Comprehensive T cell immunophenotyping and next generation sequencing from HPV-positive and -negative head and neck squamous cell carcinomas

Abstract

The success of PD-1 inhibition in achieving a clinical response in a subset of HNSCC patients emphasizes the need to better understand the immunobiology of head and neck squamous cell carcinomas (HNSCC). Immunophenotyping was performed on 30 patients with HNSCC (16 HPV-positive, 14 HPV-negative) from matched tissue from the primary tumor site, locally metastatic cervical lymph nodes (LN), uninvolved local cervical LN and peripheral blood. CD4⁺ and CD8⁺ T cell lymphocytes obtained from tissue were analyzed for expression levels of inhibitory receptors PD-1, TIM-3 and CTLA-4. Next generation sequencing (NGS) of the T cell receptor (TCR) β chain was performed on patients (n= 9) for receptor repertoire diversity and clonality analysis. HPV-negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8⁺ T cells expressing CTLA-4⁺ in tumor tissue (p= 0.0013) and peripheral blood (p= 0.0344) compared to HPV-positive patients, as well as higher expression levels of TIM-3⁺PD-1⁺ CD8⁺ T cells (p= 0.0072) compared to controls. For all patients, PD-1⁺ expression on CD8⁺ T cells—particularly in HPV-negative HNSCC cases—strongly correlated (r= 0.63, p= 0.013) to tumor size at the primary site. Top CD8⁺ TCR clones from tumor tissue significantly overlapped with circulating peripheral blood TCR clones (r= 0.946) and HPV-positive patients had frequently expanded TCR clones that were more hydrophobic—and potentially more immunogenic—then those from HPV-negative patients. Collectively, our findings demonstrate for the first time that high-stage HPV-negative HNSCC patients with primary tumors at different sites in the head and neck have elevated peripheral CTLA-4⁺CD8⁺ T cell levels, that tumor-familiar CD8⁺ T cells are detectable in peripheral blood from HNSCC patients and that the TCR from HPV-positive HNSCC patients are potentially recognizing distinctly antigenic cognate antigens. However, our findings are preliminary and need to be further confirmed in a larger patient cohort and for determination of how these factors affect patient response to immunotherapy.

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