Background: The KEYNOTE-177 trial demonstrated pembrolizumab’s superiority over first-line chemotherapy in patients with MSI-H/dMMR CRC. However, in a subgroup analysis, patients with KRAS or NRAS mutations did not show the same favorable PFS benefit with PD-1 blockade therapy (HR 1.19; CI 0.68-2.07). The impact of RAS mutations on the immunologic characteristics of the TME of MSI-H/dMMR CRCs has not been well characterized.

Methods: A retrospective review of deidentified records of patients with MSI-H/dMMR CRC tumors was conducted using next-generation sequencing (NGS) data (Tempus |xT assay, DNA-seq of 595-648 genes at 500x coverage, and full transcriptome RNA-seq). MSI-H determined by assessment of 239 loci by NGS. dMMR determined by IHC. Several immune markers were assessed, including tumor mutational burden (TMB), neoantigen tumor burden (NTB, ScanNeo), PD-L1 expression, immune infiltration, and canonical immune pathways (82 gene set signatures).

Results: A total of 463 MSI-H/dMMR CRCs were analyzed, of which 110 (24%) tumors harbored RAS mutations
(RAS mut) [KRAS: 93%, NRAS 6% and HRAS 1%}, while 353 were RAS-wild-type (RAS WT). Compared to MSI-H/dMMR RAS
WT, MSI-H/dMMR RAS mut tumors were more frequently identified in males (53% vs. 38%; P=0.005), and younger patients (median age: 57 yrs vs. 71 yrs, P<0.001). Although there were no significant differences in median TMB (40 mut/MB for both, p=0.9) or frequency of TMB-high status (>=10 mut/MB) between the two groups, RAS mut tumors had a lower tumor NTB (16 vs. 12 neoAg/Mb, P<0.001) and lower % CD8 T cell but higher % CD4 T cell infiltration (P<0.05). Significant differences were observed in genomic alterations co-occurring with RAS mut compared to RAS WT (e.g., MLH1 (23% vs. 8.8%, P<0.001), MSH6 (36% vs. 24%, P= 0.017), APC (60% vs. 20%, P<0.001), ARID1A (54% vs 30%, P<0.001), PIK3CA (36% vs 19%, P<0.001), and TP53 (19% vs. 32% vs 19%, P = 0.014). Pathway enrichment analysis identified 14 differentially expressed pathways among RAS
mut tumors. Four pathways showed significant upregulation, including Hedgehog, Wnt, TGFβ, and cancer stem cell pathways. Ten pathways of interest showed significant downregulation among RAS mut tumors. The majority (9/10) were immune related, including cytokine signaling [JAK-STAT, TGFβ, TH1], innate immune [NK cells], and adaptive immune events (CD8 T cell, Tregs)].

Conclusions:
MSI-H/dMMR CRCs harboring RAS mut exhibited overall upregulated WNT/SHH pathway activity, coupled with reduced NTB, cytokine signaling, and innate and adaptive immune events. TGFβ is pleiotropic, and different members were associated with variable modulation. Overall, these data suggest that MSI-H/dMMR CRCs harboring RAS mutations are less immunogenic and appeared to contain a TME that is less sensitive to immune checkpoint blockade than MSI-H/dMMR RAS wt CRCs.

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