Background Loss of human leukocyte antigen (HLA) class I expression and loss of heterozygosity (LOH) are common events implicated in the primary resistance of non-small cell lung cancer (NSCLC) to immunotherapy. However, there is no data on perioperative chemoimmunotherapy (ChIO) efficacy or response mechanisms in the context of HLA class I defects. Methods Baseline HLA class I tumor status (HLA-deficient (HLA-DEF) or HLA-proficient (HLA-PRO)) was determined by DNA LOH combined with immunohistochemistry for protein levels in tissue of 24 patients with NSCLC treated with perioperative nivolumab plus chemotherapy from NADIM trial (NCT03081689). We integrated HLA tumor status with molecular data (programmed death-ligand 1 (PD-L1), TMB, TCR repertoire, TILs populations, bulk RNA-seq, and spatial transcriptomics (ST)) and clinical outcomes (pathological response and survival data) to study the activity of perioperative ChIO considering HLA class I defects. Results HLA-DEF tumors comprised 41.7% of analyzed tumors and showed a desert-like microenvironment at baseline, with lower PD-L1 levels and reduced immune infiltrate. However, perioperative ChIO induced similar complete pathological response (CPR) rates in both HLA-DEF and PRO tumors (50% and 60% respectively, p=0.670), as well as 3-year survival rates: Progression-free survival (PFS) and overall survival (OS) of 70% (95% CI 32.9% to 89.2%) for HLA-DEF, and PFS 71.4% (95% CI 40.6% to 88.2%) and OS 92.9% (95% CI 59.1% to 99.0%) for HLA-PRO (log-rank PFS p=0.909, OS p=0.137). Proof-of-concept ST analysis of a CPR HLA-DEF tumor after ChIO showed a strong immune response with tertiary lymphoid structures (TLS), CD4+T cells with HLA class II colocalization, and activated CD8+T cells. Conclusions Our findings highlight the activity of perioperative ChIO, and the potential role of TLS and T-cell immune response, in NSCLC HLA-DEF tumors.

Cancer eradication and clinical outcome of immunotherapy depend on tumor cell immunogenicity, including HLA class I (HLA-I) and PD-L1 expression on malignant cells, and on the characteristics of the tumor microenvironment, such as tumor immune infiltration and stromal reaction. Loss of tumor HLA-I is a common mechanism of immune escape from cytotoxic T lymphocytes and is linked to cancer progression and resistance to immunotherapy with the inhibitors of PD-L1/PD-1 signaling. Here we observed that HLA-I loss in bladder tumors is associated with T cell exclusion and tumor encapsulation with stromal elements rich in FAP-positive cells. In addition, PD-L1 upregulation in HLA-I negative tumors demonstrated a correlation with high tumor grade and worse overall- and cancer-specific survival of the patients. These changes define common immuno-morphological signatures compatible with cancer immune escape and acquired resistance to therapeutic interventions across different types of malignancy. They also may contribute to the search of new targets for cancer treatment, such as FAP-expressing cancer-associated fibroblasts, in refractory bladder tumors.

Total or partial loss of HLA class I antigens reduce the recognition of specific tumor peptides by cytotoxic T lymphocytes favoring cancer immune escape during natural tumor evolution. These alterations can be caused by genomic defects, such as loss of heterozygosity at chromosomes 6 and 15 (LOH-6 and LOH-15), where HLA class I genes are located. There is growing evidence indicating that LOH in HLA contributes to the immune selection of HLA loss variants and influences the resistance to immunotherapy. Nevertheless, the incidence and the mechanism of this chromosomal aberration involving HLA genes has not been systematically assessed in different types of tumors and often remains underestimated. Here, we used SNP arrays to investigate the incidence and patterns of LOH-6 and LOH-15 in a number of human cancer cell lines and tissues of different histological types. We observed that LOH in HLA is a common event in cancer samples with a prevalence of a copy neutral type of LOH (CN-LOH) that affects entire chromosome 6 or 15 and involves chromosomal duplications. LOH-6 was observed more often and was associated with homozygous HLA genotype and partial HLA loss of expression. We also discuss the immunologic and clinical implications of LOH in HLA on tumor clonal expansion and association with the cancer recurrence after treatment.

Introduction: The complex interactions between tumors and the immune system are not yet fully elucidated. Expression of different T-cell subsets in peripheral blood may become a marker for non-small cell lung cancer (NSCLC). Aims and objectives: This study was undertaken to analyze T-cell expression in patients with non-small cell lung cancer (NSCLC) before surgery in comparison to age-matched healthy controls. Methods: T-cell expression was analyzed in 16 patients with NSCLC undergoing lung resection in comparison to 28 randomly selected healthy controls in the same age range. Statistical analysis: Statistical analysis was performed with the SPSS program package. Differences in T-cell subsets between groups were assessed using the Mann- Whitney U- Test. Results: We found a higher proportion of T regulatory cells (CD4/CD127low/CD25high) in cases than in controls (30.75 % vs. 17.79 % p> 0.001). The absolute number of lymphocytes was higher in the control group (26.67 vs. 14.13 p>0.002). We also detected a high proportion of fully-differentiated CD8+CD57+ cells in the T CD8 compartment, but there was no significant difference in the proportion of these cells versus the control group (p< 0.65). Results show the heterogeneity of this type of cell. Conclusions : Our data are compatible with an immunosuppressive phenotype status in the peripheral blood lymphocytes of NSCLC patients before surgery. The accumulation of a high proportion of CD8/CD57 senescent cells is probably associated with the age of the patients rather than with the tumour itself.

Total or selective loss of tumor antigen presentation caused by the loss of HLA class I (HLA-I) haplotype due to the loss of heterozygosity at chromosome 6 (LOH-HLA) may result in reduced repertoire of antigenic peptides presented to cytotoxic T-cells and is known to facilitate cancer immune evasion. LOH involving HLA genes has been linked to increased resistance to immunotherapy, cancer recurrence and poor survival after treatment with checkpoint blockade therapy. Previous studies have shown that neoadjuvant chemo-immunotherapy (CIT) is effective in NSCLC tumors with low tumor mutational burden (TMB). However, there is no data on perioperative chemo-immunotherapy efficacy or response mechanisms in the context of HLA-I defects. In the present study, using SNP arrays, we demonstrated that LOH-HLA is a common event in cancer samples with a prevalence of a copy neutral type of LOH (CN-LOH) that affects entire chromosome 6 or 15 and involves chromosomal duplications. LOH-6 and was associated with homozygous HLA genotype and partial loss of HLA-I expression. Moreover, we analyzed the baseline HLA-I expression and LOH-HLA at diagnosis in correlation with the characteristics of the tumor microenvironment (TME) and clinical outcomes in a cohort of 24 patients with NSCLC after perioperative chemo-immunotherapy (nivolumab plus chemotherapy). We integrated HLA-I tumor status with molecular data (PD-L1, TMB, TCR repertoire, TILs populations, bulk RNA-seq, spatial transcriptomics) and clinical outcomes (pathological response and survival data) to study the activity of perioperative CIT considering HLA-I defects. Baseline LOH and/or decreased HLA protein levels occurred in 41.7% of tumors and were categorized as HLA-deficient status. These tumors showed a “desert”-like tumor microenvironment at baseline, with lower PD-L1 levels and immune cell infiltrate. Despite their “cold” microenvironment, perioperative CIT induced similar complete pathological response rates, as well as 3-year progression-free survival (PFS) and overall survival (OS) rates, in both HLA-deficient and HLA-positive tumors. HLA-deficient tumors had B-cell aggregates forming tertiary lymphoid structures (TLS), CD4+T cells in a close location with HLA class II positive cells, and activated CD8+T cells. These data suggest a positive important role of TLS and the coordinated action of helper and cytotoxic T-cells in tumor rejection. All patients with TMB high and HLA-positive tumors were free of disease progression and alive at 3 years. In conclusion, our findings highlight the impact of antigen presentation status on the immune microenvironment of NSCLC, and its potential synergy with TMB as a promising prognostic biomarker for long-term survival following perioperative CIT. It can help to unveil a potential response mechanism to be therapeutically enhanced in non-responsive tumors.