Abstract 1372: Quantification of PD-L1 and PD-1 expression on tumor cells in non-small cell lung cancer (NSCLC) using non-enzymatic tissue dissociation and flow cytometry
Authors: Chargin Amanda , Morgan Rian , Sundram Uma , Ratti Navneet , Shults Keith , Patterson Bruce 
Published: July 2016

Abstract
Objective: The objective of this study was to develop a truly quantitative technology for PD-L1 expression in NSCLC. In addition, we also present a non-enzymatic technology that creates a tumor cell suspension from fresh tumor tissue so that either fine needle aspiration or fresh tissue can be used in this assay.
Methods: 4 mm punches were taken from each tumor. Non-enzymatic tissue homogenization (IncellPREP; IncellDx, Menlo Park, CA) was performed. A French technique FNA was also taken from the same tumor to create matched sample sets. Cells were labeled with antibodies directed against CD45, PD-1, PD-L1, fixed and permeabilized then stained with DAPI to identify intact, single cells, and to analyze cell cycle.
Results: Both FNA and IncellPREP generated greater than 1 million cells per mL with the IncellPREP yields being 2.5 times the number of cells per preparation compared to FNA (Mann-Whitney, p = 0.003). Comparing the IncellPREP homogenization and FNA, a strong correlation (r2-0.8) was demonstrated for expression of PD-L1. We compared PD-L1 expression by flow cytometry using a 1%a cut-off for positivity in the tumor cell population and a 1% cut-off of cells with at least 1+ intensity in immunohistochemically stained tissue sections as positive (Table 1). As demonstrated in the table, 10 of 12 lung tumor samples were concordant while 2 were discordant, one positive by flow and negative by IHC and one negative by flow and positive by IHC. PD-L1 expression by flow cytometry varied widely (1.2% to 89.4%) even in the positive concordant cases. In addition, PD-L1 expression in the aneuploid tumor population did not necessarily agree with the expression in the diploid tumor population.
Summary: Fine, unequivocal, quantification of PD-L1 on tumor and immune cells in NSCLC may allow for better prediction of response to therapies. The present study also offers a technology that can create a universal sample type from either FNA or fresh tissue. Table 1DiagnosisDNA Index TumorAneuploid IndexPD-L1 (1% CO)PD-L1% (Aneuploid)IHC (1% CO)Squamous cell carcinoma0.941.6418.9% (+)28.8%PosAdenocarcinoma0.97None1.2% (+)-NegAdenocarcinoma0.902.1886.6% (+)99.6%PosAdenocarcinoma0.961.4085.1% (+)97.0%PosSquamous cell carcinoma0.951.3494.8% (+)97.7%PosSquamous cell carcinoma1.01None13.7% (+)-PosAdenocarcinoma0.93None0.8% (-)-NegSquamous cell carcinoma0.94None0.5% (-)-PosAcinar Adenocarcinoma0.961.2122.9% (+)88.5%PosAdenocarcinoma0.87None0% (-)-NegSquamous cell carcinoma0.91None25.8% (+)-PosAdenocarcinoma0.96None0.1% (-)-Neg
Citation Format: Amanda Chargin, Rian Morgan, Uma Sundram, Navneet Ratti, Keith Shults, Bruce Patterson. Quantification of PD-L1 and PD-1 expression on tumor cells in non-small cell lung cancer (NSCLC) using non-enzymatic tissue dissociation and flow cytometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1372.