Figure S1. Principal component analysis and hierarchical clustering of the 218 BRAF mutant patient cohort;Figure S2: Gene-wise and patient-wise biclustering heatmap of the 218 BRAF mutant patient cohort and comparison of BRAF mutant subtypes obtained with and without adjusting for microsatellite instability status;Figure S3: Geneset enrichment analysis (GSEA) of BM subtypes for the four most enriched signature of the Hallmark collection and gene overlap between signatures and BM-specific genes;Figure S4: Patient-level single sample geneset enrichment analysis (ssGSEA) between BM subtypes, double BRAF wild-type KRAS wild-type (WT2) and KRAS mutant patients for the hallmark signature collection;Figure S5: Patient-averaged single sample geneset enrichment analysis (ssGSEA) between BM subtypes, double BRAF wild-type KRAS wild-type (WT2) and KRAS mutant patients for the hallmark signature collection;Figure S6: Gene overlap between selected sets of gene signatures used in this study;Figure S7: Immune landscape of the BM subtypes;Figure S8: Methylation analysis of BM subtypes using TCGA data;Figure S9: Survival and clinical characteristics of the BRAF mutant patients enrolled in the survival analysis;Figure S10: Performance of the generalized model used to classify patients into BM1 and BM2 subtypes;Figure S11: Drug responses of colorectal cancer cell lines classified into BM1 and BM2.
ARTICLE ABSTRACT
Purpose: Mutation of BRAF at the valine 600 residue occurs in approximately 10% of colorectal cancers, a group with particularly poor prognosis. The response of BRAF mutant colorectal cancer to recent targeted strategies such as anti-BRAF or combinations with MEK and EGFR inhibitors remains limited and highly heterogeneous within BRAF V600E cohorts. There is clearly an unmet need in understanding the biology of BRAF V600E colorectal cancers and potential subgroups within this population.Experimental Design: In the biggest yet reported cohort of 218 BRAF V600E with gene expression data, we performed unsupervised clustering using non-negative matrix factorization to identify gene expression–based subgroups and characterized pathway activation.Results: We found strong support for a split into two distinct groups, called BM1 and BM2. These subtypes are independent of MSI status, PI3K mutation, gender, and sidedness. Pathway analyses revealed that BM1 is characterized by KRAS/AKT pathway activation, mTOR/4EBP deregulation, and EMT whereas BM2 displays important deregulation of the cell cycle. Proteomics data validated these observations as BM1 is characterized by high phosphorylation levels of AKT and 4EBP1, and BM2 patients display high CDK1 and low cyclin D1 levels. We provide a global assessment of gene expression motifs that differentiate BRAF V600E subtypes from other colorectal cancers.Conclusions: We suggest that BRAF mutant patients should not be considered as having a unique biology and provide an in depth characterization of heterogeneous motifs that may be exploited for drug targeting. Clin Cancer Res; 23(1); 104–15. ©2016 AACR.