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PD-L1 expression heterogeneity in non-small cell lung cancer: evaluation of small biopsies reliability.

ABSTRACT: Immunotherapy with checkpoint inhibitors, allowing recovery of effector cells function, has demonstrated to be highly effective in many tumor types and represents a true revolution in oncology. Recently, the anti-PD1 agent pembrolizumab was granted FDA approval for the first line treatment of patients with advanced non-small cell lung cancer (NSCLC) whose tumors show PD-L1 expression in ? 50% of neoplastic cells and as a second line treatment for patients with NSCLC expressing PD-L1 in ?1% of neoplastic cells, evaluated with a validated assay. For the large majority of patients such evaluation is made on small biopsies. However, small tissue samples such as core biopsies might not be representative of tumors and may show divergent results given the possible heterogeneous immunoexpression of the biomarker. We therefore sought to evaluate PD-L1 expression concordance in a cohort of 239 patients using tissue microarrays (TMA) as surrogates of biopsies stained with a validated PD-L1 immunohistochemical assay (SP263) and report the degree of discordance among tissue cores in order to understand how such heterogeneity could affect decisions regarding therapy. We observed a discordance rate of 20% and 7.9% and a Cohen's ? value of 0.53 (moderate) and 0,48 (moderate) for ? 1% and ? 50% cutoffs, respectively. Our results suggest that caution must be taken when evaluating single biopsies from patients with advanced NSCLC eligible for immunotherapy; moreover, at least 4 biopsies are necessary in order to minimize the risk of tumor misclassification.

SUBMITTER: Munari E

PROVIDER: S-EPMC5685736 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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PD-L1 expression heterogeneity in non-small cell lung cancer: evaluation of small biopsies reliability.

Munari Enrico E Zamboni Giuseppe G Marconi Marcella M Sommaggio Marco M Brunelli Matteo M Martignoni Guido G Netto George J GJ Moretta Francesca F Mingari Maria Cristina MC Salgarello Matteo M Terzi Alberto A Picece Vincenzo V Pomari Carlo C Lunardi Gianluigi G Cavazza Alberto A Rossi Giulio G Moretta Lorenzo L Bogina Giuseppe G

Oncotarget 20171004 52

Immunotherapy with checkpoint inhibitors, allowing recovery of effector cells function, has demonstrated to be highly effective in many tumor types and represents a true revolution in oncology. Recently, the anti-PD1 agent pembrolizumab was granted FDA approval for the first line treatment of patients with advanced non-small cell lung cancer (NSCLC) whose tumors show PD-L1 expression in ≥ 50% of neoplastic cells and as a second line treatment for patients with NSCLC expressing PD-L1 in ≥1% of ne ...[more]

PMID: 29163815

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Project description:Background:The Dako PD-L1 immunohistochemistry (IHC) 22C3 pharmDx and the Dako 28-8 IHC pharmDx assays were approved by the US Food and Drug Administration, as a companion diagnostic test for pembrolizumab (Keytruda, Merk, Kenilworth, NJ, USA) and a complementary diagnostic test for nivolumab (Opdivo, Bristol Meyer Squibb, New York, NY, USA) in non-small cell lung cancer (NSCLC), respectively. Increased PD-L1 expression levels can be associated with greater therapeutic efficacy of pembrolizumab relative to other anti-PD-1 agents. However, in treatment decision making, little is known about which tissue (primary or metastatic lesion) should be stained by 22C3 antibody. We investigated the relationship between PD-L1 expression in primary tumors and paired metastatic lymph nodes using the 22C3 assay, and evaluated the concordance between the 22C3 and 28-8 assays. Methods:PD-L1 expression was evaluated in cells from primary tumors and paired metastatic lymph nodes using the 22C3 and 28-8 IHC assays. Total 35 patients with primary tumor and paired metastatic lymph node were enrolled into this study, and all samples were surgically resected, formalin-fixed, and paraffin-embedded NSCLC tissues. Tumor cells exhibiting complete or partial membrane staining, were considered as PD-L1 positive. On the basis of tumor proportion score (TPS), all samples were classified as no expression (TPS: <1%), low expression (TPS: 1-49%), or high expression (TPS: ?50%). Results:TPS distribution was markedly different between primary tumors and paired metastatic lymph nodes. In 22C3 IHC assay, TPS similar to that of metastatic lymph nodes was demonstrated in 10 primary tumors, and concordance rate between them was 28.6%. Concurrently, in 28-8 IHC assay, 11 primary tumors had TPS similar to that of metastatic lymph nodes, with a concordance rate of 31.4%. Conclusions:TPS concordance rates (for both 22C3 and 28-8 antibodies) between primary tumors and paired lymph nodes were low. Inter-tumor heterogeneity of PD-L1 expression is an important issue for clinical oncologists during treatment planning.

Project description:ImportanceEarly-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.ObjectiveTo 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.Design, setting, and participantsPD-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.Main outcomes and measuresPD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study.ResultsUsing 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 and relevanceObjective 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.

Project description:BackgroundProgrammed cell death ligand-1 (PD-L1) is a predictive marker of anti-PD-1/PD-L1 therapy response. Intra-tumour heterogeneity of PD-L1 expression has been reported in non-small cell lung cancer (NSCLC), but comprehensive studies regarding the determination of PD-L1 expression in different materials are still lacking. Therefore, we aimed to compare PD-L1 expression in paired tumour samples and in different specimen types.MethodsA total of 1,002 resected NSCLC specimens, 35 biopsy specimens and 54 endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) samples were performed PD-L1 immunohistochemistry (IHC) testing using the 22C3 assay. PD-L1 expression was evaluated using the tumour proportion score (TPS) and categorized into three levels: negative (TPS <1%), low expression (TPS 1-49%) and high expression (TPS ≥50%).ResultsA total of 1,002 resected NSCLC specimens, including 852 adenocarcinomas (ADCs) and 150 squamous cell carcinomas (SCCs); 35 paired biopsy and resected samples; 54 paired cell block and biopsy samples; 53 paired blocks from the same resected tissue and 49 paired primary and metastatic lesion samples were included in this study. Interestingly, high PD-L1 expression was significantly more frequent in poorly differentiated subtypes than in well-differentiated subtypes in the ADC subgroup (P<0.001). In the SCC subgroup, high PD-L1 expression was significantly more associated with the nonkeratinizing type than the keratinizing type (P=0.001). PD-L1 expression differed between cell blocks and matched biopsy specimens (discordance rate =11.1%, 6/54) and between biopsy and matched resected specimens (discordance rate =31.4%, 11/35). PD-L1 expression differed between different paraffin blocks from the same resected specimen (discordance rate =35.8%, 19/53), and the discordance rate of PD-L1 expression between primary tumours and matched lymph node metastases was 28.6% (14/49).ConclusionsDiscordant PD-L1 expression is not uncommon in NSCLC and warrants additional studies and serious consideration when interpreting PD-L1 test results. Initial negative test results may lead to repeat PD-L1 testing in additional samples or the use of a different clone if necessary.

Project description:BackgroundProgrammed cell death ligand-1 (PD-L1) and ligand-2 (PD-L2) interaction with programmed cell death protein-1 (PD-1) represent an immune-inhibiting checkpoint mediating immune evasion and is, accordingly, an important target for blockade-based immunotherapy in cancer. In non-small-cell lung cancer (NSCLC), improved understanding of PD-1 checkpoint blockade-responsive biology and identification of biomarkers for prediction of a clinical response to immunotherapy is warranted. Thus, in the present study, we systematically described PD-L1 and PD-L2 expression correlated genes in NSCLC.MethodsWe performed comparative retrospective analyses to identify PD-L1 and PD-L2 mRNA expression correlated genes in NSCLC. For this, we examined available datasets from the cancer cell line encyclopedia (CCLE) project lung non-small-cell (Lung_NSC) and the cancer genome atlas (TCGA) projects lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC).ResultsAnalysis of the CCLE dataset Lung_NSC identified expression correlation between PD-L1 and PD-L2. Moreover, we identified expression correlation between 489 genes and PD-L1, 191 genes and PD-L2, and 111 genes for both. PD-L1 and PD-L2 also expression correlated in TCGA datasets LUAD and LUSC. In LUAD, we identified expression correlation between 257 genes and PD-L1, 914 genes and PD-L2, and 211 genes for both. In LUSC, we identified expression correlation between 26 genes and PD-L1, 326 genes and PD-L2, and 13 genes for both. Only a few genes expression correlated with PD-L1 and PD-L2 across the CCLE and TCGA datasets. Expression of Interferon signaling-involved genes converged in particular with the expression correlated genes for PD-L1 in Lung_NSC, for PD-L2 in LUSC, and for both PD-L1 and PD-L2 in LUAD. In LUSC, PD-L1, and to a lesser extent PD-L2, expression correlated with chromosome 9p24 localized genes, indicating a chromosome 9p24 topologically associated domain as an important driver of in particular LUSC PD-L1 expression. Expression correlation analyses of the PD-L1 and PD-L2 receptors programmed cell death protein-1 (PD-1), Cluster of differentiation 80 (CD80), and Repulsive guidance molecule B (RGMB) showed that PD-1 and CD80 expression correlated with both PD-L1 and PD-L2 in LUAD. CD80 expression correlated with PD-L2 in LUSC.ConclusionsWe present gene signatures associated with PD-L1 and PD-L2 mRNA expression in NSCLC which could possess importance in relation to understand PD-1 checkpoint blockade-responsive biology and development of gene signature based biomarkers for predicting clinical responses to immunotherapy.

Dataset Information

PD-L1 expression heterogeneity in non-small cell lung cancer: evaluation of small biopsies reliability.

Publications

PD-L1 expression heterogeneity in non-small cell lung cancer: evaluation of small biopsies reliability.

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