Molecular pathological classification of colorectal cancer-an update

Abstract

Colorectal cancer (CRC) has a broad range of molecular alterations with two major mechanisms of genomic instability (chromosomal instability and microsatellite instability) and has been subclassified into 4 consensus molecular subtypes (CMS) based on bulk RNA sequence data. Here, we update the molecular pathological classification of CRC with an overview of more recent bulk and single-cell RNA data analysis for development of transcriptional classifiers and risk stratification methods, taking into account the marked inter-tumoural and intra-tumoural heterogeneity of CRC. The importance of the stromal and immune components or tumour microenvironment (TME) to prognosis has emerged from these analyses. Attempts to remove the contribution of the tumour microenvironment and reveal neoplastic-specific transcriptional traits involved identification of the CRC intrinsic subtypes (CRIS). The use of immunohistochemistry and digital pathology to implement classification systems are evolving fields. Conventional adenoma versus serrated polyp pathway transcriptomic analysis and characterisation of canonical LGR5+ crypt base columnar stem cell versus ANXA1+ regenerative stem cell phenotypes emerged as key properties for improved understanding of transcriptional signals involved in molecular subclassification of colorectal cancers. Recently, classification by three pathway-derived subtypes (PDS1-3) has been developed, revealing a continuum of intrinsic biology associated with biological, stem cell, histopathological, and clinical attributes.

Keywords: Colorectal cancer; Hereditary; Molecular pathological classification.

Fig. 1

Diagrammatic summary of colorectal cancer molecular pathology classification systems. The Cancer Genome Atlas (TCGA), published in 2012, used a predominantly DNA-based classification, splitting colorectal cancers (CRC) into a large group (84%) with chromosomal instability (CIN), 13% hypermutated CRC due to deficient mismatch repair (dMMR) that causes microsatellite instability (MSI), and 3% ultramutated CRC due to proofreading exonuclease domain mutations in the two polymerases POLE and POLD1. The consensus molecular subtype (CMS) classification, published in 2015, used bulk RNA sequences to classify CRC into 4 major groups, CMS1–CMS4, with a residual ‘Mixed Features’ or transitional group. CMS1 correlated strongly with the TCGA hypermutated group. The large TCGA CIN group splits into 3 CMS groups—CMS2: canonical, CMS3: metabolic, and CMS4: mesenchymal with the features shown. The Colorectal Intrinsic Subtypes (CRIS) classification, published in 2016, separated CRC epithelial neoplastic cells (without stromal and immune components) into 5 subtypes, CRIS-A to CRIS-E. Most CMS1 and CMS3 cancers fell into CRIS-A (some CMS1 were in CRIS-B); CMS2 cancers were found within CRIS-C, CRIS-D, and CRIS-E; CMS4 cancers split into CRIS-B, CRIS-C, CRIS-D, and CRIS-E. The single-cell intrinsic consensus molecular subtype (iCMS) classification of CRC, published in 2022, based on single-cell transcriptomes identified a transcriptomic dichotomy of malignant cells, resulting in two intrinsic subtypes, iCMS2 and iCMS3, that refined the earlier CMS classification. Most CMS2 and CMS3 CRCs have iCMS2 and iCMS3 epithelium, respectively, whereas iCMS3 contains the dMMR/MSI cancers and one-third of microsatellite-stable (MSS) tumours. The Pathway-Derived Subtypes (PDS) classification of CRC, published in 2023, puts colorectal cancers into 3 subtypes, PDS1–PDS3, with a small residual PDS Mixed group, based on Gene Ontology inferred pathway activation patterns, revealing a continuum of features associated with biological pathways, stem cell populations, morphological/histopathological characteristics, and clinical attributes. Abbreviations: dMMR, deficient mismatch repair; MSI, microsatellite instability; MSS, microsatellite stability; CIMP, CpG island methylator phenotype (-H, high or -L, low); SCNA, somatic copy number alteration (-H, high or -L, low); MLH1-s, silencing of MLH1 protein expression by promoter hypermethylation; BRAFm, BRAF mutation; TILs, tumour infiltrating lymphocytes; EMT, epithelial-mesenchymal transition; CBC, LGR5+ crypt base columnar stem cell; RSC, ANXA1+ regenerative stem cell; SMI, Stem Maturation Index

Fig. 2

Typical standard-of-care pathway for early stage (stage I–III) localised colonic or rectal cancers. Following the patient coming to the pathway due to bowel symptoms, screening, or referral, endoscopic biopsies are taken for histopathological diagnosis (also available for some biomarker testing, such as for KRAS mutation analysis in certain circumstances) and combined with radiological scans to determine likely cancer stage, with the key cancer and patient features being discussed at a multi-disciplinary team (MDT) meeting for making appropriate management decisions, including treatment of the patient, which may involve resection and/or adjuvant chemotherapy (upper panel) and/or neoadjuvant radiotherapy (middle panel) and/or neoadjuvant chemotherapy (lower panel), with tumour response/regression analysis made by histopathological assessment of resection specimens

Fig. 3

Comparison of colorectal serrated polyp (‘Serrated’) versus conventional adenoma (‘Conventional’) pathway progression to cancers in terms of cancer histology, cancer subtype (CMS1/4 and PDS2 versus CMS2/3 and PDS1/3), selected cancer hallmarks (immune-rich/stroma-rich versus MYC targets, canonical stem cells, proliferation, PRC targets, epithelial differentiation, and repression), and clinical relapse-free survival differences

Fig. 4

Transcriptomic analysis of stem cell populations in CRC showed variable populations of both LGR5+ /ANXA1− crypt base columnar (CBC) stem cells and LGR5-/ANXA1+ regenerative stem cells (RSC), reflecting stem cell plasticity that can respond adaptively to acute selective pressures, and this admixture can be assessed using a transcriptional molecular tool to assess the Intestinal Stem Cell Index (ISC), with CMS2 and PDS1 groups enriched for LGR5+ CBC stem cells, whereas CMS1/CMS4 and PDS2 groups are enriched for ANXA1+ RSC. Combining PDS and stem cell analyses showed that both LGR5+ CBC and ANXA1+ RSC were fast-cycling and abundant in stem-rich PDS1 and PDS2, whereas the PDS3 group was slow-cycling and stem-poor, containing more enterocytic differentiated cells as shown by the Stem Maturation Index (SMI)