Estrogen receptor (ER) and progesterone receptor (PR) status in breast carcinomas are considered validated predictive factors for selecting patients for antihormonal therapy. Published surveys have shown a significant rate of disagreement and lack of reproducibility of immunohistochemistry (IHC) results from laboratories around the world. To address these limitations IHC assays for ER and PR were developed using characterized reagents, after careful calibration of the sensitivity and specificity to match established assays previously validated in large clinical studies. The ER assay uses a cocktail of 2 mouse monoclonal antibodies (1D5 and ER-2-123) and the PR assay uses 1 mouse monoclonal antibody (PgR 1294); both are followed by a polymer-peroxidase-based detection system. All antibodies were tested for specificity by epitope mapping. The sensitivity of the new assays was calibrated to be equivalent to previously validated IHC assays followed by a comparison with the validated assays in a concordance study involving over 200 specimens. All slides were scored with the "Allred Score," also used for scoring of the original validated assays. The overall concordance between the new and the established IHC assays was nearly perfect (99%). The concordance study demonstrated greater than 98% positive agreement and 100% negative agreement of the new IHC assays with the previously validated IHC assays. This equivalence establishes the clinical validation of the assays and, as they are based on newer generation reagents and are produced and tested under stringent quality control conditions to ensure their consistency, they add additional advantages to the user and patients.
2071 Background: EGFRvIII is a mutant version of EGF receptor resulting from the genomic deletion of exons 2 through 7 and is expressed only in certain tumors. AMG 595 is an experimental therapeutic specifically targeting EGFRvIII and consists of an EGFRvIII-specific antibody conjugated to the maytansinoid antimicrotubule agent DM-1. Due to its specificity, mechanism of action, and pre-clinical activity, AMG 595 is expected to have clinical effect only in tumors expressing EGFRvIII. Since the reported prevalence of EGFRvIII in glioblastoma multiforme (GBM) is ~30%, prospective selection of patients with EGFRvIII positive tumors was desired for clinical development. An immunohistochemical (IHC) assay developed with Dako using a novel EGFRvIII antibody is currently being employed for patient selection for the AMG 595 phase I study in recurrent GBM (NCT01475006). Methods: An appropriate IHC reagent for human tissue was created using the variable region of a novel EGFRvIII-specific antibody developed using Xenomouse technology. Staining conditions were optimized using Dako pharmDx reagents, the Dako Link 48 Autostainer, and FFPE tissue sections. Confirmatory transcript analyses of adjacent sections were conducted using the NanoString platform. Results: Robust and reproducible staining for EGFRvIII was observed using archived GBM resections. Percentage of stained cells correlated with levels of EGFRvIII transcripts, and tumors without staining did not express EGFRvIII transcripts. Although some tumors exhibited homogenous staining, most were heterogeneous with varying distribution and percentages of stained tumor cells similar to literature reports of IHC analysis utilizing other EGFRvIII-specific antibodies. Tumor samples from patients entering into the AMG 595 Phase 1 study analyzed with this IHC test have displayed the predicted staining prevalence. Conclusions: The developed EGFRvIII IHC assay, approved under an Investigational Device Exemption, is currently being successfully employed to prospectively select patients in an ongoing phase I trial. Use of this well characterized IHC test will enable correlation of clinical outcome and staining characteristics to inform subsequent studies.
7561 Background: CD38 is a type II transmembrane glycoprotein expressed on normal lymphoid and myeloid cells and can be highly expressed in hematologic malignancies. An IHC prototype assay was developed to detect CD38 expression in formalin-fixed, paraffin-embedded (FFPE) tissue specimens from three relapsed or refractory non-Hodgkin’s lymphoma (NHL) subtypes: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL) for selection of patients for treatment with daratumumab in a Phase 2 clinical trial (NCT02413489). Methods: This assay is based on EnVision FLEX IHC technology using CD38, clone DAK-CD38, primary antibody that has been developed and manufactured by Agilent Technologies. The assay staining protocol was developed for Dako PT Link and Autostainer Link 48. Specificity of DAK-CD38 staining was demonstrated by Western blot analysis of cancer cell lysates, as well as IHC on both normal and cancer tissue. Assay precision and robustness were evaluated using commercially procured FFPE DLBCL, FL and MCL specimens. Results: CD38 IHC DAK-CD38 detected a broad range of CD38 expression in DLBCL, FL, and MCL specimens. FFPE specimens derived from cancer cell lines exhibited a range of IHC staining intensity and confirmed the reported expression of CD38 in the scientific literature. All precision and robustness results met acceptance criteria for both IHC intensity and percent positive cells staining. Conclusions: Our studies demonstrate that the Dako CD38 IHC DAK-CD38 assay is sensitive, specific, precise, and robust for the detection of CD38 expression in DLBCL, FL and MCL. This assay may also have the potential to be used for the detection of CD38 in additional tumor types.
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