Next generation sequencing of PD-L1 for predicting response to immune checkpoint inhibitors

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.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0489-5) contains supplementary material, which is available to authorized users.

Progress Toward Identifying Exact Proxies for Predicting Response to Immunotherapies

“…For example, in melanoma patients, there was a strong association of objective response rate to high PDL-1 RNA-seq expression, even regardless of PDL-1 IHC results. Some advantages warrant further assessing and perhaps validating PDL-1 RNA-seq as an approach that offers superior standardization and less interpretation bias than IHC (Conroy et al, 2019). Obtaining serial tumor tissue biopsies is highly challenging, amount of tissue is often insufficient, and high bleeding risk often prevents serial tissue sampling.…”

Biomarkers for Response to Immune Checkpoint Blockade

“…Technical limitations in assessing PD-L1 expression could be overcome by alternative assays, such as mRNA (messenger RNA) measurement by RNA sequencing or reverse transcriptase polymerase chain reaction (RT-PCR) (Conroy et al 2019, Erber et al 2017; however, addressing potential biological limitations will remain challenging. New approaches for evaluating PD-L1 expression that are being developed include noninvasive nuclear imaging approaches, which may allow unbiased whole-body imaging of PD-L1 expression that can be followed over time (Kumar et al 2019, Niemeijer et al 2018.…”

Modern Aspects of Immunotherapy with Checkpoint Inhibitors in Melanoma

“…Although PD-L1 overexpressing tumors showed an association with the higher response to ICI, durable responses could be also observed in PD-L1 negative tumors [90,91]. Therefore, complementary approaches are needed to improve the prognostic value of tumor PD-L1, including a dynamic monitoring of PD-L1 expression or PD-L1 RNA sequencing [92,93].…”