Abstract LB-011: Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples
Authors: O'Rourke Dennis , Wang Danyi , Cusano Maria , Sanchez-Garcia Jorge F. , Cai Ti , Scheuenpflug Juergen , Feng Zheng 
Published: August 2019

Abstract
Purpose
Programmed death ligand receptor 1, (PD-L1) is a key immune checkpoint inhibitory receptor expressed on activated tumor specific immune cells. While immunohistochemistry assays have been developed for the detection of PD-L1 expression in several cancer types for patient treatment decision, a blood-based test for PD-L1 expression evaluation would be beneficial in providing complimentary evidence for potential clinical application. This study developed a fit-for-purpose clinical biomarker assay for the detection of PD-L1 mRNA expression in blood using droplet digital polymerase chain reaction (ddPCR) methods.
Study Procedure
Taqman primer/probe assays were selected based on coverage of PD-L1 gene and were tested for linearity and efficiency using a cDNA construct. Four housekeeping genes were analyzed in positive control cell line (A549 treated with interferon gamma), negative control cell line (A549 untreated) and PAXgene blood from healthy volunteers.
Real-time quantitative PCR method was tested first, following initial experiments there were concerns with the performance of our qPCR assay prompting the switch to ddPCR which offers absolute quantification and a lower limit of detection.
Assay performance characteristics of the ddPCR assay were evaluated including linearity, limit of blank (LOB), limit of detection (LOD), and precision. Thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression.
Data Summary
Conducting linearity comparison between Taqman primer/probe kits using cDNA titrations showed no significant difference in linearity (r2=0.99) or efficiency (~95%) among the three primer/probe kits.
Housekeeping genes B2M, GUSB, RPLP0, and TBP were compared and GUSB was most consistently expressed between IFN gamma treated and untreated cells; it was selected for use thereafter.
In ddPCR, the LOB was determined to be 0 copies and the LOD was determined to be 19 copies of PD-L1. The average intra-run coefficient of variation (CV) was 7.44% and average inter-run CV was 7.70%. Analysis of healthy control samples yielded an average of 2415 PD-L1 copies with a range of 768-7510 copies. Treatment of A549 cells with IFN gamma resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA).
Conclusion
Our fit-for purpose clinical biomarker assay development efforts have demonstrated that the ddPCR assay is capable of reliably and accurately detecting PD-L1 in PAXgene blood with low limit of detection and minimal inter-and-intra run variability. Compared to real-time PCR, ddPCR is a purely quantitative and offers a much broader dynamic range and provides more sensitive detection of PD-L1 in blood. Further evaluation and comparison of cancer to healthy volunteer samples is underway for more information on assay performance and disease biology.
Citation Format: Dennis O'Rourke, Danyi Wang, Maria Cusano, Jorge F. Sanchez-Garcia, Ti Cai, Juergen Scheuenpflug, Zheng Feng. Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples [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 LB-011.