Profiling the TCR beta repertoire in liquid biopsies from NSCLC donors

Introduction: During infection and in cancer, the immune system’s response to antigen leads to changes in the T-cell repertoire. T-cell clonal expansions can be measured by sequencing the antigen-specific loci in the T-Cell Receptor Beta gene (TCRβ). In oncology research, TCRβ sequencing is being explored as a predictor for response to immunotherapy as well as IRAE (immune-related adverse events) post-immunotherapy. Recent studies have focused on two metrics, T-cell clonality and TCR convergence, as potential biomarkers. Noninvasive testing for these markers can be achieved using peripheral blood lymphocytes (PBL). In this study, PBL specimens from donors previously diagnosed with NSCLC were evaluated using TCRβ sequencing. Additionally, to model T-cell repertoire changes due to antigen stimulation, primary PBL were challenged in vitro with cytomegalovirus (CMV) antigen. Methods: Peripheral blood mononuclear cells (PBMC) from 4 healthy donors were challenged with a 1-week exposure to whole-cell lysate from CMV-infected cells or CMV pp65495-503 peptide (NLVPMVATV). T-cell repertoire perturbations were assessed using the Oncomine TCR Beta-SR Assay and Ion GeneStudio S5 Sequencer. A pp65 tetramer flow cytometry assay was used as an orthogonal method to assess clonal expansion of a subset of CMV-specific T cells. For evaluation of the assay in PBL from NSCLC donors, five whole-blood specimens were evaluated using the same sequencing workflow. Results: The TCR Beta assay identified 6,683-61,936 unique clones from 1-2 million reads per sample, and an average of 80% of the total reads were usable for TCR profiling. In the NSCLC donors, TCR convergence and clonality values were consistent with published results and ranged from 0.016-0.033 for convergence and 0.09-0.48 for clonality. In the CMV study, TCR sequencing detected the expansion of a common family of clones in 3 samples in response to pp65 peptide stimulation. This expansion corresponded to an increase in pp65 tetramer staining by flow cytometry. Interestingly, this family was not detected in the 4th sample, and an increase in tetramer staining was not observed with pp65 challenge. However, TCRβ clonality increased significantly in the CMV lysate condition for this sample. A single clonotype increased from 6.5% to 76% frequency, and this shift corresponded to increased pp65 tetramer staining by flow cytometry. Baseline TCR convergence scores ranged from 0.009-0.041 and increased 5-fold in one sample as a result of pp65 antigen stimulation. Conclusions: These data demonstrate that the TCRβ assay can detect repertoire features with high resolution using PBMC isolated from liquid biopsies. Profiling of the TCRβ repertoire using the Ion Torrent platform represents a valuable new solution given the technology’s relatively low substitution error rate. Additional studies are being pursued to evaluate the clinical utility of sequencing the immune repertoire in NSCLC patients receiving immunotherapy. Citation Format: Leisa Jackson, Benjamin Tjoa, Hestia Mellert, Gary Pestano. Profiling the TCR beta repertoire in liquid biopsies from NSCLC donors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B54.