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The evolving role of PD-L1 testing in patients with metastatic urothelial carcinoma

  • Thomas Powles
    Correspondence
    Corresponding authors at: Centre for Experimental Cancer Medicine, Barts Cancer Institute, Queen Mary University of London, Barts Health NHS Trust, London EC1A 7BE, UK (T. Powles). Harvard Medical School, Boston, MA, USA (J. Bellmunt).
    Affiliations
    Barts Health NHS Trust, London, UK
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  • Author Footnotes
    1 Address: Oncology Companion Diagnostics Unit, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
    Jill Walker
    Footnotes
    1 Address: Oncology Companion Diagnostics Unit, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
    Affiliations
    AstraZeneca, Cambridge, UK
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  • Author Footnotes
    1 Address: Oncology Companion Diagnostics Unit, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
    J. Andrew Williams
    Footnotes
    1 Address: Oncology Companion Diagnostics Unit, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
    Affiliations
    AstraZeneca, Cambridge, UK
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  • Joaquim Bellmunt
    Correspondence
    Corresponding authors at: Centre for Experimental Cancer Medicine, Barts Cancer Institute, Queen Mary University of London, Barts Health NHS Trust, London EC1A 7BE, UK (T. Powles). Harvard Medical School, Boston, MA, USA (J. Bellmunt).
    Affiliations
    PSMAR-IMIM Research Institute in Barcelona and Harvard Medical School, Boston, MA, USA
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  • Author Footnotes
    1 Address: Oncology Companion Diagnostics Unit, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
Open AccessPublished:November 11, 2019DOI:https://doi.org/10.1016/j.ctrv.2019.101925

      Highlights

      • PD-L1 has emerged as an important cancer biomarker and target for immunotherapy in UC.
      • PD-L1 is frequently expressed on tumor cells and tumor-infiltrating immune cells.
      • Diagnostic assays are available, each with different scoring cutoffs to detect PD-L1.
      • Despite lack of assay standardization, PD-L1 expression can be reproducibly scored.
      • PD-L1 testing will increasingly guide treatment and should be discussed and offered to patients with UC.

      Abstract

      Immune checkpoint inhibitors targeting the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway improve clinical outcomes in patients with locally advanced/metastatic urothelial carcinoma (UC). PD-L1 complementary or companion diagnostic assays are now available for anti–PD-1 and anti–PD-L1 antibodies and these assays enable testing at diagnosis. The role of PD-L1 testing in UC is, however, the subject of much discussion within the medical community, particularly in light of recent restrictions on recruitment of PD-L1–low patients in clinical trials of atezolizumab and pembrolizumab as first-line therapy, and the European Medicines Agency and US Food and Drug Administration limiting use of these agents as first-line therapy in cisplatin-ineligible patients to those with high PD-L1 expression. We explore the evolving evidence for PD-L1 expression testing in UC and the role of PD-L1 expression in both tumor cells and tumor-infiltrating immune cells. We review clinical data on the prognostic and predictive value of PD-L1 expression in response to anti–PD-1/PD-L1 agents as first- and second-line therapy, considering issues such as the differences among complementary diagnostic assays in terms of the type of cells scored, antibodies used, and cutoff values. We consider how PD-L1 testing fits into decision-making and the potential of emerging biomarkers in UC. We conclude that, based on the scientific rationale for its use and evidence from clinical trials, PD-L1 testing provides enriched information on the patients most likely to benefit from immune checkpoint blockade and should be routinely offered to patients with metastatic UC.

      Keywords

      Background and introduction

      Urothelial carcinomas (UC) are immunogenic tumors against which immunotherapeutic approaches have shown substantial clinical benefit [
      • Siefker-Radtke A.
      • Curti B.
      Immunotherapy in metastatic urothelial carcinoma: focus on immune checkpoint inhibition.
      ]. Despite their immunogenicity, tumors can evade the immune system by a variety of means, including altered expression of proteins that act within the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway to suppress T-cell antitumor activity [
      • Siefker-Radtke A.
      • Curti B.
      Immunotherapy in metastatic urothelial carcinoma: focus on immune checkpoint inhibition.
      ,
      • Davarpanah N.N.
      • Yuno A.
      • Trepel J.B.
      • Apolo A.B.
      Immunotherapy: a new treatment paradigm in bladder cancer.
      ,
      • Pardoll D.M.
      The blockade of immune checkpoints in cancer immunotherapy.
      ]. PD-L1 is highly expressed on the surface of activated T cells in response to inflammation or infection, acting as an “immune checkpoint” to maintain self-tolerance and modulate physiological immune responses. Tumors co-opt this pathway as a major mechanism of immune resistance, such that PD-L1 expression on tumor cells (TCs) and/or tumor-infiltrating immune cells (ICs) can play a critical role in immunosuppression and evasion of host immune responses by tumors [
      • Siefker-Radtke A.
      • Curti B.
      Immunotherapy in metastatic urothelial carcinoma: focus on immune checkpoint inhibition.
      ,
      • Davarpanah N.N.
      • Yuno A.
      • Trepel J.B.
      • Apolo A.B.
      Immunotherapy: a new treatment paradigm in bladder cancer.
      ,
      • Pardoll D.M.
      The blockade of immune checkpoints in cancer immunotherapy.
      ].
      Understanding the role of immune checkpoints in immune evasion has led to the development of therapies that target the PD-1/PD-L1 pathway. Five immune checkpoint inhibitors shown to improve outcomes in patients with locally advanced/metastatic UC have been approved [
      • Balar A.V.
      • Castellano D.
      • O'Donnell P.H.
      • Grivas P.
      • Vuky J.
      • Powles T.
      • et al.
      First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study.
      ,
      • Balar A.V.
      • Galsky M.D.
      • Rosenberg J.E.
      • Powles T.
      • Petrylak D.P.
      • Bellmunt J.
      • et al.
      Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial.
      ,
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      ,
      • Patel M.R.
      • Ellerton J.
      • Infante J.R.
      • Agrawal M.
      • Gordon M.
      • Aljumaily R.
      • et al.
      Avelumab in metastatic urothelial carcinoma after platinum failure (JAVELIN Solid Tumor): pooled results from two expansion cohorts of an open-label, phase 1 trial.
      ,
      • Powles T.
      • O'Donnell P.H.
      • Massard C.
      • Arkenau H.T.
      • Friedlander T.W.
      • Hoimes C.J.
      • et al.
      Efficacy and safety of durvalumab in locally advanced or metastatic urothelial carcinoma: updated results from a phase 1/2 open-label study.
      ,
      • Rosenberg J.E.
      • Hoffman-Censits J.
      • Powles T.
      • van der Heijden M.S.
      • Balar A.V.
      • Necchi A.
      • et al.
      Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.
      ,
      • Sharma P.
      • Callahan M.K.
      • Bono P.
      • Kim J.
      • Spiliopoulou P.
      • Calvo E.
      • et al.
      Nivolumab monotherapy in recurrent metastatic urothelial carcinoma (CheckMate 032): a multicentre, open-label, two-stage, multi-arm, phase 1/2 trial.
      ,
      • Sharma P.
      • Retz M.
      • Siefker-Radtke A.
      • Baron A.
      • Necchi A.
      • Bedke J.
      • et al.
      Nivolumab in metastatic urothelial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial.
      ]. Indications for anti–PD-L1 (durvalumab, atezolizumab, and avelumab) and anti–PD-1 (nivolumab, pembrolizumab) antibodies in UC are shown in Table 1 [

      Bavencio (avelumab) [summary of product characteristics]. Amsterdam (The Netherlands): Merck Europe B.V.; 2017. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/004338/WC500236647.pdf [accessed August 24, 2018].

      ,

      Bavencio (avelumab) injection [prescribing information]. New York (NY): EMD Serono Inc. and Pfizer Inc; 2017. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/761049s000lbl.pdf [accessed August 24, 2018].

      ,

      Imfinzi (durvalumab) [Summary of product characteristics]. Södertälje (Sweden): AstraZeneca AB; 2018. Available at: https://www.ema.europa.eu/documents/product-information/imfizi-epar-product-information_en.pdf [accessed November 14, 2018].

      ,

      Imfinzi (durvalumab) injection [prescribing information]. Wilmington (DE): AstraZeneca Pharmaceuticals; 2018. Available at: https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm555930.htm [accessed August 24, 2018].

      ,

      Keytruda (pembrolizumab) [prescribing information]. Whitehouse Station (NJ): Merck & Co., Inc.; 2018. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/125514s043lbl.pdf [accessed Dec 5, 2018].

      ,

      Keytruda (pembrolizumab) [summary of product characteristics]. Haarlem (The Netherlands): Merck Sharp & Dohme B.V.; 2018. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/003820/WC500190990.pdf [accessed Dec 5, 2018].

      ,

      Opdivo (nivolumab) injection [prescribing information]. Princeton (NJ): Bristol-Myers Squibb Company; 2018. Available at: https://packageinserts.bms.com/pi/pi_opdivo.pdf [accessed Dec 5, 2018].

      ,

      Opdivo (nivolumab) injection [summary of product characteristics]. UK: Bristol-Myers Squibb Pharma EEIG Uxbridge; 2018. Available at: https://www.ema.europa.eu/documents/product-information/opdivo-epar-product-information_en.pdf [accessed Dec 5, 2018].

      ,

      PD-L1 IHC 22C3 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2018. Available at: https://www.agilent.com/cs/library/packageinsert/public/P03951%20rev%2004.pdf [accessed Dec 3, 2018].

      ,

      PD-L1 IHC 28-8 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf15/P150025S003C.pdf [accessed Dec 5, 2018].

      ,

      Tecentriq (atezolizumab) injection [summary of product characteristics]. Grenzach-Wyhlen (Germany): Roche Pharma AG; 2017. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/004143/WC500235778.pdf [accessed Dec 5, 2018].

      ,

      Tecentriq (atezolizumab) injection [prescribing information]. South San Francisco (CA): Genentech Inc.; 2018. Available at: https://www.gene.com/download/pdf/tecentriq_prescribing.pdf [accessed Dec 5, 2018].

      ,

      VENTANA PD-L1 (SP142) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2016. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160002c.pdf [accessed Dec 5, 2018].

      ,

      VENTANA PD-L1 (SP263) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160046C.pdf [accessed Dec 5, 2018].

      ].
      Table 1Indications for anti-PD-1/PD-L1 agents in the United States and Europe and complementary or companion diagnostic assays.
      US, FDAEurope, EMAAssay
      UCOther indications
      See Prescribing Information for details of [13,15,16,18,23].
      UCOther indications
      See Summary of Product Characteristics for details of indications [12,14,17,19,22].
      PD-1
      NivolumabPatients with locally advanced or metastatic UC who have disease progression during or following platinum-containing chemotherapy or have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy

      Opdivo (nivolumab) injection [prescribing information]. Princeton (NJ): Bristol-Myers Squibb Company; 2018. Available at: https://packageinserts.bms.com/pi/pi_opdivo.pdf [accessed Dec 5, 2018].

      NSCLC; melanoma; renal cell carcinoma; classical Hodgkin lymphoma; HNSCC; colorectal cancer; hepatocellular carcinoma

      Opdivo (nivolumab) injection [prescribing information]. Princeton (NJ): Bristol-Myers Squibb Company; 2018. Available at: https://packageinserts.bms.com/pi/pi_opdivo.pdf [accessed Dec 5, 2018].

      Locally advanced unresectable or metastatic UC in adults after failure of prior platinum-containing therapy

      Opdivo (nivolumab) injection [summary of product characteristics]. UK: Bristol-Myers Squibb Pharma EEIG Uxbridge; 2018. Available at: https://www.ema.europa.eu/documents/product-information/opdivo-epar-product-information_en.pdf [accessed Dec 5, 2018].

      Melanoma; NSCLC; renal cell carcinoma; classical Hodgkin lymphoma; HNSCC

      Opdivo (nivolumab) injection [summary of product characteristics]. UK: Bristol-Myers Squibb Pharma EEIG Uxbridge; 2018. Available at: https://www.ema.europa.eu/documents/product-information/opdivo-epar-product-information_en.pdf [accessed Dec 5, 2018].

      Complementary Dx (US, FDA)

      PD-L1 IHC 28–8 pharmDx

      PD-L1 IHC 28-8 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf15/P150025S003C.pdf [accessed Dec 5, 2018].

      PembrolizumabPatients with locally advanced or metastatic UC who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (CPS ≥ 10), or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status, patients who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy

      Keytruda (pembrolizumab) [prescribing information]. Whitehouse Station (NJ): Merck & Co., Inc.; 2018. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/125514s043lbl.pdf [accessed Dec 5, 2018].

      NSCLC; melanoma; HNSCC; classical Hodgkin lymphoma; microsatellite instability high cancer

      Keytruda (pembrolizumab) [prescribing information]. Whitehouse Station (NJ): Merck & Co., Inc.; 2018. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/125514s043lbl.pdf [accessed Dec 5, 2018].

      Locally advanced or metastatic UC in adults who have received prior platinum-containing chemotherapy and in adults who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 with a combined positive score ≥ 10

      Keytruda (pembrolizumab) [summary of product characteristics]. Haarlem (The Netherlands): Merck Sharp & Dohme B.V.; 2018. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/003820/WC500190990.pdf [accessed Dec 5, 2018].

      Melanoma, NSCLC, classical Hodgkin lymphoma

      Keytruda (pembrolizumab) [summary of product characteristics]. Haarlem (The Netherlands): Merck Sharp & Dohme B.V.; 2018. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/003820/WC500190990.pdf [accessed Dec 5, 2018].

      Companion Dx (US, FDA)

      PD-L1 IHC 22C3 pharmDx
      PD-L1 IHC 22C3 pharmDx is an FDA-approved companion diagnostic used to select patients with NSCLC and gastric cancer for pembrolizumab treatment.

      PD-L1 IHC 22C3 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2018. Available at: https://www.agilent.com/cs/library/packageinsert/public/P03951%20rev%2004.pdf [accessed Dec 3, 2018].

      PD-L1
      AtezolizumabPatients with locally advanced or metastatic UC who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (PD-L1 stained IC covering ≥ 5% of the tumor area), as determined by an FDA-approved test, or are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status, or who have disease progression during or following any platinum-containing chemotherapy, or within 12 months of neoadjuvant or adjuvant chemotherapy

      Tecentriq (atezolizumab) injection [prescribing information]. South San Francisco (CA): Genentech Inc.; 2018. Available at: https://www.gene.com/download/pdf/tecentriq_prescribing.pdf [accessed Dec 5, 2018].

      NSCLC

      Tecentriq (atezolizumab) injection [prescribing information]. South San Francisco (CA): Genentech Inc.; 2018. Available at: https://www.gene.com/download/pdf/tecentriq_prescribing.pdf [accessed Dec 5, 2018].

      Patients with locally advanced or metastatic UC after prior platinum-containing chemotherapy or who are considered cisplatin ineligible and whose tumors have PD-L1 expression ≥ 5%

      Tecentriq (atezolizumab) injection [summary of product characteristics]. Grenzach-Wyhlen (Germany): Roche Pharma AG; 2017. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/004143/WC500235778.pdf [accessed Dec 5, 2018].

      NSCLC

      Tecentriq (atezolizumab) injection [summary of product characteristics]. Grenzach-Wyhlen (Germany): Roche Pharma AG; 2017. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/004143/WC500235778.pdf [accessed Dec 5, 2018].

      Complementary Dx (2L)

      VENTANA PD-L1 (SP142)

      VENTANA PD-L1 (SP142) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2016. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160002c.pdf [accessed Dec 5, 2018].

      AvelumabPatients with locally advanced or metastatic UC who have disease progression during or following platinum-containing chemotherapy or who have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy

      Bavencio (avelumab) injection [prescribing information]. New York (NY): EMD Serono Inc. and Pfizer Inc; 2017. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/761049s000lbl.pdf [accessed August 24, 2018].

      Merkel cell carcinoma

      Bavencio (avelumab) injection [prescribing information]. New York (NY): EMD Serono Inc. and Pfizer Inc; 2017. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/761049s000lbl.pdf [accessed August 24, 2018].

      Merkel cell carcinoma

      Bavencio (avelumab) [summary of product characteristics]. Amsterdam (The Netherlands): Merck Europe B.V.; 2017. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/004338/WC500236647.pdf [accessed August 24, 2018].

      No FDA-approved assay
      DurvalumabPatients with locally advanced or metastatic UC who have disease progression during or following platinum-containing chemotherapy or who have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy

      Imfinzi (durvalumab) injection [prescribing information]. Wilmington (DE): AstraZeneca Pharmaceuticals; 2018. Available at: https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm555930.htm [accessed August 24, 2018].

      NSCLCNSCLC

      Imfinzi (durvalumab) [Summary of product characteristics]. Södertälje (Sweden): AstraZeneca AB; 2018. Available at: https://www.ema.europa.eu/documents/product-information/imfizi-epar-product-information_en.pdf [accessed November 14, 2018].

      VENTANA PD-L1 (SP263)

      VENTANA PD-L1 (SP263) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160046C.pdf [accessed Dec 5, 2018].

      EMA, European Medicines Agency; FDA, US Food and Drug Administration; HNSCC, squamous cell carcinoma of the head and neck; NSCLC, non-small cell lung cancer; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand-1; UC, urothelial carcinoma.
      a See Prescribing Information for details of

      Bavencio (avelumab) injection [prescribing information]. New York (NY): EMD Serono Inc. and Pfizer Inc; 2017. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/761049s000lbl.pdf [accessed August 24, 2018].

      ,

      Imfinzi (durvalumab) injection [prescribing information]. Wilmington (DE): AstraZeneca Pharmaceuticals; 2018. Available at: https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm555930.htm [accessed August 24, 2018].

      ,

      Keytruda (pembrolizumab) [prescribing information]. Whitehouse Station (NJ): Merck & Co., Inc.; 2018. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/125514s043lbl.pdf [accessed Dec 5, 2018].

      ,

      Opdivo (nivolumab) injection [prescribing information]. Princeton (NJ): Bristol-Myers Squibb Company; 2018. Available at: https://packageinserts.bms.com/pi/pi_opdivo.pdf [accessed Dec 5, 2018].

      ,

      Tecentriq (atezolizumab) injection [prescribing information]. South San Francisco (CA): Genentech Inc.; 2018. Available at: https://www.gene.com/download/pdf/tecentriq_prescribing.pdf [accessed Dec 5, 2018].

      .
      b See Summary of Product Characteristics for details of indications

      Bavencio (avelumab) [summary of product characteristics]. Amsterdam (The Netherlands): Merck Europe B.V.; 2017. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/004338/WC500236647.pdf [accessed August 24, 2018].

      ,

      Imfinzi (durvalumab) [Summary of product characteristics]. Södertälje (Sweden): AstraZeneca AB; 2018. Available at: https://www.ema.europa.eu/documents/product-information/imfizi-epar-product-information_en.pdf [accessed November 14, 2018].

      ,

      Keytruda (pembrolizumab) [summary of product characteristics]. Haarlem (The Netherlands): Merck Sharp & Dohme B.V.; 2018. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/003820/WC500190990.pdf [accessed Dec 5, 2018].

      ,

      Opdivo (nivolumab) injection [summary of product characteristics]. UK: Bristol-Myers Squibb Pharma EEIG Uxbridge; 2018. Available at: https://www.ema.europa.eu/documents/product-information/opdivo-epar-product-information_en.pdf [accessed Dec 5, 2018].

      ,

      Tecentriq (atezolizumab) injection [summary of product characteristics]. Grenzach-Wyhlen (Germany): Roche Pharma AG; 2017. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/004143/WC500235778.pdf [accessed Dec 5, 2018].

      .
      c PD-L1 IHC 22C3 pharmDx is an FDA-approved companion diagnostic used to select patients with NSCLC and gastric cancer for pembrolizumab treatment.
      To minimize the tendency to administer questionable treatments (eg, vinflunine in UC), regulatory agencies have approved these five immune-oncology (IO) agents, often based on a small effect size, signal of activity from earlier trials, and surrogate endpoints, including the objective response rate (ORR) stratified per PD-L1. Each of these IO agents has been approved based on data from single-arm phase 2 trials. Only two have been tested in randomized trials: atezolizumab and pembrolizumab. The approval of multiple immune checkpoint inhibitors has represented a significant advance in this setting, as substantial unmet needs are present for pretreated or cisplatin-ineligible patients, where options are narrow and the possibility to complete a full course of treatment is quite low.
      However, response to anti–PD-L1 and anti–PD-1 agents is not observed in all patients, such that novel (including molecular) predictors of response would be beneficial to inform therapeutic choices [
      • Siefker-Radtke A.
      • Curti B.
      Immunotherapy in metastatic urothelial carcinoma: focus on immune checkpoint inhibition.
      ,
      • Miao D.
      • Margolis C.A.
      • Vokes N.I.
      • Liu D.
      • Taylor-Weiner A.
      • Wankowicz S.M.
      • et al.
      Genomic correlates of response to immune checkpoint blockade in microsatellite-stable solid tumors.
      ]. PD-L1 is increasingly being used as a biomarker to identify patients likely to benefit most from immune checkpoint blockade. For a biomarker to be of value, three criteria need to be met. First, it is important to understand what the biomarker is measuring, and how it is being measured. Second, clinical utility must be demonstrated. Finally, the biomarker must be practicable (ie, it should be reliable; reproducible; avoid additional, invasive procedures; be cost-effective; usable by different laboratories in different countries) [
      • Holland R.L.
      What makes a good biomarker?.
      ,
      • Ioannidis J.P.A.
      • Bossuyt P.M.M.
      Waste, leaks, and failures in the biomarker pipeline.
      ]. Based on the mechanism of action of anti–PD-L1 therapies and their observed benefit, PD-L1 expression is a logical biomarker for response in UC. Investigations into the clinical utility of PD-L1 in UC have followed a similar path in non-small cell lung cancer (NSCLC), where early studies investigated the impact of PD-L1 expression on objective response to IO therapy in all-comer populations, with later phase 3 studies using stratification or prospective selection to confirm clinical utility [
      • Büttner R.
      • Gosney J.R.
      • Guldhammer Skov B.
      • Adam J.
      • Motoi N.
      • Bloom Kenneth J.
      • et al.
      Programmed death-ligand 1 immunohistochemistry testing: a review of analytical assays and clinical implementation in non–small-cell lung cancer.
      ].
      The value of understanding PD-L1 expression has been reinforced by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) restrictions to PD-L1-high patients based on emerging data from ongoing trials [

      US Food and Drug Administration. List of cleared or approved companion diagnostic devices (in vitro and imaging tools); 2018. https://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm301431.htm [accessed Jan 3, 2018].

      ].
      Two open-label phase 3 trials are evaluating first-line anti–PD-1/PD-L1 agents with or without chemotherapy versus chemotherapy alone in previously untreated patients with locally advanced or metastatic UC: the IMvigor130 trial of atezolizumab and the KEYNOTE-361 trial of pembrolizumab [

      ClinicalTrials.gov. Study of pembrolizumab with or without platinum-based combination chemotherapy versus chemotherapy alone in urothelial carcinoma (MK-3475-361/KEYNOTE-361); 2018. https://clinicaltrials.gov/ct2/show/NCT02853305 [accessed Dec 5, 2018].

      ,

      ClinicalTrials.gov. Study of atezolizumab as monotherapy and in combination with platinum-based chemotherapy in participants with untreated locally advanced or metastatic urothelial carcinoma (IMvigor130); 2018. https://clinicaltrials.gov/ct2/show/study/NCT02807636?show_locs=Y#locn [accessed Dec 5, 2018].

      ]. Early reviews by the studies’ independent data monitoring committees suggest that patients in the checkpoint inhibitor monotherapy arms of both trials with PD-L1 low status had adverse outcomes [

      European Medicines Agency. EMA restricts use of Keytruda and Tecentriq in bladder cancer; 2018. http://www.ema.europa.eu/ema/index.jsp?curl=pages/news_and_events/news/2018/05/news_detail_002964.jsp&mid=WC0b01ac058004d5c1 [accessed Dec 5, 2018].

      ,

      US Food and Drug Administration. Keytruda (pembrolizumab) or tecentriq (atezolizumab): FDA alerts health care professionals and investigators: FDA statement - decreased survival in some patients in clinical trials associated with monotherapy; 2018. https://www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm608253.htm [accessed Dec 5, 2018].

      ]. The FDA issued an alert to health care professionals that they should be aware that the populations enrolled in the ongoing clinical trials were eligible for platinum-containing chemotherapy, and therefore differ from those enrolled in the trials that led to the accelerated approvals of the two agents [

      US Food and Drug Administration. Keytruda (pembrolizumab) or tecentriq (atezolizumab): FDA alerts health care professionals and investigators: FDA statement - decreased survival in some patients in clinical trials associated with monotherapy; 2018. https://www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm608253.htm [accessed Dec 5, 2018].

      ]. The FDA has also revised the indication of these two agents in the first-line setting. Pembrolizumab and atezolizumab are indicated for the treatment of patients with locally advanced/metastatic UC who are not eligible for cisplatin-containing chemotherapy and who have high PD-L1 expression. For pembrolizumab, this is defined as combined positive score (CPS: the percentage of PD-L1–expressing TCs and ICs relative to the total number of TCs) ≥ 10 as measured by the PD-L1 immunohistochemical (IHC) 22C3 pharmDx assay. For atezolizumab, this is defined as ICs ≥ 5% of the tumor area as measured by the VENTANA PD-L1 SP142 assay [

      US Food and Drug Administration. FDA updates prescribing information for keytruda and tecentriq; 2018. https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm617378.htm [accessed Dec 5, 2018].

      ]. Similarly, the EMA has restricted approval of pembrolizumab and atezolizumab as first-line treatment of cisplatin-ineligible patients with metastatic UC to those with high PD-L1 expression (CPS ≥ 10 for pembrolizumab and ICs ≥ 5% for atezolizumab) [

      European Medicines Agency. EMA restricts use of Keytruda and Tecentriq in bladder cancer; 2018. http://www.ema.europa.eu/ema/index.jsp?curl=pages/news_and_events/news/2018/05/news_detail_002964.jsp&mid=WC0b01ac058004d5c1 [accessed Dec 5, 2018].

      ].
      The ability of PD-L1 to predict response to therapy has been investigated in various tumor types using different PD-L1 complementary or companion diagnostic assays (Table 1), antibodies, scoring algorithms, and cutoffs to measure PD-L1 expression in TCs or ICs, or both (Fig. 1), which can lead to variability in results [
      • Diggs L.P.
      • Hsueh E.C.
      Utility of PD-L1 immunohistochemistry assays for predicting PD-1/PD-L1 inhibitor response.
      ,
      • Liu D.
      • Wang S.
      • Bindeman W.
      Clinical applications of PD-L1 bioassays for cancer immunotherapy.
      ]. For example, in advanced NSCLC, high versus low PD-L1 expression is associated with greater likelihood of treatment benefit with anti–PD-1/PD-L1 therapy, and requirements for testing for PD-L1 expression using a companion diagnostic are included in indications for pembrolizumab (expression on ≥50% of TCs for first-line therapy and ≥1% TCs for patients who have received at least one prior chemotherapy regimen) [

      Keytruda (pembrolizumab) [prescribing information]. Whitehouse Station (NJ): Merck & Co., Inc.; 2018. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/125514s043lbl.pdf [accessed Dec 5, 2018].

      ,

      Keytruda (pembrolizumab) [summary of product characteristics]. Haarlem (The Netherlands): Merck Sharp & Dohme B.V.; 2018. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/003820/WC500190990.pdf [accessed Dec 5, 2018].

      ,
      • Brody R.
      • Zhang Y.
      • Ballas M.
      • Siddiqui M.K.
      • Gupta P.
      • Barker C.
      • et al.
      PD-L1 expression in advanced NSCLC: Insights into risk stratification and treatment selection from a systematic literature review.
      ]. In contrast, in advanced renal cell carcinoma, the benefit of nivolumab in patients previously treated with anti-angiogenic agents is not linked to TC PD-L1 expression (≥1%), but it does appear to be of some predictive value in terms of response to combination therapy with ipilimumab in the frontline setting [

      Opdivo (nivolumab) injection [prescribing information]. Princeton (NJ): Bristol-Myers Squibb Company; 2018. Available at: https://packageinserts.bms.com/pi/pi_opdivo.pdf [accessed Dec 5, 2018].

      ,
      • Motzer R.J.
      • Escudier B.
      • McDermott D.F.
      • George S.
      • Hammers H.J.
      • Srinivas S.
      • et al.
      Nivolumab versus everolimus in advanced renal-cell carcinoma.
      ,
      • Motzer R.J.
      • Tannir N.M.
      • McDermott D.F.
      • Aren Frontera O.
      • Melichar B.
      • Choueiri T.K.
      • et al.
      Nivolumab plus ipilimumab versus sunitinib in advanced renal-cell carcinoma.
      ]. Thus, the predictive value of PD-L1 expression in UC cannot be extrapolated from data from other tumors; findings must be carefully evaluated in the tumor of interest.
      Figure thumbnail gr1
      Fig. 1Comparison of different PD-L1 scoring algorithms used in UC. PD-L1 testing in conjunction with pembrolizumab in UC (PharmDx 22C3) is based on the CPS of TCs and ICs with PD-L1 staining as a proportion of total TC area; with nivolumab (PharmDx 28-8), the proportion of TCs with PD-L1 staining is assessed; with durvalumab (VENTANA SP263), the proportion of ICs with PD-L1 staining within the total IC area and the proportion of TCs with PD-L1 staining is used; with atezolizumab (VENTANA SP142), the proportion of the tumor area occupied by PD-L1 stained ICs is measured. CPS, combined positive score; IC, tumor-infiltrating immune cell; PD-L1, programed cell death ligand 1; TC, tumor cell.
      In this review, we evaluate the prognostic value of PD-L1 expression levels in UC, and the rationale and evidence for PD-L1 expression testing to predict response to anti–PD-1/PD-L1 therapy. We aim to explore the relative roles of TC versus IC versus CPS PD-L1 expression in suppression of antitumor immune responses, as this may be an important consideration in understanding PD-L1 as a predictive biomarker in metastatic UC. We also consider emerging biomarkers in UC, and how PD-L1 testing might fit into future decision-making matrices.

      PD-L1 expression in UC

      PD-L1 is expressed in TCs as an adaptive response to inflammatory signals, interferon-γ (IFN-γ) in particular, produced by an active antitumor immune response; correlations between TC PD-L1 expression and lymphocytic infiltration suggest the occurrence of a negative feedback loop in which IFN-γ induces TC PD-L1 expression, which then suppresses the activity of PD-1–positive T cells [
      • Pardoll D.M.
      The blockade of immune checkpoints in cancer immunotherapy.
      ].
      PD-L1 may also be expressed by ICs in UC [
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      ,
      • Powles T.
      • Eder J.P.
      • Fine G.D.
      • Braiteh F.S.
      • Loriot Y.
      • Cruz C.
      • et al.
      MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer.
      ]. PD-L1 expression on ICs appears to be induced by two extrinsic pathways involving CD4+T cells, one IFN-γ-dependent and one IFN-γ-independent [
      • Noguchi T.
      • Ward J.P.
      • Gubin M.M.
      • Arthur C.D.
      • Lee S.H.
      • Hundal J.
      • et al.
      Temporally distinct PD-L1 expression by tumor and host cells contributes to immune escape.
      ]. The relative importance of TC versus IC PD-L1 expression may be greater in highly immunogenic tumors [
      • Juneja V.R.
      • McGuire K.A.
      • Manguso R.T.
      • LaFleur M.W.
      • Collins N.
      • Haining W.N.
      • et al.
      PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity.
      ], such as UC, which is associated with a high mutational load [
      • Siefker-Radtke A.
      • Curti B.
      Immunotherapy in metastatic urothelial carcinoma: focus on immune checkpoint inhibition.
      ,
      • Davarpanah N.N.
      • Yuno A.
      • Trepel J.B.
      • Apolo A.B.
      Immunotherapy: a new treatment paradigm in bladder cancer.
      ]. Preclinical data suggest that while TC PD-L1 expression is transient, IC expression is prolonged, so that the majority of PD-L1 in the immunosuppressive tumor microenvironment may be provided by ICs [
      • Noguchi T.
      • Ward J.P.
      • Gubin M.M.
      • Arthur C.D.
      • Lee S.H.
      • Hundal J.
      • et al.
      Temporally distinct PD-L1 expression by tumor and host cells contributes to immune escape.
      ].

      Prognostic value of PD-L1 expression in UC

      Several observational studies have examined TC PD-L1 and/or IC PD-L1 expression in UC, using different antibodies and cutoffs (Table 2) [
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      ,
      • Bellmunt J.
      • Mullane S.A.
      • Werner L.
      • Fay A.P.
      • Callea M.
      • Leow J.J.
      • et al.
      Association of PD-L1 expression on tumor-infiltrating mononuclear cells and overall survival in patients with urothelial carcinoma.
      ,
      • Boorjian S.A.
      • Sheinin Y.
      • Crispen P.L.
      • Farmer S.A.
      • Lohse C.M.
      • Kuntz S.M.
      • et al.
      T-cell coregulatory molecule expression in urothelial cell carcinoma: clinicopathologic correlations and association with survival.
      ,
      • Faraj S.F.
      • Munari E.
      • Guner G.
      • Taube J.
      • Anders R.
      • Hicks J.
      • et al.
      Assessment of tumoral PD-L1 expression and intratumoral CD8+ T cells in urothelial carcinoma.
      ,
      • Inman B.A.
      • Sebo T.J.
      • Frigola X.
      • Dong H.
      • Bergstralh E.J.
      • Frank I.
      • et al.
      PD-L1 (B7–H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression.
      ,
      • Krabbe L.M.
      • Heitplatz B.
      • Preuss S.
      • Hutchinson R.C.
      • Woldu S.L.
      • Singla N.
      • et al.
      Prognostic value of PD-1 and PD-L1 expression in patients with high grade upper tract urothelial carcinoma.
      ,
      • Nakanishi J.
      • Wada Y.
      • Matsumoto K.
      • Azuma M.
      • Kikuchi K.
      • Ueda S.
      Overexpression of B7–H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers.
      ,
      • Powles T.
      • Durán I.
      • van der Heijden M.S.
      • Loriot Y.
      • Vogelzang N.J.
      • De Giorgi U.
      • et al.
      Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.
      ,
      • Wang Q.
      • Liu F.
      • Liu L.
      Prognostic significance of PD-L1 in solid tumor: an updated meta-analysis.
      ,
      • Wang Y.
      • Zhuang Q.
      • Zhou S.
      • Hu Z.
      • Lan R.
      Costimulatory molecule B7–H1 on the immune escape of bladder cancer and its clinical significance.
      ,
      • Wu P.
      • Wu D.
      • Li L.
      • Chai Y.
      • Huang J.
      PD-L1 and survival in solid tumors: a meta-analysis.
      ,
      • Xylinas E.
      • Robinson B.D.
      • Kluth L.A.
      • Volkmer B.G.
      • Hautmann R.
      • Kufer R.
      • et al.
      Association of T-cell co-regulatory protein expression with clinical outcomes following radical cystectomy for urothelial carcinoma of the bladder.
      ,
      • Zhang B.
      • Yu W.
      • Feng X.
      • Zhao Z.
      • Fan Y.
      • Meng Y.
      • et al.
      Prognostic significance of PD-L1 expression on tumor cells and tumor-infiltrating mononuclear cells in upper tract urothelial carcinoma.
      ]. Most of these studies have assessed TC PD-L1, and, although some found no association between TC PD-L1 expression level and survival [
      • Bellmunt J.
      • Mullane S.A.
      • Werner L.
      • Fay A.P.
      • Callea M.
      • Leow J.J.
      • et al.
      Association of PD-L1 expression on tumor-infiltrating mononuclear cells and overall survival in patients with urothelial carcinoma.
      ,
      • Faraj S.F.
      • Munari E.
      • Guner G.
      • Taube J.
      • Anders R.
      • Hicks J.
      • et al.
      Assessment of tumoral PD-L1 expression and intratumoral CD8+ T cells in urothelial carcinoma.
      ], the majority of observational studies indicate worse prognosis in patients with high TC PD-L1 expression (Table 2) [
      • Boorjian S.A.
      • Sheinin Y.
      • Crispen P.L.
      • Farmer S.A.
      • Lohse C.M.
      • Kuntz S.M.
      • et al.
      T-cell coregulatory molecule expression in urothelial cell carcinoma: clinicopathologic correlations and association with survival.
      ,
      • Inman B.A.
      • Sebo T.J.
      • Frigola X.
      • Dong H.
      • Bergstralh E.J.
      • Frank I.
      • et al.
      PD-L1 (B7–H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression.
      ,
      • Krabbe L.M.
      • Heitplatz B.
      • Preuss S.
      • Hutchinson R.C.
      • Woldu S.L.
      • Singla N.
      • et al.
      Prognostic value of PD-1 and PD-L1 expression in patients with high grade upper tract urothelial carcinoma.
      ,
      • Nakanishi J.
      • Wada Y.
      • Matsumoto K.
      • Azuma M.
      • Kikuchi K.
      • Ueda S.
      Overexpression of B7–H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers.
      ,
      • Wang Y.
      • Zhuang Q.
      • Zhou S.
      • Hu Z.
      • Lan R.
      Costimulatory molecule B7–H1 on the immune escape of bladder cancer and its clinical significance.
      ,
      • Xylinas E.
      • Robinson B.D.
      • Kluth L.A.
      • Volkmer B.G.
      • Hautmann R.
      • Kufer R.
      • et al.
      Association of T-cell co-regulatory protein expression with clinical outcomes following radical cystectomy for urothelial carcinoma of the bladder.
      ,
      • Zhang B.
      • Yu W.
      • Feng X.
      • Zhao Z.
      • Fan Y.
      • Meng Y.
      • et al.
      Prognostic significance of PD-L1 expression on tumor cells and tumor-infiltrating mononuclear cells in upper tract urothelial carcinoma.
      ]. Studies looking at IC PD-L1 expression appear to support a positive prognostic association (Table 2) [
      • Bellmunt J.
      • Mullane S.A.
      • Werner L.
      • Fay A.P.
      • Callea M.
      • Leow J.J.
      • et al.
      Association of PD-L1 expression on tumor-infiltrating mononuclear cells and overall survival in patients with urothelial carcinoma.
      ,
      • Zhang B.
      • Yu W.
      • Feng X.
      • Zhao Z.
      • Fan Y.
      • Meng Y.
      • et al.
      Prognostic significance of PD-L1 expression on tumor cells and tumor-infiltrating mononuclear cells in upper tract urothelial carcinoma.
      ]. While observational studies play an important role in research, there are major methodological issues in the design and analytical phases of these studies including selection bias and confounding [
      • Boyko E.J.
      Observational research—opportunities and limitations.
      ]. Furthermore, variations in the timing, origin, and quality of the UC tissue collected are likely to be relevant.
      Table 2PD-L1 expression as a prognostic biomarker in UC.
      Study populationnAssayTC/IC cutoffKey findingsPrognostic value
      Individual studies
      Bellmunt et al.
      • Bellmunt J.
      • Mullane S.A.
      • Werner L.
      • Fay A.P.
      • Callea M.
      • Leow J.J.
      • et al.
      Association of PD-L1 expression on tumor-infiltrating mononuclear cells and overall survival in patients with urothelial carcinoma.
      Metastatic UC treated with platinum-based chemotherapy100IHC, 405.9A11 mAbTC ≥ 5%

      ICs scored as absent (0), focal (1), mild (2), moderate (3), or severe (4), with cutoff 0/1 vs 2–4
      Median OS not correlated with TC PD-L1 expression (P = 0.45)No association

      TC PD-L1 and median
      IC PD-L1 expression score 2–4 vs 0/1 correlated with longer median OS (95% CI), 23 months (12 - not reached) vs 12 months (9–16) (P = 0.04 univariate, P = 0.0007 multivariate)Positively prognostic association

      IC PD-L1 and median OS
      Boorjian et al.
      • Boorjian S.A.
      • Sheinin Y.
      • Crispen P.L.
      • Farmer S.A.
      • Lohse C.M.
      • Kuntz S.M.
      • et al.
      T-cell coregulatory molecule expression in urothelial cell carcinoma: clinicopathologic correlations and association with survival.
      UC with radical cystectomy, followed for >10 years318IHC, 5H1TC ≥ 5%TC PD-L1 ≥ 5% vs < 5% associated with increased risk of death (HR 3.18, 95% CI, 1.74–5.79; P < 0.001) in patients with organ-confined diseaseNegatively prognostic association

      TC PD-L1 and death
      Faraj et al.
      • Faraj S.F.
      • Munari E.
      • Guner G.
      • Taube J.
      • Anders R.
      • Hicks J.
      • et al.
      Assessment of tumoral PD-L1 expression and intratumoral CD8+ T cells in urothelial carcinoma.
      UC with cystectomy, median follow-up 31.5 months56IHC, 5H1TC ≥ 5%No association between PD-L1 TC ≥ 5% and OSNo association

      PD-L1 TC and OS
      Inman et al.
      • Inman B.A.
      • Sebo T.J.
      • Frigola X.
      • Dong H.
      • Bergstralh E.J.
      • Frank I.
      • et al.
      PD-L1 (B7–H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression.
      High-risk UC280IHC, 5H1TC ≥ 1%TC PD-L1 expression prognostic factor of stage progression (OR = 2.20, P = 0.012)Negatively prognostic association

      TC PD-L1 and stage progression
      Krabbe et al.
      • Krabbe L.M.
      • Heitplatz B.
      • Preuss S.
      • Hutchinson R.C.
      • Woldu S.L.
      • Singla N.
      • et al.
      Prognostic value of PD-1 and PD-L1 expression in patients with high grade upper tract urothelial carcinoma.
      High-grade upper tract UC, median follow-up 37.0 months423IHC, E1L3NTC ≥ 1%TC PD-L1 expression independent prognostic factor (multivariate analysis) in organ-confined disease for longer recurrence-free survival (HR = 0.2, 95% CI, 0.06–0.98; P = 0.046) and OS (HR = 0.3, 95% CI, 0.11–0.63; P = 0.003)Negatively prognostic association

      TC PD-L1 and recurrence-free survival and OS
      Nakanishi et al.
      • Nakanishi J.
      • Wada Y.
      • Matsumoto K.
      • Azuma M.
      • Kikuchi K.
      • Ueda S.
      Overexpression of B7–H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers.
      UC65IHC, MIH1TC ≥ 12.2%TC PD-L1 expression associated with higher frequency of post-operative recurrence (42% vs 81%, P = 0.026) and worse 5-year survival (55% vs 100%, P = 0.041)

      Negative independent prognostic factor in multivariate analysis (P = 0.021)
      Negatively prognostic association

      TC PD-L1 and post-operative recurrence and 5-year survival
      Wang et al.
      • Wang Y.
      • Zhuang Q.
      • Zhou S.
      • Hu Z.
      • Lan R.
      Costimulatory molecule B7–H1 on the immune escape of bladder cancer and its clinical significance.
      Bladder cancer50IHCTC > 10%PD-L1 expression associated with pathological grade, clinical stage and recurrence (P < 0.05)Negative prognostic association

      TC PD-L1 expression and pathological grade, clinical stage and recurrence
      OS lower in PD-L1 positive than PD-L1 negative (P = 0.02); independent prognostic factor on multivariate analysisNegatively prognostic association

      TC PD-L1 positive and OS
      Xylinas et al.
      • Xylinas E.
      • Robinson B.D.
      • Kluth L.A.
      • Volkmer B.G.
      • Hautmann R.
      • Kufer R.
      • et al.
      Association of T-cell co-regulatory protein expression with clinical outcomes following radical cystectomy for urothelial carcinoma of the bladder.
      UC with radical cystectomy and lymphadenectomy302IHCTC ≥ 5%TC PD-L1 expression associated with increased risk of mortality in organ-confined disease (HR = 1.93, 95% CI, 1.09–3.43; P = 0.02 univariate analysis)Negatively prognostic association

      TC PD-L1 and mortality risk
      Zhang et al.
      • Zhang B.
      • Yu W.
      • Feng X.
      • Zhao Z.
      • Fan Y.
      • Meng Y.
      • et al.
      Prognostic significance of PD-L1 expression on tumor cells and tumor-infiltrating mononuclear cells in upper tract urothelial carcinoma.
      Upper-tract UC162IHC, E1L3NTC ≥ 5%

      IC scored 1–3; 2 or 3 considered positive
      TC PD-L1 expression independent prognostic factor of shorter cancer-specific survival (multivariate analysis, HR = 2.572, 95% CI, 1.233–5.364; P = 0.012)Negatively prognostic association

      TC PD-L1 and cancer-specific survival
      IC PD-L1 expression independent prognostic factor of longer cancer-specific survival (multivariate analysis, HR = 0.324, 95% CI, 0.114–0.921; P = 0.034)Positively prognostic association

      IC PD-L1 and cancer-specific survival
      Meta-analyses
      Wang et al.
      • Wang Q.
      • Liu F.
      • Liu L.
      Prognostic significance of PD-L1 in solid tumor: an updated meta-analysis.
      Four studies in UC402TCWorse OS: HR = 2.24 (95% CI, 1.61–3.12); P < 0.0009Negatively prognostic association

      TC and OS
      Two studies in UC161TCNo association with PFS/DFS: HR = 1.27 (95% CI, 0.58–2.77); P = 0.548No association

      TC and PFS/DFS
      Wu et al.
      • Wu P.
      • Wu D.
      • Li L.
      • Chai Y.
      • Huang J.
      PD-L1 and survival in solid tumors: a meta-analysis.
      Four studies in UC378TCWorse 3-year OS: OR = 3.74 (95% CI, 1.14–12.32); P = 0.03Negatively prognostic association

      PD-L1 and 3-year OS
      No association with 5-year OS: OR = 2.61 (95% CI,1.00–6.85); P = 0.05No association

      PD-L1 and 5-year OS
      Clinical trial data
      Bellmunt et al. (KEYNOTE-045)
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      Metastatic UC that recurred or progressed after platinum-based chemotherapy542PharmDx 22C3CPS ≥ 10
      Combined positive score, % of PD-L1-expressing TCs and ICs expressed relative to the total number of TCs.
      PD-L1 expression appeared to be associated with poorer prognosis relative to overall population in pembrolizumab- and chemotherapy-treated patients: OS (95% CI) (months), pembrolizumab, overall 10.3 (8.0–11.8) vs 8.0 (5.0–12.3) in CPS ≥ 10 group; chemotherapy, overall 7.4 (6.1–8.3) vs 5.2 (4.0–7.4) in CPS ≥ 10 groupNegatively prognostic association

      PD-L1 and OS
      Powles et al. (IMvigor211)
      • Powles T.
      • Durán I.
      • van der Heijden M.S.
      • Loriot Y.
      • Vogelzang N.J.
      • De Giorgi U.
      • et al.
      Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.
      Metastatic UC with progression after platinum-based therapy931VENTANA SP142IC0, <1%

      IC1, ≥1% < 5%

      IC2/3, ≥5%
      IC2/3 PD-L1 expression appeared to be associated with improved ORR in comparison with ITT population in atezolizumab and chemotherapy groups: ORR (95% CI) %:

      atezolizumab, IC2/3, 23.0 (15.6–31.9) vs ITT 13.4 (10.5–16.9)

      Chemotherapy, IC2/3 21.6 (14.5–30.2) vs ITT 13.4 (10.5–16.9)
      Positively prognostic association

      IC2/3 PD-L1 and ORR
      CI, confidence interval; CPS, combined positive score; DFS, disease-free survival; HR, hazard ratio; IC, tumor-infiltrating immune cell; IHC, immunohistochemistry; ITT, intent-to-treat; OR, odds ratio; ORR, objective response rate; OS, overall survival; PD-L1, programmed cell death ligand-1; PFS, progression-free survival; TC, tumor cell; UC, urothelial carcinoma.
      Quality criteria for inclusion: minimum number, must use a validated PD-L1 test.
      a Combined positive score, % of PD-L1-expressing TCs and ICs expressed relative to the total number of TCs.
      Meta-analyses of studies in solid tumors including studies in metastatic UC that recurred or progressed after platinum-based therapy have provided mixed results with regard to the prognostic value of tumor PD-L1 expression in UC, with association between worse overall survival (OS) but no association with progression-free/disease-free survival in one analysis, and association with worse 3-year but not 5-year OS in another (Table 2) [
      • Wang Q.
      • Liu F.
      • Liu L.
      Prognostic significance of PD-L1 in solid tumor: an updated meta-analysis.
      ,
      • Wu P.
      • Wu D.
      • Li L.
      • Chai Y.
      • Huang J.
      PD-L1 and survival in solid tumors: a meta-analysis.
      ].
      Randomized, stratified clinical studies remain the gold standard when conducting research [
      • Spieth P.M.
      • Kubasch A.S.
      • Penzlin A.I.
      • Illigens B.M.
      • Barlinn K.
      • Siepmann T.
      Randomized controlled trials - a matter of design.
      ]. Analysis of data from such trials of anti–PD-1/PD-L1 therapy can provide information about the prognostic value of PD-L1 expression. Two studies that were conducted more recently than the meta-analyses referred to above provide somewhat contradictory information about prognostic value. In KEYNOTE-045, in both pembrolizumab- and chemotherapy-treated patients, PD-L1 expression with CPS ≥ 10 was associated with worse OS in comparison with the overall population (Table 2) [
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      ]. In contrast, data from IMvigor211 suggest that IC PD-L1 expression ≥5% is associated with improved ORR in comparison with the intent-to-treat population, in both atezolizumab and chemotherapy groups (Table 2) [
      • Powles T.
      • Durán I.
      • van der Heijden M.S.
      • Loriot Y.
      • Vogelzang N.J.
      • De Giorgi U.
      • et al.
      Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.
      ]. Although these findings are seemingly contradictory, it is important to note that differing PD-L1 testing approaches were used in the two trials (TCs and ICs in KEYNOTE-045 and ICs only in IMvigor211).
      Although the current evidence from these observational studies, meta-analyses, and randomized clinical trials is far from clear, it does suggest that higher IC PD-L1 expression may be linked to better prognosis in metastatic UC, potentially as a reflection of an active antitumor immune response. Higher TC PD-L1 expression appears to be linked to worse prognosis, a conclusion supported by meta-analyses including other tumor types [
      • Wang Q.
      • Liu F.
      • Liu L.
      Prognostic significance of PD-L1 in solid tumor: an updated meta-analysis.
      ,
      • Wu P.
      • Wu D.
      • Li L.
      • Chai Y.
      • Huang J.
      PD-L1 and survival in solid tumors: a meta-analysis.
      ].

      PD-L1 expression in prediction of response to anti–PD-1/PD-L1 monotherapy in metastatic UC

      Data on the correlations between clinical outcomes and TC and/or IC PD-L1 expression in metastatic UC are available from numerous clinical trials (Table 3) [
      • Balar A.V.
      • Castellano D.
      • O'Donnell P.H.
      • Grivas P.
      • Vuky J.
      • Powles T.
      • et al.
      First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study.
      ,
      • Balar A.V.
      • Galsky M.D.
      • Rosenberg J.E.
      • Powles T.
      • Petrylak D.P.
      • Bellmunt J.
      • et al.
      Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial.
      ,
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      ,
      • Patel M.R.
      • Ellerton J.
      • Infante J.R.
      • Agrawal M.
      • Gordon M.
      • Aljumaily R.
      • et al.
      Avelumab in metastatic urothelial carcinoma after platinum failure (JAVELIN Solid Tumor): pooled results from two expansion cohorts of an open-label, phase 1 trial.
      ,
      • Powles T.
      • O'Donnell P.H.
      • Massard C.
      • Arkenau H.T.
      • Friedlander T.W.
      • Hoimes C.J.
      • et al.
      Efficacy and safety of durvalumab in locally advanced or metastatic urothelial carcinoma: updated results from a phase 1/2 open-label study.
      ,
      • Rosenberg J.E.
      • Hoffman-Censits J.
      • Powles T.
      • van der Heijden M.S.
      • Balar A.V.
      • Necchi A.
      • et al.
      Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.
      ,
      • Sharma P.
      • Callahan M.K.
      • Bono P.
      • Kim J.
      • Spiliopoulou P.
      • Calvo E.
      • et al.
      Nivolumab monotherapy in recurrent metastatic urothelial carcinoma (CheckMate 032): a multicentre, open-label, two-stage, multi-arm, phase 1/2 trial.
      ,
      • Sharma P.
      • Retz M.
      • Siefker-Radtke A.
      • Baron A.
      • Necchi A.
      • Bedke J.
      • et al.
      Nivolumab in metastatic urothelial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial.
      ,

      PD-L1 IHC 22C3 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2018. Available at: https://www.agilent.com/cs/library/packageinsert/public/P03951%20rev%2004.pdf [accessed Dec 3, 2018].

      ,

      PD-L1 IHC 28-8 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf15/P150025S003C.pdf [accessed Dec 5, 2018].

      ,

      VENTANA PD-L1 (SP142) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2016. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160002c.pdf [accessed Dec 5, 2018].

      ,

      VENTANA PD-L1 (SP263) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160046C.pdf [accessed Dec 5, 2018].

      ,
      • Powles T.
      • Durán I.
      • van der Heijden M.S.
      • Loriot Y.
      • Vogelzang N.J.
      • De Giorgi U.
      • et al.
      Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.
      ], with most data coming from trials of therapies in the second-line setting. As noted earlier, interpretation of the results of these studies is complicated by the use of unique assay antibodies and particular assay formats for specific agents. Despite this, several trials have suggested that patients with PD-L1–expressing TCs and/or ICs have a greater response to PD-1/PD-L1 inhibition, while others have not demonstrated a correlation between expression and outcomes (Table 3) [
      • Siefker-Radtke A.
      • Curti B.
      Immunotherapy in metastatic urothelial carcinoma: focus on immune checkpoint inhibition.
      ,
      • Davarpanah N.N.
      • Yuno A.
      • Trepel J.B.
      • Apolo A.B.
      Immunotherapy: a new treatment paradigm in bladder cancer.
      ].
      Table 3PD-L1 expression as a biomarker predictive of response to anti-PD-1/anti-PD-L1 therapy in clinical trials in metastatic UC.
      PhasenAssayPD-L1 cutoffGroup (n)ORR (95% CI), %Predictive of response: Yes/No
      Second-line
      Atezolizumab

      IMvigor210

      NCT02108652
      • Rosenberg J.E.
      • Hoffman-Censits J.
      • Powles T.
      • van der Heijden M.S.
      • Balar A.V.
      • Necchi A.
      • et al.
      Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.
      2310VENTANA SP142

      VENTANA PD-L1 (SP142) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2016. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160002c.pdf [accessed Dec 5, 2018].

      IC ≥ 5%

      IC ≥ 1%–<5%

      IC < 1%
      Overall (310)15 (11–19)Yes

      Higher IC PD-L1 expression associated with response
      IC ≥ 5% (1 0 0)26 (18–36)
      IC ≥ 1% (2 0 7)18 (13–24)
      IC < 1% (103)8 (3–15)
      Atezolizumab

      IMvigor211

      NCT02302807
      • Powles T.
      • Durán I.
      • van der Heijden M.S.
      • Loriot Y.
      • Vogelzang N.J.
      • De Giorgi U.
      • et al.
      Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.
      3931VENTANA SP142

      VENTANA PD-L1 (SP142) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2016. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160002c.pdf [accessed Dec 5, 2018].

      IC ≥ 5%

      IC ≥ 1%–<5%

      IC < 1%
      IC ≥ 5% Atezolizumab (116)23.0 (15.6–31.9)No

      Higher IC PD-L1 expression associated with response to both atezolizumab and chemotherapy
      IC ≥ 5% Chemotherapy (118)21.6 (14.5–30.2)
      Avelumab

      JAVELIN

      NCT01772004
      • Patel M.R.
      • Ellerton J.
      • Infante J.R.
      • Agrawal M.
      • Gordon M.
      • Aljumaily R.
      • et al.
      Avelumab in metastatic urothelial carcinoma after platinum failure (JAVELIN Solid Tumor): pooled results from two expansion cohorts of an open-label, phase 1 trial.
      1b249
      242 patients with progression after platinum-based chemotherapy, seven cisplatin-ineligible patients.
      PharmDx 73–10TC ≥ 5%Overall (161)17 (11–24)Yes

      Higher TC PD-L1 expression associated with response
      TC ≥ 5% (63)24.0 (14–36)
      TC < 5% (76)13 (7–23)
      Durvalumab

      NCT01693562
      • Powles T.
      • O'Donnell P.H.
      • Massard C.
      • Arkenau H.T.
      • Friedlander T.W.
      • Hoimes C.J.
      • et al.
      Efficacy and safety of durvalumab in locally advanced or metastatic urothelial carcinoma: updated results from a phase 1/2 open-label study.
      1/2191
      182 patients with progression after platinum-based chemotherapy, nine cisplatin-ineligible patients.
      VENTANA SP263

      VENTANA PD-L1 (SP263) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160046C.pdf [accessed Dec 5, 2018].

      TC or IC ≥ 25%Overall (191)17.8 (12.7–24.0)Yes

      Lower TC/IC PD-L1 expression associated with reduced response
      TC/IC ≥ 25% (98)27.6 (19.0–37.5)
      TC&IC < 25% (79)5.1 (1.4–12.5)
      Nivolumab

      CheckMate 032

      NCT01928394
      • Sharma P.
      • Callahan M.K.
      • Bono P.
      • Kim J.
      • Spiliopoulou P.
      • Calvo E.
      • et al.
      Nivolumab monotherapy in recurrent metastatic urothelial carcinoma (CheckMate 032): a multicentre, open-label, two-stage, multi-arm, phase 1/2 trial.
      1/278PharmDx 28-8

      PD-L1 IHC 28-8 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf15/P150025S003C.pdf [accessed Dec 5, 2018].

      TC ≥ 1%Overall (78)24.4 (15.3–35.4)No

      Response not associated with higher TC PD-L1 expression
      TC ≥ 1% (25)24.0 (9.4–45.1)
      TC < 1% (42)26.2 (13.9–42.0)
      Nivolumab

      CheckMate 275
      • Sharma P.
      • Retz M.
      • Siefker-Radtke A.
      • Baron A.
      • Necchi A.
      • Bedke J.
      • et al.
      Nivolumab in metastatic urothelial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial.
      2265PharmDx 28-8

      PD-L1 IHC 28-8 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2017. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf15/P150025S003C.pdf [accessed Dec 5, 2018].

      TC ≥ 1% or TC ≥ 5%Overall (265)19.6 (15.0–24.9)No

      Response not associated with TC PD-L1 expression

      Therapy provided meaningful benefit irrespective of PD-L1 expression

      No optimal PD-L1 expression cutoff
      TC ≥ 5% (81)28.4 (18.9–39.5)
      TC ≥ 1% (122)23.8 (16.5–32.3)
      TC < 1% (143)16.1 (10.5–23.1)
      Pembrolizumab

      KEYNOTE-045

      NCT02256436
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      3542PharmDx 22C3

      PD-L1 IHC 22C3 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2018. Available at: https://www.agilent.com/cs/library/packageinsert/public/P03951%20rev%2004.pdf [accessed Dec 3, 2018].

      CPS ≥ 10
      Combined positive score, % of PD-L1-expressing TCs and ICs expressed relative to the total number of TCs.
      Pembrolizumab (270)
      ORR by PD-L1 expression status is not provided. Among patients with CPS ≥ 10, pembrolizumab offered significant benefits over chemotherapy in terms of median OS (see text for details).
      21.1 (16.4–26.5)No

      Benefit of pembrolizumab over chemotherapy was not associated with CPS PD-L1 expression level
      Chemotherapy (272)11.4 (7.9–15.8)
      First-line
      Atezolizumab

      IMvigor210

      NCT02108652
      • Balar A.V.
      • Galsky M.D.
      • Rosenberg J.E.
      • Powles T.
      • Petrylak D.P.
      • Bellmunt J.
      • et al.
      Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial.
      2119VENTANA SP142

      VENTANA PD-L1 (SP142) assay [prescribing information]. Tucson (AZ): Ventana Medical Systems, Inc; 2016. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160002c.pdf [accessed Dec 5, 2018].

      IC ≥ 5%

      IC ≥ 1%–<5%

      IC < 1%
      Overall (119)23 (16–31)No

      No statistically significant enrichment of response by IC PD-L1 expression
      IC ≥ 5% (32)28 (14–47)
      IC ≥ 1% (80)24 (15–35)
      IC ≥ 1%–<5% (48)21 (10–35)
      IC < 1% (39)21 (9–36)
      Pembrolizumab

      KEYNOTE-052

      NCT02335424
      • Balar A.V.
      • Castellano D.
      • O'Donnell P.H.
      • Grivas P.
      • Vuky J.
      • Powles T.
      • et al.
      First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study.
      2370PharmDx 22C3

      PD-L1 IHC 22C3 pharmDx [prescribing information]. Carpinteria (CA): Dako North America; 2018. Available at: https://www.agilent.com/cs/library/packageinsert/public/P03951%20rev%2004.pdf [accessed Dec 3, 2018].

      CPS ≥ 10Overall (370)
      Centrally assessed objective response; in the validation set (n = 265), ORR (95% CI) was 39% (28–50) in 80 patients with CPS ≥ 10, 20 (14–28) in 139 patients with a CPS of ≥ 1–< 10, and 11 (4–24) in 46 patients with a CPS < 1.
      24 (20–29)Yes

      CPS ≥ 10 associated with higher frequency of response
      CPS ≥ 10 (110)38 (29–48)
      CI, confidence interval; CPS, combined positive score; DFS, disease-free survival; HR, hazard ratio; IC, tumor-infiltrating immune cell; IHC, immunohistochemistry; ITT, intent-to-treat; OR, odds ratio; ORR, objective response rate; OS, overall survival; PD-L1, programmed cell death ligand-1; TC, tumor cell; UC, urothelial carcinoma.
      Ordered according to trial design: from control arms of randomized trials (eg, Imvigor211, KN045, to single arm or observational studies).
      a 242 patients with progression after platinum-based chemotherapy, seven cisplatin-ineligible patients.
      b 182 patients with progression after platinum-based chemotherapy, nine cisplatin-ineligible patients.
      c Combined positive score, % of PD-L1-expressing TCs and ICs expressed relative to the total number of TCs.
      d ORR by PD-L1 expression status is not provided. Among patients with CPS ≥ 10, pembrolizumab offered significant benefits over chemotherapy in terms of median OS (see text for details).
      e Centrally assessed objective response; in the validation set (n = 265), ORR (95% CI) was 39% (28–50) in 80 patients with CPS ≥ 10, 20 (14–28) in 139 patients with a CPS of ≥ 1–< 10, and 11 (4–24) in 46 patients with a CPS < 1.

      Atezolizumab

      In IMvigor210, a phase 2 trial of atezolizumab, higher levels of IC PD-L1 expression (VENTANA SP142 assay), ICs ≥ 5% versus ICs < 1%, were associated with ORRs of 26% versus 8% (Table 3). In contrast, TC PD-L1 expression was low, and did not show an association with objective response [
      • Rosenberg J.E.
      • Hoffman-Censits J.
      • Powles T.
      • van der Heijden M.S.
      • Balar A.V.
      • Necchi A.
      • et al.
      Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.
      ].
      In the phase 3, randomized controlled trial, IMvigor211, enrolling more than 900 patients (Table 3), the study design was based on the hypothesis that the efficacy of atezolizumab would be associated with IC PD-L1 expression; the primary endpoint of improvement in OS versus chemotherapy in patients with ≥5% IC PD-L1 expression was not met (median OS [95% confidence interval (CI)] was 11.1 [8.6–15.5] months with atezolizumab and 10.6 [8.4–12.2] months with chemotherapy, hazard ratio (HR) [95% CI] 0.87 [0.63–1.21], P = 0.41), precluding further statistical analysis. Overexpression of PD-L1 on ICs was associated with a more favorable outcome with both atezolizumab and chemotherapy, consistent with a positive prognostic value of higher IC PD-L1 expression, as discussed above [
      • Bellmunt J.
      • Mullane S.A.
      • Werner L.
      • Fay A.P.
      • Callea M.
      • Leow J.J.
      • et al.
      Association of PD-L1 expression on tumor-infiltrating mononuclear cells and overall survival in patients with urothelial carcinoma.
      ]. However, the IC PD-L1 biomarker enriched for responses regardless of treatment arm, negating predictive value for response to anti–PD-L1 therapy [
      • Powles T.
      • Durán I.
      • van der Heijden M.S.
      • Loriot Y.
      • Vogelzang N.J.
      • De Giorgi U.
      • et al.
      Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.
      ].
      More recently, the IMvigor130 trial, which investigated atezolizumab alone or in combination with standard chemotherapy showed a progression-free survival (PFS) advantage for the chemotherapy + immune therapy regardless of biomarker expression. However, the monotherapy arm appeared to out-perform chemotherapy in biomarker positives. As no formal statistical testing has occurred owing to the trial design, conclusions cannot be drawn. Atezolizumab is, however, still FDA-approved for the indication of IMvigor211 (which was intended to be a confirmatory trial).

      Avelumab

      In a pooled analysis of patients with metastatic UC (post-platinum or cisplatin-ineligible) treated with avelumab in the phase 1 JAVELIN solid tumor study, TC PD-L1 expression ≥ 5% (PharmDx 73–10 assay) was associated with an ORR of 24%, versus 13% with TC PD-L1 < 5% (Table 3) [
      • Patel M.R.
      • Ellerton J.
      • Infante J.R.
      • Agrawal M.
      • Gordon M.
      • Aljumaily R.
      • et al.
      Avelumab in metastatic urothelial carcinoma after platinum failure (JAVELIN Solid Tumor): pooled results from two expansion cohorts of an open-label, phase 1 trial.
      ]. This association was weaker than in an earlier analysis of 44 patients from this study, in which ORR was 54% with TC PD-L1 ≥ 5% versus 4% with TC PD-L1 < 5% [
      • Apolo A.B.
      • Infante J.R.
      • Balmanoukian A.
      • Patel M.R.
      • Wang D.
      • Kelly K.
      • et al.
      Avelumab, an anti-programmed death-ligand 1 antibody, in patients with refractory metastatic urothelial carcinoma: results from a multicenter, phase Ib study.
      ].

      Durvalumab

      In a planned analysis of the UC cohort in a phase 1/2 study of durvalumab in solid tumors (Study 1108), where PD-L1 expression status was based on a cutoff of expression on ≥25% of TCs or ICs (VENTANA SP263 assay), ORR was 17.8% overall (95% CI, 12.7–24.0), 27.6% (95% CI, 19.0–37.5) in the subgroup with high PD-L1 expression, and 5.1% (95% CI, 1.4–12.5) in the subgroup with PD-L1 expression on <25% of TCs and ICs (Table 3) [
      • Powles T.
      • O'Donnell P.H.
      • Massard C.
      • Arkenau H.T.
      • Friedlander T.W.
      • Hoimes C.J.
      • et al.
      Efficacy and safety of durvalumab in locally advanced or metastatic urothelial carcinoma: updated results from a phase 1/2 open-label study.
      ].

      Nivolumab

      In CheckMate 032, a phase 1/2, trial of nivolumab in patients with metastatic UC with progression or recurrence after platinum-based therapy, with a cutoff of TC PD-L1 expression ≥ 1% (PharmDx 28-8 assay), no association of ORR with PD-L1 was observed (Table 3) [
      • Sharma P.
      • Callahan M.K.
      • Bono P.
      • Kim J.
      • Spiliopoulou P.
      • Calvo E.
      • et al.
      Nivolumab monotherapy in recurrent metastatic urothelial carcinoma (CheckMate 032): a multicentre, open-label, two-stage, multi-arm, phase 1/2 trial.
      ].
      In the phase 2 CheckMate 275 trial of nivolumab as second-line therapy, TC PD-L1 expression ≥ 5% was associated with a numerically higher ORR of 28% more than the 16% rate in patients with TC PD-L1 < 1% (Table 3). Nevertheless, the authors concluded that ORR in all PD-L1 subgroups compared favorably with historical ORRs (10%) in this setting [
      • Sharma P.
      • Retz M.
      • Siefker-Radtke A.
      • Baron A.
      • Necchi A.
      • Bedke J.
      • et al.
      Nivolumab in metastatic urothelial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial.
      ].

      Pembrolizumab

      Trials of pembrolizumab have used the PD-L1 IHC 223 pharmDX assay, with cutoffs based on CPS [
      • Balar A.V.
      • Castellano D.
      • O'Donnell P.H.
      • Grivas P.
      • Vuky J.
      • Powles T.
      • et al.
      First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study.
      ,
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      ]. In KEYNOTE-045, a phase 3 trial in which pembrolizumab was compared with chemotherapy in post-platinum metastatic UC, pembrolizumab was associated with significantly improved median OS versus chemotherapy (10.3 vs 7.4 months; P = 0.002). Among patients with CPS ≥ 10, median OS was significantly longer in patients treated with pembrolizumab (8.0 months, 95% CI, 5.0–12.3) than in patients treated with chemotherapy (5.2 months, 95% CI, 4.0–7.4; HR 0.57, P = 0.005) [
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      ]. Pembrolizumab monotherapy with the 22C3 biomarker has transformed first-line therapy in UC.

      First-line therapy

      Data on the predictive value of PD-L1 testing in patients treated with anti–PD-1/PD-L1 agents as first-line therapy for metastatic UC are available from two completed single-arm, phase 2 trials, IMvigor210 and KEYNOTE-052. In these trials, PD-L1 testing was with the same assay as that used for each agent in the second-line setting [
      • Balar A.V.
      • Castellano D.
      • O'Donnell P.H.
      • Grivas P.
      • Vuky J.
      • Powles T.
      • et al.
      First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study.
      ,
      • Balar A.V.
      • Galsky M.D.
      • Rosenberg J.E.
      • Powles T.
      • Petrylak D.P.
      • Bellmunt J.
      • et al.
      Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial.
      ]. In the IMvigor210 trial of atezolizumab in 119 cisplatin-ineligible patients, overall ORR was 23%, but responses occurred in all IC PD-L1 expression groups (Table 3). Median OS was 12.3 months (95% CI 6.0–not estimable) in patients with IC PD-L1 expression ≥ 5%, and 19.1 months (95% CI 9.8–not estimable) in patients with IC PD-L1 expression < 5%. Unlike in the second-line setting, no statistically significant enrichment of response according to PD-L1 expression was observed [
      • Balar A.V.
      • Galsky M.D.
      • Rosenberg J.E.
      • Powles T.
      • Petrylak D.P.
      • Bellmunt J.
      • et al.
      Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial.
      ]. However, the small sample size and exploratory nature of biomarker analysis were limitations of this analysis. In the KEYNOTE-052 trial of pembrolizumab in 370 patients, the highest ORR was in patients with CPS ≥ 10 (Table 3). However, responses were observed across all categories of PD-L1 expression [
      • Balar A.V.
      • Castellano D.
      • O'Donnell P.H.
      • Grivas P.
      • Vuky J.
      • Powles T.
      • et al.
      First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study.
      ]. Thus, PD-L1 expression cutoff of CPS ≥ 10 had differing value in predicting response to pembrolizumab in the first- and second-line settings. Although CPS ≥ 10 enriched for response to pembrolizumab when used as first line, benefit of pembrolizumab as second-line therapy was independent of PD-L1 expression in terms of CPS ≥ 10 in patients treated post-platinum therapy.
      As described above, early review of data from the ongoing phase 3 trials of pembrolizumab (KEYNOTE-361) and atezolizumab (IMvigor130) evaluated these agents with or without chemotherapy versus chemotherapy alone in patients with locally advanced or metastatic UC. Unpublished results have indicated that patients in the checkpoint inhibitor monotherapy arms of both trials with PD-L1 low status (CPS < 10 for pembrolizumab or IC PD-L1 < 5% for atezolizumab) struggled compared with patients who received cisplatin- or carboplatin-based chemotherapy [

      ClinicalTrials.gov. Study of pembrolizumab with or without platinum-based combination chemotherapy versus chemotherapy alone in urothelial carcinoma (MK-3475-361/KEYNOTE-361); 2018. https://clinicaltrials.gov/ct2/show/NCT02853305 [accessed Dec 5, 2018].

      ,

      ClinicalTrials.gov. Study of atezolizumab as monotherapy and in combination with platinum-based chemotherapy in participants with untreated locally advanced or metastatic urothelial carcinoma (IMvigor130); 2018. https://clinicaltrials.gov/ct2/show/study/NCT02807636?show_locs=Y#locn [accessed Dec 5, 2018].

      ,

      European Medicines Agency. EMA restricts use of Keytruda and Tecentriq in bladder cancer; 2018. http://www.ema.europa.eu/ema/index.jsp?curl=pages/news_and_events/news/2018/05/news_detail_002964.jsp&mid=WC0b01ac058004d5c1 [accessed Dec 5, 2018].

      ,

      US Food and Drug Administration. Keytruda (pembrolizumab) or tecentriq (atezolizumab): FDA alerts health care professionals and investigators: FDA statement - decreased survival in some patients in clinical trials associated with monotherapy; 2018. https://www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm608253.htm [accessed Dec 5, 2018].

      ]. Published data from these trials will be needed to establish any predictive value of high PD-L1 expression, but these early results suggest that low PD-L1 expression is associated with lack of response to anti–PD-1/PD-L1 monotherapy [

      European Medicines Agency. EMA restricts use of Keytruda and Tecentriq in bladder cancer; 2018. http://www.ema.europa.eu/ema/index.jsp?curl=pages/news_and_events/news/2018/05/news_detail_002964.jsp&mid=WC0b01ac058004d5c1 [accessed Dec 5, 2018].

      ,

      US Food and Drug Administration. Keytruda (pembrolizumab) or tecentriq (atezolizumab): FDA alerts health care professionals and investigators: FDA statement - decreased survival in some patients in clinical trials associated with monotherapy; 2018. https://www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm608253.htm [accessed Dec 5, 2018].

      ].
      In summary, in phase 2 studies in the first-line setting, high PD-L1 expression in terms of CPS ≥ 10 was associated with better outcomes [
      • Balar A.V.
      • Castellano D.
      • O'Donnell P.H.
      • Grivas P.
      • Vuky J.
      • Powles T.
      • et al.
      First-line pembrolizumab in cisplatin-ineligible patients with locally advanced and unresectable or metastatic urothelial cancer (KEYNOTE-052): a multicentre, single-arm, phase 2 study.
      ], while the predictive value of higher IC PD-L1 expression remains unclear (note that the number of patients in IMvigor 210 was small [119]) [
      • Balar A.V.
      • Galsky M.D.
      • Rosenberg J.E.
      • Powles T.
      • Petrylak D.P.
      • Bellmunt J.
      • et al.
      Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial.
      ]. Current ongoing phase 3 studies [

      ClinicalTrials.gov. Study of pembrolizumab with or without platinum-based combination chemotherapy versus chemotherapy alone in urothelial carcinoma (MK-3475-361/KEYNOTE-361); 2018. https://clinicaltrials.gov/ct2/show/NCT02853305 [accessed Dec 5, 2018].

      ,

      ClinicalTrials.gov. Study of atezolizumab as monotherapy and in combination with platinum-based chemotherapy in participants with untreated locally advanced or metastatic urothelial carcinoma (IMvigor130); 2018. https://clinicaltrials.gov/ct2/show/study/NCT02807636?show_locs=Y#locn [accessed Dec 5, 2018].

      ] suggest low PD-L1 expression (CPS < 10 or IC ≥ 5%) is associated with adverse outcomes to anti–PD-1/PD-L1 agents in comparison with chemotherapy [

      European Medicines Agency. EMA restricts use of Keytruda and Tecentriq in bladder cancer; 2018. http://www.ema.europa.eu/ema/index.jsp?curl=pages/news_and_events/news/2018/05/news_detail_002964.jsp&mid=WC0b01ac058004d5c1 [accessed Dec 5, 2018].

      ,

      US Food and Drug Administration. Keytruda (pembrolizumab) or tecentriq (atezolizumab): FDA alerts health care professionals and investigators: FDA statement - decreased survival in some patients in clinical trials associated with monotherapy; 2018. https://www.fda.gov/Safety/MedWatch/SafetyInformation/SafetyAlertsforHumanMedicalProducts/ucm608253.htm [accessed Dec 5, 2018].

      ].
      There appear to be differences in the clinical utility of PD-L1 as a biomarker in untreated and platinum-pretreated metastatic UC. It is possible that PD-L1 expression is changed after first-line treatment, with chemotherapy acting as an immunomodulator that increases PD-L1 expression in ICs and priming for response to anti–PD-1/PD-L1 therapy [
      • Davarpanah N.N.
      • Yuno A.
      • Trepel J.B.
      • Apolo A.B.
      Immunotherapy: a new treatment paradigm in bladder cancer.
      ,
      • McDaniel A.S.
      • Alva A.
      • Zhan T.
      • Xiao H.
      • Cao X.
      • Gursky A.
      • et al.
      Expression of PDL1 (B7–H1) before and after neoadjuvant chemotherapy in urothelial carcinoma.
      ].
      Although questions remain about the predictive value of PD-L1 expression, the available knowledge from PD-L1 testing can inform treatment choices. In the second-line setting, anti–PD-1/PD-L1 therapy may still be an appropriate option in PD-L1–low patients, due to the low efficacy of other treatment options. In the first-line setting, chemotherapy may, however, be a better option in PD-L1–low patients [

      ClinicalTrials.gov. Study of pembrolizumab with or without platinum-based combination chemotherapy versus chemotherapy alone in urothelial carcinoma (MK-3475-361/KEYNOTE-361); 2018. https://clinicaltrials.gov/ct2/show/NCT02853305 [accessed Dec 5, 2018].

      ,

      ClinicalTrials.gov. Study of atezolizumab as monotherapy and in combination with platinum-based chemotherapy in participants with untreated locally advanced or metastatic urothelial carcinoma (IMvigor130); 2018. https://clinicaltrials.gov/ct2/show/study/NCT02807636?show_locs=Y#locn [accessed Dec 5, 2018].

      ]

      PD-L1 as a biomarker: issues for consideration

      Biomarker expression in general may change owing to the dynamic nature of the tumor microenvironment [
      • Siefker-Radtke A.
      • Curti B.
      Immunotherapy in metastatic urothelial carcinoma: focus on immune checkpoint inhibition.
      ]; as IFN-γ induces PD-L1 expression, PD-L1, is a dynamic biomarker that is present at sites of active inflammation, such that biopsy may miss the relevant overexpression [
      • Patel S.P.
      • Kurzrock R.
      PD-L1 expression as a predictive biomarker in cancer immunotherapy.
      ]. As with other biopsy biomarkers, intratumoral heterogeneity of PD-L1 expression may also affect PD-L1 testing due to incomplete sampling and differential expression. This is another potential reason why response to immunotherapy is seen in some apparently PD-L1–low patients [
      • Patel S.P.
      • Kurzrock R.
      PD-L1 expression as a predictive biomarker in cancer immunotherapy.
      ,
      • Aggen D.H.
      • Drake C.G.
      Biomarkers for immunotherapy in bladder cancer: a moving target.
      ]. PD-L1 expression status may differ in primary tumor and metastatic sites [
      • Pichler R.
      • Heidedgger I.
      • Fritz J.
      • Danzl M.
      • Sprung S.
      • Zegler B.
      • et al.
      PD-L1 expression in bladder cancer and metastasis and its influence on oncological outcome after cystectomy.
      ]. In addition, there may be intra-patient heterogeneity of expression between different metastatic locations, as has been observed in melanoma and NSCLC [
      • Kluger H.M.
      • Zito C.R.
      • Barr M.L.
      • Baine M.K.
      • Chiang V.L.
      • Sznol M.
      • et al.
      Characterization of PD-L1 expression and associated T-cell infiltrates in metastatic melanoma samples from variable anatomic sites.
      ,
      • Madore J.
      • Vilain R.E.
      • Menzies A.M.
      • Kakavand H.
      • Wilmott J.S.
      • Hyman J.
      • et al.
      PD-L1 expression in melanoma shows marked heterogeneity within and between patients: implications for anti-PD-1/PD-L1 clinical trials.
      ,
      • McLaughlin J.
      • Han G.
      • Schalper K.A.
      • Carvajal-Hausdorf D.
      • Pelekanou V.
      • Rehman J.
      • et al.
      Quantitative assessment of the heterogeneity of PD-L1 expression in non-small-cell lung cancer.
      ]. The timing of sampling in relation to treatment is likely to be important, given that chemotherapy may affect PD-L1 expression in UC [
      • McDaniel A.S.
      • Alva A.
      • Zhan T.
      • Xiao H.
      • Cao X.
      • Gursky A.
      • et al.
      Expression of PDL1 (B7–H1) before and after neoadjuvant chemotherapy in urothelial carcinoma.
      ]. Clinical trials have used varying samples for PD-L1 assay. For example, in the KEYNOTE-045 trial of pembrolizumab, archival tumor samples and newly obtained core or excisional biopsy samples were permitted, with no restrictions on the age of the archival samples or the number of intervening therapies received after the sample was obtained [
      • Bellmunt J.
      • de Wit R.
      • Vaughn D.J.
      • Fradet Y.
      • Lee J.L.
      • Fong L.
      • et al.
      Pembrolizumab as second-line therapy for advanced urothelial carcinoma.
      ], whereas in CheckMate 032, biopsy specimens were fresh or archived within 3 months of the start of treatment with nivolumab [
      • Sharma P.
      • Callahan M.K.
      • Bono P.
      • Kim J.
      • Spiliopoulou P.
      • Calvo E.
      • et al.
      Nivolumab monotherapy in recurrent metastatic urothelial carcinoma (CheckMate 032): a multicentre, open-label, two-stage, multi-arm, phase 1/2 trial.
      ]. The differing expression of PD-L1 in TCs and ICs, dynamic changes, and intratumoral heterogeneity may mean that there are benefits to using both TCs and ICs to obtain the most comprehensive picture of PD-L1 status.
      As evident in Table 2 and Fig. 1, there is a lack of standardization between available PD-L1 assays. As noted previously, some measure PD-L1 in TCs, some in ICs, and some use both, while different complementary assays use different antibodies. This lack of standardization is an obstacle to evaluating the strength of PD-L1 as a biomarker [
      • Davarpanah N.N.
      • Yuno A.
      • Trepel J.B.
      • Apolo A.B.
      Immunotherapy: a new treatment paradigm in bladder cancer.
      ]. There is evidence, however, for concordance and reproducibility among assays, indicating that PD-L1 expression can be reliably scored in UC. In a study of 235 UC samples comparing four antibody clones (ie, 22C3, 28-8, SP142, E1L3N), the overall results were highly concordant despite some heterogeneity in staining, suggesting diagnostic equivalence between these assays [
      • Tretiakova M.
      • Fulton R.
      • Kocherginsky M.
      • Long T.
      • Ussakli C.
      • Antic T.
      • et al.
      Concordance study of PD-L1 expression in primary and metastatic bladder carcinomas: comparison of four commonly used antibodies and RNA expression.
      ]. Similar analytical performance was found with VENTANA SP263, pharmDx 22C3, and pharmDx28-8 assays in a study of 335 tumor biopsy samples from patients with UC, but although the VENTANA SP142 assay had similar performance to the other assays in terms of IC staining, it was less sensitive for TC staining [
      • Zajac M.
      • Scott M.
      • Ratcliffe M.
      • Sharpe A.H.
      • Scorer P.W.
      • Barker C.
      • et al.
      A study of PD-L1 diagnostic assay concordance in urothelial carcinoma.
      ]. The Blueprint study in lung cancer has shown similar results [
      • Hirsch F.R.
      • McElhinny A.
      • Stanforth D.
      • Ranger-Moore J.
      • Jansson M.
      • Kulangara K.
      • et al.
      PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the Blueprint PD-L1 IHC assay comparison project.
      ,
      • Tsao M.S.
      • Kerr K.M.
      • Kockx M.
      • Beasley M.B.
      • Borczuk A.C.
      • Botling J.
      • et al.
      PD-L1 immunohistochemistry comparability study in real-life clinical samples: results of Blueprint phase 2 project.
      ].
      Different assays and different algorithms may lead to differences (ie, limited overlap) in the patient populations that would be classified as PD-L1 high versus PD-L1 low/negative and impact treatment results (Fig. 2). In a study staining archival UC tumor samples, the VENTANA SP263 (TC/IC ≥ 25%) and pharmDx 22C3 (CPS ≥ 10) assays classified 35% and 52% of samples, respectively, as PD-L1 high, with an overlap of 32% (ie, samples were classified as PD-L1 high by both assays). In contrast, the VENTANA SP142 assay (ICs ≥ 5% tumor area) classified only 5.7% of samples as PD-L1 high, with an overlap with the VENTANA SP263 assay of 5.4% [
      • Rijnders M.
      • van der Veldt A.A.M.
      • Zuiverloon T.C.M.
      • Grunberg K.
      • Thunnissen E.
      • de Wit R.
      • et al.
      PD-L1 antibody comparison in urothelial carcinoma.
      ].
      Figure thumbnail gr2
      Fig. 2Selected differences between populations by assay/algorithm in UC specimens. Figure shows differences between the populations selected as PD-L1 high between VENTANA SP263 (TC/IC ≥ 25%) versus: (A) PharmDx 22C3 (CPS ≥ 10); (B) PharmDx 28–8 (TC ≥ 1%); (C) VENTANA SP142 (IC ≥ 5%). CPS, combined positive score; IC, tumor-infiltrating immune cell; OPA, overall percentage agreement; NPA, negative percentage agreement; PPA, positive percentage agreement; TC, tumor cell. aOPA, PPA, NPA (95% confidence interval [CI]). For each metric, lower boundary of 95% CI was calculated with no upper bound using the Clopper-Pearson method.

      Emerging biomarkers in metastatic UC

      Tumor mutation burden

      PD-L1 has been extensively studied in UC, but emerging biomarkers may be of value independently or in combination with PD-L1 [
      • Aggen D.H.
      • Drake C.G.
      Biomarkers for immunotherapy in bladder cancer: a moving target.
      ,
      • Zhu J.
      • Armstrong A.J.
      • Friedlander T.W.
      • Kim W.
      • Pal S.K.
      • George D.J.
      • et al.
      Biomarkers of immunotherapy in urothelial and renal cell carcinoma: PD-L1, tumor mutational burden, and beyond.
      ]. Tumor mutation burden (TMB; reflective of increased neoantigen burden) is a potential predictive biomarker in UC independent of PD-L1; TMB was associated with response to atezolizumab as first- and second-line therapy, but was not prognostic, and selected a different patient population from IHC PD-L1 assay [
      • Balar A.V.
      • Galsky M.D.
      • Rosenberg J.E.
      • Powles T.
      • Petrylak D.P.
      • Bellmunt J.
      • et al.
      Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial.
      ,
      • Rosenberg J.E.
      • Hoffman-Censits J.
      • Powles T.
      • van der Heijden M.S.
      • Balar A.V.
      • Necchi A.
      • et al.
      Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial.
      ,
      • Powles T.
      • Durán I.
      • van der Heijden M.S.
      • Loriot Y.
      • Vogelzang N.J.
      • De Giorgi U.
      • et al.
      Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.
      ]. Combining TMB and PD-L1 assessment appeared to increase predictive value: in IMvigor211, median OS in patients with high TMB and IC PD-L1 expression ≥ 5% was 17.8 months (95% CI 9.7–not estimable) in those treated with atezolizumab in comparison with 10.6 months (8.2–14.3) in those treated with chemotherapy [
      • Powles T.
      • Durán I.
      • van der Heijden M.S.
      • Loriot Y.
      • Vogelzang N.J.
      • De Giorgi U.
      • et al.
      Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.
      ]. Challenges to the use of TMB include difficulties in standardization, tumor evolution over time, and lack of assessment of the immune microenvironment [
      • Aggen D.H.
      • Drake C.G.
      Biomarkers for immunotherapy in bladder cancer: a moving target.
      ].

      Gene expression profiles

      Targeted IC gene expression profiles, quantifying chemokines, cytokines, or cell surface proteins may delineate an inflamed tumor microenvironment more fully than a single marker such as PD-L1 [
      • Aggen D.H.
      • Drake C.G.
      Biomarkers for immunotherapy in bladder cancer: a moving target.
      ]. In CheckMate 275, higher values in a 25-gene IFN-γ signature were associated with response to nivolumab and higher TC PD-L1 expression [
      • Sharma P.
      • Retz M.
      • Siefker-Radtke A.
      • Baron A.
      • Necchi A.
      • Bedke J.
      • et al.
      Nivolumab in metastatic urothelial carcinoma after platinum therapy (CheckMate 275): a multicentre, single-arm, phase 2 trial.
      ]. Similarly, a four-gene IFN-γ signature was associated with response to durvalumab in the UC cohort of Study 1108 [
      • Higgs B.W.
      • Morehouse C.A.
      • Streicher K.
      • Brohawn P.Z.
      • Pilataxi F.
      • Gupta A.
      • et al.
      Interferon gamma messenger RNA signature in tumor biopsies predicts outcomes in patients with non-small cell lung carcinoma or urothelial cancer treated with durvalumab.
      ]. Multiple gene panels are available, but there is no standardized commercially available panel, and their utility will need to be validated in prospective clinical trials; cost may also be an issue [
      • Aggen D.H.
      • Drake C.G.
      Biomarkers for immunotherapy in bladder cancer: a moving target.
      ,
      • Zhu J.
      • Armstrong A.J.
      • Friedlander T.W.
      • Kim W.
      • Pal S.K.
      • George D.J.
      • et al.
      Biomarkers of immunotherapy in urothelial and renal cell carcinoma: PD-L1, tumor mutational burden, and beyond.
      ].
      Examination of tumors from patients with metastatic UC treated with atezolizumab has shown that lack of response to this agent is associated with a signature of transforming growth factor (TGF)-β signaling in fibroblasts, particularly in tumors showing exclusion of CD8+T cells from the tumor (a common phenotype in metastatic UC). In a non-clinical model of this immune-excluded phenotype, combination treatment with anti–PD-L1 and TGF-β-blocking antibodies reduced stromal TGF-β signaling, facilitated T-cell penetration into the tumor center, and provoked anti-tumor immunity and tumor regression [
      • Mariathasan S.
      • Turley S.J.
      • Nickles D.
      • Castiglioni A.
      • Yuen K.
      • Wang Y.
      • et al.
      TGFbeta attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells.
      ]. More studies will be needed to demonstrate the utility and practicality of using TGF-β as a biomarker.
      The Cancer Genome Atlas (TCGA) RNA sequencing of muscle-invasive UC has identified five expression subtypes, luminal, luminal-papillary, luminal infiltrated, basal/squamous, and a poor-survival neuronal type, that may stratify response to different treatments [
      • Robertson A.G.
      • Kim J.
      • Al-Ahmadie H.
      • Bellmunt J.
      • Guo G.
      • Cherniack A.D.
      • et al.
      Comprehensive molecular characterization of muscle-invasive bladder cancer.
      ]. As with IFN-γ gene panels, multiple gene cluster assays have been used for TCGA subtyping, making standardization an issue. Additionally, TCGA subtyping in patients treated with immunotherapy has been limited to small patient numbers. As a result, TCGA subtyping appears to have low negative predictive value for immunotherapy [
      • Aggen D.H.
      • Drake C.G.
      Biomarkers for immunotherapy in bladder cancer: a moving target.
      ]. However, individual TCGA subtypes, in particular the luminal or luminal papillary subtypes, may be most suitable for targeted therapies. For example, the luminal papillary subtype is characterized by aberrations in FGFR3, and several ongoing studies are investigating the efficacy of FGFR inhibitors in patients with mutations or translocations in FGFR3 [
      • Ibrahim T.
      • Gizzi M.
      • Bahleda R.
      • Loriot Y.
      Clinical development of FGFR3 inhibitors for the treatment of urothelial cancer.
      ]. Erdafitinib (a pan-FGFR tyrosine kinase inhibitor) has been tested in a phase 2 trial (n = 99) in patients with FGFR DNA alterations (FGFR2 or 3). Confirmed response rates were 40% with median PFS and OS of 5.5 months (95% CI 4.2–6.0) and 13.8 months (95% CI 9.8–NA), respectively (median follow-up: 11 months) [
      • Loriot Y.
      • Necchi A.
      • Park S.H.
      • Garcia-Donas J.
      • Huddart R.
      • Burgess E.
      • et al.
      Erdafitinib in locally advanced or metastatic urothelial carcinoma.
      ]. In these select patients, erdafitinib was approved by the FDA. Data in combination with immune therapy are awaited.
      Given the limitations of evaluation of biopsies derived from primary tumors and on clinical staging, interest is growing in non-invasive liquid biopsies, which may be more reflective of the current phenotypic/genotypic status of disease, an example is circulating tumor (ct) DNA [
      • Riethdorf S.
      • Soave A.
      • Rink M.
      The current status and clinical value of circulating tumor cells and circulating cell-free tumor DNA in bladder cancer.
      ]. Detection of specific mutations in ctDNA has the potential to identify patients likely to respond to certain therapeutic approaches [
      • Todenhofer T.
      • Struss W.J.
      • Seiler R.
      • Wyatt A.W.
      • Black P.C.
      Liquid biopsy-analysis of circulating tumor DNA (ctDNA) in bladder cancer.
      ]. Changes in variant allele frequencies (VAFs) in ctDNA are being investigated as an early marker of response to treatment: in an analysis of patients with metastatic UC treated with durvalumab, decrease in VAF was correlated with response while patients with progressive disease showed no changes in VAF [
      • Todenhofer T.
      • Struss W.J.
      • Seiler R.
      • Wyatt A.W.
      • Black P.C.
      Liquid biopsy-analysis of circulating tumor DNA (ctDNA) in bladder cancer.
      ,
      • Kuziora M.
      • Higgs B.W.
      • Brohawn P.Z.
      • Raja R.
      • Bais C.
      • Ranade K.
      Association of early reduction in circulating tumor DNA (ctDNA) with improved progression-free survival (PFS) and overall survival (OS) of patients (pts) with urothelial bladder cancer (UBC) treated with durvalumab (D).
      ]. Blood-based TMB is another emerging biomarker that has shown promise in predicting response to second-line atezolizumab in NSCLC [
      • Gandara D.
      • Kowanetz M.
      • Mok T.
      • Rittmeyer A.
      • Fehrenbacher L.
      • Fabrizio D.
      • et al.
      Blood-based biomarkers for cancer immunotherapy: tumor mutational burden in blood (bTMB) is associated with improved atezolizumab (atezo) efficacy in 2L+ NSCLC (POPLAR and OAK).
      ].

      Discussion points

      For a biomarker to be of value, it is important to understand what it is measuring, and how it is being measured; clinical utility must be demonstrated; and the biomarker must be practicable. For PD-L1, there is a clearly defined target and strong rationale for its use. At present, it is not clear whether TCs or ICs represent the best cells to test, but there may be justification for measuring expression levels of PD-L1 in both. In terms of clinical utility, PD-L1 expression, in general, appears to correlate with improved response in patients with UC treated with anti–PD-1/PD-L1 monotherapy [
      • Davarpanah N.N.
      • Yuno A.
      • Trepel J.B.
      • Apolo A.B.
      Immunotherapy: a new treatment paradigm in bladder cancer.
      ]. Low–PD-L1 expression may help to identify patients who are more likely to have poor response to second-line monotherapy and who might benefit from the opportunity to receive novel therapies or combination therapy [
      • Davarpanah N.N.
      • Yuno A.
      • Trepel J.B.
      • Apolo A.B.
      Immunotherapy: a new treatment paradigm in bladder cancer.
      ]. PD-L1 expression testing may be particularly important in the first-line setting to select the most appropriate therapy, although at present the data are preliminary. Overall, the results in the platinum refractory and previously untreated setting are, at best, inconclusive and inconsistent. However, results from first-line clinical trials should be able to provide clarification.
      The efficacy of immunotherapy combinations is also under investigation. The role of PD-L1 expression in prediction of efficacy for such combinations is, at present, unclear. The data supporting the PD-L1 biomarker in immune combinations (with cytotoxic T-lymphocyte-associated antigen 4) were expected to show that the combinations would be effective in biomarker negatives, but, this does not appear to be the case [
      • Low J.L.
      • Walsh R.J.
      • Ang Y.
      • Chan G.
      • Soo R.A.
      The evolving immuno-oncology landscape in advanced lung cancer: first-line treatment of non-small cell lung cancer.
      ]. Data on ipilimumab and nivolumab do show enrichment in biomarker positives in single-arm trials. Additional research is ongoing.
      There is currently a lack of standardization among available PD-L1 assays and cutoffs, such that patient populations identified as “PD-L1 high” can differ when different assays and algorithms are used; this represents an obstacle to evaluating the strength of PD-L1 as a biomarker. There is, however, also concordance across some assays, which is important where multiple assays are available, and indicates that PD-L1 expression can be reproducibly scored in UC. With strong evidence of practicability and utility in predicting response, PD-L1 is currently the most advanced biomarker, and should be routinely offered to patients with metastatic UC. However, PD-L1 is far from a perfect biomarker, and, as new biomarkers emerge, they are likely to be added into a matrix system to more accurately define which patients are most likely to respond to immunotherapies. For example, additional tests may identify patients who respond to IO therapy, regardless of PD-L1 expression, refining its role as a selective biomarker. In the future, monitoring of PD-L1 expression on circulating tumor cells could provide a minimally invasive means of longitudinal monitoring of real-time, on-treatment PD-L1 status that may overcome some of the limitations of current PD-L1 assay methods that rely on archival samples that do not reflect the current state of the cancer or require fresh, invasive tissue biopsy [
      • Anantharaman A.
      • Friedlander T.
      • Lu D.
      • Krupa R.
      • Premasekharan G.
      • Hough J.
      • et al.
      Programmed death-ligand 1 (PD-L1) characterization of circulating tumor cells (CTCs) in muscle invasive and metastatic bladder cancer patients.
      ].
      As the science of biomarkers progresses, PD-L1 testing may become embedded within the testing paradigm in UC alongside other emerging biomarkers, especially given the recent FDA and EMA restrictions on the first-line use of atezolizumab in combination with chemotherapy in “unfit” patients with low levels of PD-L1 expression as based on the recent IMvigor 130 trial. However, in the same trial, PD-L1 testing was shown to be important in monotherapy in the first-line use [
      • Grande E.
      • Galsky M.
      • Arranz Arija J.A.
      • De Santis M.
      • Davis I.D.
      • De Giorgi U.F.F.
      • et al.
      IMvigor130: efficacy and safety from a phase 3 study of atezolizumab (atezo) as monotherapy or combined with platinum-based chemotherapy (PBC) vs placebo + PBC in previously untreated locally advanced or metastatic urothelial carcinoma (mUC).
      ]. The role of biomarkers is unknown as drugs are moved earlier in the disease and contradictory results are sometimes observed from small data sets. Randomized trials are awaited.

      Disclosures

      Thomas Powles: Advisory role with Genentech, MSD, Pfizer, GSK, BMS, Roche, Astellas, OncoGenex, AZ, Pfizer. Lecture fees from Pfizer, MSD, GSK, Novartis, Astellas. Research funding from Roche, Pfizer, Novartis, AZ.
      Jill Walker: Employee and shareholder of AstraZeneca.
      J. Andrew Williams: Employee of and recipient of research funding from AstraZeneca. Former employee of and recipient of research funding from Genentech.
      Joaquim Bellmunt: Advisory role with Genentech, MSD, Pfizer, GSK, BMS, Pierre-Fabre, Sanofi Aventis, Astellas, OncoGenex, Janssen, Inovio, Bioclin. Lecture fees from Pfizer, MSD, GSK, Novartis, Pierre-Fabre, Astellas. Research funding from Takeda, Pfizer, Novartis, Sanofi Aventis.

      Acknowledgments

      Medical writing support, which was in accordance with Good Publication Practice (GPP3) guidelines, was provided by Sherri Baber of Parexel (Hackensack, NJ) and was funded by AstraZeneca.

      Author contributions

      AstraZeneca conforms to the guidelines set forth by GPP3, the International Committee of Medical Journal Editors for Authorship (ICMJE), and the International Society of Medical Publication Professionals (ISMPP). All authors were involved in the study design, data analysis/interpretation, drafting of the article, and final approval.

      Role of the funding source

      This manuscript was funded by AstraZeneca LP (Wilmington, DE). The sponsor participated in manuscript review and the decision to submit the paper for publication.

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