Immune microenvironmental heterogeneity according to tumor DNA methylation phenotypes in microsatellite instability-high colorectal cancers

Abstract

The detailed association between tumor DNA methylation, including CpG island methylation, and tumor immunity is poorly understood. CpG island methylator phenotype (CIMP) is observed typically in sporadic colorectal cancers (CRCs) with microsatellite instability-high (MSI-H). Here, we investigated the differential features of the tumor immune microenvironment according to CIMP status in MSI-H CRCs. CIMP-high (CIMP-H) or CIMP-low/negative (CIMP-L/0) status was determined using MethyLight assay in 133 MSI-H CRCs. All MSI-H CRCs were subjected to digital pathology-based quantification of CD3 + /CD8 + /CD4 + /FoxP3 + /CD68 + /CD204 + /CD177 + tumor-infiltrating immune cells using whole-slide immunohistochemistry. Programmed death-ligand 1 (PD-L1) immunohistochemistry was evaluated using the tumor proportion score (TPS) and combined positive score (CPS). Representative cases were analyzed using whole-exome and RNA-sequencing. In 133 MSI-H CRCs, significantly higher densities of CD8 + tumor-infiltrating lymphocytes (TILs) were observed in CIMP-H tumors compared with CIMP-L/0 tumors. PD-L1 TPS and CPS in CIMP-H tumors were higher than in CIMP-L/0 tumors. Next-generation sequencing revealed that, compared with CIMP-L/0 tumors, CIMP-H tumors had higher fractions of CD8 + T cells/cytotoxic lymphocytes, higher cytolytic activity scores, and activated immune-mediated cell killing pathways. In contrast to CIMP-L/0 tumors, most CIMP-H tumors were identified as consensus molecular subtype 1, an immunogenic transcriptomic subtype of CRC. However, there were no differences in tumor mutational burden (TMB) between CIMP-H and CIMP-L/0 tumors in MSI-H CRCs. In conclusion, CIMP-H is associated with abundant cytotoxic CD8 + TILs and PD-L1 overexpression independent of TMB in MSI-H CRCs, suggesting that CIMP-H tumors represent a typical immune-hot subtype and are optimal candidates for immunotherapy in MSI-H tumors.

Keywords: Colorectal carcinoma; CpG Island methylation; DNA methylation; Microsatellite instability; Mismatch repair; Tumor immunology.

Fig. 1

Comparison of pathology-based TIME features between CIMP subgroups of MSI-H CRCs. a Comparison of CD8⁺ TIL densities between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs at IM (left) and CT (right) areas. b Representative photomicrographs of CD8 IHC in CIMP-H and CIMP-L/0 MSI-H CRCs. Note the higher density of CD8⁺ TILs in the CIMP-H tumor, compared with the CIMP-L/0 tumor (scale bar, 200 μm). c Comparison of PD-L1 IHC scores, including TPS (left) and CPS (right), between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs. d Representative photomicrographs of PD-L1 IHC in CIMP-H and CIMP-L/0 MSI-H CRCs. Note the diffuse strong expression pattern of PD-L1 in the CIMP-H tumor, compared with the CIMP-L/0 tumor (scale bar, 100 μm). Abbreviations: TIME, tumor immune microenvironment; CIMP, CpG island methylator phenotype; CIMP-H, CIMP-high; CIMP-L/0, CIMP-low/negative; MSI-H, microsatellite instability-high; CRCs, colorectal cancers; TIL, tumor-infiltrating lymphocyte; IM, invasive margin; CT, center of tumor

Fig. 2

Comparison of transcriptome-based immuno-molecular features between CIMP subgroups of MSI-H CRCs. a Comparison of immune cell types and immune-related scores between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs. The combined p-value is computed using the Fisher method. Colors indicate significance and enrichment: Red signifies significantly enriched cell types and scores in CIMP-H tumors, while blue signifies significantly enriched cell types and scores in CIMP-L/0 tumors. Gray indicates insignificant cell types and scores. b Comparison of cytolytic activity scores between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs. c Comparison of frequencies of CMS1 between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs. d GSEA-based comparison of 50 cancer hallmarks between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs. The dot size corresponds to the gene counts associated with gene ontology terms, while the color gradient from purple to red indicates the adjusted p-value. e GSEA-based comparison of biological process terms between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs. Abbreviations: CIMP, CpG island methylator phenotype; CIMP-H, CIMP-high; CIMP-L/0, CIMP-low/negative; MSI-H, microsatellite instability-high; CRCs, colorectal cancers; CMS1, consensus molecular subtype 1; GSEA, gene set enrichment analysis; DEG, differentially expressed gene; GO, gene ontology

Fig. 3

Comparison of genomic features between CIMP subgroups of MSI-H CRCs. a Comparison of TMB between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs. b The mutational landscape comparison between CIMP-H and CIMP-L/0 subgroups of MSI-H CRCs. Genes include known major oncogenes or tumor suppressor genes in CRC. Alteration types were denoted by color and shape. Abbreviations: CIMP, CpG island methylator phenotype; CIMP-H, CIMP-high; CIMP-L/0, CIMP-low/negative; MSI-H, microsatellite instability-high; CRCs, colorectal cancers; TMB, tumor mutational burden; ns, not significant

Fig. 4

Graphical summary of this study