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65 PD-L1 by RNA next generation sequencing: comparison with PD-L1 IHC 22C3 and association with survival benefit from pembrolizumab with or without chemotherapy in non-small cell lung cancer
  1. Yong Hee Lee1,
  2. Grace Dy2,
  3. Paul DePietro1,
  4. Jeffrey Conroy1,
  5. Sarabjot Pabla1 and
  6. Mary Nesline1
  1. 1OmniSeq, Buffalo, NY, USA
  2. 2Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA


Background PD-L1 immunohistochemistry (IHC) testing is suboptimal for predicting patient clinical benefit for checkpoint inhibition, while PD-L1 liquid biopsy is not clinically validated and lacks sensitivity, underscoring the need to include PD-L1 testing in more robust, tissue-efficient, comprehensive, scalable next generation sequencing (NGS) tests.

Methods To assess comparability and efficacy of PD-L1 testing by NGS with IHC, we identified NSCLC patients treated by first-line pembrolizumab alone (n=54) or pembrolizumab + chemotherapy (n=49) whose tumors underwent companion diagnostic PD-L1 testing by IHC antibody 22C3 testing (high≥50%; low=1–49%, or negative=0% tissue proportion score), and also by RNA-seq, as part of a comprehensive immune profiling panel. PD-L1 expression by RNA-seq, was measured as a percentile rank, with ≥75 considered ‘high’, and <75 considered ‘not high’, based on comparison to a reference population and normalized to a value of 1–100. All testing was performed in a CLIA certified laboratory prior to treatment initiation (any line) at Roswell Park Comprehensive Cancer Center (June 2017-March 2019, with a minimum of 1 year of follow up). Assay equivalence was assessed by proportion analysis using Fisher exact test comparing IHC versus to RNA-seq, and Bonferroni pairwise post-hoc analysis of IHC (high vs. low, high vs. negative, low vs. negative) with RNA-seq (high vs. not high). A Cox regression model evaluated associations between IHC and RNA-Seq with OS from first dose of pembrolizumab.

Results More than 75% of IHC high cases were classified as high by RNA-Seq for both treatment groups (p<0.001). Post-hoc pairwise comparisons showed PD-L1 IHC and RNA-Seq ‘high’ results were significantly associated with each other, and PD-L1 IHC low/negative results were associated with RNA-seq ‘not high’ results. In the pembrolizumab monotherapy group, RNA-seq high was associated with improved survival for pembrolizumab compared to RNA-seq not high status (HR=3.96; CI=1.22–12.87; p=0.02), while PD-L1 IHC high status was not associated with survival benefit in this group (p=0.63). In the pembrolizumab + chemotherapy group, as expected, neither IHC (high versus low), nor RNA-seq (high versus not high) status was associated with survival benefit (p=0.81 and p=0.76, respectively). These findings are consistent with our previous work demonstrating PD-L1 RNA-seq was predictive of CPI response in multiple tumor types.

Conclusions PD-L1 status by RNA-seq and IHC appear to be comparable. Unlike PD-L1 IHC however, PD-L1 RNA-seq high status versus not high status is associated with greater survival benefit, indicating PD-L1 by NGS may have utility for pembrolizumab selection.

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See:

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