RNA-Seq-Based Molecular Classification Analyses in Colorectal Cancer and Synchronous Adenoma

SIMPLE SUMMARY: The study focuses on colorectal cancers (CRC) and their molecular subtypes (CMS) based on gene expression. A revised classification system, iCMS, incorporates epithelial status, microsatellite instability, and fibrosis. The research uses iCMS to investigate the connection between CRC and adenomas, examining gene expression and cell types. An in silico method called CiberSortx estimates cell proportions, with a random forest model classifying CMS classes. Results suggest most CRCs are CMS2 or CMS3, while a novel subtype, iCMS2-F/iCMS3-F, is proposed due to enrichment in myofibroblasts. The study highlights the potential of iCMS and in silico methods for CRC and adenoma analysis. ABSTRACT: Colorectal cancers (CRC) are classified into consensus molecular subtypes (CMS) based on gene expression profiles. The revised classification system iCMS was proposed by considering intrinsic epithelial status, microsatellite instability (MSI), and fibrosis. This study aimed to provide molecular evidence for the adenoma–carcinoma sequence concept by examining CRC and synchronous adenomas using iCMS. Epithelial CMS cell proportion was estimated using CiberSortx, an in silico cell fractionation method that included CMS cell types among the reference cell types. A random forest (RF) model estimated the posterior probabilities of CMS classes, which were compared with the CiberSortx results. Gene expression profiles of the published iCMS signature panel were retrieved from our dataset and subjected to heatmap clustering for classification. Bulk RNA sequencing data were collected from 29 adenocarcinomas and 11 adenoma samples. CiberSortx showed all CRC contained either CMS2 or CMS3 as the major epithelial cancer cell type. The RF model classified approximately half of the CRC as CMS4, whereas CMS4 was hardly detected by CiberSortx. Because they were enriched with myofibroblasts as per the CiberSortx classification, we tentatively designated them as iCMS2-F/iCMS3-F. iCMS coupled with the application of an in silico cell fractionation method can provide the molecular dissection of CRC and adenoma.