Analysis of genomic and immune microenvironment differences in Chinese populations: Revealing potential mechanisms of poor immunotherapy outcomes in patients with EGFR mutation and ALK fusion non-small cell lung cancer.

8565 Background: Non-small cell lung cancer (NSCLC) patients with EGFR mutations or ALK fusions often exhibit suboptimal responses to immunotherapy, yet the genomic and immunological basis for this remains poorly understood. This study aims to elucidate the genomic and immune microenvironment differences in NSCLC patients harboring EGFR mutations or ALK fusions that may contribute to their distinct immunotherapy responses. Methods: We analyzed tumor specimens from 12,528 NSCLC patients using a comprehensive next-generation sequencing (NGS) panel targeting 733 genes. Of these, 191 patients also underwent multiplex fluorescence immunohistochemistry (mIHC) analysis to assess the immune microenvironment. Subgroups included patients with EGFR mutations, ALK fusions, both EGFR mutations and ALK fusions, and those wild-type for both EGFR and ALK. We compared these groups for DNA damage response (DDR) gene mutation frequencies, tumor mutational burden (TMB), intratumoral heterogeneity (ITH), and immune microenvironment. Results: Among the cohort, 6389 (51%) harbored EGFR mutations and 300 (2.4%) had ALK fusions. Patients with EGFR mutations or ALK fusions exhibited significantly lower DDR mutation frequencies compared to their wild-type counterparts (P<0.001). TMB levels showed a gradient: wild-type > EGFR mutation > EGFR mutation with ALK fusion > ALK fusion (P< 2.2e−16), with wild-type patients having markedly higher TMB than any other group (P< 2.22e−16, P=0.0047, P< 2.22e−16). Conversely, ITH was lowest in wild-type patients and progressively higher with ALK fusions (P=0.017), EGFR mutations (P< 2.22e−16), and combined mutations (P< 2.22e−16). Lower ITH was associated with higher immunogenic neoantigen production, correlating with improved immunotherapy responses. Immunohistochemical analysis revealed that EGFR-mutant tumors had significantly fewer M1 tumor-associated macrophages (CD68+ HLA-DR+) (P<0.001)within the tumor parenchyma and reduced CD4+ T cell (P=0.013) infiltration in the tumor stroma compared to wild-type. Conclusions: Our findings suggest that low TMB, high ITH, and a suppressed immune microenvironment characterize EGFR-mutant and ALK-fusion NSCLC, potentially undermining their immunotherapy efficacy. These genomic and immunological signatures, particularly ITH and immune cell distribution, might be critical factors affecting the immunotherapy responsiveness of these patient subsets, warranting further investigation into tailored therapeutic strategies.