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Comprehensive Evaluation of Programmed Death-Ligand 1 Expression in Primary and Metastatic Prostate Cancer

Open ArchivePublished:March 22, 2018DOI:https://doi.org/10.1016/j.ajpath.2018.02.014
      Antibodies targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-L1) interaction have shown clinical activity in multiple cancer types. PD-L1 protein expression is a clinically validated predictive biomarker of response for such therapies. Prior studies evaluating the expression of PD-L1 in primary prostate cancers have reported highly variable rates of PD-L1 positivity. In addition, limited data exist on PD-L1 expression in metastatic castrate-resistant prostate cancer (mCRPC). Here, we determined PD-L1 protein expression by immunohistochemistry using a validated PD-L1–specific antibody (SP263) in a large and representative cohort of primary prostate cancers and prostate cancer metastases. The study included 539 primary prostate cancers comprising 508 acinar adenocarcinomas, 24 prostatic duct adenocarcinomas, 7 small-cell carcinomas, and a total of 57 cases of mCRPC. PD-L1 positivity was low in primary acinar adenocarcinoma, with only 7.7% of cases showing detectable PD-L1 staining. Increased levels of PD-L1 expression were noted in 42.9% of small-cell carcinomas. In mCRPC, 31.6% of cases showed PD-L1–specific immunoreactivity. In conclusion, in this comprehensive evaluation of PD-L1 expression in prostate cancer, PD-L1 expression is rare in primary prostate cancers, but increased rates of PD-L1 positivity were observed in mCRPC. These results will be important for the future clinical development of programmed cell death protein 1/PD-L1–targeting therapies in prostate cancer.
      Antibodies that block the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) interaction currently are approved by the US Food and Drug Administration for multiple tumor types, including melanoma, non–small-cell lung cancer, kidney cancer, bladder cancer, and several other cancers.
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      Materials and Methods

       In Silico Expression Analyses

      In silico expression analyses were performed using publicly available data sets. In brief, raw data for GSE35988
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      The mutational landscape of lethal castration-resistant prostate cancer.
      and processed data for Stand Up To Cancer metastatic prostate cancer
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      Integrative clinical genomics of advanced prostate cancer.
      were obtained from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo) and cBioPortal version 1.12.1
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      The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.
      (http://www.cbioportal.org), respectively, and processed as previously described.
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       Western Blot Analysis

      Western blot analysis confirming the specificity of PD-L1 antibodies were performed as described previously.
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      Anti–PD-L1 antibody (rabbit monoclonal clone sp263, ref 790-4905; Ventana Medical Systems, Inc., Tucson, AZ) was used at a 1:300 dilution.

       Patient Cohorts

      PD-L1 expression was examined using three distinct patient cohorts. The first was a collection of primary prostatic tumors from the Johns Hopkins Hospital (n = 539) represented on 14 tissue microarrays (TMAs); the second was a collection of core biopsy specimens and surgical resection specimens from men with mCRPC from the Johns Hopkins Hospital (n = 28); and the third was a collection of rapid-autopsy tumor samples from men who had died of mCRPC at the University of Michigan (n = 29) represented on two TMAs (Supplemental Table S1).
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      The Johns Hopkins Hospital mCRPC cohort (n = 28) comprised two sample sets. The first set comprised core biopsy specimens of metastatic lesions (n = 9) obtained at baseline from mCRPC patients who were enrolled in a prospective clinical trial investigating the oral Hedgehog pathway inhibitor vismodegib.
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      Details on anatomic location and treatment history for these cases are summarized in Supplemental Table S2. All patients provided written informed consent before undergoing a biopsy, and both studies were conducted under Johns Hopkins' Institutional Review Board–approved protocols.

       Immunohistochemistry

      Based on detailed antibody validation and previously published antibody comparisons,
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      A quantitative comparison of antibodies to programmed cell death 1 ligand 1.
      the rabbit monoclonal clone SP263 (ref 790-4905; Ventana Medical Systems, Inc.) was used on the entire study cohort. A staining protocol, using SP263, was developed on an automated instrument (BenchMark Ultra; Ventana Medical Systems, Inc.). Staining was performed on 4- to 5-μm–thick, formalin-fixed, paraffin-embedded tissue sections from various tumors. Human placenta and tonsil tissue were used for optimization and as positive controls. Slides were deparaffinized, hydrated, and heat-induced antigen retrieval was performed with a high pH buffer (CC1; Ventana Medical Systems, Inc., 56 minutes). PD-L1 antibody (rabbit monoclonal clone SP263, ref 790-4905; Ventana Medical Systems, Inc.) was applied for 16 minutes at room temperature and staining was developed with the Opti-View detection system as per the manufacturer's instructions using diaminobenzidine chromogen (Ventana Medical Systems, Inc.). Slides were counterstained with hematoxylin, dehydrated, and coverslipped. For additional selected cases two additional antibodies were used. Tissue controls (placenta and lymphoid tissue) as well as cell line controls (SR and SN12C) were used with all staining batches.

       Scoring

      Biopsy and resection specimens were evaluated by conventional light microscopy. TMA slides were visualized and annotated using TMAJ software version 3.15 (Tissue Microarray Core Facility, Johns Hopkins University, Baltimore, MD)
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      on images acquired on an Aperio Scanscope AT Turbo scanner (Leica Biosystems, Buffalo Grove, IL) at a magnification of ×20. Note that all scoring was performed by two pathologists (G.G., M.C.H.), and all positive cases were reviewed by an expert genitourinary pathologist (A.M.D.). All three pathologists were blinded to the clinical characteristics of the specimens. Adjacent hematoxylin and eosin–stained sections were reviewed in cases of uncertain tumor involvement. Any PD-L1–specific immunoreactivity on malignant cells (≥1%) was considered positive.

       Microsatellite Instability Analysis

      Mismatch repair status was determined in two primary tumor cases that showed high PD-L1 expression using the Microsatellite Instability Analysis System v1.2 (MSI Multiplex Kit; Promega, Madison, WI) and the ABI 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA). Selected microsatellite sequences that are particularly prone to errors in the setting of mismatch repair were evaluated as described previously.
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      • Duffy S.M.
      • Goldberg R.M.
      • la Chapelle de A.
      • Koshiji M.
      • Bhaijee F.
      • Huebner T.
      • Hruban R.H.
      • Wood L.D.
      • Cuka N.
      • Pardoll D.M.
      • Papadopoulos N.
      • Kinzler K.W.
      • Zhou S.
      • Cornish T.C.
      • Taube J.M.
      • Anders R.A.
      • Eshleman J.R.
      • Vogelstein B.
      • Diaz L.A.
      PD-1 blockade in tumors with mismatch-repair deficiency.

      Results

      To establish robust and reproducible controls for the PD-L1 immunolabeling assay integrated copy number and in silico expression analyses were performed on previously published data from the NCI-60 cell line panel (Figure 1A).
      • Cerami E.
      • Gao J.
      • Dogrusoz U.
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      • Sumer S.O.
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      • Jacobsen A.
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      • Heuer M.L.
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      • Schultz N.
      The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.
      • Reinhold W.C.
      • Sunshine M.
      • Liu H.
      • Varma S.
      • Kohn K.W.
      • Morris J.
      • Doroshow J.
      • Pommier Y.
      CellMiner: a web-based suite of genomic and pharmacologic tools to explore transcript and drug patterns in the NCI-60 cell line set.
      The human lymphoblastic cell line SR showed high levels of PD-L1 (CD274) mRNA expression and showed no copy number alteration of the CD274 locus, in contrast to the human renal cell carcinoma cell line SN12C, which showed copy number loss of the CD274 locus with associated reduced CD274 mRNA expression. Several studies have extensively validated commercially available PD-L1–specific antibodies for immunohistochemistry and showed favorable sensitivity and specificity profiles of clone SP263.
      • Gaule P.
      • Smithy J.W.
      • Toki M.
      • Rehman J.
      • Patell-Socha F.
      • Cougot D.
      • Collin P.
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      • Neumeister V.
      • Rimm D.L.
      A quantitative comparison of antibodies to programmed cell death 1 ligand 1.
      • Sunshine J.C.
      • Nguyen P.L.
      • Kaunitz G.J.
      • Cottrell T.R.
      • Berry S.
      • Esandrio J.
      • Xu H.
      • Ogurtsova A.
      • Bleich K.B.
      • Cornish T.C.
      • Lipson E.J.
      • Anders R.A.
      • Taube J.M.
      PD-L1 expression in melanoma: a quantitative immunohistochemical antibody comparison.
      Based on these prior reports, the specificity of SP263 was characterized further by Western blot analysis of placental tissue and SR and SN12C cell line lysates and identified bands at the predicted molecular weight of PD-L1 in placenta and SR lysates. Corroborating the predictions from in silico expression analyses, lysates from SN12C showed no immunoreactivity (Figure 1B). The specificity of the antibody for immunohistochemical analysis of formalin-fixed, paraffin-embedded tissues was confirmed further by immunostaining formalin-fixed, paraffin-embedded cell blocks containing SR and SN12C cells, showing strong membranous immunoreactivity in SR cells and absence of signal in SN12C cells (Figure 1C).
      Figure thumbnail gr1
      Figure 1Validation of programmed death-ligand 1 (PD-L1) antibody used in the study. A: Integrated in silico analysis of NCI60 cell lines shows high-level PD-L1 expression in the lymphoblastic cell line SR and absence of PD-L1 expression in the renal cell carcinoma cell line SN12C. B: Western blot analysis of placental tissue lysate and SR and SN12C cell line lysates probed with anti–PD-L1 (SP263) shows immunoreactivity only in the predicted molecular weight range of PD-L1. C: Formalin-fixed, paraffin-embedded SN12C and SR cells show absence and strong membranous immunoreactivity for PD-L1, respectively. IHC, immunohistochemistry. Original magnification, ×20. WB, western blot.
      Previous studies evaluating the expression of PD-L1 in primary prostate carcinoma yielded highly variable results.
      • Baas W.
      • Gershburg S.
      • Dynda D.
      • Delfino K.
      • Robinson K.
      • Nie D.
      • Yearley J.H.
      • Alanee S.
      Immune characterization of the programmed death receptor pathway in high risk prostate cancer.
      • Gevensleben H.
      • Dietrich D.
      • Golletz C.
      • Steiner S.
      • Jung M.
      • Thiesler T.
      • Majores M.
      • Stein J.
      • Uhl B.
      • Müller S.
      • Ellinger J.
      • Stephan C.
      • Jung K.
      • Brossart P.
      • Kristiansen G.
      The immune checkpoint regulator PD-L1 is highly expressed in aggressive primary prostate cancer.
      • Ness N.
      • Andersen S.
      • Khanehkenari M.R.
      • Nordbakken C.V.
      • Valkov A.
      • Paulsen E.-E.
      • Nordby Y.
      • Bremnes R.M.
      • Donnem T.
      • Busund L.-T.
      • Richardsen E.
      The prognostic role of immune checkpoint markers programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) in a large, multicenter prostate cancer cohort.
      To determine the prevalence of PD-L1 expression in primary prostate carcinoma, the expression of PD-L1 was evaluated by immunohistochemistry in a total of 539 primary prostate cancers represented by 6137 tissue cores on 14 TMAs (Table 1). Of 508 primary prostatic adenocarcinomas, 39 (7.7%) showed PD-L1 expression as defined by detectable membranous PD-L1 immunoreactivity in 1% or more of the total cellularity of the lesion. Fourteen cases (2.8%) showed immunoreactivity in 5% or more of cells. Two primary adenocarcinomas showed PD-L1 expression in more than 50% of cancer cells (Figure 2, Supplemental Figures S1 and S2). Given the recently described association between PD-L1 expression and mismatch-repair–deficient carcinomas, the microsatellite instability status was determined in these two lesions and no evidence of microsatellite instability was found (Supplemental Figures S1 and S2; Supplemental Tables S3 and S4). There was a trend for higher PD-L1 expression in higher-grade groups (P = 0.08, t-test), and in particular high PD-L1 expression (≥5%) was associated with Gleason patterns 4 and 5. In addition to conventional adenocarcinoma, the PD-L1 expression also was evaluated in a cohort of prostatic duct adenocarcinomas (n = 24) and small-cell carcinomas (n = 7), and immunoreactivity was observed in 4 of 24 prostatic duct adenocarcinomas (16.7%) and 3 of 7 (42.9%) small-cell carcinomas (Supplemental Figure S3). In addition to PD-L1 expression detected in neoplastic cells and tumor-associated immune cells, focal PD-L1 positivity was observed frequently in benign atrophic glands, in particular, in association with a chronic immune infiltrate, as seen previously (Figure 2).
      • Martin A.M.
      • Nirschl T.R.
      • Nirschl C.J.
      • Francica B.J.
      • Kochel C.M.
      • van Bokhoven A.
      • Meeker A.K.
      • Lucia M.S.
      • Anders R.A.
      • DeMarzo A.M.
      • Drake C.G.
      Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.
      Table 1Overview of Clinicopathologic Characteristics of PD-L1–Positive and PD-L1–Negative Tumors
      Tumor typeCases, nPD-L1PD-L1+
      n%n%
      Primary tumors
       Acinar adenocarcinoma50846992.3397.7
      Grade group
      1449.437.7
      213929.61538.5
      312426.4410.3
      46614.125.1
      59620.51538.5
      Stage
      T217938.21641.0
      T3A19441.41230.8
      T3B8117.3923.1
      T40012.6
      Lymph node status
      N043292.13589.7
      N1337.037.7
       Prostatic duct adenocarcinoma242083.3416.7
       Small cell carcinoma7457.1342.9
      Distant metastases (mCRPC)
       Rapid autopsies292069931
       Biopsies281967.9932.1
      Figure thumbnail gr2
      Figure 2Summary of observed programmed death-ligand 1 (PD-L1) immunoreactivity in primary and metastatic prostate cancer. A: Bar graph showing relative frequency (%) of lesions with detectable PD-L1 expression. B: Representative micrograph of primary prostate carcinoma showing no PD-L1 expression. C and D: Representative micrograph of metastatic prostate cancer with moderate- and high-level PD-L1 expression. E: Focal immunoreactivity in benign atrophic prostate epithelium associated with chronic inflammatory infiltrate. Original magnification, ×20 (B–E).
      To evaluate the expression of PD-L1 in the setting of mCRPC, PD-L1 immunoreactivity was determined in mCRPC samples procured by rapid autopsy studies (n = 29), surgical resection specimens (n = 10), and biopsy specimens (n = 18). Note that the majority of patients for whom biopsy or resection specimens were available were treated previously with androgen-deprivation therapy including abiraterone acetate or enzalutamide (Supplemental Table S2). Independent of tumor site, CRPC metastases showed greatly increased incidence of PD-L1 expression, with more than 31% of cases showing PD-L1 positivity (≥1% of tumor cells) and up to 11% showing PD-L1 immunoreactivity in ≥5% of tumor cells (Figure 2 and Supplemental Tables S1 and S2). Importantly, despite different sampling methods of tissue acquisition (core biopsy, surgical resection, post-mortem rapid autopsy) the rate of PD-L1 positivity did not vary (Figure 2). In the autopsy cohort, for which multiple metastatic sites from each case were available for analysis, a high level of heterogeneity was observed in PD-L1 expression in different anatomic sites (Supplemental Table S1). In addition, patchy PD-L1 immunostaining of the neoplastic cell compartment was observed in lesions with high PD-L1 expression with strongly labeled focal cancer cell nests located adjacent to PD-L1–negative cells (Supplemental Figure S4). These immunohistochemical in situ studies were corroborated by re-analysis from publicly available RNA sequencing studies, showing PD-L1 transcript expression in a subset of mCRPC (Supplemental Figure S5).

      Discussion

      Antibody-mediated blockade of the PD-1/PD-L1 axis is effective in multiple solid tumor types.
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      Prior studies have documented only limited or no therapeutic activity of PD-1–blocking therapies in mCRPC; however, several clinical trials currently are investigating the use of such therapies in prostate cancer further. PD-L1 expression on tumor cells has been shown to be associated with response to anti–PD-1 therapies
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      and subsequently has been established as a predictive biomarker that is clinically useful in non–small-cell lung cancer.
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      Although a small subset of tumors show constitutive up-regulation of PD-L1 expression owing to genomic alterations involving the PD-L1 gene locus,
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      the majority of cancers (including prostate cancer) likely show PD-L1 up-regulation as an adaptive response to changes in the immune microenvironment.
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      In general, it has been noted that the reproducibility of biomarker studies evaluating PD-L1 expression has been challenging owing to high interlaboratory and interobserver variation. The use of divergent, often insufficiently validated, antibodies and nonstandardized scoring systems has complicated the interpretation and comparison of previously published studies. These analytic differences likely have contributed to discordant results regarding PD-L1 expression in multiple tumor types. Several groups have assessed the expression of PD-L1 in primary prostate cancer specimens using different monoclonal antibodies, and the rate of PD-L1 positivity varies greatly between different studies. For instance, Gevensleben et al
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      The immune checkpoint regulator PD-L1 is highly expressed in aggressive primary prostate cancer.
      reported up to 61.7% of cases showing moderate to strong PD-L1 expression in a large TMA-based cohort. This report also suggested a strong association between high PD-L1 expression and early biochemical recurrence. Although the investigators performed Western blot and flow cytometry–based specificity control experiments, immunohistochemistry-based validation experiments were not performed and this antibody eventually was discontinued by the manufacturer. Another study reported an even higher rate of PD-L1 positivity of 92% in primary prostate cancer, with 59% of cases showing high PD-L1 expression in tumor cells.
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      Using two broadly validated antibody clones, three recent studies documented tumor-cell–specific PD-L1 expression in 18 of 130 (14%), 2 of 25 (8%), and 3 of 20 (11%) cases, respectively, in treatment-naive primary prostate cancer.
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      Expression of PD-L1 in hormone-naïve and treated prostate cancer patients receiving neoadjuvant abiraterone acetate plus prednisone and leuprolide.
      • Baas W.
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      • Robinson K.
      • Nie D.
      • Yearley J.H.
      • Alanee S.
      Immune characterization of the programmed death receptor pathway in high risk prostate cancer.
      • Martin A.M.
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      • Lucia M.S.
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      • Drake C.G.
      Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.
      It is important to note that although some antibodies were validated using cell line models and immunoblotting, nonspecific off-target reactivity in human tissues may not be excluded. Therefore, careful cross-comparisons of different PD-L1 antibodies in well-defined case cohorts are necessary to establish the reproducibility of immunohistochemical assays used to detect PD-L1 expression.
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      Quantitative assessment of the heterogeneity of PD-L1 expression in non-small-cell lung cancer.
      • Reck M.
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      • Gottfried M.
      • Peled N.
      • Tafreshi A.
      • Cuffe S.
      • O'Brien M.
      • Rao S.
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      • Rangwala R.
      • Brahmer J.R.
      KEYNOTE-024 Investigators
      Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer.
      With increasing mechanistic insights into the molecular basis of response to PD-1/PD-L1 axis blockade, additional biomarkers are being explored that allow for a more accurate prediction of treatment response, at least in a limited subset of patients. One such emerging biomarker for response to immunotherapy is the total number of mutations present in a tumor specimen, that is, the tumor mutational burden.
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      PD-1 blockade in tumors with mismatch-repair deficiency.
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      Along these lines, high response rates to PD-1–targeting therapies were observed in cancers deficient in mismatch repair, which are known to contain exceptionally high numbers of somatic mutations.
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      PD-1 blockade in tumors with mismatch-repair deficiency.
      In the selected cohort, two primary prostate cancer cases with remarkably high PD-L1 expression were identified. These cases did not show evidence of microsatellite instability (Supplemental Figures S1 and S2; Supplemental Tables S1 and S2); however, recent evidence suggests that microsatellite instability PCR using the contemporary markers developed for colorectal carcinoma may be suboptimal for the detection of mismatch repair–deficiency prostate cancers.
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      MSH2 loss in primary prostate cancer.
      It therefore remains to be seen which genetic, epigenetic, or microenvironmental factors contribute to the high-level expression of PD-L1 in a very small subset of primary prostate cancers.
      Interestingly, a recent study showed that prostatic duct adenocarcinoma, which is morphologically distinct from the more common acinar adenocarcinoma of the prostate, frequently shows mismatch repair gene alterations and associated hypermutation phenotypes.
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      Mismatch repair deficiency may be common in ductal adenocarcinoma of the prostate.
      PD-L1 expression therefore was evaluated in a cohort of 24 prostatic duct adenocarcinomas and 5 of 24 cases (21%) showed detectable PD-L1 immunolabeling.
      Based on prior in vitro data, it was suggested that blockade of the androgen axis and conversion to castration resistance may be associated with increased expression of PD-L1.
      • Bishop J.L.
      • Sio A.
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      PD-L1 is highly expressed in enzalutamide resistant prostate cancer.
      Results from a neoadjuvant trial in primary prostate cancer, however, suggested a trend toward lower PD-L1 expression in men receiving neoadjuvant hormonal therapy.
      • Calagua C.
      • Russo J.
      • Sun Y.
      • Schaefer R.
      • Lis R.
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      • Loda M.
      • Taplin M.-E.
      • Balk S.P.
      • Ye H.
      Expression of PD-L1 in hormone-naïve and treated prostate cancer patients receiving neoadjuvant abiraterone acetate plus prednisone and leuprolide.
      To date, however, there are no comprehensive data published on PD-L1 expression in mCRPC. To address this knowledge gap, both biopsy as well as surgical resection specimens of men with mCRPC who were treated with contemporary androgen-deprivation therapy, with the majority of patients receiving enzalutamide or abiraterone acetate, were analyzed (Supplemental Table S2). Importantly, in this cohort of treatment-refractory mCRPC, low-level expression (1% to 5% of tumor cells positive) in 6 of 18 cases (33%) and somewhat higher level expression (>5% of tumor cells positive) in 2 of 18 cases (11%) showing PD-L1 immunolabeling in >5% of tumor cells was found. This finding indicates that PD-L1 expression levels appear to be increased in mCRPC compared with hormone-naive primary carcinoma. In addition, using a cohort of surgical resection specimens of metastases from men with mCRPC for which diagnostic slides sampling an entire cross-section of the lesion were available, a similar distribution of PD-L1 positivity with low-level expression (1% to 5% of cells positive) in three of nine cases (33%) and >5% of tumor cell positivity in one of nine cases was observed. These data suggest that at least in this cohort of similarly treated metastatic lesions, no differences in PD-L1 expression levels were observed between core biopsy and surgical resection material.
      This study had a few limitations. First, the majority of cases evaluated in this study were represented by two to four tissue cores on TMAs. Given the heterogeneity and often limited focal expression of PD-L1 in tumor cells, it is likely that the frequency estimates of PD-L1 positivity may represent an underestimation. Indeed, a high level of intratumor heterogeneity in PD-L1 expression was seen in cases of resected distant metastases (Supplemental Figure S4). In addition, the majority of TMAs were constructed from archival formalin-fixed, paraffin-embedded tissues, and it is unclear if long-term storage of tissue material may result in a loss of PD-L1 immunoreactivity. However, in this study, TMAs containing recently collected formalin-fixed, paraffin-embedded tissues were included, which showed no difference in the level of PD-L1 immunoreactivity compared with older archival tissues (data not shown). Importantly though, a large number of recent biopsy specimens from distant soft-tissue metastases were included, which are most representative of tissue samples used for clinical decision making. Immunoreactivity was scored in epithelial cancer cells and signals from tumor-associated immune cells were not counted. Neoplastic epithelial cells were identified based on histomorphologic features and co-immunolabeling was not performed to identify individual cell lineages. It is important to note that based on these criteria the vast majority of immunostaining was restricted to cancer cells. However, it cannot be excluded that a small fraction of PD-L1 immunoreactivity was contributed by immune cells in close proximity to cancer cells. Because of technical challenges, biopsy specimens from bony lesions were not included. It therefore remains to be shown if metastases to the bone show a different PD-L1 expression pattern.
      In conclusion, this study presents the most comprehensive evaluation of PD-L1 expression in both primary and metastatic prostate cancer. Furthermore, this study documents that PD-L1 expression is present in a small subset of primary prostate cancer, and is increased in mCRPC.

      Supplemental Data

      • Supplemental Figure S1

        No evidence of microsatellite instability (MSI) in case 1531. A: Micrograph of programmed death-ligand 1 (PD-L1) immunostaining showing a high-level of PD-L1 expression. B: Microsatellite marker profile for case 1531 shows stable microsatellites (Supplemental Table S3). Original magnification, ×20.

      • Supplemental Figure S2

        No evidence of microsatellite instability (MSI) in case 15749. A: Micrograph of programmed death-ligand 1 (PD-L1) immunostaining showing a high-level of PD-L1 expression in the case. B: Microsatellite marker profile for case 15749 shows stable microsatellites (Supplemental Table S4). Original magnification, ×20.

      • Supplemental Figure S3

        Representative micrographs of small-cell (A and B) and prostatic duct carcinoma (C and D) showing focal programmed death-ligand 1 (PD-L1) immunoreactivity (B and D). Original magnification, ×20.

      • Supplemental Figure S4

        Intratumoral heterogeneity of programmed death-ligand 1 (PD-L1) expression. Representative micrograph of a lung metastasis resection specimen showing patchy PD-L1 immunoreactivity with focally strong PD-L1 expression (arrows) in neoplastic cells and low-level/absence of PD-L1 immunolabeling in adjacent tumor cells (arrowheads).

      • Supplemental Figure S5

        mRNA expression of programmed death-ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) in castrate-resistant prostate cancer (CRPC). Waterfall plot shows expression distribution of CD274 (PD-L1) and CD279 (PD-1) in two publicly available data sets containing expression information of mCRPC patients: University of Michigan

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