Background: The BRAFV600E mutation is associated with the hypermethylator phenotype CIMP, which can also
lead to the MSI-H phenotype. BRAFV600E mutation and MSI-H/dMMR status seem to be biologically intertwined; however, the impact of coexisting BRAFV600E mutations on the TME and immune-metabolomic features of MSI-H/dMMR CRC tumors is not well characterized.

Methods:
A retrospective review of deidentified records of patients with MSI-H/dMMR CRC tumors was conducted using next-generation sequencing data (Tempus |xT assay: DNA-seq of 595-648 genes at 500x coverage, and full transcriptome RNA-seq). Several immune markers of tumor immunogenicity in BRAF wild-type (BRAFwt) vs. V600E-mutated (BRAFV600E) tumors were assessed, including tumor mutational burden (TMB), neoantigen tumor burden (NTB, ScanNeo), PD-L1 expression, immune infiltration, and canonical immuno-metabolomic pathways (82 geneset signatures).

Results:
A total of 459 MSI-H/dMMR CRC tumors were analyzed, of which 123 (27%) tumors harbored BRAFV600E mutations, and 336 (72%) were BRAFwt. MSI-H/dMMR BRAFV600E tumors were more frequently identified in females (69% vs. 55%; P=0.01), non-Hispanic or non-Latino (100% vs. 73%; P<0.001), and older patients (median age: 76 yrs vs. 62 yrs; P<0.001). Compared to BRAFWT, BRAFV600E tumors exhibited significantly higher TMB (Median: 49 mut/MB vs. 36 mut/MB; P <0.001) and were more frequently associated with TMB-High status (>10 mut/MB; 100% vs. 95%; P=0.008); however, no significant differences were observed with tumor NTB, immune score, or T cell infiltration (CD4 or CD8). NK cell infiltration was higher in the BRAFV600E cohort (<0.001). When compared to BRAFWT tumors, BRAFV600E tumors harbored a greater frequency of mutations in MSH6 (42% vs. 20%), B2M (33% vs. 16%), BRCA2 (31% vs. 12%), ATM (23% vs. 12%), and TP53 (30% vs. 19%) but a lower frequency of MSH2 (3.3% vs. 11%), all P<0.001. Pathway enrichment analysis identified 10 significantly altered signaling pathways, most of which related to stromal/immune cell signaling and metabolism. Five were upregulated among BRAFV600E tumors: glycerophospholipid, galactose, cyclin-dependent cell signaling; Nucleotide, and TH1 inflammation. Five pathways were downregulated (Wnt, Notch, TH17 inflammation, amino sugar, and cancer stem cell signaling).

Conclusions: MSI-H/dMMR BRAFV600E CRCs undergo broad metabolic reprogramming (e.g., glycerophospholipid galactose, nucleotide). A rise in lipid metabolism, particularly with NK inflammation, suggests that BRAFV600E mutated tumors may be associated with a non-classical immune component. BRAFV600E and BRAFwt CRCs exhibited similar NTB and T cell infiltration, suggesting that both are likely to benefit from immune checkpoint inhibitors.

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