Immune marker profiling and PD-L1, PD-L2 expression mechanisms across non-small cell lung cancer mutations.

Abstract

9076 Background: PD-1/PD-L1 axis inhibitors have been proven effective, especially in patients expressing Programmed Death Ligand 1 (PD-L1). Their clinical efficacy in patients with epidermal growth factor receptor (EGFR) activating mutations is still unclear, while KRAS mutations seem to be associated with high response rates. In this study we investigated the expression of PD-L1, PD-L2 and Tumor Infiltrating Lymphocyte (TIL) status as a function of mutation status in Non-Small Cell Lung Cancer (NSCLC). Methods: We used the AQUA method of quantitative fluorescence (QIF) to compare PD-L1 and PD-L2 expression and to characterize TILs populations and their activation status in over 150 NSCLC patient tumors with known mutation status. EGFR activation was assessed in situ using the proximity ligation assay (PLA) for EGFR and GRB2 and T cell activation was assessed using a novel multiplexed QIF assay including CD3, Granzyme B and Ki67. Results: PD-L1 tumor and stroma expression was significantly lower in EGFR mutant compared to KRAS mutant (p = 0.009) and EGFR/KRAS Wild Type (p < 0.0001) tumors, while they had a higher frequency of PD-L2 expression. Conversely, KRAS mutants had significantly lower PD-L2 tumor and stroma expression but they were also more inflamed with higher CD4+, CD8+ and CD20+ TILs. Subgroup analysis of patients by their TILs activation status revealed that EGFR mutants had a very high frequency of inactive TILs even though lymphocytes were present in the tumor microenvironment. In contrast, in KRAS mutants, when TILs were present, they were almost always active. Finally, we find that PLA-defined activated EGFR correlated with increased PD-L1 expression in EGFR mutants but not in EGFR WT, while TIL activation was associated with higher PD-L1 in EGFR/KRAS WT. Conclusions: Our findings are consistent with the unique biology of EGFR mutant tumors. The high frequency of inactive TILs could explain the low immune therapy response rates in this patient group. Similarly, in this group, the reason PD-L1 expression fails to predict response may be due to expression as a result of constitutive oncogenic signaling rather than immune signaling, which would be associated with high PD-L1 levels and TILs activation.

Clinical and Molecular Characteristics Associated With Survival Among Patients Treated With Checkpoint Inhibitors for Advanced Non–Small Cell Lung Carcinoma

“…Other recent insights that may help to further elucidate the mechanisms of resistance include the finding that EGFR mutant tumors are associated with a high frequency of inactive tumor-infiltrating lymphocytes even though lymphocytes are present in the tumor microenvironment. 19 The finding that high CD73 expression on NSCLC and other tumors is associated with low PD-L1 expression and low densities of CD8 + tumor-infiltrating lymphocytes 20 may also provide an explanation given that EGFR activation is thought to induce CD73 expression. One hypothesis raised is that in the EGFR mutant tumors with overexpression of CD73, which is also associated with reduced expression of interferon gamma messenger RNA signature, 21 CD73 results in immunosuppression via decreased T-cell activation and effector function and hence reduced benefit from checkpoint inhibitor therapies.…”

“…Unlike EGFR mutant tumors, tumor-infiltrating lymphocytes are frequently present in the microenvironment of KRAS mutant tumors and are almost always active. 19 Mutations in STK11 or LKB1 and TP53 tumor suppressor genes commonly co-occur in KRAS mutant NSCLC. 22 Loss of TP53 function is associated with an increase in expression of PD-L1 23 and an increase in mutation burden.…”

Correlation between PD-L1 expression and clinicopathological characteristics of non-small cell lung cancer: A real-world study of a large Chinese cohort

“…Approximately 80% of lymphocytes are T cells expressing various activation antigens. Most prior studies evaluating TILs in lung cancer applied IHC (CD4, CD8, and CD3) to differentiate IC subsets and to assess their density, distribution and localization (21,30). Here, we evaluated stromal TILs localized only in the invasive margin by commonly used H&E staining methods.…”

Efficacy of nivolumab in pre-treated non-small-cell lung cancer patients harbouring KRAS mutations

“…Preclinical and clinical evidences suggested that KRAS -positive NSCLC seems to gain more benefit from immunotherapy. First of all, KRAS -mutant tumours are characterised by the presence of CD8 + lymphocyte infiltrates in the tumour microenvironment (TME), 20 while a significant association between KRAS mutations and PD-L1 expression has been observed in lung adenocarcinoma. 21,22 Coelho et al 23 have recently demonstrated that oncogenic RAS signalling upregulated tumour PD-L1 expression stabilising the PD-L1 transcript in KRAS -mutant adenocarcinoma, thus providing an additional mechanism whereby KRAS -positive tumours respond to PD-1/PD-L1 inhibitors.…”