| Pancreatic cancer is a disease characterized with insidious onset and rapid progression,of which the treatment effect and prognosis of most patients are poor.According to the data reported in 2020,pancreatic cancer is the fourth leading cause of tumor death and ranks the second most common malignant tumor in digestive system.It is estimated that by 2030,the fatality rate of pancreatic cancer will rank the second among tumors,following lung cancer.Due to the insidious onset of pancreatic cancer and the difficulty of early diagnosis,most patients with pancreatic cancer are advanced or locally advanced at the time of diagnosis,with less than 25% of patients eligible for up-front surgery.The incidence rate of pancreatic cancer is about 5.1/100,000 people,which is relatively low compared to other malignant tumors,thus the benefits of large-scale population screening are limited.Therefore,targeting high-risk populations for pancreatic cancer for targeted screening can improve the positive detection rate and reduce the waste of medical resources.The risk factors for pancreatic cancer can be divided into non-hereditary factors,which depend on the impact of lifestyle and environment on the human body,and hereditary factors,which are closely related to individual’s genetic background.A germline mutation study of10,389 cases of sporadic pancreatic cancer revealed that nearly 15% of pancreatic cancer patients carried pathogenic germline mutations,ranking the third among malignant tumors surpassing breast cancer.The latest data released by the Pancreatic Cancer Case-Control Consortium shows that the heritability of pancreatic cancer may be twice as high as previously predicted,reaching 21.2%.Another study reported that nearly half of people at high risk of pancreatic cancer carry germline mutations in genetic susceptibility genes.Because the pathogenic germline mutations are of significant importance in the clinical treatment and family management of pancreatic cancer,and the next-generation sequencing has provided a simpler and more accurate detection method for identifying patients with germline mutations in susceptible genes,the latest National Comprehensive Cancer Network clinical guidelines propose that all newly diagnosed pancreatic cancer patients should undergo risk assessment of hereditary syndromes and also consider gene germline mutation detection even without an obvious family history.With the development of NGS technology and its wide application in clinical practice,a large amount of clinical sample sequencing data has been accumulated by plenty of study groups,laying a solid foundation for germline research in a large population.In addition,various academic alliances have established large databases of sequencing data from populations,such as the Genome Aggregation Database(gnom AD)and the 1000 Genomes(1000G),which collect and organize various whole exome sequencing(WES)and whole genome sequencing(WGS)data.Unlike somatic mutations,germline mutations have ethnic heterogeneity.Most of the existing research and sequencing data focus on Europeans,which cannot fully indicate the characteristics of germline mutations in Chinese population.Therefore,the establishment of a large Chinese pancreatic cancer germline mutation research cohort is of great importance for studying the germline mutation characteristics and the genetic background characteristics of pancreatic cancer in Chinese populations.In addition,there are a large number of variants of uncertain significance(VUS)in germline mutations that need to be further explored in order to clarify their pathogenicity,which is of great significance for disease risk management of patients and the clinical application of gene sequencing technology.Based on the domestic and abroad current research status of germline mutations in pancreatic cancer,we established a cohort of 2,944 healthy Chinese individuals and a cohort of 1,123 Chinese pancreatic cancer patients.For the first time,we depicted the landscape of germline mutations(including VUS)in pancreatic cancer in the Chinese population at multiple omics levels,including genomics,transcriptomics,and epigenetics.We identified,for the first time,a high-frequency mutation of Cystic fibrosis transmembrane conductance regulator(CFTR)in Chinese pancreatic cancer patients and analyzed the impact of CFTR on the prognosis of pancreatic cancer based on clinical data.Finally,we validated the tumorsuppressive function of CFTR in vitro and the effect of VUS in CFTR on its function.Part 1: Landscape of Germline Mutations of Chinese Pancreatic Cancer PatientsObjective:To setup the pipeline of calling,filtering,and annotating germline mutations;to depict the landscape of germline mutations in the Chinese population;to identify instances of "twohit" events in mutated genes;to analyze the correlation between deleterious germline mutations and clinical pathology;to compare germline mutations in pancreatic cancer with those in other types of cancer.Methods:1.Retrospectively collect patients from our center’s database who meet the inclusion and exclusion criteria and establish a prospective study cohort.Patients will be categorized into discovery,validation,and functional validation cohorts based on the type of sequencing data.2.Process the sequencing data using the standard pipelines of GATK and Var Scan2 to detect mutations.Establish filtering and annotation processes for germline mutations and analyze copy number variations using Control Free C and GISTIC software.Identify loss of heterozygous(LOH)events by integrating mutation and copy number variation results.3.Statistical analysis was performed using R software(Version 4.1.0).The χ2 or Fisher’s exact test was used for categorical data comparison between groups,and the t-test or the Mann-Whitney U test for the quantitative data comparison between groups.Differences with a two-sided p-value < 0.05 were considered statistically significant.Results:1.A research cohort for pancreatic cancer germline research in the largest population in China was established,of which 1,123 patients with pancreatic cancer and organoid samples and 2,499 healthy individuals were enrolled,including a discovery cohort(N=389,comprising 108 WGS and 281 WES),a validation cohort(N=665,PANEL sequencing),and a functional validation cohort(N=69,organoid samples,WGS data),and a healthy population cohort(N=2,944).2.The landscape of germline mutation in Chinese population was depicted and 6 highfrequency mutated pathways were identified,of which the CFTR is the most frequently mutated gene.Thirty-five LOH events among 26 tumor suppressor genes were also detected.3.The results revealed that patients carrying deleterious germline mutations and CFTR germline mutations were younger(age of patients with deleterious germline mutations vs.age of patients without deleterious germline mutations: 60.00 vs.62.00,p = 0.025;age of patients with CFTR germline mutations vs.age of patients without CFTR germline mutations:56.00 vs.62.00,p = 0.033),and had a higher proportion of family history of tumors(with deleterious germline mutations vs.without deleterious germline mutations: 27.63% vs.15.69%,p = 0.002;with CFTR germline mutations vs.without CFTR germline mutations:42.42% vs.24.32%,p = 0.019)and a higher proportion of personal history of cancer(with deleterious germline mutations vs.without deleterious germline mutations: 10.53% vs.4.58%,p = 0.015;with CFTR germline mutations vs.without CFTR germline mutations:15.15% vs.5.83%,p = 0.047).Moreover,female patients accounted for a higher proportion of CFTR germline mutation carriers(57.58% vs.38.44%,p = 0.027),and a higher proportion of chronic pancreatitis history(18.18% vs.2.98%,p < 0.001).4.Compared with 12 other types of tumors,the mutation frequency of CFTR was the highest in pancreatic cancer at 3.9%,followed by cervical cancer and endometrial cancer at3.8% and 3.6%,respectively,while the mutation frequencies of CFTR in other types of tumors were all less than 2%.Conclusion:CFTR was identified as a high-frequency germline mutation gene in pancreatic cancer,with a mutation frequency of 3.9%,and patients with deleterious germline mutations and CFTR mutations are younger and have a higher proportion of family/personal tumor history,and the proportion of female patients and patients with chronic pancreatitis history in patients carrying CFTR germline mutations is higher.These findings set the direction for further research.Part 2: Expression Characteristics of CFTR and its Correlation with the Prognosis of Pancreatic Cancer PatientsObjective:Combined with the pathological and prognostic information data of clinical patients,the expression of CFTR in pancreatic cancer tissues and paracancerous tissues was analyzed,the correlation between CFTR expression and pancreatic cancer prognosis was elucidated,and its possible regulatory mechanism was explored,as well as the effects of promoter methylation and germline mutations on CFTR expression.Methods:1.The RNA sequencing data of tumor/paracancerous tissue of 108 patients with pancreatic cancer were analyzed to obtain the expression of CFTR,and the patients were divided into high and low groups according to the median expression of CFTR,and the prognosis difference between the two groups was analyzed.In order to avoid the influence of tumor purity,organoid sequencing data were also analyzed.The results were validated using the sequencing data of bulk tissue sequencing and laser capture microdissection(LCM)samples in public databases.2.Immunohistochemical staining was applied to analyze the expression of CFTR in tumor/paracancerous tissues of pancreatic cancer patients,and quantitative analysis was performed by software.Patients were divided into high and low groups according to the median expression of CFTR,and the prognosis difference between the two groups was analyzed.3.The effects of CFTR promoter methylation and chromatin accessibility on the expression of CFTR were analyzed by methylation-specific PCR(MSPCR)and assays for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq)using organoid and cell line samples.Results:1.By comparing the CFTR RNA expression and immunohistochemistry quantification results of tumor and paracancerous tissues,it was found that CFTR expression in tumor tissues was downregulated.2.By comparing the differences in clinical features of patients with different CFTR RNA expression and immunohistochemistry quantification results between tumors,it was found that patients with high CFTR expression had better prognosis [high vs.low expression,bulk tissue sequencing(overall survival): 22.9(14.0-31.8)vs.15.9(11.1-20.7),p > 0.05;organoid(disease-free survival): 13.8(9.5-29.0)vs.3.3(5.9-18.7),p < 0.05;public database bulk tissue sequencing(overall survival): 23.3(3.6-43.0)vs.16.5(12.0-21.0),p > 0.05;public database LCM samples(overall survival): 21.9(13.6-30.2)vs.6.6(3.7-9.5),p < 0.01],with more pronounced results in purer organoid and LCM samples.Similar results were obtained by immunohistochemistry quantification analysis [overall survival: high vs.low expression = 27.7(22.9-32.5)vs.15.0(8.3-21.7),p < 0.05].3.Methylation-specific PCR and ATAC-seq revealed that the CFTR promoter region exhibited a state of hypermethylation and lower chromatin accessibility in organoid samples with low CFTR expression.4.By comparing the RNA expression and immunohistochemistry quantification data of CFTR wild-type samples and mutation-type samples,it was found that germline mutations could cause downregulation of CFTR expression.Conclusion:The study found that CFTR expression is correlated with the prognosis of pancreatic cancer patients.CFTR has the characteristics of tumor suppressor genes in pancreatic cancer,and the regulation of CFTR expression is influenced by epigenetics.Its specific function needs to be further studied.Part 3: Tumor Suppressive Function of CFTR and its Mutant IsoformsObjective:The tumor suppressive function of CFTR in pancreatic cancer was determined by in vitro experiments,and the impact of different mutation subtypes on its function was clarified.Methods:1.PANC03.27 and SU86.86 CFTR knock-down cell lines,as well as ASPC1 and PANC-1 CFTR overexpression cell lines were constructed,and 9 CFTR mutation overexpression stable transfer strains were constructed using PANC-1 cell lines.2.Identify transfection results by q PCR and Western blotting assay.3.The proliferation activity of pancreatic cancer cells was evaluated by CCK-8 and colony formation assays.The migration/invasion capacity of pancreatic cancer cells was assessed,and apoptosis and cell cycle of pancreatic cancer cells were detected by flow cytometry.Results:1.CFTR knockdown/overexpression cell lines and 9 mutant overexpression stable cell lines were successfully constructed and identified.2.CFTR overexpression led to decreased proliferation,migration/invasion capacity of pancreatic cancer cell lines,and promoted apoptosis of pancreatic cancer cell lines,resulting in more cells arrested in the G0-G1 phase.On the contrary,CFTR knock-out promoted proliferative activity of pancreatic cancer cell in vitro.3.Compared with CFTR overexpression cell lines,the inhibitory effect of 9 mutant CFTR cell lines on cell proliferation were relatively weakened.Conclusion:CFTR can inhibit the proliferation,migration,and invasion of pancreatic cancer cells,and promote apoptosis in vitro,exhibiting tumor suppressive functions.However,the inhibitory ability of CFTR on pancreatic cancer cells is weakened after mutation. |
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