Abstract
BackgroundPD-L1 immunohistochemistry (IHC) has been traditionally used for predicting clinical responses to immune checkpoint inhibitors (ICIs). However, there are at least 4 different assays and antibodies used for PD-L1 IHC, each developed with a different ICI. We set to test if next generation RNA sequencing (RNA-seq) is a robust method to determine PD-L1 mRNA expression levels and furthermore, efficacy of predicting response to ICIs as compared to routinely used, standardized IHC procedures.MethodsA total of 209 cancer patients treated on-label by FDA-approved ICIs, with evaluable responses were assessed for PD-L1 expression by RNA-seq and IHC, based on tumor proportion score (TPS) and immune cell staining (ICS). A subset of serially diluted cases was evaluated for RNA-seq assay performance across a broad range of PD-L1 expression levels.ResultsAssessment of PD-L1 mRNA levels by RNA-seq demonstrated robust linearity across high and low expression ranges. PD-L1 mRNA levels assessed by RNA-seq and IHC (TPS and ICS) were highly correlated (p < 2e-16). Sub-analyses showed sustained correlation when IHC results were classified as high or low by clinically accepted cut-offs (p < 0.01), and results did not differ by tumor type or anti-PD-L1 antibody used. Overall, a combined positive PD-L1 result (≥1% IHC TPS and high PD-L1 expression by RNA-Seq) was associated with a 2-to-5-fold higher overall response rate (ORR) compared to a double negative result. Standard assessments of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) showed that a PD-L1 positive assessment for melanoma samples by RNA-seq had the lowest sensitivity (25%) but the highest PPV (72.7%). Among the three tumor types analyzed in this study, the only non-overlapping confidence interval for predicting response was for “RNA-seq low vs high” in melanoma.ConclusionsMeasurement of PD-L1 mRNA expression by RNA-seq is comparable to PD-L1 expression by IHC both analytically and clinically in predicting ICI response. RNA-seq has the added advantages of being amenable to standardization and avoidance of interpretation bias. PD-L1 by RNA-seq needs to be validated in future prospective ICI clinical studies across multiple histologies.
Highlights
progressive disease (PD)-L1 immunohistochemistry (IHC) has been traditionally used for predicting clinical responses to immune checkpoint inhibitors (ICIs)
For samples #3 and #4, PD-L1 transcript detection values ranged from 0 to < 450 absolute reads, demonstrating a positive linear correlation (R2 > 0.98) for clinical specimens expressing low-to-moderate PD-L1 levels. These results demonstrate that detection of PD-L1 mRNA levels in formalin-fixed paraffin-embedded (FFPE) samples by RNA sequencing (RNA-seq) is consistent across a dynamic range of expression, and that PD-L1 transcripts can be reliably quantified by a continuous variable of absolute transcript reads down to values approaching background
Data analysis To demonstrate the linearity of PD-L1 mRNA detection, coefficient of determination (R2) was calculated for the absolute reads generated across various library dilutions
Summary
PD-L1 immunohistochemistry (IHC) has been traditionally used for predicting clinical responses to immune checkpoint inhibitors (ICIs). Five trial-evaluated immunohistochemistry (IHC) assays for the assessment of CD274 (best known as programmed death ligand-1, PD-L1) expression in formalin-fixed paraffin-embedded (FFPE) samples have been developed as companion and complementary diagnostics alongside immune checkpoint inhibitors (ICIs) targeting PD-L1 and its main receptor (programmed cell death 1, PDCD1, best known as PD-1) [1,2,3,4,5] While these tests measure PD-L1 protein levels, they differ by antibody clone, staining platform, and scoring system. The use of FFPE archival tumor tissues with non-standardized fixation and storage methods may be a source of unpredictable and unintended results for adequate PD-L1 antigen retrieval, potentially increasing the heterogeneity of IHC intensity, extent and topography of staining All these factors complicate the use of PD-L1 status as assessed by IHC for predicting patient clinical response to ICIs [13, 14]
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