Abstract 4582: Capturing T cell receptor clones from circulating free DNA in cancer patients with solid malignancies

Introduction Understanding the tumor microenvironment is challenging in situations where a tumor biopsy is unavailable. In these situations, liquid biopsies provide a less invasive alternative that can be collected at multiple timepoints throughout treatment. In particular, analyses of circulating free DNA (cfDNA) from plasma samples can distinguish genomic alterations from shed tumor DNA (ctDNA) within the cfDNA population. In this study, we aim to better understand the origin of cfDNA as well as its potential in understanding the intratumoral immune infiltration by developing and validating a strategy for identifying T cell receptor β (TCR β) rearrangements in the cfDNA from patients with solid malignancies. Methods We devised a target enrichment approach for high-throughput DNA sequencing that employs a hybridization capture strategy to bait germline variable (V) and joining (J) gene sequences in the TCR β loci. The target sequences are proximal to rearrangement sites flanking the CDR3, the principal site of contact for recognition specificity with foreign antigens presented by the major histocompatibility complex (MHC). We controlled for amplification bias by tagging DNA fragments with unique molecular indices (UMI). We validated our approach on dilutions of T cell lines in non-T cell lines, compared T cell levels determined from our method to those derived from flow cytometry, and compared data complexity at DNA input levels from 100 ng to 2 ng. Finally, we analyzed 27 pre-treatment plasma samples from cancer patients with solid malignanices, including quantification of total cfDNA concentrations, analyzing somatic genomic alterations using whole exome sequencing (WES), and employing our method for capture of TCR β rearrangements. Results Results indicate we are able to capture T cell rearrangements from cfDNA in cancer patient plasma. T cell rearrangements were identified in more than 25% of samples (7/27). Interestingly, the total cfDNA levels were not correlated to the number of T cell rearrangements detected, but all samples with detectable somatic alterations in the cfDNA (3/27) were also those with T cells identified. These preliminary results suggest a connection between apoptosis of lymphocytes and solid tumors that shed DNA into the bloodstream, and demonstrate potential utility as liquid biopsy for identifying tumor infiltrating lymphocytes. Citation Format: Lara McGrath, Elizabeth Maloney, Daniel Stetson, Tristan J. Lubinski, Rory Kirchner,