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Clinical Trial
. 2016 Oct 8;11(1):95.
doi: 10.1186/s13000-016-0545-8.

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

Marlon C Rebelatto  1 , Anita Midha  2 , Amita Mistry  3 , Constantine Sabalos  3 , Nicole Schechter  3 , Xia Li  4 , Xiaoping Jin  4 , Keith E Steele  4 , Paul B Robbins  4 , John A Blake-Haskins  4 , Jill Walker  5
Affiliations
Clinical Trial

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

Marlon C Rebelatto et al. Diagn Pathol. .

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.

Trial registration: ClinicalTrials.gov: NCT01693562.

Keywords: Assay; Diagnostic; Durvalumab; HNSCC; Immunohistochemistry; Immunotherapy; MEDI4736; NSCLC; PD-L1.

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Figures

Fig. 1
Fig. 1
PD-L1 expression in adenocarcinoma cell lines labeled with the PD-L1 (SP263) antibody. Cell staining and flow cytometry results. Photomicrographs of cell lines stained with the PD-L1 (SP263) antibody (20X): MCF7 (negative control, no PD-L1 staining), HCT116 (low level of PD-L1 staining), ACHN (medium PD-L1 staining), MDA-MB231 (high-level PD-L1 staining), LOX (high-level PD-L1 staining). Corresponding fluorescence intensities achieved by flow cytometry with the same cell lines labeled with the PD-L1 (SP263) antibody. Ctrl control, PD-L1 programmed cell death ligand-1
Fig. 2
Fig. 2
Western blot analyses of cell lysates labeled with the PD-L1 (SP263) antibody. Western blot analysis of cell lysates from adenocarcinoma cell lines loaded onto an SDS-PAGE gel and labeled with the PD-L1 (SP263) antibody. The antibody labeled a single band of the appropriate molecular weight for fully glycosylated PD-L1 (~55 kDa). Relative levels of PD-L1 immunoreactivity matched the known relative PD-L1 mRNA expression levels for these cell lines (H820 > MDA-MB231 > H1975 > Calu-3). PD-L1 programmed cell death ligand-1
Fig. 3
Fig. 3
PD-L1 (SP263) antibody staining in tumor tissue samples. Photomicrographs of PD-L1 (SP263) staining in NSCLC and HNSCC tissue, demonstrating the range of PD-L1 staining intensity and frequency obtained with the PD-L1 (SP263) assay on NSCLC (a-d) and HNSCC (e-h) samples. a, e negative tumor cells (20X), positive immune cells; b, f (10X): low tumor staining; c, g (10X): moderate tumor staining; d, h (10X,20X): high tumor staining. HNSCC head and neck squamous cell carcinoma, NSCLC non-small cell lung cancer, PD-L1 programmed cell death ligand-1
Fig. 4
Fig. 4
ROC analysis on confirmed objective response (per RECIST v1.1 per investigator). Analysis provided the rationale for the choice of tumor membrane score and 25 % cut-off. AUC area under the curve, NSCLC non-small cell lung cancer, RECIST Response Evaluation Criteria In Solid Tumors, ROC receiver operating characteristic, TM tumor membrane
Fig. 5
Fig. 5
Relationship between ORR (per RECIST v1.1 per investigator) and tumor membrane score (logistic regression). *Based on N = 155 NSCLC patients enrolled prior to enrichment. CI confidence interval, NSCLC non-small cell lung cancer, ORR objective response rate, RECIST Response Evaluation Criteria In Solid Tumors
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