Background: Conventional colorectal cancer (CRC) develops following the adenoma-carcinoma sequence of mutational events. We aimed to characterize the heterogenicity of mutational alterations and immune cell infiltrates in colorectal polyp subtypes obtained from patients undergoing colonoscopy. We examined relationships of these molecular features with histopathology variables used for disease risk assessment.
Methods: Polyp-, adjacent-, healthy-mucosal biopsies and blood were collected from 78 patients during colonoscopy at Royal North Shore Hospital. Indications for colonoscopy included rectal bleeding, FOBT-positive, scheduled surveillance, family history of bowel neoplasia. Patients with familiar syndromes or inflammatory bowel disease were excluded. Molecular profiling included whole-exome sequencing (WES) targeting 200X depth, and multiplexed IHC immune cell phenotyping.
Results:
It’s been well-known that the crosstalk between Wnt/β-catenin and RTK-RAS pathways is a key event in tumorigenesis of CRC, which was observed in WES data of five adenocarcinoma samples collected in our study as well as in TCGA COAD/READ. Based on this knowledge, we investigated oncogenic/likely oncogenic variants in Wnt/β-catenin, RTK-RAS and TP53 pathways and defined a high-risk group (15 polyps) by looking for polyps that carried multiple variants in any two of three pathways. The frequencies of high-risk polyps with co-occurring oncogenic mutations in WNT/RTK-RAS, RTK-RAS/TP53 or WNT/TP53 pathways were 10.6%, 6.1% and 6.1% respectively. Strikingly we found six small polyps (3-6mm) representing high-risk molecular characteristics which would be categorized as no-risk in conventional surveillance guideline. Two out of those six were small hyperplastic polyps harboring oncogenic variants in TP53, BRAF, PTPRT, ATR and RNF43.
Molecular profiling revealed that APC, KRAS, BRAF, ROS1, TP53, and CTNNB1, were the top 6 driver genes most frequently mutated in colorectal polyps. Mean mutational burden of high-risk polyps was 4.17/Mb significantly higher than the mean 3.13/Mb in low-risk group. Mutation signature analysis showed four prevalent signatures: SBS1, SBS5, SBS10b and SBS42. One polyp with SBS10b signature had POLE variant, exhibiting highest TMB 6.52/Mb. There’s no significant difference of TMB between different polyp type groups.
Immunophenotyping of polyps showed CD3+ T cell density was significantly increased compared to paired normal mucosa. In contrast, significant decreases in CD68+ macrophage and CD11c+ dendritic cell densities occurred in polyp tissue. Small high-risk conventional polyps displayed significantly reduced macrophage infiltration compared to low-risk conventional polyps.
Conclusion: In-depth WES and molecular characterization provides a comprehensive view of genetic background in early colorectal polyps. This information may be useful to identify at-risk disease which would be missed by conventional histopathology review.