigenetic-related gene mutations further in predicting responsiveness to ICI therapy in MSS-CRC, we collected a clinical cohort of 89 MSS-CRC patients who had received PD-1 mAb-based treatment. Table 1 A B FIGURE 1 Mutational landscape of Epigenetic-related genes associated with MSS-CRC cases from the TCGA and HMUCH cohorts. shows the baseline patient characteristics based on epigeneticrelated gene status. Of the 89 patients, 24 had Epigenetic_Mut, and 65 had Epigenetic_Wt. Using RECIST version 1.1, we evaluated the patients' best overall responses. Compared to Epigenetic_Wt, Epigenetic_Mut had a significantly higher ORR ( Figure 4A, 37.50% (9/24) vs. 15.38% (10/65), Fisher's exact test P = 0.039). As for DCR, the rate was 66.67% (16/24) in patients with epigenetic-related gene mutations from ICI treatment but only 36.92% (24/65) in patients without epigenetic-related gene mutations ( Figure 4B, Fisher's exact test P = 0.017). As expected, PFS was greatly improved in patients with epigenetic-related gene mutations compared to those without epigenetic-related gene mutations in this cohort ( Figure 4C, mPFS:6.00 vs. 3.17 months, Log_rank P = 0.002, HR = 0.4778), and ICI treatment also had a greater benefit on OS in the Epigenetic-Mut group than that in the Epigenetic-Wt group. (Figure 4D, mOS: 10.80 vs. 6.07 months, Log_rank P = 0.003, HR = 0.4279). In addition, we screened 9 genes with high mutation frequency from all epigenetic-related genes, whose predictive value has been demonstrated in other solid tumors, including ARID1A, ATRX, KMT2A/B/C/D, and TET1/2/3. The results showed that MSS-CRC with these gene mutations had more considerable ORR (Supplementary Table 4
The abundance of immune cells in tumor tissue and peripheral blood of patients with or without epigenetic-related gene mutation
We explored the densities of CD8 + and FOXP3 + cells in MSS-CRC samples with different epigenetic-related gene statuses using IHC. Of the 34 MSS-CRC samples, 10 had epigenetic gene mutations. Further, we captured representative images of CD8 + cells and FOXP3 + cells from three samples. The first patient had an ARID1A mutation (ARID1A Frame_Shift_Del), and the second patient had a KMT2D mutation (KMT2D Nonsense_mutation). Both samples showed increased CD8 + cell density and decreased FOXP3 + cell density in tumor tissues (Figures 5A, B). However, in the third patient, who did not present any epigenetic-related gene mutations, the density of CD8 + lymphocytes was lower, and the density of FOXP3 + lymphocytes was higher than that of the other two ( Figure 5C). CD8 + and FOXP3 + cell densities were counted in 38 patients (Epigenetic_Mut, N = 10; Epigenetic_Wt, N = 28), and from this we discovered that CD8 + cell density increased in the Epigenetic_Mut group ( Figure 5D) and that the FOXP3 + cell density decreased in the Epigenetic_Mut group ( Figure 5E). Furthermore, the ratio of CD8/FOXP3 cells in the Epigenetic_Mut group was significantly higher than that in the Epigenetic_Wt group ( Figure 5F). Next, we collected peripheral blood from 12 patients with MSS-CRC (3 with epigenetic-related gene mutations) and measured the proportion of CD8 + PD1 + T cells and CD3 -CD56 + CD16 + NK cells by flow cytometry. We found that both the proportion of CD8 + PD1 + T cells and CD3 -CD56 + CD16 + NK cells was higher in the Epigenetic_Mut group ( Figures 6A, B).
Discussion
Although ICI-based combination therapies have shown certain effectiveness in pMMR/MSS CRC, especially in combination with antiangiogenic agents (Lenvatinib or Regorafenib) that resulted in an ORR of 20-30% (6, 33), most patients still cannot benefit from the combination therapy because of the high heterogeneity of pMMR/MSS CRC. Recently, the MAYA phase II trial (NCT03832621) showed that MSS-CRC patients with silenced MGMT could benefit from ICIs combined with temozolomide treatment (34). This trial showed 36% for 8-month PFS, 42% for ORR, and 18.4 months for the median OS. Therefore, screening the MSS CRC patients with active anti-tumor immune response may be the key to improving the efficacy of immunotherapy. However, the predictive biomarkers for ICI therapy in MSS-CRC patients are limited. pMMR/MSS CRC is a cold tumor that contains few neoantigens and either no or inactive TILs (35). Meanwhile, CRC is a multilayered heterogeneous disease with specific treatment challenges and opportunities (36). Additionally, Previous studies have reported that epigenetic-related gene mutations affect both the tumor microenvironment and efficacy of ICIs (24)(25)(26). Mechanistically, epigenetic modification can reshape the tumor microenvironment by affecting genomic instability and enhancing the immunogenicity of tumor cells. First, epigenetic modification can affect the DNA damage repair response by regulating the accessibility of chromatin. Studies have shown that epigenetic-related gene mutations can lead to increased TMB in tumor cells, such as ARID1A and KMT2D. ARID1A specifically has a 6.7% mutation rate in MSS-CRC (22) and may increase the instability of the genome by adjusting the MMR pathway (21,37). Mutations in the KMT2D gene are common in cancer patients, and their deficiency can increase the levels of genomic DNA damage and TMB, as well as increase transcription instability. Clinical studies have shown that individuals with mutations in genes from the KMT family are more likely to benefit from ICI therapy (24,25). Furthermore, epigenetic-related gene mutation enhances the immunogenicity of tumor cells. Accounting for 5%-10% of genomic DNA sequences, human endogenous retroviruses (ERVs) are remnants of the evolution of germline integrations of exogenous infectious retroviruses (38,39). These exogenous genes are not expressed in healthy tissues other than germ cells but are often abnormally expressed in tumors with epigenetic regulation defects. Here, neoantigen expression increases immunogenicity and triggers an innate immune response against tumors (40,41). Recently, genome-wide technologies have revealed frequent mutations in epigenetic modifier genes, particularly in cancers (42). It is therefore necessary to analyze systematically the immune activity and the effect of immunotherapy in MSS-CRC patients with epigenetic regulation impairment.
In our study, we systematically analyzed 68 epigeneticrelated genes from 13 pathways involved in chromatin regulatory processes in MSS-CRC samples. The mutation rate of epigenetic-related genes in the TCGA cohort was 18.35%. This mutation frequency was higher than that of any previous marker in the population, such as POLE or DDR mutations, and was closer to the potential benefit ratio in MSS-CRC clinical trials. ARID1A, KMT2C, RSF1, CHD9, PBRM1, and ATRX were the most mutated genes in the TCGA cohort, accounting for approximately 75% of the epigenetic-related gene-mutated MSS-CRC patients. This is consistent with previous reports, and ARID1A is thus a marker gene that should be investigated in clinical practice.
Using bioinformatics algorithms, we also assessed whether the MSS-CRC samples with epigenetic-related gene mutations from TCGA had better immune activity, including immune signatures, tumor-infiltrating lymphocytes, and expression of immune checkpoints and key genes. Furthermore, we validated our bioinformatic findings using immunohistochemical analyses of CD8 + and FOXP3 + cells from a cohort of MSS-CRC patients, and similar results were obtained at the histopathological level. In the Epigenetic _Mut group, CD8 + cells were higher and FOXP3 + cells were lower. The Epigenetic _Mut group also had a higher proportion of CD8/FOXP3 cells than the Epigenetic_Wt group. The VOLTAGE trial demonstrated that among MSS-CRC patients receiving ICIs as neoadjuvant treatment, patients with an elevated CD8/FOXP3 cell ratio were more likely to achieve pathologic complete response (pCR), suggesting that the CD8/FOXP3 cell ratio may be a predictor for ICI therapy efficacy (43). Finally, we validated the predictive power of epigeneticrelated gene mutations in the HMUCH cohort of 89 MSS CRC patients who received immunotherapy and discovered that patients with epigenetic mutations were more likely to benefit from ICI-based combination therapy and had better clinical outcomes. These preliminary results demonstrate that epigenetic-related gene mutations can predict the response to ICIs in MSS-CRC patients.
This study has several limitations, including the validation cohort coming from a single-center, the small size of the cohort, and the lack of validation in other populations. This is because ICI-based regimens have not been recommended by any clinical guidelines for MSS-CRC. Numerous patients included in this study experienced the failure of standard treatment, and the treatment compliance and completeness of the clinical information in many of these patients, were not ideal. Additionally, since the genetic information in the HMUCH cohort was obtained from clinical testing, transcriptomic data were lacking. Thus, our TCGA cohort findings could not be validated. Instead, we performed immunohistochemical staining analysis of pathological sections to validate the immune activation status of the Epigenetic _Mut group, but a largerscale validation remains necessary. Furthermore, the application of ICIs in MSS-CRC has not been standardized, and most patients enrolled in our study were patients who had experienced multiple failed lines of treatment, bringing considerable heterogeneity to the population of this study. Therefore, future prospective studies with larger cohort studies are needed.
Conclusion
In conclusion, our data suggest that identifying epigeneticrelated gene mutations might help select the right immunotherapy for MSS-CRC patients and can be used as a biomarker to predict ICI therapy effectiveness. Importantly, the status of epigenetic-related gene mutations is highly accessible from clinical genetic testing, although it is often overlooked by clinicians. Further exploration of the molecular mechanisms underlying the increased effectiveness in specific MSS-CRC patients and prospective clinical trials are therefore warranted. Tumors with epigenetic-related genes mutation had significantly higher levels of intra-tumoral CD8 + lymphocytes than tumors with wild-type epigenetic-related genes. (E) Tumors with epigenetic-related genes mutation had significantly lower levels of intra-tumoral FOXP3 + lymphocytes than tumors with wild-type epigenetic-related genes. (F) The Epigenetic_Mut group had a higher CD8/FOXP3 cell ratio than the Epigenetic_Wt group.
FIGURE 6
Proportion of CD8 + PD1+T cells and NK cells in the peripheral blood of patients with or without epigenetic-related gene mutations. (A) The Epigenetic_Mut group had a higher proportion of CD8 + PD1 + T cells compared to the Epigenetic_Wt group in peripheral blood. (B) The Epigenetic_Mut group had a higher proportion of NK cells compared to Epigenetic_Wt group in peripheral blood.
Data availability statement
The data presented in the study are deposited in the Genome Sequence Archive in National Genomics Data Center repository (https://ngdc.cncb.ac.cn/gsa-human), accession number GSA-Human: HRA003408.
Ethics statement
The study was approved by the Ethics Committee of Harbin Medical University Cancer Hospital (Harbin, China). Informed consents were obtained from all patients before surgery and during the experimental procedures.
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Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors
Background Chimeric antigen receptor T (CAR-T) cell therapy has made significant advances for hematological malignancies but encounters obstacles in the treatment of solid tumors mainly due to tumor immunosuppressive microenvironment. Methods Immunohistochemistry analysis was performed to examine the cellular expression of nectin cell adhesion molecule-4 (Nectin4) and fibroblast activation protein (FAP) in a variety of malignant solid tumors. Then, we engineered the fourth-generation Nectin4-targeted CAR-T (Nectin4-7.19 CAR-T) and FAP-targeted CAR-T (FAP-12 CAR-T) cells to evaluate their safety and efficacy in vitro and in vivo. Results In our study, we firstly demonstrated the aberrant overexpression of Nectin4 on both primary and metastatic solid tumors and FAP on cancer-associated fibroblasts. Then, we found that our fourth-generation Nectin4-7.19 CAR-T cells expressed IL-7 and CCL19 efficiently and exhibited superior proliferation, migration, and cytotoxicity compared to the second-generation Nectin4 CAR-T cells, while FAP-12 CAR-T cells exerted their ability of targeting both murine and human FAP effectively in vitro. In a fully immune-competent mouse model of metastatic colorectal cancer, lymphodepletion pretreated mice achieved complete remission with human Nectin4-targeted murine CAR-T (Nectin4 mCAR-T) cells. In the NSG mouse model of lung metastases, Nectin4-7.19 CAR-T cells eradicated metastatic tumors and prolonged survival in combination with FAP-12 CAR-T cells. Conclusions These findings showed that Nectin4-7.19 CAR-T cells had potential therapeutic efficacy and exerted a synergistic role with FAP-12 CAR-T cells, further demonstrating that Nectin4 and FAP were able to serve as promising targets for safe and effective CAR-T therapy of malignant solid tumors.
Introduction
In recent years, chimeric antigen receptor (CAR) technology has revolutionized cancer therapy, particularly in blood cancers (1)(2)(3)(4). However, CAR-T therapy for malignant solid tumors remains challenging owing to tremendous phenotypic heterogeneity, inefficient proliferation and short persistence of CAR-T cells, and immunosuppressive microenvironment in tumor stroma where inhibitory checkpoints lead to T-cell dysfunction, factors like adenosine and reactive oxygen species inhibit T cells, immunosuppressive cells like regulatory T cells and myeloid-derived suppressor cells promote tumor growth and inhibit T-cell activity, and cancer-associated fibroblasts (CAFs) deposit extracellular matrix to limit T-cell penetration and recruit other immunosuppressive cells (5)(6)(7)(8)(9).
Nectin cell adhesion molecule 4 (Nectin4) is a type I transmembrane protein whose extracellular domain is composed of three Ig-like domains (V-C-C type), participating in the formation and maintenance of adhesion junctions together with cadherin. Nectin4 is ubiquitously expressed in human embryonic cells but hardly in normal adult tissues, while it is highly expressed on the surface of malignant solid tumors such as urothelial cancer, ovarian cancer, and melanoma, playing key roles in various aspects of tumor progression like proliferation, angiogenesis, epithelial-to-mesenchymal transition, metastasis, DNA repair, tumor relapse, and poor prognosis of these epithelial malignancies (10)(11)(12)(13). Enfortumab vedotin, an antibody-conjugated drug targeting Nectin4, has shown unprecedented response rates in locally advanced or metastatic urothelial carcinoma with a tolerable safety in a phase I clinical trial (NCT02091999) and a phase II clinical trial (NCT03219333), and is undergoing phase III clinical trial (NCT03474107) to demonstrate a survival benefit (14-16). Thus, the U.S. Food and Drug Administration granted accelerated approval to Padcev (enfortumab vedotin-ejfv), a Nectin4-directed antibody and microtubule inhibitor conjugate, being indicated for the treatment of adult patients with locally advanced or metastatic urothelial cancer who had previously received a PD-1/PD-L1 inhibitor and a platinum-containing chemotherapy (17). While a growing number of studies have indicated that Nectin4 may be regarded as a potential target for cancer immunotherapy (18,19), no study so far has reported the use of Nectin4-targeted CAR-T cells for clinical therapy of malignant solid tumors. Thus, our phase I study (NCT03932565) has been ongoing to examine the safety and feasibility of Nectin4-7.19 CAR-T cells in patients with Nectin4positive malignant solid tumors.
CAFs are the major components of tumor-associated stroma, forming a highly tumorigenic and immunosuppressive microenvironment (20,21). Fibroblast activation protein (FAP), a type II serine protease with dual specificity of dipeptidyl peptidase and gelatinase activities, is expressed on CAFs in a majority of malignant solid tumors but rarely on fibroblasts in normal tissues, making it an attractive immunotherapeutic target (22,23).
Here, our study showed that Nectin4-7.19 CAR-T cells displayed significant anti-tumor activity in vitro and in vivo and were not likely to cause unacceptable on-target off-tumor toxicities. Furthermore, the combination of Nectin4-7.19 CAR-T cell therapy and FAP-12 CAR-T cell therapy exhibited synergistic anti-tumor effects and thus may be a promising double-pronged approach for patients with Nectin4-positive malignant solid tumors.
Cell lines
The HEK-293T cell line was purchased from the American Type Culture Collection (Cat#ACS-4500); ABC-1, HT1376, and A549 cell lines were purchased from Cobioer (Nanjing, China); the MDA-MB-453 cell line was a gift from Dr. Haihua Gu (Wenzhou Medical University); and the MC38 cell line was a gift from Dr. Jindan Wang (Wenzhou Medical University). All cells were maintained in DMEM supplemented with 10% heat-inactivated FBS in 5% CO 2 at 37°C. Then, ABC-1, A549, and MC38 cells were transduced with lentivirus encoding the Firefly-Luciferase-GFP gene to generate Luc. ABC-1, Luc. A549, and Luc. MC38 cells; MC38 cells were transduced with lentivirus encoding the human Nectin4-Firefly-Luciferase-GFP gene to generate hNectin4-Luc. MC38 cells; HEK-293T cells were respectively transduced with lentivirus encoding the human FAP-Firefly-Luciferase-GFP gene or the murine FAP-Firefly-Luciferase-GFP gene to generate hFAP-Luc. 293T or mFAP-Luc. 293T cells. All of these transduced cells were sorted by flow cytometry.
Generation of CAR constructs and mCAR constructs
Nectin4 CAR consisted of a single-chain variable fragment (scFv) derived from an antibody (24,25) against human Nectin4, a human CD8 leader signal, a human 4-1BB co-stimulatory domain, and a human CD3z activation domain (4), while Nectin4-7.19 CAR was a tandem construct encoding Nectin4 CAR, interleukin (IL)-7, and CCL19 with two 2A peptide sequence (26,27). FAP CAR was constructed by an scFv derived from an anti-FAP antibody (28) with a human 4-1BB and CD3z, while FAP-12 CAR was constructed with FAP CAR and interleukin (IL)-12 by 2A polypeptide strategy. These CARs were cloned into the pLenti-vector to obtain the recombinant plasmids. To construct the human Nectin4-targeted secondgeneration murine CAR (Nectin4 mCAR), the anti-human Nectin4 scFv was fused with the murine CD8a hinge region and transmembrane, the murine intracellular domain of 4-1BB, and murine CD3z (29). Then, the mCAR was cloned into upstream of an IRES-GFP marker in the MSCV retroviral plasmid pMIGR1.
T-cell isolation and transduction
To isolate human T cells, peripheral blood mononuclear cells were extracted from whole blood of healthy donors by Ficoll density gradient centrifugation. T cells were enriched with Dynabeads ® Human T-Activator CD3/CD28 (Thermo Fisher Scientific, USA) and followed by stimulation for 24-36 h in the X-Vivo medium (Lonza, CH) supplemented with 50 IU/ml recombinant human interleukin (IL)-2 (PeproTech, USA) and then transduced with the lentiviral particles at multiplicity of infection (MOI) = 40. Mouse T cells isolated from spleen and lymph nodes of C57BL/6 mice by the Pan T Cell Isolation Kit II (Miltenyi Biotec) were activated with Dynabeads ® Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific) and recombinant murine IL-2 (ProSpec) for 48 h, and then infected with retroviral particles at MOI = 10.
Cytokine secretion analysis
Enzyme-linked immunosorbent assay (ELISA) was used to quantify the concentration of cytokines and chemokines. Culture supernatant of CAR-T cells was collected and then detected by an IL-7 ELISA kit (Mutiscience) and a CCL19 ELISA kit (NeoBioscience, Shenzhen, CN), respectively.
Proliferation analysis
CAR-T cells were labeled with CellTrace ™ CFSE (Thermo Fisher Scientific) and co-cultured with tumor cells at an Effect/ Target ratio of 1:1 in a 24-well plate without the addition of external cytokines for 5 days and then analyzed using a flow cytometer with 488-nm excitation and emission filters appropriate for fluorescein to assess the proliferation of CAR-T cells.
Migration analysis
Chemotaxis on T cells was measured with a transwell (Corning, USA) with a 5-µm pore permeable membrane insert. Untransduced T cells labeled with CellTrace ™ CFSE were added to the upper chamber of the transwell, and the 5-day CAR-T cell culture supernatant without any cytokines was collected and 400 ml was added to the lower chamber. After 2 h of incubation, untransduced T cells migrating into the lower chamber were observed with a fluorescence microscope and pictures from three horizons were taken at random (30).
Cytotoxicity analysis
The xCELLigence RTCA MP instrument (Acea Biosciences Inc, CA, USA) was utilized for the assessment of CAR-T cell-mediated cytotoxicity (31). Briefly, 1 × 10 4 tumor cells were seeded on each well of an E-Plate 16 (Acea Biosciences) and grew until their adherence. Then, CAR-T cells were added into each unit at different Effect/Target ratios, with media or 2.5% Triton-X 100 (Solarbio, Beijing, CN) as negative or positive controls. Each group consisted of three replicate wells and the impedance signals (Cell index) were recorded for a duration of 24-48 h. Electrical impedance was quantified every 15 min by the use of the RTCA DP Analyzer.
In the luciferase bioluminescence technique, tumor cells expressing luciferase reporter were plated into a 96-well plate (32). T cells were added with different Effect/Target ratios after target cells adhered onto the well. Media and 2.5% Triton-X 100 were regarded as a negative control (K min ) and a positive control (K max ), respectively. Each group consisted of three replicate wells. After 12 h co-incubation, cells were centrifuged and the supernatant was removed. Then 200 ml of serum-free DMEM medium containing 0.5 mM D-luciferin (MedChemExpress, Shanghai, CN) was added to each well, and the fluorescence intensity was measured by Luminometric Measurement on a microplate reader after 10 min. The fluorescence intensity value K of each well was counted, and the killing efficiency was equal to (
Animal experiments
In the metastatic colorectal cancer model, the fully immunecompetent male 6-to 8-week-old C57BL/6 mice (Charles River, Beijing, China) were inoculated subcutaneously (s.c.) at the right flank with 1.0 × 10 6 hNectin4-Luc. MC38 cells on Day 0. To evaluate the dose dependence of Nectin4 mCAR-T cells, 5.0 × 10 6 untransduced mouse T (mUTD) cells and different doses of hNectin4 mCAR-T cells were injected intravenously (i.v.) on Day 10 after randomization of mice (N = 6 mice per group). To improve the anti-tumor efficacy, cyclophosphamide (CPA) at 100 mg/kg was administered intraperitoneally (i.p.) 3 days before the infusion of 5.0 × 10 6 mUTD or hNectin4 mCAR-T cells (N = 6 mice per group). The tumor volumes of the mice were recorded every 2 days and calculated as length × (width) 2 × 0.5. In the metastatic lung cancer model, the severely immunodeficient male 6-to 8-week-old NSG mice (NOD-Prkdc em26Cd52 Il2rg em26Cd22 /Nju) (GemPharmatech Co, Ltd, Nanjing, China) were injected i.v. with 1.0 × 10 6 Luc. ABC-1 cells on Day 0. After randomization (N = 3 mice per group), mice were treated i.v. with different doses of CAR-T cells on Day 7. Treatment with untransduced T (UTD) cells served as a negative control. Tumor progression was confirmed regularly by BLI using a Xenogen IVIS imaging system (PerkinElmer, Shanghai, CN), and the intensity of the signal was measured as total photon/second/cm 2 /steradian (p/s/cm 2 /sr). At the end of the experiment, mice were euthanized and tissues were resected for HE staining. All mice were bred and housed under SPF conditions in the Animal Center of Wenzhou Medical University. All mouse experiments were approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University and performed in accordance with relevant institutions and national guidelines and regulations.
Immunohistochemistry
Tumor tissues were obtained from patients at the Sixth Affiliated Hospital of Wenzhou Medical University to detect the expression of Nectin4 on tumor cells and FAP on CAFs. All informed consents were obtained from all included patients, and a supportive grant obtained from the Ethics Committee of the Sixth Affiliated Hospital of Wenzhou Medical University. For FAP staining, sections were blocked with 20% normal goat serum (Sigma, USA) in PBS for 30 min at room temperature and stained with 5 mg/ml primary mouse anti-human FAP antibody (Clone: EPR20021, Abcam) at 4°C overnight. The corresponding peritumoral normal tissues served as negative controls. The sections were rewarmed at 37°C for at least 45 min and incubated in the secondary antibody enhancement solution at room temperature for 20 min and then the secondary goat F (ab) anti-mouse IgG H&L (HRP) antibody (Abcam) at 37°C for 30 min. For Nectin4 staining, sections were stained with 10 mg/ ml primary goat anti-human Nectin4, affinity-purified polyclonal antibody (Catalog # AF2659, R&D Systems) at 4°C overnight. Then, the sections were stained with the secondary biotinylated rabbit anti-goat IgG antibody (Abcam) at 37°C for 30 min and incubated in Streptavidin-Biotin Complex at 37°C for 30 min. Then, the sections were developed with SignalStain ® DAB Substrate Kit, counterstained with hematoxylin (Biocare Medical, Shanghai, CN) for 90 s, dehydrated with ethanol, clarified with xylene, and then examined under an optical microscope (Olympus, Japan).
Statistical analysis
Data were analyzed as mean ± SD by t-test. Survival curve was analyzed by Kaplan-Meier curves and log-rank test. p-values < 0.05 were considered statistically significant. All experiments were repeated at least three times. All statistical analyses were performed with GraphPad Prism v6.0 (GraphPad Prism, USA).
High expression of Nectin4 on malignant solid tumors and FAP on CAFs
Firstly, immunohistochemistry (IHC) analysis was performed to examine the cellular expression of Nectin4 in a variety of tumor biopsies. We found that not only common tumors such as lung, ovarian, and gastrointestinal cancers as previously reported (12,24,(33)(34)(35), but also glioma, leiomyosarcoma, liposarcoma, gingival carcinoma, nasopharyngeal carcinoma, and laryngocarcinoma highly expressed Nectin4 ( Figure 1A and Figure S1). Furthermore, Nectin4 was also overexpressed on metastatic cancers ( Figure 1B), especially bone-metastasized triple-negative breast cancer (TNBC), which was without the expression of estrogen receptor, progesterone receptor and proto-oncogene Her-2, indicating that Nectin4 could be used as a good therapeutic target for both primary and metastatic tumors.
It has been shown that FAP is overexpressed in tumorassociated stromal cells of epithelial tumors and its expression is related to advanced stages, worse prognosis, and poor survival. We found that FAP was overexpressed not only on CAFs of epithelial cancers ( Figures S2 and S3), but also on mesenchymal cells of sarcomas ( Figure 1A).
Nectin4-7.19 CAR-T cells exhibited superior proliferation and lower differentiation
We constructed the human Nectin4-targeted secondgeneration CAR and fourth-generation CAR, designated Nectin4 CAR and Nectin4-7.19 CAR, respectively ( Figure 2A). Flow cytometric analysis showed that the cell-surface expression of CAR in Nectin4-7.19 CAR-T cells was almost equivalent to that in Nectin4 CAR-T cells ( Figure 2B). There was no significant difference in CAR expression between CD4 + and CD8 + T subsets in both Nectin4 CAR-T and Nectin4-7.19 CAR-T cells ( Figure 2C). However, the proportion of the (Naïve + T SCM ) subpopulation was higher in Nectin4-7.19 CAR-T cells than that in Nectin4 CAR-T cells, particularly in CD8 + T subsets (Figures 2D, E).
Then, we verified that Nectin4-7.19 CAR-T cells could produce IL-7 ( Figure 2F) and CCL19 efficiently ( Figure 2G). CCL19 secreted from Nectin4-7.19 CAR-T cells had chemotactic capacity to recruit more T cells ( Figure 2H). As IL-7 has been shown to enhance the proliferation and viability of T cells (36), we investigated the absolute number and found that the proliferation of Nectin4-7.19 CAR-T cells was substantially stronger than that of Nectin4 CAR-T cells (Figure 2I), and their cell viability remained well ( Figure 2J). Furthermore, after being stimulated by Nectin4-positive ABC-1 cells, Nectin4-7.19 CAR-T cells divided faster than Nectin4 CAR-T cells, indicating the specific antigen-driven proliferation ( Figure 2K).
Nectin4-7.19 CAR-T cells exhibited efficient cytotoxicity in vitro
Flow cytometric analysis showed that high-level expression of Nectin4 was present on the surface of various tumor cell lines ( Figure 3A). Then, we performed different assays to verify the specific cytotoxicity of Nectin4 CAR-T cells in vitro through the xCELLigence RTCA label-free technology and found that the coincubation of Nectin4 CAR-T cells with ABC-1, HT1376, and MDA-MB-453 cells could cause an immediate and timedependent decrease in cell index within 4 h, respectively, but not CD19 CAR-T cells ( Figure 3B), demonstrating that Nectin4 CAR-T cells efficiently executed specific cytolysis against Nectin4-positive tumor cells and exhibited better cytotoxicity at the gradually increasing appropriate ratio of Effect/Target. To compare the cytotoxicity between Nectin4 CAR-T and Nectin4-7.19 CAR-T cells, we generated Luc. ABC-1 cells to express luciferase ( Figure 3C) and observed that Nectin4 CAR-T and Nectin4-7.19 CAR-T cells exhibited equivalent oncolytic potentiality against Nectin4-positive Luc. ABC-1 cells ( Figure 3D), but not Nectin4-negative Luc. A549 cells ( Figure S4). Intriguingly, the expression of several immunological checkpoints on Nectin4-7.19 CAR-T cells were lower than those on Nectin4 CAR-T cells ( Figure 3E).
Nectin4 mCAR-T cells exerted antitumor effects on metastatic colorectal cancer in fully immune-competent mice
Preclinical studies have been limited by the use of xenograft models that do not adequately recapitulate the immune system of a clinically relevant host, so we developed the Nectin4 mCAR-T cells to determine its anti-tumor effects in a fully immune-competent mouse model of metastatic colorectal cancer. Firstly, we constructed the mCAR with the anti-human Nectin4 scFv and used pMIGR1-mCAR-IRES-GFP retrovirus to transfect mouse T cells to prepare Nectin4 mCAR-T cells (Figures 4A, B). Then, we found that Nectin4 mCAR-T cells specifically recognized human Nectin4 and exhibited efficient cytotoxicity against hNectin4-Luc. MC38, but not Luc. MC38 cells (Figures 4C, D). Accordingly, the secretion of IFN-g in CD4 + or CD8 + T subsets was higher in Nectin4 mCAR-T cells than those in mUTD cells ( Figure 4E and Figure S5).
In order to explore the anti-tumor effect of Nectin4 mCAR-T therapy in vivo, C57BL/6 mice were subcutaneously inoculated with hNectin4-Luc. MC38 cells and treated with increasing doses of Nectin4 mCAR-T cells intravenously. Compared with the mice treated with mUTD cells, Nectin4 mCAR-T therapy at low dosage had no significant anti-tumor effect, but prolonged survival and even cured two mice without recurrence at high dosage ( Figures 4F, G). For the purpose of improving survival, we then performed lymphodepletion with CPA before CAR-T therapy and found that only the mice treated with CPA and Nectin4 mCAR-T cells dramatically lessened tumor burden and achieved a complete remission without recurrence for more than 60 days, confirming that CAR-T therapy in combination with chemotherapy may be a promising strategy for malignant solid tumors (Figures 4H, I).
Nectin4-7.19 CAR-T therapy displayed significant anti-tumor activity without on-target off-tumor toxicity for metastatic lung cancer in mice
The severely immunodeficient mice were intravenously injected with Luc. ABC-1 cells expressing a GFP-firefly luciferase fusion protein ( Figure 3C and Figure S6) and then treated with Nectin4-7.19 CAR-T cells ( Figure 5A). Adoptive transfer with Nectin4-7.19 CAR-T cells could significantly eliminate metastases ( Figures 5B, C), leading to a long-term survival ( Figure 5D). However, one mouse treated with Nectin4-7.19 CAR-T cells suffered a relapse on Day 42 and finally died on Day 65.
To assess the potential on-target off-tumor toxicity of Nectin4-7.19 CAR-T therapy, we excised and examined susceptible murine organs from euthanized mice. No obvious pathological changes were detected in the organs ( Figure S7), and no weight loss or abnormal behavior was observed in mice treated with Nectin4-7.19 CAR-T cells ( Figure 5E).
Combination of Nectin4-7.19 CAR-T cell therapy and FAP-12 CAR-T cell therapy showed synergistic effects in the mouse model of lung metastasis
To explore if FAP-12 CAR-T cells targeting CAFs could collaborate with Nectin4-7.19 CAR-T cells to enhance the anti-tumor efficacy, we constructed FAP-targeted CAR ( Figures 6A, B) and found that there was no significant difference in phenotypic composition between FAP CAR-T and FAP-12 CAR-T cells ( Figure 6C). Then, we generated hFAP-Luc. 293T and mFAP-Luc. 293T cells to verify the efficient cytotoxicity of FAP CAR-T cells against both murine and human FAP in vitro (Figures 6D, E) and found that FAP-12 CAR-T cells exhibited a slightly stronger specific cytotoxicity than FAP CAR-T cells (Figures 6F, G). In addition, we found that FAP-positive tumor stroma appeared in the ABC1 lung cancer of the NSG mouse model ( Figure S8), and our previous study has proven the safety and effectiveness of FAP-targeted CAR-T cells in this mouse model (37). Then, Luc. ABC-1 cells were intravenously injected into mice to establish a metastasis lung cancer mouse model ( Figure 6H). The mice were given different therapeutic regimens ( Figure 6I). After several weeks, the combination of Nectin4-7.19 CAR-T cells and FAP-12 CAR-T cells had the most effective anti-tumor effects ( Figure 6J) and survival advantages compared to each monotherapy alone ( Figure 6K). To evaluate the safety of monotherapy or combination therapy with CAR-T cells, we verified that there were no weight losses or other obvious adverse events ( Figure 6L and Figure S9).
Discussion
So far, there are more than 1,000 ongoing CAR-T therapy clinical trials, most of which are for recurrent/refractory hematological tumors. As for malignant solid tumors, an increasing number of studies have been devoted to searching for tumor-associated antigens, but only few clinical trials conducted have shown promising results, due to severe side effects and toxicities (38). Here, we described the characterization of our fourth-generation Nectin4-7.19 CAR-T and FAP-12 CAR-T cells, which were shown to possess potent proliferation, migration, and cytotoxicity in vitro and significant anti-tumor effect in vivo. Recent reports have revealed the correlation between variations in the function of T-cell subpopulation and efficacy of CAR-T cell immunotherapy (39). T S C M from a CD45RA + CD45RO + T population expressing CCR7 and CD62L possesses higher effectiveness and persistence against tumors than T CM (40). Both CD8 + and CD4 + T subsets exhibit synergistic anti-tumor CAR-T activities, as CD4 + cells are conducive to developing CD8 + memory functions (41,42). Our data showed that expression of CAR in the CD4 + T subset was equal to that in the CD8 + subset, and the proportion of the T SCM subpopulation in the CD8 + T subset of Nectin4-7.19 CAR-T was higher than that of Nectin4 CAR-T cells, but there was no difference between FAP CAR-T and FAP-12 CAR-T cells, which may be related to IL-7 function in retaining the subpopulation of T SCM (43).
After trafficking to the tumor site and encountering their cognate antigen, T cells undergo rapid expansion to attain the appropriate numbers relative to the tumor burden. As previously reported, CCL19 could enhance recruitment and activation of CCR7-positive antigen-presenting cells and T cells by dendritic cell-and stromal cell-based intratumoral delivery (26,30,44), and IL-7 could stimulate proliferation of lymphocytes and maintain their survival and homeostasis (45). Furthermore, IL-7 signaling could prevent the exhaustion of T cells through a variety of mechanisms including downregulation of PD-1 expression (46). Accordingly, our Nectin4-7.19 CAR-T cells could reduce the expression of immunological checkpoints, such as PD-1, CTLA-4, TIM-3, and LAG-3, for the protection of CAR-T cells from exhaustion. Localized delivery of one or two scFvs from checkpoint blockers by CAR-T cells could enhance anti-tumor efficacy in vivo with minimal systematic toxicity (47). Thus, we are going to construct the fourth-generation CAR-T cells to secrete a PD-1-or/and CTLA-4-blocking scFv together with IL-7 and CCL19, which may maximize the efficiency of CAR-T therapy for malignant solid tumors.
Enfortumab vedotin (ASG-22ME), an antibody-drug conjugate targeting Nectin4, has demonstrated a clinically significant response rate with a manageable and tolerable safety profile in patients with locally advanced or metastatic urothelial carcinoma and thus received FDA approval based on phase I/II data, representing an alternative to established thirdline chemotherapies with vinflunine, paclitaxel, or docetaxel (15,48). In our study, we established Nectin4-targeted CAR-T cells based on the safety and efficacy of Nectin4 as a therapeutic target in the clinic and confirmed its capability and security in vivo. this may not predict an absence of toxicity in humans, since human Nectin4-targeted CAR-T cells had no cross-reaction with murine Nectin4 (24). Our phase I study (NCT03932565) addressing this issue has been ongoing to examine the safety and feasibility of Nectin4-7.19 CAR-T cells in patients with Nectin4-positive malignant solid tumors. We found that hemorrhagic rash and rash desquamation occurred due to the high expression of Nectin4 in skin tissues, but the symptoms were resolved without special treatment, and no severe CRS or neurotoxicity was observed. Therefore, Nectin4-7.19 CAR-T therapy is a promising treatment for malignant solid tumors. Previous studies have found that cancer cells initiate and sustain the activation of CAFs while CAFs support the growth, motility, and invasion of cancer cells (49,50). Targeting FAP with antibodies, vaccines, or pharmacological agents could lessen tumor progression in several preclinical animal models (51,52). Nowadays, there were some preclinical studies on the use of FAP-targeted CAR-T cells to eliminate CAFs to inhibit tumor growth and enhance host immunity without serious side effects (53). A recently described CAR-T therapy was to modulate tumor stroma by CAR-T cells secreting IL-12, which was deposited in the targeted tumor lesion to attract innate immune cells toward tumor cells that were invisible to CAR-T cells (54). Hence, we engineered the FAP-12 CAR-T cells and validated their biological function in vitro. As tumor stroma could express murine FAP in desmoplastic human lung cancer xenografts (55), and our FAP-12 CAR-T cells could target both human and murine FAP, the combination of Nectin4-7.19 CAR-T cell therapy and FAP-12 CAR-T cell therapy for metastatic lung cancer in mice exerted a synergistic anti-tumor effect without any toxicities.
In conclusion, the delivery of Nectin4-7.19 CAR-T therapy may be a feasible strategy for Nectin4-positive malignant solid tumors. Furthermore, the combination of Nectin4-7.19 CAR-T cell therapy and FAP-12 CAR-T cell therapy will be a promising synergistic approach to co-target Nectin4-positive tumor cells and FAP-positive CAFs. However, it is necessary to further confirm the safety of this combination therapy in our phase I study due to the toxicities that may be attributed to the secreted cytokines or offtarget effects.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was reviewed and approved by Laboratory Animal Ethics Committee of Wenzhou Medical University.
Author contributions
FL, JG, YG, XJX and AZ designed this study. FL, SZ, and YH performed most of the experiments. CW, TX, XYX, TZ, LS, SK, and JZ assisted with the experiments. FL and JG analyzed and interpreted the data and wrote the manuscript. AZ, YG, and JZ assisted with the data analysis and modified the manuscript. All authors contributed to the article and approved the submitted version.
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Prognostic significance of CDC20 expression in malignancy patients: A meta-analysis
Background Cell Division Cycle Protein 20(CDC20) is reported to promote cancer initiation, progression and drug resistance in many preclinical models and is demonstrated in human cancer tissues. However, the correlation between CDC20 and cancer patients’ prognosis has not yet been systematically evaluated. Therefore, this present meta-analysis was performed to determine the prognostic value of CDC20 expression in various malignancy tumors. Methods A thorough database search was performed in EMBASE, PubMed, Cochrane Library and Web of Science from inception to May 2022. Stata14.0 Software was used for the statistical analysis. The pooled hazard ratios(HRs) and their 95% confidence intervals (95% CIs) were used to analysis of overall survival (OS), recurrence-free survival (RFS), distant-metastasis free survival (DMFS). Qualities of the included literature were assessed by JBI Critical appraisal checklist. Egger’s test was used to assess publication bias in the included studies. Results Ten articles were selected, and 2342 cancer patients were enrolled. The cancer types include breast, colorectal, lung, gastric, oral, prostate, urothelial bladder cancer, and hepatocellular carcinoma. The result showed strong significant associations between high expression of CDC20 and endpoints: OS (HR 2.52, 95%CI 2.13-2.99; HR 2.05, 95% CI 1.50-2.82, respectively) in the multivariate analysis and in the univariate analysis. Also, high expression of CDC20 was significantly connected with poor RFS (HR 2.08, 95%CI 1.46-2.98) and poor DMFS (HR 4.49, 95%CI 1.57-12.85). The subgroup analysis was also performed, which revealed that CDC20 upregulated expression was related to poor OS in non-small cell lung cancer (HR 2.40, 95% CI 1.91-3.02). Conclusions This meta-analysis demonstrated that highly expressing CDC20 was associated with poor survival in human malignancy tumors. CDC20 may be a valuable prognostic predictive biomarker and a potential therapeutic target in various cancer parents.
Introduction
Cancer is a common cause of morbidity and mortality throughout the world (1). Worldwide, an estimated 19.3 million new cancer cases and almost 10.0 million cancer deaths occurred in 2020 (2). In spite of desperate development of new remedies in recent years, the prognosis of cancer remains bleak (3). Therefore, detection of new biomarkers related to the progression of cancer is essential for improving clinical outcomes.
It is known that CDC20 consists of 499 amino acids with WD40 repeats at its C-terminus for protein binding, serving as the substrate recognizing subunit of Anaphase Promoting Complex (APC) (4). CDC20 has been found to play critical roles in regulating timely cell cycle progression in both the G2 and M phases (5). Multiple studies from various groups have demonstrated that CDC20 targets several key substrates including Securin, Cyclin B1, Cyclin A, Nek2A, p21 and Mcl-1 for degradation to govern cell cycle progression (6)(7)(8)(9)(10)(11). Moreover, some studies show that p53, Mad2, RASSF1A and APC15 could inhibits tumors cell growth through regulation of CDC20 (12)(13)(14)(15). CDC20 have been proven to have kinds of functions, including regulation of cell cycle,and regulation of apoptosis. Mounting evidence has revealed that CDC20 plays an oncogenic role in human tumorigenesis, and increased CDC20 expression is associated with clinical progression in human cancers, such as poor differentiation and poor recurrence-free survival rates (16,17). However, it remains unclear whether CDC20 is associated with a worse outcome across solid cancer patients. These conflicting results may be due to the small sample size among individual studies and limitation of current technology.
Therefore, we present a meta-analysis evaluating the prognostic value of CDC20 over-expression in solid tumor. The purpose of this study was to estimate the correlation of CDC20 over-expression with survival in solid tumors, thereby shed more light on the development of CDC20 targeted therapy and prognostic prediction.
Search strategy
This study followed the Preferred Reporting Items for Systematics Reviews and Meta-Analysis (PRISMA) (18). We utilized a systematic search based on the PubMed, Embase, Web of Science and Cochrane Library from the establishment of data to May 2022. The search terms included ("cell division cycle protein 20" or "CDC20") and ("neoplasms" or "tumors" or "cancers" or "carcinoma" or "malignancies") and ("prognosis" or "survival"). All potentially eligible studies were retrieved, and their bibliographies were carefully scanned to identify other eligible studies and extra studies were identified by a hand search of the references cited in the original studies. When multiple studies of the same patient population were identified, we included the published report with the largest sample size. The above search process was done by two reviewers independently.
Inclusion criteria
To be eligible for inclusion in this meta-analysis and data extraction, studies had to: (1) Patients were pathologically diagnosed with any type of malignancy. (2) The expression levels of CDC20 were identified in tissues samples. (3) Patients were classified into negative and positive expression or low and high expression group in line with the CDC20 of expression levels, the connection between expressing level of CDC20 and survival results was examined. (4) HR and 95% confidence intervals (CI) for survival times were computed by included articles which can provide enough data or survival curves. (5) Officially published and English-written literatures.
Exclusion criteria
Exclusion criteria were: (1) Duplicated articles; (2) literatures published as letters, editorials, abstracts, reviews, case reports and export opinions; (3) experiments performed in vitro or in vivo, but not based on patients; (4) insufficient data about survival analysis; (5) the follow-up duration was shorter than 3 years.
Data extraction
Two reviewers extracted related data from the articles independently and came to an agreement on the following items. Original data of elementary demographic characteristics (authors of article; year of publication; detection method; age; region; CDC20 level; the category of carcinoma; follow-up duration; endpoints; the Joanna Briggs Institute (JBI) score) were exhaustively extracted from included literature involving Kaplan-Meier curves, test words and tables. In term of endpoints, OS, RFS and DMFS were considered as terminal events. For some articles, HR can be directly obtained; for the studies in which survival data are presented only with K-M curves, Tierney's method was employ was used to calculate the HR and 95% CI (19).
Methodological assessment
Two investigators individually assessed qualities of all enrolled studies by utilizing the JBI (20). The JBI Critical appraisal checklist for observational cohort study included 11 items. We regarded the included studies with at least 15 score as high-quality in methodology. And if the score were less than 15, those articles were considered as low-quality studies.
Statistical analysis
Our quantitative calculation was conducted based on Stata Software 14.0. We applied pooled HRs (high/low) along with its related 95% CIs to evaluate the association between the prognostic value and the expression levels of CDC20 in different malignancies. By utilizing Cochran's Q and I 2 statistics, the heterogeneity of enrolled literatures can be evaluated precisely. Additionally, Chi square-based Chochran Q test and I 2 test were calculated to determine the heterogeneity among these articles (21). Heterogeneity was considered insignificant when p>0.10 or I 2 < 50%, and then fixed-effects was employed to pool the HRs and 95% CIs; Otherwise, a fixedeffects model was used.
In order to explore the source of heterogeneity, we also performed subgroup analysis and meta-regression. Furthermore, sensitivity analysis was implemented to confirm the steadiness of collected results. Finally, we assessed publication bias by means of utilizing Egger's test. What's more, if the p-value is no more than 0.05, the results above all can be regarded as statistical significance.
Characteristics of studies
Eventually, ten studies (22-31) involving a total of 2342 patients were used for the meta-analysis (Figure 1). The included studies are summarized in Table 1. Two studies evaluated lung cancer, two studies evaluated breast cancer, and one each evaluated colorectal cancer, hepatocellular carcinoma, gastric cancer, oral cancer, urothelial bladder cancer, prostate cancer. The studies were performed in six countries (People's Republic of China, Finland, United King, Gandra, Korea, Japan) and published prior to May 2022.
Relationship between CDC20 expression level and OS of malignancy patients
There were eight studies that reported OS data with multivariate analysis. The relevant results showed that CDC20 overexpression in human tumor tissues was associated with a decrease in survival among malignancy patients (HR 2.52, 95% CI 2.13-2.99, p <0.001) in the multivariate analysis and (HR 2.05, 95% CI 1.50-2.82, p <0.001) in the univariate analysis (Figures 2A, B). There was slight heterogeneity among the eight studies mentioned (P =0.18, I 2 = 30.8%). Subgroup analyses were conducted as different factors including type of cancer (lung cancer or other cancers), follow-up duration (over 60 or less than 60 months), country (China or other country), and the pooled HRs for OS were shown in Figures 3A-C. The results of subgroup analysis showed that the high expression of CDC20 was associated with poor OS of malignancy patients ( Table 2).
Relationship between the expression of CDC20 and OS of lung cancer patients
Additionally, the prognosis role of the expression levels of CDC20 in lung cancer was assessed systemically. The results suggested that elevated CDC20 level implicated an unfavorable OS in lung cancer (HR 2.40, 95% CI 1.91, 3.02, p <0.001) (Figure 4). The flow chart of the selection process in our meta-analysis.
Relationship between CDC20 expression level and RFS, DMFS of malignancy patients
Among the included studies, two research estimated the relevance between CDC20 expression level and RFS, DMFS, respectively. The results showed that CDC20 increasingly expression was significantly related with poor RFS (HR 2.08, 95% CI 1.46, 2.98, p <0.001) ( Figure 5A) and DMFS (HR 4.49, 95% CI 1.57, 12.85, p <0.001) ( Figure 5B). However, we did not perform subgroup analysis for other types of cancer because of the insufficient numbers of trials included.
Sensitivity analysis
In order to assess the impacts of single study on the total outcomes, sensitivity analysis was conducted. AS to OS, our result of sensitivity analysis revealed that all the outcomes could not influence consequences remarkably, which means that the outcomes of OS were stable. The list of pooled HRs and 95% CIs after excluding single study one by one indicated the robustness of our results (Figures 6A, B). Furthermore, the sensitivity analysis RFS ( Figure 6C) and DMFS ( Figure 6D) identified that each included study influenced outcomes greatly, which suggested that the results of RFS and DMFS were not stable because of the limited number of studies included in each analysis. Thus, more related studies were needed to explore the effects of CDC20 on RFS and DMFS in human malignancy.
Publication bias
By Egger's test, we systemically assessed publication bias of all above included studies. The result of Egger's test (p =0.664) ( Figures 7A, B) about OS revealed that there existed no significant publication bias among enrolled documents. We didn't perform the publication bias of RFS and DMFS because of no more than four studies included in each analysis.
Discussion
The results of the study illustrated that elevated CDC20 expression indicated unfavorable prognosis OS of various malignancy patients, which was consistent with Wang et al.'s study (32). Our study further found that high CDC20 expression was connected with poor RFS and DMFS in malignancies.
FIGURE 4
Forest plot of meta-analysis of the relationship between CDC20 expression and OS of lung cancer patients. What's more, we did more subgroup analyses stratified by follow-up time, type of cancers, different countries, respectively. Then we discovered that high expressing CDC20 was related to poor OS in lung cancer. The above conclusions appear to be rational and understandable in line with the current agreement that as a chief cancer promoter, CDC20 can promote the abnormal growth and tumorigenesis of different kinds of tumors, such as lung cancer, which can serve as a promoter for regulating the progression of G1/S transition and the survival of cancer cells (10,33). What's more, Zheng et al. had demonstrated that besides the initiation, the presence of CDC20 is essential for tumor maintenance (34), which jointly contributes to the unfavorable prognosis in patients with elevated CDC20 expression level. Furthermore, Gao et al. and Li et al. showed that CDC20 could be useful for the treatment of osteosarcoma and might be a promising solution for the treatment of osteosarcoma with some chemotherapeutics insensitivity (35,36). Notably, creasing studies found that CDC20 played a critical role in hematological malignancies as a prognostic factor and therapeutic target (37). Thus, CDC20 is likely to act as a prognosis factor for the occurrence, maintenance, drug resistance of malignancy tumors.
Furthermore, we also explored the association between the CDC20 expressing levels with the prognostic value among various cancers. But just on account of the restricted amounts of selected research, we only evaluated the prognostic value of CDC20 in lung cancer. And the result showed that higher CDC20 level implicated an unfavorable OS in lung cancer. Moreover, although we found that there existed sight heterogeneity among non-lung cancer group, it presented significant correlation between the elevated expression levels of CDC20 and the poor OS of non-lung cancer (HR 2.67, 95%CI 2.08-3.44, p <0.001). Based on the above, we believe that the prognostic role of CDC20 in diverse cancers is significant. With regard to RFS, DMFS, disease free survival (DFS) and progression-free survival (PFS), these are all essential parameters reflecting the procession of malignancy. As all the included studies did not present any data about DFS and PFS, we just made analysis about RFS and DMFS. The outcomes of this meta-analysis revealed that higher CDC20 level implicated a poor RFS and DMFS in tumor patients. What's more, due to the fact that only two researched were enrolled to appraise the connection among CDC20 expressing levels and RFS, DMFS respectively, more researched are essential to investigate the connection about CDC20 and the development of cancer.
Except for the encouraging results, there are several limitations among this quantitative meta-analysis. First, there was a risk of publication bias, as some studies with small sample sizes or negative results may not have been published. Second, there may be a certain publication bias within some of the included studies, as any negative results are less likely to have been reported. Finally, the results cannot fully represent all solid tumors and hematological malignancies since the types of cancer covered by included trials are incomplete, and further clinical trials are needed to explore.
Conclusions
In sum up, this meta-analysis suggested that higher expressing levels of CDC20 was correlative to poor prognosis of OS, RFS and DMFS among difference kinds of malignancy patient. In brief, our current study is the most comprehensive meta-analysis that systemically explores the incontrovertible evidence of the prognosis value of CDC20 in various malignancy patients. More related works still need to improve the understanding of CDC20 expression and prognosis in difference cancer types.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Author contributions
FX and GX designed this study. FX and XY contributed to the literature search, review, and data extraction. FX and GX conducted the statistical analyses. FX and XY contributed to the manuscript drafting. FX and GX contributed to the manuscript revision. XY offered the funding. All authors accepted the eventual manuscript. All authors read and approved the final manuscript.
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Development and validation of a nomogram to predict liver metastasis for pancreatic ductal adenocarcinoma after radical resection
Objectives This study aimed to develop and externally validate a nomogram for predicting liver metastasis after radical resection in patients with pancreatic ductal adenocarcinoma (PDAC). Methods A total of 247 PDAC patients who underwent radical resection were retrospectively reviewed from January 2015 to March 2022 at Ningbo Medical Centre Lihuili Hospital Eastern Section, and used as a training cohort to develop the nomogram. 83 PDAC patients from the Ningbo Medical Centre Lihuili Hospital Xingning Section were enrolled as the validation cohort. The postoperative liver metastasis was recorded during the follow-up, and the liver metastasis-free survival was defined as the time from operation to the date of liver metastasis diagnosis or death. The nomogram was established based on independent prognostic factors selected by LASSO and multivariate Cox regression model. The performance was assessed using the concordance index (C-index) and calibration curves. The receiver operating characteristic (ROC) curve and decision curve analysis (DCA) were used to determine the clinical utility of the nomogram model. Results From the training cohort of 247 patients, a total of 132 patients developed liver metastasis during the follow-up, the 1-, 2- and 3- year liver metastasis-free survival were 52.4%, 43.5% and 40% respectively. The LASSO and multivariate Cox regression analysis indicated that postoperative CA125 (hazard ratio [HR] = 1.007, p <0.001), tumor differentiation (HR = 1.640, p = 0.010), tumor size (HR = 1.520, p = 0.029), lymph node ratio (HR = 1.897, p = 0.002) and portal/superior mesenteric/splenic vein invasion degree (PV/SMV/SV) (HR = 2.829, p <0.001) were the independent factors of liver metastasis. A nomogram with independent factors was developed and the C-index was 0.760 (95% confidence interval [CI], 0.720-0.799) and 0.739 (95% CI, 0.669-0.810) in the training and validation cohorts, respectively. The areas under curve (AUC) of the nomogram at 1-, 2- and 3-year were 0.815, 0.803 and 0.773 in the training cohort, and 0.765, 0.879 and 0.908 in the validation cohort, respectively, higher than those in TNM stage. Decision curve analysis (DCA) analysis revealed that the nomogram model provided superior net benefit in clinical utility. Liver metastasis-free survival curves showed a significant discriminatory ability for liver metastasis risk based on the nomogram (p <0.001). Conclusions The nomogram showed high accuracy in predicting liver metastasis for PDAC after radical resection, and may serve as a clinical support tool to guide personalized and prescient intervention.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is the 12th most common malignancy and the 7th leading cause of cancer mortality, as one of the most intractable malignant neoplasms worldwide (1). Due to its extremely aggressive nature, radical resection is the only chance for long-term survival for patients with PDAC. However, even after radical resection, most patients still have tumor recurrence or metastasis, resulting in 5-year survival of only 12% to 27%, negatively affecting the curative nature of the operation and the prognosis of PDAC patients (2,3).
Liver metastasis has the worst prognosis among all recurrence patterns, the median OS is significantly shorter than that of other recurrence patterns (15.4 months vs 17.7-39.6 months) (4). Meanwhile, liver metastasis accounts for the largest proportion of all recurrence patterns, up to 35%-40% of patients (5). Postoperative liver metastasis in patients with PDAC may present a unique biologic characteristic and always indicates a poor prognosis, constituting a key cohort worthy of further study (6). Several stage systems have been used to estimate the overall survival or recurrence-free survival (7,8), however considering the absence of a prognostic model specifically for liver metastasis after radical resection, it was necessary to develop a predictive model for liver metastasis with an unfavorable prognosis.
In the present study, we developed and externally validated a nomogram to predict the liver metastasis for PDAC after radical resection, which has not been reported in previous studies, aimed to explore the patients with a high risk of liver metastasis after radical resection and potentially assist in clinical.
Materials and methods Patients
The retrospective study consisted of 247 patients who underwent radical pancreatic cancer resection between January 2015 and March 2022 at Ningbo Medical Centre Lihuili Hospital Eastern Section, Ningbo University. The inclusion criteria were as follows: (1) pathology confirmed PDAC, (2) integrated intraoperative and clinical data, (3) enhanced CT/MR performed within 1 month before the operation, and (4) negative final margins with no residual tumor based on pathology. The exclusion criteria were as follows: (1) death within 30 days after the operation, (2) complications with other malignancies, and (3) failure to evaluate the vascular invasion degree from the preoperative imagines or during the operation. To examine the generalizability of the model, the external validation cohort consisted of 83 PDAC patients who underwent radical resection and met the above criteria at Ningbo Medical Centre Lihuili Hospital Xingning Section between January 2016 and August 2021. The study was approved by the ethics committee of Ningbo Medical Center Lihuili Hospital (Approval number: KY2021PJ263). All research procedures complied with the relevant guidelines and regulations. Informed consent was obtained from all patients before inclusion. We confirmed that this study was conducted following the Declaration of Helsinki.
Assessment of the vascular invasion degree
To assess portal vein/superior mesenteric vein (PV/SMV) and splenic vein (SV) invasion, we recorded the PV/SMV/SV invasion condition in each patient during the operation, evaluated by the chief surgeon. We also review the PV/SMV/SV invasion on preoperative images, evaluated by two radiologists (Supplementary Figure S1). The degree of PV/SMV/SV invasion was assessed as follows (9): (1) PV/SMV/SV without tumor abutment or invasion, (2) PV/SMV/SV invasion <180°, (3) PV/ SMV/SV invasion >180°.
For most patients, the intraoperative evaluation of vascular invasion was usually consistent with preoperative CT imaging evaluation, if there was a difference, the intraoperative evaluation was prevail.
Liver metastasis and follow up
Liver metastasis-free survival was defined as the time from operation to the date of liver metastasis diagnosis, death or the last follow-up. The liver metastasis is essentially a particular pattern of tumor recurrence, so the liver metastasis-free survival is a bit like the term recurrence-free survival (RFS), and we concentrated on liver metastasis in this study. The diagnosis of liver metastasis and other recurrence patterns was based on imaging studies, and rarely tissue confirmation. Information regarding liver metastasis was obtained at regular follow-up.
Patients were followed up until September 2022, and all patients were followed up for more than 6 months unless they died. The median follow-up time of patients from the Ningbo Medical Centre Lihuili Hospital Eastern Section and the Xingning Section were 15.0 (range 3-78) months and 19.0 (range 3-77) months, respectively. In general, patients had at least 1 follow-up by imaging study (CT, MRI or PET/CT) and tumor biomarkers every 3 months for the first year after the operation and then every 3-6 months after the first year. Followup was performed in the outpatient clinic or via phone call.
Study variables and operation
The following clinicopathological variables were analyzed: demographic data, biochemical tests, tumor markers, pathological features, vascular invasion degree, operative and adjuvant treatment characteristics. The preoperative biochemical and tumor markers test were performed within 7 days before the radical resection, and postoperative tumor markers were measured at the first follow-up. The lymph node ratio was defined as the proportion of positive lymph nodes in the total examined lymph node. The disease stage was evaluated according to the American Joint Committee on Cancer (AJCC) 8th edition and the 7th edition Japanese Pancreas Society (JPS) derived from tumor-node-metastasis (TNM) staging system (10, 11). Adjuvant chemotherapy was routinely recommended and started within 3 months after the operation if conditions permit.
Resectability evaluation and synchronous liver metastasis exclusion were performed by a multidisciplinary team, based on CT and MRI. Surgical methods included pancreaticoduodenectomy and distal pancreatectomy, resected tissues were pathologically examined in frozen and final sections to confirm negative surgical margins. According to preoperative imaging studies and intraoperative exploration, if the tumor invaded, PV/SMV resection and reconstruction were performed in pancreaticoduodenectomy, invaded SV along with the pancreatic body/tail and spleen resection was performed in distal pancreatectomy.
Statistical analysis
Continuous variables were presented as mean with standard deviation or median with range, categorical variables were presented as frequencies with percentages. Survival curves were calculated using the Kaplan-Meier method and the Logrank test. Optimal features were selected using the least absolute shrinkage and selection operator (LASSO) regression, and factors with nonzero coefficients were identified and selected. Independent prognostic factors of liver metastasis were identified by univariate and multivariate Cox proportional hazards regression. Subsequently, a nomogram was developed to predict the probability of 1-, 2-, and 3-year liver metastasisfree survival rates after the operation. The performance was evaluated based on the discriminating ability (discrimination) and accuracy of point estimates of the survival function (calibration) with 1000 time bootstraps, and to calculate a relatively corrected concordance index (C-index). The area under curves of the receiver operating characteristic (ROC) curves were calculated and compared with TNM stage, to validate the nomogram model performance. The clinical utility of the nomogram was investigated using the decision curve analysis (DCA), by quantifying the net benefits along with the increase in threshold probabilities. Each patient had a total risk score for risk stratification of liver metastasis according to the nomogram model. Patients were divided into different risk groups (low-; moderate-; high-) with the cut-off points automatically calculated using X-tile software (version 3.6.1; Yale University, New Haven, CT, USA) (12), and further applied to the validation cohort, and the respective Kaplan-Meier curves were constructed.
Patients characteristics in the training and validation cohorts
The training cohort consisted of 247 patients who underwent pancreatic cancer resection and had histologically confirmed PDAC at Ningbo Medical Centre Lihuili Hospital Eastern Section, Ningbo University between January 2015 and March 2022. A total of 132 patients developed liver metastasis during the follow-up, and the 1-, 2-and 3-year liver metastasisfree survival were 52.4%, 43.5% and 40% respectively. The validation cohort consisted of 83 eligible patients who underwent radical resection at the Ningbo Medical Centre Lihuili Hospital Xingning Section between January 2016 and August 2021, a total of 46 patients developed liver metastasis, the 1-, 2-and 3-year liver metastasis-free survival were 56.6%, 45.0% and 43.5%, respectively. All clinicopathological characteristics of patients in the training and validation cohorts were summarized ( Table 1). The patients with liver metastasis may be accompanied by other patterns of recurrence, the specific recurrence patterns of postoperative liver metastasis were summarized (Table 2). There was no difference in overall survival between the patients with only-liver metastasis (14.0 months, 95%CI, 11.323-16.677) and the patients with other multiple recurrence (12.0 months, 95%CI, 1.653-22.347, p=0.871).
Prognostic factors selection with LASSO analysis in the training cohort LASSO regression was performed for all 34 clinicopathological characteristics to select the prognostic factors of liver metastasis ( Figures 1A, B). The neoadjuvant chemotherapy was not an independent prognostic factor of liver metastasis after the operation (HR=1.468, 95%CI, 0.881-2.447, p=0.141). The analysis indicated that postoperative CA125, total examined lymph node number, tumor differentiation, lymphovascular invasion, capsule invasion, tumor size, lymph node ratio and PV/SMV/SV invasion degree were associated with liver metastasis after the operation. All significant factors selected from the LASSO regression were further included in the multivariable Cox analysis, and showed that postoperative CA125 (hazard ratio [HR] = 1.007, p <0.001), tumor differentiation (HR = 1.640, p = 0.010), tumor size (HR = 1.520, p = 0.029), lymph node ratio (HR = 1.897, p = 0.002) and PV/ SMV/SV invasion degree (HR = 2.829, p <0.001) were the independent factors for liver metastasis (Table 3).
Construction and validation of nomogram for liver metastasis-free survival prediction
As shown in Figure 2, the nomogram was established based on the independent factors of liver metastasis. PV/SMV/SV invasion degree and postoperative CA125 level were the largest contributions to liver metastasis prediction, followed by tumor differentiation and lymph node ratio. The calibration curves showed high agreement between predicted and actual liver Table 4).
Clinical utility of the nomogram DCA analysis revealed that the nomogram model could provide superior net benefits and exhibited a wider range of threshold probabilities than the AJCC and JPS stage system in both training and validation cohorts ( Figure 5). Patients were divided into three different risk groups based on the total risk scores calculated by the nomogram models, to validate the predictive abilities of the nomogram for liver metastasis after the operation. The optimal cut-off points were auto-calculated by Xtile software. The risk scores calculated divide patients into the low-risk group (<99.6), moderate-risk group (99.6-160.1) and high-risk group (>160.1). The liver metastasis-free survival rates were calculated in three groups, the results showed a significant discriminatory ability for liver metastasis risk based on the nomogram risk scores ( Figure 6).
Discussion
In the present study, we developed and externally validated a nomogram model based on clinicopathological and vascular invasion characteristics, which could be used to predict liver metastasis in patients with PDAC after radical resection. The nomogram model showed superior performance in predicting liver metastasis, with C-indexes of 0.760 (95% CI, 0.720-0.799) and 0.739 (95% CI, 0.669-0.810) in the training and validation cohorts, respectively. As the prognosis of PDAC patients with liver metastasis after radical resection is significantly poor, and currently there is no specifical model for predicting liver metastasis, the present nomogram provided an intuitive and utility tool for guiding the personalized and rational choice of prescient intervention, which is of increased clinical significance.
Liver metastasis is an important feature of PDAC after radical resection, which accounts for the largest proportion and the poorest prognosis among all recurrence patterns, resulting in an increase in mortality (5,13). Previous study demonstrated that specific patterns of PDAC recurrence result in different survival outcomes, the post progression survival of patients with liver metastasis (4.7months) or multiple-site recurrence (7.2months) had significantly worse when compared to patients with local recurrence (9.7months) or lung metastasis (15.4 months, p<0.001) (4). Hishinuma et al. (14) reported that local recurrence is rarely the direct cause of death, instead most patients died of liver metastasis, based on 27 patient autopsies. Previous reports have shown that more than 40% of PDAC patients develop liver metastasis after radical resection (4, 15), similar to the results of this study, but we further focused on liver metastasis throughout the follow-up period, to obtain accurate liver metastasis-free survival in each patient, for developing a more precise and prognostic nomogram model. So, we introduced the term of liver metastasis-free survival, which is a bit like the term recurrence-free survival (RFS), since the liver metastasis is essentially a particular pattern of tumor recurrence, and we B A only concentrate on liver metastasis during follow-up, for the nomogram development. Morever, the patients with postoperative liver metastasis may also be accompanied by other patterns of recurrence, and we found that there was no significant difference in overall survival between the patients with only-liver metastasis and patients with multiple recurrence, highlighting the malignancy of liver metastasis and the importance of this nomogram.
In the process of developing our nomogram, PV/SMV/SV invasion degree is an important factor, which is not easily Nomogram for predicting the 1-, 2-and 3-year liver metastasis-free survival in PDAC patients after the operation. The nomogram was established in the training group, with postoperative CA125, tumor differentiation, tumor size, lymph node ratio and PV/SMV/SV invasion degree.
measurable as other clinicopathological variables, needed an intuitive and standard classification to define the different invasion degrees. Nakao et al. (16) based on the narrowing of vascular invaded by the tumor, suggested four types of vascular invasion degree: normal, unilateral narrowing, bilateral narrowing and complete obstruction. However this classification has limited capacity in predicting prognosis. Shen et al. (17) reported four types to indicate the relationship between vein and tumor: type 1 (absent), type 2 (mild deformity), type 3 (tethering or stenosis >1/2) and type 4 (obstruction or embolus), this classification can accurately predict the prognosis and similiar to ours. According to the degree of the tumor abutment or invasion, we classified into PV/ SMV/SV without invasion, invasion <180°, and invasion >180°, considering both the SMV and SV belong to the portal vein circulatory system, this classification could combine the pancreatic head and body/tail cancer, evaluating the invasion degree in a simple and duplicatable way. As the close adjacent anatomical relationship between the pancreas and PV/SMV/SV, these veins are a common site of direct tumor involvement, but the impact on the prognosis is not clear (18)(19)(20). In the present study, PV/SMV/SV invasion was a significant independent risk factor for liver metastasis, 83.7% of patients with vascular invasion >180°developed liver metastasis after radical resection. The "circulating tumor cell (CTC)" hypothesis may explain: that the tumor cells invading the PV/SMV/SV were The calibration curves for predicting liver metastasis-free survival at 1 year (A), 2 years (B) and 3 years (C) in the training cohort, and those at 1 year (D), 2 years (E) and 3 years (F) in validation cohorts, respectively.
B C D E F
A FIGURE 4 ROCs of nomogram, AJCC and JPS for predicting liver metastasis-free survival at 1 year (A), 2 years (B) and 3 years (C) in the training cohort, and those at 1 year (D), 2 years (E) and 3 years (F) in validation cohorts, respectively.
likely to enter portal vein circulation and metastasize to liver (21,22). Tien et al. (23) detected the CTCs in portal vein blood obtained during the operation, and found that patients with positive CTCs tended to develop liver metastasis after the operation, supporting the above hypothesis. Postoperative CA125 level is another independent risk factor of liver metastasis, increased CA125 level after radical resection was an important feature of high PDAC tumor burden and distant metastasis tendency, which indicated the poor curative effect of the operation. Previous study suggested that serum CA125 levels were the most strongly associated with early distant metastasis after pancreatectomy, when compared with other tumor markers such as CA199, CEA, CA242 and CA724. High CA125 levels was consistent with the expression of a "drive" metastasis associated gene signature, which may be the reason for CA125 highly sensitive to liver metastasis (24). Xu et al. (25,26) also reported that postoperative CA125 level can better predict the prognosis when compared with preoperative tumor markers. Moreover, poor tumor differentiation was associated with liver metastasis as well, in this study, the probabilities of liver metastasis were 35.3%, 50% and 59.5% in the high, moderate and poor tumor differentiation, respectively.
A previous large sample study supported our result, indicating that poor differentiation of tumor could promote infiltration and invasion, and contribute to liver metastasis (5). The "intriguing hypothesis" may explain: that poorly differentiated tumors highly expressed epidermal growth factor and E-cadherin, enhanced the ability of liver metastasis (27). Apart from the above risk factors, the nomogram model also covered several risk factors including lymph node ratio and tumor size. Compared with positive lymph node number, the lymph node ratio is a more valuable prognostic indicator, also associated with liver metastasis after radical resection (28,29). Furthermore, we found that preoperative neoadjuvant chemotherapy was not associated with liver metastasis, which is a regrettable result. We believe that selective bias is the cause: the patients in cohort of neoadjuvant chemotherapy tend to have bigger tumor size and worse vascular invasion degrees, these undesirable tumor characteristic may lead to postoperative liver metastasis, leading to negative result of neoadjuvant chemotherapy.
Compared with the previous traditional nomograms for survival and recurrence prediction, our model can predict liver metastasis after radical resection more specifically and accurately, for early intervention of this unfavorable DCA curves for predicting 1-year liver metastasis-free survival based on nomogram as compared with 8th AJCC and 7th JPS stage system in the training cohort (A) and the validation cohort (B). metastasis. The nomogram achieved a C-index of 0.760 and 0.739 in the training and external validation cohorts, respectively, and the calibration curve indicated the precisely predictive ability of the nomogram in prediction. The present nomogram showed higher AUC and better performance in predicting liver metastasis, when compared with the TNM stage system of 8th AJCC and 7th JPS (10, 11). In addition, DCA analysis indicated that the nomogram could augment net benefits and expose a wider range of threshold probabilities by risk stratification in the prediction of liver metastasis. Furthermore, we calculated the nomogram risk score and compared the liver metastasis-free survival rates, the results showed a significant discriminatory ability for liver metastasis risk based on the nomogram. Liver metastasis possibly represents a unique biological subtype of PDAC (6), personalized follow-up and intervention was needed for the patients with a high nomogram risk score. Randomized clinical trials confirmed that several gemcitabine-based chemotherapies were effective in preventing postoperative liver metastasis and prolonging survival (30). Masayuki et al. (31) reported that hepatic artery infusion chemotherapy can observably increase intrahepatic drug concentration and eliminate tumor metastatic lesions. Additionally, hepatectomy for PDAC patients with postoperative liver metastasis has been proven successful in improving survival (32).
The present study had several limitations. First, liver metastasis was generally based on imaging studies, the tiny hepatic nodules were difficult to identify as metastasis or cyst, limiting the accuracy of the liver metastasis dignosis date. Second, the specific adjuvant chemotherapy regimen after the operation were not included in the variable, making the cohorts relatively heterogenous. In future, a study especially for the patients with/ without systemic adjuvant treatment will be established, to explore the effect of systemic adjuvant treatment, as an upgrade to the present nomogram. Third, some differences exist between the training and validation cohorts, but in general, the two cohorts are basically balanced, and the C-index were 0.760 and 0.739, indicating the nomogram has good consistency. Furthermore, a A B FIGURE 6 Kaplan-Meier curve analysis. Liver metastasis-free survival curves were stratified by the model risk score in the training cohort (A) and the validation cohort (B).
large sample of prospective cohorts is still needed, to further confirm the predictive value.
In conclusion, we developed and externally validated a nomogram to predict liver metastasis after radical resection in patients with PDAC. The nomogram based on clinicopathological characteristics showed great accuracy in predictive performance, and provided an intuitive and utility tool to guide personalized and prescient intervention for patients with a potential risk of liver metastasis.
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: [email protected].
Ethics statement
The study was approved by the ethics committee of Ningbo Medical Center Lihuili Hospital (Approval number: KY2021PJ263). All research procedures complied with the relevant guidelines and regulations. Informed consent was obtained from all patients before inclusion. We confirmed that this study was conducted following the Declaration of Helsinki.
Author contributions
JT and CL proposed and designed the study. SW, W J and SM collected the data. JT and SM analyzed the data, interpreted the results, and drafted the article. All authors contributed to the article and approved the submitted version.
Funding
Funded by Ningbo medical and health brand discipline (PPXK2018-03).
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