Abstract 3664: A workflow to evaluate PD-L1 protein expression on circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC)
Authors: Renier Corinne , Wilkerson Charles L. , Hur SJ Claire , Park Da Eun Rachel , Lemaire Clementine A. , Matsumoto Melissa , Carroll James , Crouse Steve , Goldman Jonathan W. , Garon Edward B. , Carlo Dino Di , Christen-Sollier Elodie 
Published: August 2018

Abstract
Introduction
Many tumors evade immune surveillance by deploying immunosuppressive mechanisms and co-opting immune checkpoint pathways, such as PD-1/PD-L1. PD-1/PD-L1 pathway blockade is a highly promising therapy and has elicited durable antitumor responses in a variety of cancers. Challenges in advancing immunotherapies lie in patient stratification and monitoring therapy. Evaluation of PD-L1 expression is generally regarded as an inclusion criterion for clinical trials. Current methods rely on the analysis of excisional biopsies, which are invasive and carry associated medical risks. Isolation and analysis of CTCs is a noninvasive alternative that may provide, in real-time, more representative information on tumor biomarkers, and allow the monitoring of treatment effectiveness over time. Here, we present an integrated workflow for the analysis of PD-L1 protein expression on CTCs from metastatic NSCLC patients isolated with the Vortex Biosciences VTX-1 liquid biopsy system.
Methods
Cell lines expressing various level of PD-L1 and white blood cells prepared from healthy donors were used to optimize the PD-L1 immunostaining assay. Several PD-L1 antibodies were titrated, and their specificity estimated with isotype controls. PD-L1 expression quantification was evaluated using two independent methods i) Zen 2 imaging (Zeiss) and ii) CellProfiler software. Clinical validation was performed on blood samples collected from a cohort of metastatic NSCLC patients (stage IV) with known PD-L1 tumor status. CTCs were isolated using the VTX-1 Liquid Biopsy System, stained (PD-L1, CK, CD45, CD11b, DAPI) and classified, then PD-L1 expression was quantified.
Results
We have developed a PD-L1 immunofluorescent assay that shows specificity, sensitivity, and very good intra-assay repeatability. The two methods evaluated for the quantification of PD-L1 expression provided equivalent data and could be used interchangeably. The PD-L1 assay was further validated on CTCs isolated from a small cohort of metastatic NSCLC patients. Preliminary data shows heterogeneity in PD-L1 expression levels across CTCs from the same patient. Interestingly, among PD-L1 positive CTCs, both traditional CK+/PD-L1+ and CK-/PD-L1+ cells were observed. To move toward a more automated workflow for immunostaining, imaging, and fluorescence signal analysis, methods to collect CTC on glass slides were evaluated, with cell recovery ranging from 33 to 98%.
Conclusion
We demonstrated the feasibility of evaluating PD-L1 protein expression on NSCLC's CTCs isolated with the VTX-1 Liquid Biopsy System. While further clinical validation is warranted, this test could represent a simple, non-invasive method to help identify patients most likely to respond to PD-1/PD-L1 immunotherapies. Future work will focus on compatibility of the assay with autostainers and fully automated imaging and analysis platforms.
Citation Format: Corinne Renier, Charles L. Wilkerson, SJ Claire Hur, Da Eun Rachel Park, Clementine A. Lemaire, Melissa Matsumoto, James Carroll, Steve Crouse, Jonathan W. Goldman, Edward B. Garon, Dino Di Carlo, Elodie Christen-Sollier. A workflow to evaluate PD-L1 protein expression on circulating tumor cells (CTCs) from non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3664.