Degrees of H2AX phosphorylation correlate with unique features of the intratumoral immune microenvironment in colorectal carcinomas

Background: The phosphorylated form of the histone H2AX (γH2AX), a sensor of DNA double-strand breaks (DSB), can serve as a biomarker of DNA damage and therapy response. This study aimed to evaluate the association between γH2AX expression and pathological, molecular, and immune features in colorectal cancer (CRC). Patients and methods: Levels of γH2AX were assessed by immunohistochemistry in a cohort of 198 CRCs, alongside immune-related markers (CD3 and CD8). A sub-cohort of 65 CRCs (26 γH2AX- and 39 γH2AX+) underwent RNA extraction and gene expression profiling using the IO360 Nanostring Panel to infer immune cell composition. Overall survival data were analyzed for exploratory correlations. Results: γH2AX+ CRCs (155/198, 78%) were significantly associated with higher stage and tumor grade (P < .01). A lower γH2AX prevalence was found in MMR-deficient tumors (64%) compared to MMR-proficient cases (81%, P = .05). γH2AX+ tumors showed increased CD3+ cell infiltration in the overall population (P = .038) and in MMR-proficient CRCs (P = .028). Gene expression analysis revealed higher T-cell counts (P < .01) and reduced B-cell abundance (P < .01) in γH2AX+ CRCs. Unsupervised clustering identified 3 immune subgroups with differential γH2AX accumulation. Cluster #3, enriched in γH2AX+ tumors, displayed increased CD8+ T-cells and conferred the best survival outcome. Conclusion: Elevated γH2AX expression correlates with MMR proficiency, aggressive histopathologic features, and a distinctive immune-active microenvironment in CRC. These findings may support γH2AX as a marker of immune-modulated CRC subgroups with potential prognostic and therapeutic relevance.