Abstract
Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/μL with a range of 768-7510 copies/μL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection.
Highlights
Programmed cell death ligand-1 (PD-L1) has been identified as an efficacious target to develop immune checkpoint inhibitors and has drastically evolved the treatment paradigm for advanced cancers.PD-L1 expression detected by immunohistochemistry (IHC) in tumor tissue has been used as a predictive biomarker for therapeutic response to PD-1/PD-L1 blockade treatment in various cancers such as non-small cell lung cancer (NSCLC), [1,2]
TBP shows a slight difference in expression, while GUSB and RPLP0 showed almost no difference in expression, with similar effects when analyzed in healthy volunteer PAXgene blood samples
To better assess the reproducibility of both Droplet digital PCR (ddPCR) and qPCR for use in detection of PD-L1 in PAXgene blood, we evaluated reproducibility by both inter-assay and intra-assay variation. qPCR Relative expression was defined as expression relative to A549 untreated sample normalized to the GUSB gene whereas qPCR Absolute is derived from a standard curve
Summary
Programmed cell death ligand-1 (PD-L1) has been identified as an efficacious target to develop immune checkpoint inhibitors and has drastically evolved the treatment paradigm for advanced cancers. PD-L1 expression detected by immunohistochemistry (IHC) in tumor tissue has been used as a predictive biomarker for therapeutic response to PD-1/PD-L1 blockade treatment in various cancers such as non-small cell lung cancer (NSCLC), [1,2]. Given the dynamic interplay between the PD-1/PD-L1 pathway and the tumor microenvironment, as well as the heterogeneity of immune milieu among different tumor types (hot tumor vs cold tumor), it has been recognized that tissue (especially core needle) biopsy may not accurately reflect the entirety of the tumor microenvironment. Invasive tissue biopsies provide limited information to reflect the tumor heterogeneity, and it is not clinically feasible to collect multiple tumor biopsies for longitudinal monitoring of therapeutic efficacy and disease progression
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