Abstract 683: Quantification of rare PD-L1 expressing leukocytes and CTCs in peripheral blood of cancer patients
Authors: Jendrisak Adam , Byun Jiyun , Suraneni Mahipal , Lu David , Krupa Rachel , Orr Sarah , Graf Ryon , Wang Yipeng , Landers Mark , Dittamore Ryan 
Published: July 2017

Abstract
Background: Expression of PD-L1 on tumor tissue is associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. Additionally, expression of PD-L1 on infiltrating T cells is associated with immune exhaustion. Currently, PD-L1 analysis as well as measures of infiltrating T cells are examined in surgically removed or biopsied samples, usually taken long before clinical decision points. Additionally, given tumor heterogeneity, metastatic lesions are likely to be under-sampled. We sought to examine expression of PD-L1 on circulating tumor cells (CTCs) and leukocytes cell populations with a non-invasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy.
Methods: Blood samples from cancer patients were received and shipped to Epic Sciences. Contrived blood samples were also developed utilizing spike-in of cancer cell line controls into healthy blood samples. RBCs were lysed, and nucleated cells plated onto glass slides utilizing the Epic Sciences process. Slides are stained with an IF cocktail (CK, DAPI, CD45, PD-L1) and scanned. Approximately 3 million nucleated cells are examined through advanced Digital Pathology pipelines to detect rare CTCs (CK+/-, cancer morphology, CD45-) and leukocytes (CK-, CD45+, leukocyte morphology) and analyze for PD-L1 expression.
Results: A limit of detection of 1 cell/mL of blood was analytically validated. Using Epic’s rare cell detection platform, we could detect a wide range of PD-L1+ leukocyte counts in a subset of peripheral blood of cancer patients, observed as low as 0.003% (110 of 3,192,263) of total leukocytes. PD-L1+ CTCs were observed in the presence of both high and low counts of PD-L1+ leukocyte populations.
Conclusions: Epic Sciences CTC platform’s low limit of detection coupled with ability to archive patient blood samples allowed precise quantification of PD-L1 expression on CTCs and leucocytes retrospectively. In addition, we have previously demonstrated the platform’s ability to detect leucocyte subtypes such as CD3, CD4 and CD8 cells, which will allow us to further characterize PD-L1 expression in T-lymphocytes and other immune cell types. Development of a liquid biopsy based platform that is capable of simultaneously measuring immune biomarkers in CTCs as well as leucocytes will allow real time assessment of response to immune checkpoints inhibitors.
Citation Format: Adam Jendrisak, Jiyun Byun, Mahipal Suraneni, David Lu, Rachel Krupa, Sarah Orr, Ryon Graf, Yipeng Wang, Mark Landers, Ryan Dittamore. Quantification of rare PD-L1 expressing leukocytes and CTCs in peripheral blood of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2017-683