Analytical Validity of Deep Sequencing to Characterize Tissue-Infiltrating Lymphocytes

• Published in: Blood 2012-11, Vol.120 (21), p.3286-3286
• Main Authors: Johnson, David Scott, Löhr, Andrea, Howell, Walter Mathias, Hsu, Andro, Negrin, Robert S., Meyer, Everett H.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V120.21.3286.3286

Abstract 3286 Lymphocytes infiltrate most human tissues, comprising a critical component of the adaptive immune response. Infiltrating lymphocyte populations have previously been associated with outcomes for a variety of clinical issues, including cancer, alloimmunity, and autoimmunity. Quantitative detection of lymphocyte infiltration has potential as a clinical biomarker of disease activity. Furthermore, locally infiltrating disease-associated clones can be monitored longitudinally in peripheral blood, enabling less invasive follow-up. Deep sequencing of lymphocyte variable regions is an ideal candidate for clinical characterization of tissue-infiltrating lymphocytes. Tissue biopsies produce low genomic DNA yields generally and also contain only a small percentage of cells that have undergone variable region recombination. In this study, we first performed two analytical validity experiments to determine the sensitivity of T cell receptor (TCR) repertoire sequencing, and then made measurements of infiltrating lymphocytes in non-small cell lung carcinomas (NSCLCs), gastrointestinal (GI) biopsies, and cerebrospinal fluid (CSF). The first analytical validity experiment was designed to determine whether we could accurately characterize rare T cells in a background of cells with unrearranged TCRs. We therefore spiked genomic DNA from three unique T cell clones into background unrearranged human genomic DNA derived from immortal lung cancer carcinoma cells. The dilution series was designed such that the high end of the range surveyed the equivalent of 40,000 T cells, and the low end of the range surveyed the equivalent of 1,200 T cells. We then performed commercial TCR repertoire sequencing (GigaMune Rep-Seq™). The second analytical validity experiment was designed to determine reproducibility and accuracy across a dilution series. Here, we spiked two TCR clones into blood genomic DNA from a normal human individual. These DNA mixes were created at clonal cell to normal blood genomic DNA ratios of 1:1000, 1:100, 4:100, and 1:10. Then, we subjected these DNA mixtures to commercial TCR repertoire sequencing in triplicate. Finally, as proof-of-principle, we used deep TCR sequencing to characterize tissue-infiltrating lymphocytes and peripheral blood from patients with NSCLC, multiple sclerosis (MS), and graft-versus-host disease (GVHD). In the two analytical validity experiments, we detected all expected clones, at quantities as low as 50 copies per reaction. In t