Abstract 2219: Evaluation of PD-L1 expression on circulating tumor cells (CTCs) in patients with advanced urothelial carcinoma of the bladder
Authors: Bergmann Sonja , Coym Anja , Amsberg Gunhild von , Janning Melanie , Soave Armin , Rink Michael , Pantel Klaus , Riethdorf Sabine 
Published: August 2019

Abstract
Introduction: Immune checkpoint therapies with PD-1/PD-L1 inhibitors show durable responses in a subset of patients with metastatic urothelial carcinoma of the bladder (UCB). However, PD-L1 expression in tumor biopsies does not correlate with response to PD-1/PD-L1 inhibitors in all patients. In addition, determination of PD-L1 expression in tumor tissue has not been harmonized to date and biopsies from metastatic sites are often hardly accessible. Thus, a reliable predictive biomarker is still urgently needed. In this study, we perform detection of PD-L1 on circulating tumor cells (CTCs) in blood samples of patients with advanced UCB.
Experimental procedures: Blood samples of 93 non-metastatic (M0) and 33 metastatic (M1) UCB patients were analyzed using the CellSearch® system. Following EpCAM-based enrichment, CK+/DAPI+/CD45- cells were identified as CTCs using the CTC-Kit. For PD-L1 detection, the CellSearch® CXC-Kit (optional fluorescence channel: PE) proved more suitable than the CellSearch® CTC-Kit (optional fluorescence channel: FITC). After enumeration of CTCs by the CTC-Kit, for nine CTC-positive patients (M1) a second blood sample was further analyzed for PD-L1 protein expression using the CXC-Kit. Specificity of the PE-labelled anti-PD-L1-antibody (clone E1L3N) was validated by Western blot analysis, IF and FACS.
Results: Enumeration of CTCs by the CellSearch® CTC-Kit revealed significantly higher CTC-positivity in M1 than in M0 patients (51.5% vs. 22.6%, p=0.004 for ≥1 CTCs/7.5 mL blood; 30.3% vs. 3.2%, p&lt;0.001 for ≥5 CTCs/7.5 mL blood). Also mean absolute CTC numbers were significantly higher in M1 than M0 patients (p&lt;0.001). PD-L1 detection in the CellSearch® system was established using five UCB cell lines with different PD-L1 expression, according to Western blot analysis and FACS, which were spiked into whole blood of healthy donors. Using the CXC-Kit, ≥1 PD-L1+ CTCs were detected in 77.8% (7/9) of CTC-positive M1 patients. Thus, PD-L1 expression levels presented with intra- and inter-patient heterogeneity. The follow-up analysis of one patient who received anti-PD-L1 therapy showed substantial increases in CTC numbers as well as in the ratio and intensity of PD-L1+ CTCs in conjunction with disease progression under immunotherapy. The patient died within one month after the follow-up blood draw.
Conclusion: This is the first study demonstrating the feasibility to detect and monitor PD-L1 expression on CTCs of advanced UCB patients in the CellSearch® system. The established assay is readily available for clinical application and should be implemented in future clinical trials to evaluate its relevance for predicting response to immune checkpoint inhibitors.
Citation Format: Sonja Bergmann, Anja Coym, Gunhild von Amsberg, Melanie Janning, Armin Soave, Michael Rink, Klaus Pantel, Sabine Riethdorf. Evaluation of PD-L1 expression on circulating tumor cells (CTCs) in patients with advanced urothelial carcinoma of the bladder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2219.