Developing a new urine cell flow cytometry analysis of PD-L1 expression and DNA content for diagnosis of bladder cancer.

60 Background: Bladder cancer is the fifth most common cancer in the United States. PD-1/PD-L1, a pathway used by cancer cells to evade immune response, correlates with bladder cancer severity and has emerged as a target in bladder cancer treatment. Chromosomal instability is also a prominent feature associated with the development of bladder cancer. A method for unbiased analysis of PD-L1 expression and DNA content in cells from urine samples promises to be a new test for diagnosis of bladder cancer. Methods: To evaluate the PD-L1 expression and DNA content, we developed a 4-color flow assay. Cells in voided urine samples were pelleted, fixed in incellMAX (IncellDx Inc.) and stained with antibodies against pan-cytokeratin (CK), CD45, PD-L1 and a cell cycle dye. The stained samples were analyzed by a flow cytometer alongside stained control cells. Results: Fifty bladder cancer patient and 15 normal donor urine samples were collected and tested with this assay. We could distinguish epithelial cells (pan-CK+) and white blood cells (WBCs, CD45+) in urine samples and obtain PD-L1 expression and DNA content information simultaneously from these cell populations. The patient samples showed a significantly higher percentage of WBCs with substantial PD-L1 expression (P < 0.001). The percentage of PD-L1 positive epithelial cells was not distinguishable between normal donor and patient samples. However increased post G1 epithelial cells ( > 5%) were observed in a majority of bladder cancer patients, with around 25% of samples showing a DNA index above 1.05. Conclusions: We developed a flow cytometry-based method to investigate PD-L1 and DNA content simultaneously in cells from urine samples that could provide us with a new method to accurately identify bladder cancer patients through urine testing.