Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non–Small-Cell Lung Cancer

Abstract

Importance Early phase trials with monoclonal antibodies targeting PD-1/PD-L1 have demonstrated durable clinical responses in patients with NSCLC, however, current assays for the prognostic/predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression. Objective In this study, we demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF), and compare results obtained using two different PD-L1 antibodies. Design PD-L1 was measured using two rabbit monoclonal antibodies (E1L3N and SP142) in 49 NSCLC whole tissue sections and a corresponding tissue microarray 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. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin/eosin stained slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinico-pathological features were determined. Setting NSCLC resections were all performed at Yale New Haven Hospital. Participants NSCLC resection cases from 2011–2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank in Yale Pathology based on tissue availability. Main Outcome Measure PD-L1 expression discordance or heterogeneity using DAB and QIF was the main outcome measure selected prior to performing the study. Results Using chromogenic IHC, both antibodies showed fair to poor concordance. QIF 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 <0.001). Assessment of 588 serial section fields of view by QIF showed discordant expression at a frequency of 25%. Expression of PD-L1 using both E1L3N and SP142 was correlated with high TILs (p = 0.007 and p = 0.021). Conclusions Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent inter-assay 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 underway.

Programmed Death Ligand-1 Immunohistochemistry: Friend or Foe?

“…There are limited data on comparative studies published on assays that were not validated in trials, so they are of limited value, although they might forecast what we can expect when comparing the companion or complementary diagnostic assays. 17,18 The data generated from clinical trials validate both the efficacy of these drugs and the predictive performance of each allied biomarker assay as it was performed during each of the trials. In this era of evidence-based medical practice, to what extent can we afford to deviate from what was validated in the trials while there is no evidence to support doing so?…”

“…A recent study comparing 2 commercially available clones, both developed as LDTs, based upon a SP142 clone (Spring Bioscience, Pleasanton, California) and the E1L3N clone (Cell Signaling Technologies, Boston, Massachusetts), demonstrated significant differences in staining performance. 18 Even if the very extensive technical validation that would be required to create an LDT were completed, there is still no guarantee of equivalent clinical predictive performance.…”

Programmed Death Ligand-1 Immunohistochemistry— A New Challenge for Pathologists: A Perspective From Members of the Pulmonary Pathology Society

“…Given that the FDA-approved Dako 22C3 companion diagnostic became available only in October of 2015 and that the 22C3 clone cannot be purchased apart from the approved kit, many laboratories have already technically validated other readily available and far less costly commercial antibodies as laboratory-developed tests (LDTs). Efforts to compare the performance of 2 commonly used antibody clones, E1L3N from Cell Signaling Technology and Ventana-SP142 (anticipated to serve as part of the companion diagnostic for atezolizumab), have demonstrated fair to poor concordance 16 ; however, these studies are limited by outdated antibody optimization techniques and the lack of a clear clinical gold standard in the form of response data. The importance of this latter issue cannot be understated.…”

“…Sampling errors, tumoral heterogeneity, or testing of tissue obtained at diagnosis rather than at time of progression or relapse may underestimate or overestimate the percentage of neoplastic cells showing PD-L1 expression. 16,19 There is evidence across different tumor types that mutational signatures, neoantigen burden, and expression of other checkpoint inhibitors may predict response to immunotherapies. 20 It is quite possible that in the future another assay may supersede PD-L1 IHC as the biomarker of choice, assuming these markers can be validated in samples of past or future trials involving these immunotherapy drugs.…”

Atezolizumab in non-small cell lung cancer: the era of precision immuno-oncology

“…This demonstrated that PD-L1 expression level is useful in enriching a population who would gain more from atezolizumab, but at the same time, proved itself as an imperfect biomarker in dichotomizing patients in treatment selection. The finding of observing response in tumors with low PD-L1 expression is further complicated by the high degree of intratumoral heterogeneity in NSCLC, which renders the PD-L1 expression level detected in biopsy specimens unrepresentative of whole tumor sample (8).…”