Abstract 1301: Inter- and intra-tumoral immune and genomic heterogeneity in patients with metastatic melanoma
Authors: Reuben Alexandre , Cooper Zachary A. , Roh Whijae , Cao Yu , Austin-Breneman Jacob , Jiang Hong , Amaria Rodabe N. , Chen Pei-Ling , Tetzlaff Michael T. , Chin Lynda , Futreal Andrew , Davies Michael A. , Wargo Jennifer A. 
Published: August 2015

Abstract
There have been significant advances in the treatment of metastatic melanoma via the use of targeted and immunotherapy, however a significant proportion of patients still progress on treatment. Intense research efforts to better understand resistance are underway, and multiple molecular resistance mechanisms to targeted therapy have been identified. Appreciation of intratumoral genetic heterogeneity as a contributor to resistance to therapy is growing, although immune heterogeneity in multiple solid tumors within a same patient has been less well characterized. The goal of the present study is to better understand the leukocyte infiltrate in multiple melanoma tumors within the same patient at the time of disease progression, with the potential to identify actionable strategies to overcome resistance to therapy. In this study, we prospectively evaluated 8 tumors from 4 patients on either targeted or immunotherapy. Tissues were evaluated by polychromatic flow cytometry, complemented with immunohistochemical (IHC) analysis of tissue sections. Specifically, we utilized 5 distinct flow cytometry panels comprising 35 antibodies and an 11-marker IHC antibody panel to better understand the intratumoral leukocyte composition as well as presence of immune checkpoint molecules PD-1 and PD-L1. Results demonstrate significant immune heterogeneity between different melanoma tumors within the same patient in the majority of patients studied. Of note, there were significant differences in CD4+, CD8+, and regulatory T cells (p&lt;0.05) with similar activation markers but differences in T cell memory markers. Furthermore, deep TCR sequencing data revealed that T cell populations infiltrating distinct tumors within the same patient presented different specificities, as, in aggregate, ∼92% of T cell clones detected were restricted to one tumor. This inter-tumoral leukocyte and T cell heterogeneity within the same patient may correspond to differential mutations as determined by whole exome sequencing and this analysis is currently underway. Together, these data suggest that there may be significant immune heterogeneity between tumors within a single patient with metastatic melanoma. This has important clinical implications, as a single tumor biopsy sample may not be representative of the immune profile of multiple tumors within the same individual. One could contemplate that this may account for variable responses to therapy, however this is a hypothesis that needs to be tested carefully in a much larger data set. Nonetheless, these findings may have significant implications for the treatment of melanoma and other cancers.
Citation Format: Alexandre Reuben, Zachary A. Cooper, Whijae Roh, Yu Cao, Jacob Austin-Breneman, Hong Jiang, Rodabe N. Amaria, Pei-Ling Chen, Michael T. Tetzlaff, Lynda Chin, Andrew Futreal, Michael A. Davies, Jennifer A. Wargo. Inter- and intra-tumoral immune and genomic heterogeneity in patients with metastatic melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1301. doi:10.1158/1538-7445.AM2015-1301