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 tumor-infiltrating 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 progression-free 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.

Background: Around 50% of cutaneous melanomas harbor the BRAF^V600E mutation and can be treated with BRAF inhibitors. DNA carrying this mutation can be released into circulation as cell-free BRAF^V600E (cfBRAF^V600E). Droplet digital PCR (ddPCR) is an analytically sensitive technique for quantifying small concentrations of DNA. We studied the plasma concentrations of cfBRAF^V600E by ddPCR in patients with melanoma during therapy with BRAF inhibitors. Methods: Plasma concentrations of cfBRAF^V600E were measured in 8 controls and 20 patients with advanced melanoma having the BRAF^V600E mutation during treatment with BRAF inhibitors at baseline, first month, best response, and progression. Results: The BRAF^V600E mutation was detected by ddPCR even at a fractional abundance of 0.005% in the wild-type gene. Agreement between tumor tissue BRAF^V600E and plasma cfBRAF^V600E was 84.3%. Baseline cfBRAF^V600E correlated with tumor burden (r = 0.742, P < 0.001). cfBRAF^V600E concentrations decreased significantly at the first month of therapy (basal median, 216 copies/mL; Q1–Q3, 27–647 copies/mL; first response median, 0 copies/mL; Q1–Q3, 0–49 copies/mL; P < 0.01) and at the moment of best response (median, 0 copies/mL; Q1–Q3, 0–33 copies/mL; P < 0.01). At progression, there was a significant increase in the concentration of cfBRAF^V600E compared with best response (median, 115 copies/mL; Q1–Q3, 3–707 copies/mL; P = 0.013). Lower concentrations of basal cfBRAF^V600E were significantly associated with longer overall survival and progression-free survival (27.7 months and 9 months, respectively) than higher basal concentrations (8.6 months and 3 months, P < 0.001 and P = 0.024, respectively). Conclusions: cfBRAF^V600E quantification in plasma by ddPCR is useful as a follow-up to treatment response in patients with advanced melanoma.

Anti-programmed death-1 (PD-1) treatment for advanced non-small-cell lung cancer (NSCLC) has improved the survival of patients. However, a substantial percentage of patients do not respond to this treatment. We examined the use of DNA methylation profiles to determine the efficacy of anti-PD-1 treatment in patients recruited with current stage IV NSCLC. In this multicentre study, we recruited adult patients from 15 hospitals in France, Spain, and Italy who had histologically proven stage IV NSCLC and had been exposed to PD-1 blockade during the course of the disease. The study structure comprised a discovery cohort to assess the correlation between epigenetic features and clinical benefit with PD-1 blockade and two validation cohorts to assess the validity of our assumptions. We first established an epigenomic profile based on a microarray DNA methylation signature (EPIMMUNE) in a discovery set of tumour samples from patients treated with nivolumab or pembrolizumab. The EPIMMUNE signature was validated in an independent set of patients. A derived DNA methylation marker was validated by a single-methylation assay in a validation cohort of patients. The main study outcomes were progression-free survival and overall survival. We used the Kaplan-Meier method to estimate progression-free and overall survival, and calculated the differences between the groups with the log-rank test. We constructed a multivariate Cox model to identify the variables independently associated with progression-free and overall survival. Between June 23, 2014, and May 18, 2017, we obtained samples from 142 patients: 34 in the discovery cohort, 47 in the EPIMMUNE validation cohort, and 61 in the derived methylation marker cohort (the T-cell differentiation factor forkhead box P1 [FOXP1]). The EPIMMUNE signature in patients with stage IV NSCLC treated with anti-PD-1 agents was associated with improved progression-free survival (hazard ratio [HR] 0·010, 95% CI 3·29 × 10-4-0·0282; p=0·0067) and overall survival (0·080, 0·017-0·373; p=0·0012). The EPIMMUNE-positive signature was not associated with PD-L1 expression, the presence of CD8+ cells, or mutational load. EPIMMUNE-negative tumours were enriched in tumour-associated macrophages and neutrophils, cancer-associated fibroblasts, and senescent endothelial cells. The EPIMMUNE-positive signature was associated with improved progression-free survival in the EPIMMUNE validation cohort (0·330, 0·149-0·727; p=0·0064). The unmethylated status of FOXP1 was associated with improved progression-free survival (0·415, 0·209-0·802; p=0·0063) and overall survival (0·409, 0·220-0·780; p=0·0094) in the FOXP1 validation cohort. The EPIMMUNE signature and unmethylated FOXP1 were not associated with clinical benefit in lung tumours that did not receive immunotherapy. Our study shows that the epigenetic milieu of NSCLC tumours indicates which patients are most likely to benefit from nivolumab or pembrolizumab treatments. The methylation status of FOXP1 could be associated with validated predictive biomarkers such as PD-L1 staining and mutational load to better select patients who will experience clinical benefit with PD-1 blockade, and its predictive value should be evaluated in prospective studies. "Obra Social" La Caixa, Cellex Foundation, and the Health and Science Departments of the Generalitat de Catalunya.

There is a medical need for diagnostic biomarkers in lung cancer. We evaluated the diagnostic performance of complement activation fragments. We assessed complement activation in four bronchial epithelial and seven lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; bronchoalveolar lavage supernatants from patients with lung cancer (n = 50) and nonmalignant respiratory diseases (n = 22); and plasma samples from advanced (n = 50) and early lung cancer patients (n = 84) subjects with inflammatory lung diseases (n = 133), and asymptomatic individuals enrolled in a lung cancer computed tomography screening program (n = 190). Two-sided P values were calculated by Mann–Whitney U test. Lung cancer cells activated the classical complement pathway mediated by C1q binding that was inhibited by phosphomonoesters. Survival was decreased in patients with high C4d deposition in tumors (hazard ratio [HR] = 3.06; 95% confidence interval [CI] = 1.18 to 7.91). C4d levels were increased in bronchoalveolar lavage fluid from lung cancer patients compared with patients with nonmalignant respiratory diseases (0.61±0.87 vs 0.16±0.11 µg/mL; P < .001). C4d levels in plasma samples from lung cancer patients at both advanced and early stages were also increased compared with control subjects (4.13±2.02 vs 1.86±0.95 µg/mL, P < 0.001; 3.18±3.20 vs 1.13±0.69 µg/mL, P < .001, respectively). C4d plasma levels were associated with shorter survival in patients at advanced (HR = 1.59; 95% CI = 0.97 to 2.60) and early stages (HR = 5.57; 95% CI = 1.60 to 19.39). Plasma C4d levels were reduced after surgical removal of lung tumors (P < .001) and were associated with increased lung cancer risk in asymptomatic individuals with (n = 32) or without lung cancer (n = 158) (odds ratio = 4.38; 95% CI = 1.61 to 11.93). Complement fragment C4d may serve as a biomarker for early diagnosis and prognosis of lung cancer.