Abstract 1310: Programmed death ligand-1 (PD-L1) heterogeneity in non-small cell lung cancer (NSCLC)
Authors: McLaughlin Joseph , Schalper Kurt A. , Carvajal-Hausdorf Daniel E. , Pelekanou Vasiliki , Velcheti Vamsidhar , Haack Herbert , Silver Matthew R. , Herbst Roy , LoRusso Patricia , Rimm David L. 
Published: August 2015

Abstract
Early phase I trials with monoclonal antibodies targeting PD-1/PD-L1 have demonstrated durable clinical responses in patients with NSCLC. However, the prognostic/predictive role of tumor PD-L1 expression has not been determined. Current assays for evaluation of PD-L1 protein expression are not standardized. Here, we demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF), and compare results obtained using three PD-L1 antibodies targeting the intracellular (IC) and extracellular (EC) domains.
We measured PD-L1 protein expression using two antibodies against the IC domain (E1L3N [Cell Signaling Technology] and SP142 [Spring Biosciences Inc.]) and one antibody binding the EC domain (E1J2J [Cell Signaling Technology]) in 49 NSCLC whole tissue sections and a TMA with the same 49 cases. Mel624 cells stably transfected with PD-L1, as well as Mel624 parental cells and human term placenta were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA® method of QIF. For IHC, PD-L1 positivity was determined using previously reported cut-points for tumor (1%, 5%, and 50%) and stroma (5%). IHC inter-assay concordance was evaluated using kappa coefficient. AQUA scores were measured in an average of 28.6 fields of view per case or by TMA at two-fold redundancy. Linear regression coefficients (R2) were used to compare antibody QIF scores. Tumor-infiltrating lymphocytes were scored in hematoxylin/eosin stained slides using current consensus guidelines.
Chromogenic IHC assays showed fair to poor concordance (kappa 0.124 - 0.552) for all cut-points. QIF showed PD-L1 immunopositivity was heterogeneous for all three PD-L1 antibodies. R2 values were lower when E1J2J (EC) was compared to E1L3N and SP142 (IC) (R2 = 0.090 and 0.079), while coefficients were higher when the IC antibodies were compared (R2 = 0.658). E1L3N and SP142 significantly correlated with high TILs (p = 0.007 and p = 0.021) but E1J2J did not (p = 0.281). E1J2J significantly correlated with older age (p = 0.038), but no other clinicopathological feature. SP142 significantly correlated with lymph node positive status (p = 0.030) Objective determination of PD-L1 protein levels in NSCLC reveals significant heterogeneity within tumors. There is significant inter-assay variability, even between assays detecting the same protein domain. This could be due to different antibody affinities, cross reactivity, or distinct target epitopes. Efforts to determine the clinical value of these observations are underway.
Citation Format: Joseph McLaughlin, Kurt A. Schalper, Daniel E. Carvajal-Hausdorf, Vasiliki Pelekanou, Vamsidhar Velcheti, Herbert Haack, Matthew R. Silver, Roy Herbst, Patricia LoRusso, David L. Rimm. Programmed death ligand-1 (PD-L1) heterogeneity in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1310. doi:10.1158/1538-7445.AM2015-1310