Spatial-Temporal Heterogeneity And Clonal Evolution For Prostate Cancer In China

Background:Prostate cancer is the most common malignant tumor in men worldwide,which exhibits extensive inter-and intra-tumor heterogeneity.Inter-tumor heterogeneity can be manifested as significant differences in epidemiology and clinical behavior between different races,as well as differences in tumor genomic alterations among different populations with distinct genetic backgrounds.Due to the impact of these heterogeneities on the diagnosis,prognosis,and treatment response of prostate cancer,it is necessary for clinicians to consider both population and genomic information in order to achieve better personalized management of prostate cancer.In addition,intratumoral heterogeneity of prostate cancer is even more pronounced,manifested not only in differences at the genomic,epigenomic,and phenotypic levels within individual patients,but also in differences during the evolution of prostate cancer in different spatial and temporal contexts.For instance,the multifocality of primary tumors,castration resistance resulting from therapy resistance,and progression towards lethal metastatic tumors represent the complexity of spatiotemporal heterogeneity driving tumor clonal evolution and posing challenges to clinical intervention and treatment of prostate cancer.Therefore,investigating inter-tumor heterogeneity of prostate cancer in the population’s genomic landscape,unraveling intratumoral heterogeneity and clonal evolution across different temporal and spatial progression,is of paramount research value for a deeper understanding of the origin and development of prostate cancer,guiding clinical treatment decisions,as well as monitoring tumor progression and drug resistance.Objectives:The objectives of this study encompass three aspects,each corresponding to one of the three sections of the main text.Firstly,to compare the frequencies of driver gene mutations in Chinese and Western populations with prostate cancer,and understand the interpopulation genomic heterogeneity of prostate cancer.Secondly,to delineate the spatial tumor heterogeneity during the metastatic spread of prostate cancer,and to construct the clonal evolution process at the genomic and epigenomic levels.Thirdly,to describe the temporal heterogeneity in the development of castration-resistant prostate cancer,to identify the driving events in the evolution from primary tumors to resistant tumors,and to preliminarily explore the clinical value of using clonal evolution principles to monitor the progression of prostate cancer.Methods:In this study,we analyzed the heterogeneity and clonal evolution of prostate cancer based on multi-omics sequencing of prostate tumor tissue and utilized bioinformatics analysis.Specifically,we conducted the following steps:1.The first part of our study was based on the first Chinese prostate cancer genome and epigenome project(Chinese Prostate Cancer Genome and Epigenome Atlas,CPGEA).We included 208 Chinese prostate cancer patients who underwent radical prostatectomy,and performed genome and epigenome sequencing on both the primary tumor tissue and paired normal tissue.We also integrated data from 13 Western prostate cancer cohorts and compared the frequencies of driver mutations in the two different populations.The whole genome sequencing data was analyzed using the GATK pipeline.The cancer cell fraction(CCF)and d N/d S ratio were used to explore the evolutionary role and selection pressure of the Chinese-specific FOXA1 mutation in prostate cancer origin.Immunohistochemistry and Sanger sequencing were used for FOXA1 mutation validation.The analysis of DNA methylation data was conducted using software such as Bismark,and we identified methylation variation patterns in prostate cancer.Correlation analysis was performed to investigate the interaction between epigenetic and genetic alterations during tumor evolution.2.The second part of this study tracked the clinical course of an advanced and metastatic prostate cancer patient.After the patient’s death,metastatic tumor tissue was obtained through warm autopsy,and multiple omics sequencing,including whole-genome,methylation,and RNA sequencing,were performed.Analysis of somatic mutations and copy number variations was performed using Mutect2,Strelka2,and FACETS.DNA methylation data were analyzed using software such as Bismark and methyl Kit.We utilized Py Clone algorithm to cluster mutations based on the CCF,and constructed the genomic clone evolution tree for prostate cancer metastasis using Clon Evol.Euclidean distance matrices were calculated based on the methylation level of CpG sites,and an epigenetic evolution tree was constructed using the ape package in Rsoftware.Spearman correlation analysis was used to evaluate the consistency between genomic and epigenomic evolution.Finally,we designed shRNA gene knockdown vectors and conducted scratch and Transwell assays to investigate the function of CDKN1B gene in prostate cancer metastasis.3.In the third section,we included five patients with prostate cancer.we performed whole-exome sequencing(WES)on 14 specimens from this five locally relapsed patients before and after androgen-deprivation therapy,and performed whole-exome sequencing.The basic analysis and somatic mutation detection of the sequencing data were the same as in the first and two sections.Py Clone and Clon Evol software were used for the clone evolution analysis of tumor castration resistance.The fishplot package in R was used for visualization.Circulating tumor DNA(ctDNA)in plasma was utilized to explore the application of clone evolution principles for monitoring the progression of advanced prostate cancer.We included two late-stage patients with low PSA and high risk to construct the tumor clone evolution tree.By selecting personalized sites for clonal/subclonal mutations and designing ctDNA probes,we monitored tumor progression in real-time with clinical course.Results:In this study,we analyzed the population heterogeneity of driver mutations and highfrequency mutations in prostate cancer at multiple levels,including gene mutations,copy number variations,and fusion genes,among Chinese and Western populations.We found that FOXA1 gene mutations are a genomic alteration specific to Chinese individuals,which are mostly clonal changes in the origin of prostate cancer and are positively selected during tumor evolution.Combined with multi-omics data,we observed that the global genomic DNA methylation abnormalities in prostate cancer are associated with the malignancy degree and exhibit a co-evolution phenomenon with genomic changes during tumor progression.Based on metastatic tumor samples obtained from warm autopsy,we comprehensively described the intratumor heterogeneity and clonal evolution in prostate cancer metastasis,further confirming the co-evolution of epigenomics and genomics during tumor metastasis.We identified hotspot mutations in the CDKN1B gene,which may be an important driver event promoting prostate cancer metastasis,and verified its role in prostate cancer metastasis through multi-cohort integrated data and cell experiments.Additionally,we found that tumors undergo an evolutionary bottleneck effect after androgen deprivation therapy,manifested as a reduction in genetic diversity.By constructing the clonal evolution of prostate cancer resistant to castration,we identified potential driver events for tumor progression and resistance to therapy.Finally,we preliminarily demonstrated that multitimepoint ctDNA detection based on clonal evolution can assist in determining the progression of prostate cancer.Conclusion:Here,we comprehensively described the tumor heterogeneity among populations and the spatial-temporal heterogeneity within tumors of prostate cancer,at three clinical stages of primary tumor origin,metastasis,and castration resistance.We identified FOXA1 mutation as a major and unique variation in Chinese prostate cancer,providing potential therapeutic strategies for precision diagnosis and treatment of prostate cancer in the Chinese population.Moreover,we observed the co-evolution of the genomic and epigenomic changes,providing data support for the multi-omics and comprehensive mechanisms underlying the origin and metastasis of prostate cancer.We also identified hotspot mutations in CDKN1B associated with prostate cancer metastasis and recurrence,which may serve as important molecular targets for intervention of tumor metastasis.Finally,our study provided a new theoretical basis for exploring the mechanism of castration resistance in prostate cancer.The application of the principle of clonal evolution in ctDNA monitoring also offers a new approach to evaluate patient responses and progression.