Abstract 2338: BRAF inhibition is associated with increased clonality of tumor infiltrating lymphocytes.
Authors: Cooper Zachary A. , Frederick Dennie T. , Sullivan Ryan J. , Lawrence Donald P. , Piris Adriano , Flaherty Keith T. , Fisher David A. , Wargo Jennifer A. 
Published: August 2014

Abstract
There have been significant advances in the past few years with regard to BRAF-targeted therapy for metastatic melanoma, however the majority of patients have disease progression within 6 to 7 months. We have previously shown that BRAF inhibition in melanoma in vitro is associated with an increase in melanoma antigens and increased reactivity to antigen specific T cells. More recently, we corroborated these findings in vivo in patients with melanoma undergoing treatment with BRAF inhibitors, demonstrating an increase in melanoma antigens and a significant increase in CD8+ T cells following treatment. However, the nature of this immune response to BRAF inhibition is poorly understood. The goal of the present studies is to better define the T cell infiltrate, so that we can develop a therapeutic strategy to leverage their presence.
To do this, we performed tumor biopsies in patients with metastatic melanoma undergoing treatment with BRAF inhibitors at two time-points: pre-treatment (day 0), and on-treatment (day 10-14). Biopsies were also taken at time of progression when applicable. DNA was isolated and the CDR3 regions of rearranged TCR beta chain genes within the tumor were sequenced and analyzed using the immunoSEQ platform by Adaptive Biotechnologies. Results demonstrated a significant increase in the clonality in the tumor infiltrating lymphocytes in 7 of the 8 patients in on-treatment vs. pre-treatment samples, suggesting that treatment with a BRAF inhibitor may induce an antigen-specific T cell response. We also sought to determine if the increase in T cells associated with BRAF inhibition is related to an infiltration of new T cell clones or a proliferation of existing clones within the tumor. To study this, we analyzed the total T cell clones in these patient samples with regard to their presence or absence in the pre-treatment compared to on-treatment samples. Results demonstrate that over 80% of the individual clones detected after initiation of BRAF-targeted therapy are new clones, suggesting that this therapy is associated with an influx of TIL into the tumors. However, if attention is focused only on the most prevalent clones rather than the total clones, there is a dichotomous relationship between change in clonal populations and magnitude of tumor regression. Namely, those who have persistence of the same dominant clones had a good treatment response, whereas those who had a change in the dominant clonal populations had a poor response. Taken together, this data suggest that though BRAF inhibition in melanoma results in infiltration of new TIL, response to treatment is dependent on a pre-existing population of TIL clones. This data has important clinical implications, and may ultimately help predict who is likely to derive the greatest benefit from BRAF-targeted therapy. A better understanding of this response may also guide us in developing rational combination therapy in the treatment of melanoma.
Citation Format: Zachary A. Cooper, Dennie T. Frederick, Ryan J. Sullivan, Donald P. Lawrence, Adriano Piris, Keith T. Flaherty, David A. Fisher, Jennifer A. Wargo. BRAF inhibition is associated with increased clonality of tumor infiltrating lymphocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2338. doi:10.1158/1538-7445.AM2013-2338