Molecular Characterization of Somatic Alterations in Dukes’ B and C Colorectal Cancers by Targeted Sequencing

Abstract

Despite global progress in research, improved screening and refined treatment strategies, colorectal cancer (CRC) remains as the third most common malignancy. As each type of cancer is different and exhibits unique alteration patterns, identifying and characterizing gene alterations in CRC that may serve as biomarkers might help to improve diagnosis, prognosis and predict potential response to therapy. With the emergence of next generation sequencing technologies (NGS), it is now possible to extensively and rapidly identify the gene profile of individual tumors. In this study, we aimed to identify actionable somatic alterations in Dukes’ B and C in CRC via NGS. Targeted sequencing of 409 cancer-related genes using the Ion AmpliseqTM Comprehensive Cancer Panel was performed on genomic DNA obtained from paired fresh frozen tissues, cancer and normal, of Dukes’ B (n = 10) and Dukes’ C (n = 9) CRC. The sequencing results were analyzed using Torrent Suite, annotated using ANNOVAR and validated using Sanger sequencing. A total of 141 somatic non-synonymous sequence variations were identified in 86 genes. Among these, 64 variants (45%) were predicted to be deleterious, 38 variants (27%) possibly deleterious while the other 39 variants (28%) have low or neutral protein impact. Seventeen genes have alterations with frequencies of ≥10% in the patient cohort and with 14 overlapped genes in both Dukes’ B and C. The adenomatous polyposis coli gene (APC) was the most frequently altered gene in both groups (n = 6 in Dukes’ B and C). In addition, TP53 was more frequently altered in Dukes’ C (n = 7) compared to Dukes’ B (n = 4). Ten variants in APC, namely p.R283∗, p.N778fs, p.R805∗, p.Y935fs, p.E941fs, p.E1057∗, p.I1401fs, p.Q1378∗, p.E1379∗, and p.A1485fs were predicted to be driver variants. APC remains as the most frequently altered gene in the intermediate stages of CRC. Wnt signaling pathway is the major affected pathway followed by P53, RAS, TGF-β, and PI3K signaling. We reported the alteration profiles in each of the patient which has the potential to affect the clinical decision. We believe that this study will add further to the understanding of CRC molecular landscape.

Actionable Potentials of Less Frequently Mutated Genes in Colorectal Cancer and Their Roles in Precision Medicine

“…6%-20% [20,[38][39][40][41] Sensitive to mTOR inhibitors rapamycin in breast cancer cell line with the loss of FBXW7 and deletion or mutation of PTEN [42]. Preclinical Mutated CRC cell lines are less sensitive to regorafenib and sorafenib [45].…”

“…Until today, FBXW7 has been constantly recognized as one of the less commonly mutated genes in CRC, accounting for approximately 6% to 15% of all cases [20,39,41,98]. The mutational range of FBXW7 in CRC is somewhat peculiar, with over 70% of missense single nucleotide variations affecting amino acids in the substrate-binding sites, and the most common mutational hotspots are at the two important arginine residues at the position 465 and 479 (Arg 465 and Arg 479 ) [39,99].…”

Alteration in the Immune Microenvironment Based on APC Status in MSS/pMMR Colon Cancer Data Retrieved from TCGA

“…Other major routes for colorectal cancer development account for another 15% to 20% of the cases [13]. Typical manifestations of this pathway are precursor sessile adenoma, wild-type APC (APC-wt), BRAF mutation, characteristic CpG island methylation phenotype, poor differentiation, and mucosa histology [14]. Here re report on studies of the effect of APC status on the immune microenvironment of MSS colon cancer, aiming to identify its TIMT and determine whether it might be a potential predictor for immunotherapy e cacy in these patients.…”

Genome-Wide Open Chromatin Methylome Profiles in Colorectal Cancer

“…CRC can occur through the accumulation of multiple genetics and epigenetics changes. Research has shown that somatic mutation in APC, BRAF, KRAS, PIK3CA and TP53 [2,3] have been frequently observed in CRC and are considered the drivers of CRC formation. Despite many kinds of research on molecular alterations involved in CRC pathogenesis, the existing knowledge remains inadequate for early diagnosis and prognosis assessment.…”