Research On Molecular Biomarker And Associated Clinical Significance In Colorectal Cancer

This study mainly focused on the gene mutation detection, adjuvant chemotherapy prognosis associated molecular biomarkers evaluation and novel biomarker screening with high-throughput sequencing in colorectal cancer (CRC), which is a common type of malignant tumor in China. The contents included two parts:Part 1 Research on targeted therapy associated molecular biomarker detection and clinical significance in colorectal cancerWith the era of precision medicine appears, individual therapy based on gene molecular classification has been widely used. Critical genes in the downstream of the EGFR signaling pathway play significant roles in the tumorogenesis and progression of CRC, such as PIK3CA, NRAS, KRAS and BRAF. Reported studies have shown that only KRAS/NRAS wild type could benefit from targeted therapy, and gene mutant status of PIK3CA or BRAF would also affect the treatment effect and prognosis. Adjuvant chemotherapy for early stage colon cancer patients after surgery could obtain survival benefit, while rare gene biomarkers were capable and incontrovertible for prognosis evaluation, then gene mutation detection and associated prognosis research were important for clinical practice. The study contents were as followed:Chapter 1. Gene mutation spectrum of PIK3CA and NRAS in Chinese CRC patients. Direct sequencing was used for mutation detection in PIK3CA (exon 9 and 20) and NRAS (codon 12,13 and 61) in 676 Chinese CRC patients, in order to obtain the gene mutants distribution in CRC and the association between patients’clinicopathological characteristics. The PIK3CA gene mutation frequency was 9.9%(62/625),7.0% (45/643) in exon 9 and 2.7%(17/636) in exon 20; 4.2%(26/621) tumors harbored NRAS gene mutants, and the frequency for codon 12,13 and 61 was 1.27%(8/630). 0.48%(3/630) and 2.02%(13/643) respectively. We discovered PIK3CA gene mutation was more common in colon tumors compared to rectal tumors(15.1% vs 6.1%, P<0.001), NRAS gene mutants differed in different tumor stages (P=0.03), and NRAS mutation occurred more frequently in distant metastasis tumors (12.2% vs 3.3%, P= 0.01).Chapter 2. Gene mutation detection and adjuvant chemotherapy associated study for colon cancer. In this study we collected 124 stage II and 104 stage Ⅲ colon cancer tissue samples, direct sequencing was used for gene mutation detection in KRAS (codon 12,13 and 61), BRAF (exon 11 and 15), PIK3CA (exon 9 and 20) and NRAS (codon 12,13 and 61), survival data was collected for analysis. KRAS mutants were found in 37.89%(86/227) cases,26.87%(61/227) in codon 12,6.61%(15/227) in codon 13 and 3.54%(8/226) in codon 61. The status of BRAF mutations was detected in all samples,7.02%(16/228) tumors harbored a BRAF mutation,1.75%(4/228) in exon 11 and 5.26%(12/228) in exon 15. V600E was the major subtype in BRAF mutation (43.75%,7/16). PIK3CA mutations were detected in 13.18%(29/220) tumors,10.45%(23/220) in exon 9 and 2.7%(6/221) in exon 20. NRAS mutations existed only in stage Ⅱ colon cancer,0.89%(2/224) tumors harbored a NRAS mutation, of which were G12D in codon 12 and Q61R in codon 61. KRAS and BRAF mutations were more frequent in older patients (P<0.05), PIK3CA exon 9 (E545K) mutation was significantly associated with tumor recurrence (10.7% vs.0.0%, P=0.031) in stage Ⅲ patients. In the survival analysis, PIK3CA (E545K) mutant status was prognostic for OS (P=0.044), while a significant trend for DFS only in stage III (P=0.051). No other significant associations between gene mutations and patients’ clinicopathological characteristics.Part 2. Screening and verification of chemotherapy effects and prognosis associated genes in colon cancer based on high throughput sequencingThe tumorogenesis and progression of cancer is a complex multifactor and multistep procedure, involving a myriad of gene mutations accumulation together with internal and external environment. The next generation sequencing (NGS) provided an insight for tumor investigation from genomics aspect. In patients with adjuvant chemotherapy after surgery, no suitable biomarkers available for treatment effects and prognosis evaluation. We have collected 20 stage Ⅲ colon cancer samples with different prognosis for whole exome sequencing (WES) sequencing, and verified the screening different genes, in order to investigate the potential biomarker for prognosis evaluation. A total of 6 gene SNPs (CD97, ZNF568, WARS2, APPL2, C9orf117, ZNF443) were confirmed in different groups after Sequenom test (P<0.05), and ZNF568 (rs547483) appeared a significant trend in different groups (P=0.08621) during the verification in a larger cohort. WARS2 (rs3790549, CC) was higher in the non-smokers for stage Ⅲ patients (62.5% vs 35.3%, P=0.041),WARS2 (rs3790549, CC) and C9orf117 (rs497632, AC) were higher in the non-smokers for stage Ⅱ-Ⅲ patients (P<0.05), and WARS2 (rs3790549, CC) differed in different T stage tumors (P=0.023)The research mentioned above has built the PIK3CA and NRAS gene mutation spectrum in Chinese CRC, associations between KRAS, BRAF, PIK3CA, NRAS gene mutation and clinicopathological characteristics were analyzed, together with the evaluation of prognostic value. Meanwhile, WES was utilized for screening novel gene biomarkers for prognosis assessment in colon cancer adjuvant chemotherapy. These results provided theory evidence for a better understanding of Chinese CRC and the associated clinical practice.