Immunotherapies inhibiting the Programmed Death-1 PD-1 axis can result in dramatic responses and durable benefit in patients with non-small cell lung cancer NSCLC. However, the overall response rate is only 20-30 and there is no clearly-defined biomarker that predicts which patients are most likely to benefit. Moreover, patients with NSCLC and brain metastases represent a population for which there are limited treatment options, and these patients are typically excluded from immunotherapy clinical trials or require local therapy prior to study enrollment. Therefore we are conducting a trial of the PD-1 inhibitor pembrolizumab MK-3475 in patients with NSCLC and untreated brain metastases. The objective of this proposal is to study the immunophenotypic characteristics of primary lung tumors, brain metastases and extra-cerebral metastases with the goal of determining the variability across sites, and to study tumor- and blood-based biomarkers to establish predictors of immunotherapy benefit. We hypothesize that identifying biomarkers predictive of benefit to immunotherapy in patients with NSCLC and brain metastases will result in improved patient outcomes. We have made progress towards these goals in several areas over the last year. We have optimized the assays that will be used to study the immunophenotypic characteristics of the paired tumor samples. This is a necessary step prior to moving forward with the goals for year two of this grant. Additionally, we have compiled the cohort of paired tumor samples and have begun reviewing them to determine which samples have sufficient tumor to include in the study. We have accrued patients with NSCLC and untreated brain metastases to the clinical trial with pembrolizumab and have obtained both blood and tumor tissue samples from these patients. As this is a career development grant, the PI has had the opportunity to learn the laboratory skills necessary to complete this project.