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A6157 - A Pilot Study of Endobronchial Ultrasound Guided Transbronchial Needle Aspiration for Immunophenotyping the Lung Cancer Tumor Microenvironment
Author Block: E. Moon, S. O'Brien, J. Thompson, S. Kim, K. Ma, D. DiBardino, A. Lanfranco, A. Vachani, A. Haas, S. Albelda; Pulmonary, Allergy, and Critical Care, Hospital of the University of Pennsylvania, Philadelphia, PA, United States.
Rationale: Lung cancer still remains the leading cause of cancer deaths worldwide. Immunotherapy has significantly impacted lung cancer treatment. Checkpoint blockade of immunosuppressive surface receptors expressed on tumor infiltrating lymphocytes (TILs) (e.g. PD1) and adoptive transfer of engineered T cells (ACT) are two promising immunotherapies. Although they have demonstrated remarkable results in melanoma and leukemia, respectively, they face significant hurdles in lung cancer that prevent universal success. Traditionally, peripheral blood or surgically resected lung cancer specimens have been utilized to conduct immunophenotyping studies to understand these hurdles. More efficient/accurate and minimally invasive methods are needed to sample actual tumor in advanced stage lung cancer patients. We demonstrate that endobronchial ultrasound transbronchial needle aspiration (EBUS TBNA) is such a method, and provides tissue with better viability than resected tumors subjected to more traditional digestion methods. Methods: Surgically resected lung cancer tumors were aspirated using a 22G EBUS needle on the back table of the operating room after portions for clinical care were taken. Samples were collected in conical tubes containing PBS, on ice. The remaining tumor was mechanically disaggregated and digested using a published protocol (Quatromoni et al., 2015). The EBUS aspirate and tumor digest were both subjected to flow cytometry using the following staining panel: live/dead viability dye, CD3, CD8, CD4 ,CD45RO, CD62L, PD1. Samples were also cultured on plates coated with 0.5ug/ml of anti-CD3 with brefeldin/monensin overnight, then subjected to intracellular staining for IFNg/IL2/TNFa. Results: Samples were tested for: 1) viability of CD3+ populations, 2) frequency of CD8 vs. CD4, 3) differentiation subsets (CD45RO/CD62L), 4) intracellular cytokine staining. Among live/CD3+ cells, CD4/CD8 frequencies were similar, differentiation subset frequencies were similar. However, EBUS aspirates had about two-fold greater TIL viability than the tumor digests. Additionally, tumor digest TILs had significantly lower intracellular cytokine staining than EBUS TILs. Time from tissue acquisition to analysis was significantly shorter for EBUS vs. tumor digest samples. Conclusions: As the field of immunotherapy for lung cancer continues to expand, safe/efficient methods to accurately immunophenotype the TME are crucial. EBUS TBNA sampling may be such a method. Traditionally, resected tumors have been processed using manual disaggregation and enzymatic digestion which can be harsh on TIL populations. In this study we demonstrated that EBUS TBNA TILs are more viable and have greater intracellular cytokine staining in response to anti-CD3 restimulation overnight, while frequencies of CD4/CD8 and differentiation subsets are similar.