Mutational Profiling Of A Long-term Surviving Stage Ⅲ Colorectal Cancer Patient Using High Throughput Next-Generation Sequencing

Colorectal cancer(CRC)ranks the third most common malignant cancer associated with the gastrointestinal tract.In both developed and developing countries,colorectal cancer ranks the second most common cause of cancer-related mortalities in men and women and stage Ⅲ patients account for one-third of all reported cases.Typically,stageⅢ colorectal patients have a poor prognosis and about 30%to 50%of patients inevitably experience tumor relapse manifesting as loco-regional recurrence,distant metastasis,or metachronous lesions within 5-years of treatment.The focus of this research work is to use Next-generation sequencing techniques and bioinformatics tools to explore the mutational landscape of a long-term surviving stage Ⅲ colorectal cancer patient and compare the resulting genomic data with The Cancer Genome Atlas(TCGA)database.In this study,we report the case of a 40-year old female patient from the Chinese Han population.The patient had colorectal cancer related symptoms including abdominal discomfort,tenesmus and severe back pain,and was admitted to the First Affiliated Hospital of Anhui Medical University in October,2008.The size of her tumor was 3cm×3cm,and the carcinoma had invaded the serosa layer,covering 3/4 of the intestine tube.She was diagnosed with a stage Ⅲ colorectal cancer after examination.The patient presented a good prognosis with over 8-year survival after curative surgery and adjuvant therapy with Oxaliplatin and Huaier granules,a traditional Chinese medicine.Using the whole genome sequencing data,we profiled the germline and somatic mutations and obtained an all-inclusive data of the genomic alterations.We identified 194 germline mutations in the patient.After filtering with public databases,20 of the mutated genes were found to overlap in the Cancer Gene Census(CGC),and Cancer Predisposition Genes(CPG)databases.Of the 20 overlapped genes,BAP1,EXT1,CDH1,PTPN11,KIT were predicted as deleterious.We applied three variant callers to call somatic variants and recorded a total of 111 somatic variants in the exonic regions.The variants were filtered with publicly available datasets to select the mostinformative ones.Totally,7 gene mutations;AR1D1B,BAZ1A,BRCA2,CTNND1,MUC16,MUC4,NCOA2 with perceived potential impact on colorectal cancer development were identified.The Gene Ontology(GO)enrichment analysis revealed that some of the somatic mutated genes were enriched in the O-glycan processing,Golgi lumen,and Protein O-linked glycosylation processes with perceived influence on colorectal cancer development.We investigated the relative contributions of the six SNV-classes(C>A,C>G,C>T,G>A,G>C,G>T)and their flanking bases to the somatic variants of the tumor sample and found 6 predominant mutation signatures:signatures 12(33%),1(18%)3(14%),16(12%),5(8%)and 9(7%)that characterized colorectal cancer progression in the patient.We identified somatic structural variations such as copy number variants and complex genomic rearrangements in the patient using FACETS and DELLY.Totally,1996 structural events,including 1367 translocations,143 inversions,306 tandem duplications and 180 deletions were obtained with DELLY;however,only in-frame events and fusions with distance over 50 Kb between two genes were kept.Ultimately,we reported 53 significant structural events including 31 genes.FACETS identified 107 large segments with copy number alternations,including 34 duplications,12 loss of function and 69 hemizygous.Then,we kept genes which were only alternated in coding sequences and got 34 genes with loss of heterozygosity and 1 with duplication.To investigate the similarities and differences between the patient’s genome and those of conventional stage Ⅲ patients,we compared the genomic alterations observed in our patient with those of stage Ⅲ colorectal cancer patients reported in The Cancer Genome Atlas Network(TCGA).Mutations in APC,TP53,KRAS,SMAD4,FBXW7 and PIK3CA defined as drivers in TCGA patients were not recorded in our study.However,mutations in MUC4,MUC16,ARID1B,BAZ1A,BRCA2,CTNND1 and NCOA2 rarely reported in TCGA patients were predominant in our patient.Additionally,we observed loss of heterozygosity in POLE,RET,BMPR1A,NCOA4 and 30 other genes in contrast to deletion and amplification events recorded in TCGA patients.Overall,we produced a genomic mutation profile of a long-term surviving colorectal cancer patient.We also identified recurrent and rare mutations that could provide a valuable resource for further study into the alterations that characterize advanced colorectal cancer.The data obtained may be useful to design clinical therapy for personalized medicine.