BackgroundProgrammed Death Ligand 1 (PD-L1) is an immune modulating protein expressed on the surface of various inflammatory cells, including T Cells, B Cells, dendritic cells, and macrophages. PD-L1 represents an important diagnostic target; expression of PD-L1 on the surface of tumor cells, or within tumor-associated immune cells, is an important predictor of likely response to targeted therapies. In this study, we describe the optimization of immunohistochemistry (IHC) assays using two PD-L1 antibodies (SP263 and E1L3N) and compare the performance of the optimized assays.MethodsFully automated immunohistochemical assays were optimized for the VENTANA PD-L1 (SP263) Rabbit Monoclonal Antibody and the PD-L1 (E1L3N®) XP® Rabbit mAb using instruments and detection chemistries from Ventana Medical Systems, Inc. (“SP263 assay” and “E1L3N assay,” respectively). Tissue microarrays (TMAs) containing formalin fixed paraffin embedded (FFPE) non-small cell lung cancer (NSCLC) cases were used for the optimization and comparison staining. H scores were used for membrane scoring whereas percent positivity was used for tumor-associated immune cell scoring.ResultsOne-hundred NSCLC TMA case cores each stained with the SP263 and E1L3N assays were evaluated by two pathologists in a blinded study. Analysis of these specimens showed that the SP263 assay was more sensitive and had a wider dynamic range than the E1L3N assay. For sensitivity, many cases were found to be negative for membrane staining with the E1L3N assay, but had measurable staining with the SP263 assay. Dynamic range was demonstrated by the SP263 assay having well-distributed H scores while the E1L3N assay had a significantly higher proportion of cases clustered in the lowest H score bins. For tumor-associated immune cell staining, the two assays identified similar amounts of cells staining in each case, but the SP263 assay gave overall darker staining.ConclusionSince PD-L1 status is important for targeted therapies, having a specific and accurate diagnostic test is crucial for identifying patients who could benefit from these treatments. Due to its staining intensity, scoring range, and pathologist preference, the SP263 IHC assay has been deemed superior to the E1L3N IHC assay. Future clinical utility remains to be determined.Electronic supplementary materialThe online version of this article (doi:10.1186/s13000-016-0494-2) contains supplementary material, which is available to authorized users.

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A biomarker-based patient selection strategy, coupled with the co-development of a companion diagnostic, is key to the successful development of molecularly targeted cancer therapeutics. IMGN853 is a Folate Receptor Alpha (FRα)-targeting antibody-drug conjugate (ADC) consisting of an anti-FRα antibody linked to DM4, a highly cytotoxic maytansinoid, via a cleavable disulfide linker. FRα is a glycosyl-phosphatidylinositol-linked membrane protein commonly over-expressed in several solid tumor types including epithelial ovarian cancer (EOC), endometrial cancer and lung adenocarcinoma, with limited expression in normal tissues. Previously reported preclinical data demonstrated regression or significant inhibition of tumor growth by the conjugate in tumor models with clinically relevant levels of FRα expression. IMGN853 is currently in a phase I clinical trial for safety and tolerability in patients with FRα-positive solid tumors. Preliminary evidence of clinical activity has been observed in patients receiving IMGN853 treatment. Immunohistochemistry (IHC) is one of the most widely adopted techniques in clinical labs for semi-quantitative determination of protein expression in different cellular compartments. We have developed an IHC assay to support the FRα-expression based patient selection strategy in the clinical development of IMGN853. To this end, a novel murine monoclonal antibody with high specificity for human FRα was generated. This antibody, 353-2.1, is compatible with formalin fixed and paraffin embedded (FFPE) tissue samples and can recognize the same pool of FRα molecules as IMGN853, making it an ideal candidate for a companion diagnostic for IMGN853. The antibody was used to develop and optimize an assay that covers a broad dynamic range of FRα expression, allowing discrimination among high, moderate and low levels of expression. Data from tissue microarrays that cover a broad range of normal and tumor tissue types, as well as additional prevalence data in selected tumor types, were used to validate this assay. They also demonstrate that the FRα-expression data generated using this assay are consistent with previous findings. This clinical FRα IHC assay has been successfully used to measure FRα expression in more than 100 patient tumor samples submitted for testing in the ongoing IMGN853 phase I trial. A summary of FRα staining pattern and expression level data from these patients will be presented, as well as the prevalence in each tumor type. Citation Format: Jianhua Zhao, Alyssa LaBelle, Olga Ab, Krista Acosta, Al Yates, Yinghui Zhou, Angela Romanelli. Development and application of an immunohistochemistry-based clinical assay for evaluating Folate Receptor Alpha (FRα) expression in a phase I clinical trial of IMGN853. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3400A. doi:10.1158/1538-7445.AM2015-3400A