No abstract

Background Although EGFR mutant tumors exhibit low response rates to immune checkpoint blockade overall, some EGFR mutant tumors do respond to these therapies; however, there is a lack of understanding of the characteristics of EGFR mutant lung tumors responsive to immune checkpoint blockade. Patients and methods We retrospectively analyzed de-identified clinical and molecular data on 171 cases of EGFR mutant lung tumors treated with immune checkpoint inhibitors from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, University of California Los Angeles, and Dana Farber Cancer Institute. A separate cohort of 383 EGFR mutant lung cancer cases with sequencing data available from the Yale Cancer Center, Memorial Sloan Kettering Cancer Center, and The Cancer Genome Atlas was compiled to assess the relationship between tumor mutation burden and specific EGFR alterations. Results Compared with 212 EGFR wild-type lung cancers, outcomes with programmed cell death 1 or programmed death-ligand 1 (PD-(L)1) blockade were worse in patients with lung tumors harboring alterations in exon 19 of EGFR ( EGFR Δ19 ) but similar for EGFR L858R lung tumors. EGFR T790M status and PD-L1 expression did not impact response or survival outcomes to immune checkpoint blockade. PD-L1 expression was similar across EGFR alleles. Lung tumors with EGFR Δ19 alterations harbored a lower tumor mutation burden compared with EGFR L858R lung tumors despite similar smoking history. Conclusions EGFR mutant tumors have generally low response to immune checkpoint inhibitors, but outcomes vary by allele. Understanding the heterogeneity of EGFR mutant tumors may be informative for establishing the benefits and uses of PD-(L)1 therapies for patients with this disease.

Purpose: To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non-small cell lung cancer (NSCLC). Experimental Design: Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment. Results: PD-1, LAG-3, and TIM-3 were detected in tumorinfiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progressionfree survival. Conclusions: PD-1, LAG-3, and TIM-3 have distinct tissue/ cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.

Programmed death ligand 1 (PD-L1) is expressed in tumor cells and immune cells, and both have been associated with response to anti-PD-1 axis immunotherapy. Here, we examine the expression of PD-L1 to determine which cell type carries the predictive value of the test.Experimental Design: We measured the expression of PD-L1 in multiple immune cells with two platforms and confocal microscopy on three retrospective Yale NSCLC cohorts (425 nonimmunotherapy-treated cases and 62 pembrolizumab/nivolumab/atezolizumab-treated cases). The PD-L1 level was selectively measured in different immune cell subsets using two multiplexed quantitative immunofluorescence panels, including CD56 for natural killer cells, CD68 for macrophages, and CD8 for cytotoxic T cells.Results: PD-L1 was significantly higher in macrophages in both tumor and stromal compartment compared with other immune cells. Elevated PD-L1 in macrophages was correlated with high PD-L1 level in tumor as well as CD8 and CD68 level (P < 0.0001). High PD-L1 expression in macrophages was correlated with better overall survival (OS; P ¼ 0.036 by cell count/P ¼ 0.019 by molecular colocalization), while high PD-L1 expression in tumor cells was not.Conclusions: In nearly 500 non-small cell lung cancer (NSCLC) cases, the predominant immune cell type that expresses PD-L1 is CD68 þ macrophages. The level of PD-L1 in macrophages is significantly associated with the level of PD-L1 in tumor cells and infiltration by CD8 þ T cells, suggesting a connection between high PD-L1 and "hot" tumors. In anti-PD-1 axis therapytreated patients, high levels of PD-L1 expression in macrophages are associated with longer OS and may be responsible for the predictive effect of the marker.