Fine needle aspirate flow cytometric phenotyping characterizes immunosuppressive nature of the mesothelioma microenvironment

Abstract

With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials.

Potential Diagnostic and Prognostic Role of Microenvironment in Malignant Pleural Mesothelioma

“…Specific immunophenotypic markers on tissue microarrays or fine-needle aspirate are currently investigated to better characterize the immuneenvironment of MPM. 20,24 The present study for the first time assessed a comprehensive and multiparametric analysis of the immune infiltrate of either pleural fluid or pleural tissue, performed in parallel with the routine diagnostic procedures. Based on flow cytometry assays, our experimental model allows one to recover viable cells, coupling the phenotypic analysis with functional assays of adaptive immunity.…”

“…3,[19][20][21][22][23] Lymphocyte activating 3 (LAG-3) and T cell immunoglobulin and mucin domain 3 (TIM-3) have been detected on CD4 þ and CD8 þ TILs or in formalinfixed paraffin embedded samples. 24,25 Despite the high interpatient and intrasample variability, both LAG-3 and TIM-3 contribute to the functional exhaustion of TILs. 26 Furthermore, cytotoxic T-lymphocyte associated protein 4 (CTLA-4) has been investigated as a potential therapeutic target in MPM, but the CTLA-4 inhibitor Tremelimumab did not show higher efficacy than placebo in a double blind phase II trial, raising concerns about the role of this immune checkpoint in MPM-induced immunosuppression.…”

“…28 Some studies reported the same immunosignature in the MPM pleural effusion and in the peripheral blood, whereas others did not, raising concerns about peripheral blood as a biologic surrogate that reliably reproduces the MPM immunoenvironment. 4,24 Immune cells can be continuously exchanged between pleural cavity and tumor tissue, and/or between peripheral blood and pleural environment. This dynamic immunoenvironment leads to an inhomogeneous distribution of immune cells within MPM tissue.…”

Novel immunotherapy clinical trials in malignant pleural mesothelioma

“…Multiple studies have demonstrated the prognostic role of T and B lymphocytes and macrophages (12)(13)(14)(15). Other investigators have published data showing the presence of immunosuppression in MPM through analysis of T-cell inhibitory receptors (16) and chemokines, such as C-C motif chemokine ligand 2 (CCL2), which is a factor in the protumor M2 macrophage recruitment (17). Recently available multi-color immunofluorescence techniques have allowed investigators to study the distribution and co-localization of immunostimulatory, as well as immunosuppressive cells within the tumor microenvironment on a single slide.…”

“…Checkpoint interactions lead to immune tolerance of tumors and several checkpoint inhibitors that modulate this function are currently being studied (50). High levels of PD-1 on CD4+ and CD8+ T cells have been shown in MPM (16). Also, PD-L1+ MPM tumors were found to have increased expression of PD-1 on their associated CD4+ and CD8+ T cells in comparison with PD-L1-tumors (51).…”