Next generation sequencing of PD-L1 for predicting response to immune checkpoint inhibitors.
BACKGROUND: PD-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. METHODS: A 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. RESULTS: Assessment 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. CONCLUSIONS: Measurement 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.
Conroy, JM; Pabla, S; Nesline, MK; Glenn, ST; Papanicolau-Sengos, A; Burgher, B; Andreas, J; Giamo, V; Wang, Y; Lenzo, FL; Bshara, W; Khalil, M; Dy, GK; Madden, KG; Shirai, K; Dragnev, K; Tafe, LJ; Zhu, J; Labriola, M; Marin, D; McCall, SJ; Clarke, J; George, DJ; Zhang, T; Zibelman, M; Ghatalia, P; Araujo-Fernandez, I; de la Cruz-Merino, L; Singavi, A; George, B; MacKinnon, AC; Thompson, J; Singh, R; Jacob, R; Kasuganti, D; Shah, N; Day, R; Galluzzi, L; Gardner, M; Morrison, C
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