Abstract A62: Immune and T cell receptor heterogeneity in distinct tumors within individual melanoma patients
Authors: Reuben Alexandre , Amaria Rodabe N. , Haymaker Cara , Forget Marie Andree , Bernatchez Chantale , Chen Pei-ling , Tetzlaff Michael , Davies Michael A. , Dwyer Karen C. , Wargo Jennifer A. , Cooper Zachary A. 
Published: October 2015

Abstract
There have been significant advances in the treatment of melanoma in the past several years through the use of molecularly targeted therapy and immune checkpoint blockade, however the majority of patients still progress on therapy. There is a critical need to understand the differential responses to these types of therapy. Significant genomic heterogeneity has been described in multiple lesions within the same patient, which may contribute to the development of resistance to therapy. However, immune heterogeneity within multiple tumors from the same patient has not been well characterized. The goal of the present study is to better understand heterogeneity in the immune infiltrate from multiple melanoma tumor sites in the same patient in the setting of treatment, with the underlying hypothesis that differences in immune infiltrate may account for differential responses to therapy. In these studies, we prospectively evaluated 8 tumors from 4 patients during the course of therapy on targeted therapy or immunotherapy. Tissues were evaluated by polychromatic flow cytometry in combination with immunohistochemical analysis of tissue sections. We utilized a 35-marker flow cytometry panel (with 5 leukocyte subsets) to perform a deep analysis of the leukocyte composition with a focus on 1) T cells/regulatory T cells, 2) natural killer/T/B cells, 3) tumor associated macrophages and fibroblasts, 4) dendritic cells, and 5) polymorphonuclear leukocytes. In addition, we performed sequencing of the CDR3 region of the T cell receptor (TCR) β gene to gain insight into differences in T cell clonality between distinct tumors within the same patient. Results demonstrate significant inter-tumoral immune heterogeneity in the majority of patients studied (3 of 4). There were significant differences in CD4+, CD8+, and regulatory T cells (p&lt;0.05) between distinct tumors within the same patient. In addition, CDR3 sequencing of the TCR β gene demonstrated significant differences in T cell repetoire and differences in dominant clones in all patients studied. Together, these data suggest that there is significant intra-patient immune heterogeneity between metastatic melanoma tumors from different sites on targeted therapy or immunotherapy. This may have important clinical implications, as a single tumor biopsy sample may not be representative of the immune profile of multiple tumors within the same individual. It is provocative to contemplate that this could account for variable responses to therapy, although this hypothesis requires further testing in a much larger data set. Nonetheless, these findings have potential significant implications for the treatment of melanoma and other cancers.
Citation Format: Alexandre Reuben, Rodabe N. Amaria, Cara Haymaker, Marie Andree Forget, Chantale Bernatchez, Pei-ling Chen, Michael Tetzlaff, Michael A. Davies, Karen C. Dwyer, Jennifer A. Wargo, Zachary A. Cooper. Immune and T cell receptor heterogeneity in distinct tumors within individual melanoma patients. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A62.