Urine Cell Flow Cytometry Analysis of PD-L1 Expression and DNA Content For Bladder Cancer

Document Type

Conference Proceeding

Publication Date

2-2019

Publication Title

Journal of Clinical Oncology

Abstract

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 to unbiasedly analyze PD-L1 expression and DNA content in cells from urine samples will help us better understand bladder cancer. Methods: To evaluate the PD-L1 expression and DNA content, we developed a 4-color flow assay. Cells in urine samples were pelleted, fixed/permeabilized (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. 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. In addition, a comparison of urine collection fixatives showed that incellMAX-fixed samples had the best single cell recovery and DNA content measurement, as shown by lower cell cycle dye staining variability (lower rCV). Statistically significant differences were found between cancer patients and normal samples. Conclusions: We developed a flow cytometry-based method to investigate PD-L1 and DNA content simultaneously in cells from urine samples. Comparing urine samples from bladder cancer patients and normal yielded statistically significant differences that could provide valuable information for bladder cancer patient management.

Volume

37

Issue

4 Suppl

First Page

466

Comments

American Society of Clinical Oncology (ASCO) Genitourinary Cancers Symposium, February 13-16, 2019, San Francisco, CA  and

American Society of Clinical Oncology and Society for Immunotherapy of Cancer (ASCO-SITC) Clinical Immuno-Oncology Symposium, February 28- March 2, 2019, San Francisco, CA

Last Page

466

DOI

10.1200/JCO.2019.37.7_suppl.466

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