title: Development of a programmed cell death ligand-1 immunohistochemical assay validated for analysis of non-small cell lung cancer and head and neck squamous cell carcinoma.
abstract: BACKGROUND
  A high-quality programmed cell-death ligand 1 (PD-L1) diagnostic assay may help predict which patients are more likely to respond to anti-programmed cell death-1 (PD-1)/PD-L1 antibody-based cancer therapy. Here we describe a PD-L1 immunohistochemical (IHC) staining protocol developed by Ventana Medical Systems Inc. and key analytical parameters of its use in formalin-fixed, paraffin-embedded (FFPE) samples of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC).
  
  
  METHODS
  An anti-human PD-L1 rabbit monoclonal antibody (SP263) was optimized for use with the VENTANA OptiView DAB IHC Detection Kit on the automated VENTANA BenchMark ULTRA platform. The VENTANA PD-L1 (SP263) Assay was validated for use with FFPE NSCLC and HNSCC tissue samples in a series of studies addressing sensitivity, specificity, robustness, and precision. Samples from a subset of 181 patients from a Phase 1/2 study of durvalumab (NCT01693562) were analyzed to determine the optimal PD-L1 staining cut-off for enriching the probability of responses to treatment. The scoring algorithm was defined using statistical analysis of clinical response data from this clinical trial and PD-L1 staining parameters in HNSCC and NSCLC tissue. Inter-reader agreement was established by three pathologists who evaluated 81 NSCLC and 100 HNSCC samples across the range of PD-L1 expression levels.
  
  
  RESULTS
  The VENTANA PD-L1 (SP263) Assay met all pre-defined acceptance criteria. For both cancer types, a cut-off of 25 % of tumor cells with PD-L1 membrane staining of any intensity best discriminated responders from nonresponders. Samples with staining above this value were deemed to have high PD-L1 expression, and those with staining below it were deemed to have low or no PD-L1 expression. Inter-reader agreement on PD-L1 status was 97 and 92 % for NSCLC and HNSCC, respectively.
  
  
  CONCLUSIONS
  These results highlight the robustness and reproducibility of the VENTANA PD-L1 (SP263) Assay and support its suitability for use in the evaluation of NSCLC and HNSCC FFPE tumor samples using the devised ≥25 % tumor cell staining cut-off in a clinical setting. The clinical utility of the PD-L1 diagnostic assay as a predictive biomarker will be further validated in ongoing durvalumab studies.
  
  
  BACKGROUND
  ClinicalTrials.gov: NCT01693562.
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20878, USA. RebelattoM@medimmune.com.
authors.forename: Marlon C
authors.initials: MC
authors.lastname: Rebelatto
authors.affiliation: AstraZeneca, Alderley Park, Macclesfield, UK.
authors.forename: Anita
authors.initials: A
authors.lastname: Midha
authors.affiliation: Ventana Medical Systems Inc., Tucson, AZ, USA.
authors.forename: Amita
authors.initials: A
authors.lastname: Mistry
authors.affiliation: Ventana Medical Systems Inc., Tucson, AZ, USA.
authors.forename: Constantine
authors.initials: C
authors.lastname: Sabalos
authors.affiliation: Ventana Medical Systems Inc., Tucson, AZ, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Schechter
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20878, USA.
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20878, USA.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Jin
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20878, USA.
authors.forename: Keith E
authors.initials: KE
authors.lastname: Steele
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20878, USA.
authors.forename: Paul B
authors.initials: PB
authors.lastname: Robbins
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD, 20878, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Blake-Haskins
authors.affiliation: AstraZeneca, Cambridge, UK.
authors.forename: Jill
authors.initials: J
authors.lastname: Walker
mesh_terms: D000465:Algorithms; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000627:therapeutic use; D019540:Area Under Curve; D060890:B7-H1 Antigen / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology; D014408:Biomarkers, Tumor / Q000032:analysis*; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry* / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000737:chemistry* / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D045744:Cell Line, Tumor; D005404:Fixatives; D005557:Formaldehyde; D057809:HEK293 Cells; D006258:Head and Neck Neoplasms / Q000737:chemistry* / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D059021:Laboratory Proficiency Testing; D008175:Lung Neoplasms / Q000737:chemistry* / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D015588:Observer Variation; D016612:Paraffin Embedding; D011237:Predictive Value of Tests; D012372:ROC Curve; D015203:Reproducibility of Results; D000077195:Squamous Cell Carcinoma of Head and Neck; D016707:Tissue Fixation / Q000379:methods; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D060890:B7-H1 Antigen; D014408:Biomarkers, Tumor; C423236:CD274 protein, human; D005404:Fixatives; D005557:Formaldehyde; C000613593:durvalumab
keywords: Assay; Diagnostic; Durvalumab; HNSCC; Immunohistochemistry; Immunotherapy; MEDI4736; NSCLC; PD-L1