Impact of RAS/BRAF V600E mutations on the tumor immune microenvironment in mismatch repair deficient /microsatellite instability colorectal cancers

Abstract Purpose: Routine testing for dMMR/MSI in colorectal cancers (CRCs) is recommended for Lynch Syndrome screening, prognosis, and treatment guidance. In the metastatic setting, RASBRAF mutations guide treatment decisions. The impact of these mutations on tumor immune microenvironment (TiME) in MSI/dMMR CRC are not known. Experimental Design: Retrospective analysis of 448 patients with stage I-IV MSI/dMMR CRC profiled using next-generation sequencing (NGS) (Tempus xT DNA-seq of 595-648 genes at 500x coverage and Tempus xR whole-exome capture RNA-seq). MSI status was determined by assessing 44 or 239 loci using NGS. dMMR was determined using immunohistochemistry (IHC). Tumor mutational burden (TMB), tumor neoantigen burden (NTB), PD-L1, immune infiltration, and canonical immune pathways (76 gene set signatures) were analyzed. Results: The median age at diagnosis was 67 years (range 21-86), 59% female, and 70% stage III-IV. Among the 448, 100 (22%) harbored RAS mutations (RASmut), 119 (27%) a BRAFV600E mutation, and 229 (51%) were double wild-type (RASwt, BRAFwt). RASmut exhibited lower NTB (median 12 vs. 15 vs. 16, p=0.003) and PD-L1 (3.6% vs. 13% vs. 24%, p<0.001) than BRAFV600E and wild-type tumors, respectively. The RASmut TiME had lower overall inflammation and fewer infiltrating CD8+ T-cells than wild-type or BRAFV600E tumors. In contrast, BRAFV600E tumors exhibited hyperproliferative characteristics associated with broad metabolic reprogramming but similarly inflamed TiME compared to wild-type tumors. Conclusions: Our data suggest that MSI/dMMR CRCs harboring RASmut are less immunogenic and TiME contains a lower inflammatory profile than wild-type or BRAFV600E tumors. Further analysis and validation are required to confirm these findings.