Increased neutrophil infiltration and lower prevalence of tumor mutation burden and microsatellite instability are hallmarks of RAS mutant colorectal cancers.
Authors: Lou Emil , Baca Yasmine , Xiu Joanne , Nelson Andrew , Subramanian Subbaya , Salem Mohamed E. , Beg Muhammad Shaalan , Fontana Elisa , Diab Maria , Philip Philip Agop , Goldberg Richard M. , Pandey Ritu , Arkenau Tobias , Sun Weijing , Lenz Heinz-Josef , Shields Anthony Frank , El-Deiry Wafik S. , Korn Wolfgang Michael 
Published: June 2021

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Background: The tumor microenvironment (TME) of colorectal cancers (CRC) is modulated by oncogenic drivers such as KRAS. The TME comprises a broad landscape of immune infiltration. How tumor genomics associates with the immune cell landscape is less known. We aim to characterize immune cell types in RAS wild-type (WT) and mutant (MT) CRC, and to examine the prevalence of immuno-oncologic (IO) biomarkers (e.g. tumor mutation burden (TMB), PD-L1, MSI-H/dMMR) in these tumors. We performed genomic and transcriptomic analysis to confirm associations of mutant RAS with immune infiltration of the TME conducive to metastasis vs. potential response to immunotherapies. Methods: A total of 7,801 CRC were analyzed using next-generation sequencing on DNA (NextSeq, 592 Genes and WES, NovaSEQ), RNA (NovaSeq, whole transcriptome equencing) and IHC (Caris Life Sciences, Phoenix, AZ). MSI/MMR was tested by FA, IHC and NGS. TMB-H was based on a cut-off of > 10 mutations per MB). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). Significance was determined by X2 and Fisher-Exact and p adjusted for multiple comparisons (q) was <0.05. Results: Mutant KRAS was seen in 48% of mCRC tumors; NRAS in 3.7%, HRAS in 0.1%. The distribution was similar in patients < or >= than 50 yrs. In MSS tumors, there was a significantly higher neutrophil infiltration in KRAS MT (median cell fraction 6.6% vs. 5.9%) and NRAS MT (6.9%) overall and also when individual codons were studied. B cells, M2 macrophages, CD8+ T cells, dendritic cells and fibroblasts were lower in KRAS mutant tumors; B cells and M1 macrophages are lower in NRAS (q<0.05). dMMR/MSI-H was significantly more prevalent in RAS WT (9.1%) than in KRAS (2.9%) or NRAS MT (1.8%) tumors, and highest in HRAS MT tumors (60%, q<0.05).TMB-H was more prevalent in RAS WT (11%) than KRAS (5.8%) or NRAS (5.1%) MT, and highest in HRAS MT tumors (70%, all q<0.05). In MSS tumors, KRAS MT tumors showed more TMB-H than WT (3.1% vs. 2.1%, q<0.05), especially in KRAS non 12/13/61 mutations (5.5%, vs. 2.1%, q<0.05) and G12C (4.4%, p<0.05). PD-L1 expression was studied: in MSS tumors, KRAS-G12D (10.4%) and G13 MT (11.8%) showed higher mutation rates than RAS WT tumors (q<0.05). Conclusions: KRAS & NRAS mutations are associated with increased neutrophil abundance, with codon specific differences, while HRAS shows no difference. Overall CD8+ T cells and B cells are less abundant in KRAS & NRAS mutants; substantial variability was seen amongst different protein changes. RAS mutations were more prevalent overall than generally reported, but did not vary by age. These results demonstrate significant differences in the TME of RAS mutant CRC that identify variable susceptibilities to immuno-oncologic agents, and provide further detailed characterization of heterogeneity between RAS variants, at the molecular as well as immunogenic levels.