Colorectal cancer intrinsic subtypes predict chemotherapy benefit, deficient mismatch repair and epithelial-to-mesenchymal transition

Abstract

In most colorectal cancer (CRC) patients, outcome cannot be predicted because tumors with similar clinicopathological features can have differences in disease progression and treatment response. Therefore, a better understanding of the CRC biology is required to identify those patients who will benefit from chemotherapy and to find a more tailored therapy plan for other patients. Based on unsupervised classification of whole genome data from 188 stages I-IV CRC patients, a molecular classification was developed that consist of at least three major intrinsic subtypes (A-, B- and C-type). The subtypes were validated in 543 stages II and III patients and were associated with prognosis and benefit from chemotherapy. The heterogeneity of the intrinsic subtypes is largely based on three biological hallmarks of the tumor: epithelial-to-mesenchymal transition, deficiency in mismatch repair genes that result in high mutation frequency associated with microsatellite instability and cellular proliferation. A-type tumors, observed in 22% of the patients, have the best prognosis, have frequent BRAF mutations and a deficient DNA mismatch repair system. C-type patients (16%) have the worst outcome, a mesenchymal gene expression phenotype and show no benefit from adjuvant chemotherapy treatment. Both A-type and B-type tumors have a more proliferative and epithelial phenotype and B-types benefit from adjuvant chemotherapy. B-type tumors (62%) show a low overall mutation frequency consistent with the absence of DNA mismatch repair deficiency. Classification based on molecular subtypes made it possible to expand and improve CRC classification beyond standard molecular and immunohistochemical assessment and might help in the future to guide treatment in CRC patients.

Keywords: EMT; chemotherapy benefit; colorectal cancer; mismatch repair; molecular subtypes.

© 2013 UICC.

Figures

Figure 1

Single sample predictor for molecular subtype classification. (a) Unsupervised hierarchical clustering of the development cohort shows three distinct CRC intrinsic subtypes. (b) Gene signatures specific for A-type (32 genes), B-type (53 genes) and C-type (102 genes) CRC. Yellow indicates relative up-regulation and blue down-regulation of the genes across the 188 development samples. (c) A single sample classifier for identification of the three CRC subtypes.

Figure 2

CRC subtypes are associated with MSI and dMMR phenotypes. (a) Readout of the MSI/dMMR signature for the three CRC molecular subtypes. The symbols represent the binary MSI/dMMR (crosses) or MSS/pMMR (triangles) calls based on the signature indexes that are plotted on the x-axis. (b) Mutation frequency in the cancer kinome (615 genes) across the three subtypes: 23 A-type, 37 B-type and 13 C-type samples.

Figure 3

Molecular subtypes association with EMT characteristics. (a) Relative gene expression levels of eight mesenchymal and five epithelial marker in the three molecular subtypes. Significant levels are indicated for differential expression between the subtypes (pairwise Student’s t-test). (b) Read-out of the EMT signature by Loboda et al. across the molecular subtypes. Positive signature indexes are representative for a mesenchymal phenotype. (c) Receiver operating curve of C-type classification using the Loboda signature indexes. (d) Molecular subtype association of the epithelial and mesenchymal genes as presented by Loboda et al. Genes are shown according to the original clustering in Ref.. Genes without a subtype indication showed no statistical significant differential expression.

Figure 4

Prognostic value of molecular subtypes in stages II and III. Kaplan–Meier survival analysis of the three molecular subtypes in the validation cohort for (a) DMFS of all stages II and III validation samples, (b) cancer-related OS of all stages II and III, (c) in MSI patients (MSI status is based on the hospital MSI testing or, if not available, on the previously reported MSI gene signature, see Methods section for details) and (d) BRAF wildtype patients. Survival curves of A-, B- and C-type samples are indicated in blue, gray and orange, respectively.

Figure 5

Molecular subtypes differ for their response to chemotherapy. Kaplan–Meier survival analysis (OS) between patients (validation cohort) treated with and without chemotherapy for (a) A-type, (b) B-type and (c) C-type CRC. (d) Adjuvant chemotherapy benefit for the subtypes as measured by the difference in 5-year OS. (e) Gene expression boxplots of two proliferation markers KI67 and AURKA across the three subtypes. p-Values indicate the significance of differential expression between C-type and A–B-types.

Figure 6

Classification model of CRC by A-, B- and C- subtypes. Classification model that discriminate three distinct subtypes: MMR-deficient epithelial (A-type), proliferative epithelial (B-type) and mesenchymal (C-type). A simplified model is shown for the main clinical and molecular characteristics of each of the three subtypes: baseline prognosis, 5FU-based chemotherapy response, epithelial or mesenchymal-like phenotypes, microsatellite status (MSI or MSS), MMR phenotype (deficient or proficient) and the associating BRAF mutation status and the tumor’s proliferation rate as measured by MKI67 and AURKA expression levels. This representation is a simplified model focused on the core-characteristics of each subtype.