Genome-wide Next Generation Sequencing And Methylation Chip Analysis In Identification For Individualized Molecular Biomarkers Among Coloredal Cancer Patients

Colorectal cancer is one of the leading causes of mortality worldwide. The frequency and poor prognosis in patients with metastasis colorectal cancer (mCRC) emphasizes the need for better markers usable for both treatment and prognosis. Genome wide analysis studies have identified sequence mutations causing loss-of-function that are associated with disease occurrence and severity. Epigenetic modifications, such DNA methylation, have also been implicated in many cancers but have yet to be examined in the East Asian population of colorectal cancer patients. Here we described an approach of identifying whole genomic variants in exon regions of key genes. Meanwhile, we used genome-wide methylation chip to analysis the characteristic methylation profiles of colorectal cancer patients. Part one:"Druggable" alterations of somatic mutation in exon region detected by Ion Torrent in metastasis colorectal cancer patientsMethods:Formalin-fixed paraffin embedded (FFPE) metastasis colorectal cancer patients biopsy tissue were collected. Ion AmpliSeqTM Cancer Panel was designed to detect739COSMIC mutations in604loci from46oncogenes and tumor suppressor genes. We applied the Ion Personal Genome Machine(?)(PGMTM) System to run the whole reaction by using only as little as lOng of input DNA. Samples were then analyzed by the Ampliseq Variant Caller plug-in within the newest version of the Ion Torrent Suite Software. Ingenuity Pathway Software was used to do pathway analysis. Cox regression analysis was tested regarding the potential relationship between the alteration numbers and the clinical factors including response rate, disease free survival and overall survival, etc.Results:(1) Among10specimens, we identified65genetic alterations in24genes after excluding the germline mutations according the dbSNP database.35%of those alterations were also present in the COSMIC database (Catalogue of Somatic Mutations in Cancer). No clinical factor was found to be significantly associated with the alteration numbers in patients by univariate analysis.(2) Notably, there are11genes including the expected APC, BRAF, KRAS, PIK3CA and TP53that were mutated in at least2samples. Pathway analysis identified "Colorectal Cancer Metastasis Signaling" as the top mutated canonical pathway. This analysis further revealed mutated genes in the classic signal pathways of Wnt, PI3K/AKT, and TGF-beta/SMAD as significantly present.(3) Interestingly,90%of CRCs (9out of10) harbored at least one "druggable" alteration (range from1-6)that has been linked to a clinical treatment option or is currently being investigated in clinical trials of new targeted therapies. Those pairs of drug and targeted genes are vemurafenib to BRAF; cetuximab to EGFR; palifermin to FGFR2; pazopanib to FGFR3; AEE788to KDR and BEZ235to PIK3CA.Part two:Using Illumina HumanMethylation27Chip based platform to detect the methylation profiles and the significant abrant methylated genes of colorectal cancer patientsMethods:Biopsies of tumors and matched non-cancerous tissue types were obtained from colorectal cancer patients. Genomic DNA was isolated and subjected to the bisulphite conversion method for comparative DNA methylation analysis on the Illumina Infinium HumanMethylation27BeadChip.Results:(1) A total of258and74genes were found to be differentially methylated in the two tumor tissues, respectively, as compared to the individual’s matched control tissue. Interestingly, the three genes that exhibited hypermethylation in their promoter regions, CMTM2, ECRG4, and SH3GL3, are known to be significantly associated with colorectal cancer in previous studies.(2) Using heatmap cluster analysis, eight hypermethylated and10hypomethylated genes were identified as significantly differentially methylated genes in the tumour tissues.Conclusions:(1) DNA deep sequencing of key oncogenes and tumor suppressors enables identification of "druggable" mutations for individual colorectal cancer patients. Our study reveals the Next Generation Sequencing’ great advantage and hopeful perspective in the era of individualized medicine.(2) Genome-wide methylation profiling facilitates the rapid and simultaneous analysis of cancerous cells which may help to identify methylation markers with high sensitivity and specificity for diagnosis and prognosis. Our results show the promise of the microarray technology in identification of potential methylation biomarkers for colorectal cancers.