title: Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non-Small-Cell Lung Cancer.
abstract: OBJECTIVE
  Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression.
  
  
  OBJECTIVE
  To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies.
  
  
  METHODS
  PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non-small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined.
  
  
  METHODS
  PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study.
  
  
  RESULTS
  Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02).
  
  
  CONCLUSIONS
  Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.
authors.affiliation: Department of Medical Oncology, Yale University School of Medicine, New Haven, Connecticut2Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Joseph
authors.initials: J
authors.lastname: McLaughlin
authors.affiliation: Department of Biostatistics, Yale University School of Public Health, New Haven, Connecticut.
authors.forename: Gang
authors.initials: G
authors.lastname: Han
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Schalper
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Daniel
authors.initials: D
authors.lastname: Carvajal-Hausdorf
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Pelekanou
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Jamaal
authors.initials: J
authors.lastname: Rehman
authors.affiliation: Department of Solid Tumor Oncology, Cleveland Clinic, Cleveland, Ohio.
authors.forename: Vamsidhar
authors.initials: V
authors.lastname: Velcheti
authors.affiliation: Department of Medical Oncology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Roy
authors.initials: R
authors.lastname: Herbst
authors.affiliation: Department of Medical Oncology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: Patricia
authors.initials: P
authors.lastname: LoRusso
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: David L
authors.initials: DL
authors.lastname: Rimm
mesh_terms: D000368:Aged; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D060890:B7-H1 Antigen / Q000032:analysis* / Q000276:immunology; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000276:immunology; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry* / Q000276:immunology / Q000473:pathology; D005260:Female; D005455:Fluorescent Antibody Technique / Q000592:standards*; D006801:Humans; D007150:Immunohistochemistry / Q000592:standards*; D008175:Lung Neoplasms / Q000737:chemistry* / Q000473:pathology; D008297:Male; D015588:Observer Variation; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012189:Retrospective Studies; D046888:Tissue Array Analysis / Q000592:standards
chemical_list: D000911:Antibodies, Monoclonal; D060890:B7-H1 Antigen; D014408:Biomarkers, Tumor; C423236:CD274 protein, human