HLA class-I antigen presentation machinery (APM) alterations mediate immune evasion in lung cancer brain metastases.

e14014 Background: Although ~30% of patients with lung cancer brain metastasis (LBM) achieve intracranial response with immune checkpoint inhibitors (ICI), most have primary resistance, and those who initially respond develop rapid disease progression. The biological factors contributing to this unfavorable clinical scenario are poorly understood and could include distinct immunologic properties of tumors colonizing the brain. Using annotated tumor/patient cohorts, spatially resolved protein analysis, and in vitro models, we investigated the role of HLA class-I APM defects and interferon (IFN)-γ pathway in LBM. Methods: Using multiplex quantitative immunofluorescence, we measured the levels and spatial distribution of HLA class-I APM markers (β2M, tapasin, calreticulin, ERp57, TAP1, and TAP2), IFN-γ signaling (pSTAT1 and IRF-1), and tumor-infiltrating lymphocytes (TILs) (CD8, CD4, and FOXP3) in 56 LBM, 41 primary lung tumors (PLT), and 10 lung-extracranial metastases (ECM) represented in tissue microarrays. A subset of paired LBM/PLT samples from the same patients were also included (n=8). The spatial analysis of the markers was conducted using fluorescence co-localization and single-cell segmentation strategies. Associations between the markers, clinicopathological characteristics, and survival were studied. Protein expression of β2M and IFN-γ-sensitive markers (total STAT1, pSTAT1, IRF1, and PD-L1) before and after treatment with human recombinant IFN-γ was assessed using Western blot in cultured primary lung H2030 adenocarcinoma cells and their in vivo selected brain metastatic counterpart H2030-BrM3. Results: LBM showed significantly lower β2M, TAP2, and tapasin protein levels than PLT. However, LBM also showed higher expression of pSTAT1 and comparable levels of IRF-1 relative to PLT. Similar results were seen in paired LBM/PLT samples, but not in ECM samples. Tumors with low β2M showed reduced CD8+, CD4+, and FOXP3+ TILs. In LBM no significant association between β2M and IFN-γ pathway markers or major clinicopathologic and molecular patient/tumor features were found. LBM samples from patients progressing after radiotherapy and/or chemotherapy showed significantly lower β2M levels. Reduced β2M expression was associated with shorter overall survival in PLT but not in LBM. The brain metastatic H2030-BrM3 cell line showed lower β2M expression both at baseline and after IFN-γ treatment compared with their parental H2030 counterpart. Conclusions: HLA class-I APM downregulation is a prominent feature of LBM, is independent of local adaptive/inducible IFN-γ stimulation, and is associated with unfavorable clinical features. These findings support that HLA class-I APM deficiency plays a major role in immune evasion and immunotherapy resistance in patients with LBM.