Urine Derived Lymphocytes Demonstrate Marked Differences in the Expression of Immune Checkpoint Molecules PD1 and NKG2A between non-muscle Invasive and Muscle Invasive Bladder Cancer.
John R. Heard, MS, Andrew Charap, BS, John Sfakianos, MD.
Icahn School of Medicine at Mount Sinai, New York, NY.
Intro/Objective: Lymphocytes in the urine of patients with muscle invasive bladder cancer (MIBC) have been found to be representative of those in the tumor microenvironment. Given this relationship, we hypothesized that analysis of immune checkpoint molecules on these cells may reveal differences between the microenvironment of non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). The checkpoint molecule PD1 is the target of numerous monoclonal antibody treatments for MIBC and its role in suppression of the immune response to cancer has been well studied. The novel checkpoint molecule NKG2A is an inhibitory receptor found on NK and T cells and NKG2A blocking monoclonal antibodies are currently in clinical trials for other cancer types. We sought to better understand the immune modulatory landscape of NMIBC and MIBC by studying urine derived lymphocyte expression of PD1 and NKG2A. Methods: In this study, urine was obtained from patients diagnosed with bladder cancer prior to receiving treatment. Patients were sorted into disease groups based on the pathology results from staging cystoscopy (NMIBC n = 4, MIBC n = 6). Urine was collected via void at office visit or catheterization prior to cystoscopy or cystectomy. On the same day as collection, urine derived cells were isolated by centrifugation, treated with ACK lysis buffer for removal of erythrocytes, stained, and analyzed via flow cytometry. Statistical analysis between groups was calculated using unpaired 2-tailed student's t-test. Results: There were significant decreases in the ratio of both NK cells and T cells to total urine cells for the MIBC group (p<0.05, p<0.05). The percentage of CD4+ and CD8+ T cells expressing PD1 was significantly higher in the MIBC group (p<0.05, p<0.005). The NMIBC group was found to have an increased percentage of CD8+ T cells expressing NKG2A (p<0.05). Analysis of lymphocytes expressing both NKG2A and PD1 found a significant increase in this phenotype among CD4+ and CD8+ T cells in the MIBC group compared to NMIBC (p<0.05, p<0.01). Conclusion: We conclude that it is feasible to detect differences in the urine lymphocyte composition and immune checkpoint profile between NMIBC and MIBC. Our analysis revealed increased expression of inhibitory marker PD1 on T cells from the urine of MIBC patients, suggesting the tumor microenvironment in MIBC is more inhibitory and contains more dysfunctional T cells then NMIBC. Conversely, the inhibitory receptor NKG2A, whose role in tumor immunology is still under investigation, was expressed by fewer CD8+ T cells in MIBC compared to NMIBC. However, CD4+ and CD8+ T cells expressing both PD1 and NKG2A were present in a higher proportion for the MIBC group. These differences may reflect disparities in the mechanism of immune modulation by the tumor microenvironment between these two diseases.
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