Performance of the cobas EZH2 mutation test on clinical samples from non-Hodgkin lymphoma patients.

OBJECTIVE: To present the technical verification and clinical validation of the companion diagnostic assay, cobas® EZH2 Mutation Test (cobas EZH2 Test), targeting gain-of-function EZH2 mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). The focus is on patient clinical samples proving that the test met the performance criteria required for FDA approval of a companion diagnostic test. DESIGN: Epizyme, Inc., Eisai Co., Ltd., and Roche Molecular Systems, Inc., collaborated to develop the cobas EZH2 Test on an RT-PCR platform. The assay design needed to detect the gain-of-function EZH2 mutations found in FL and DLBCL indications. Thus, the test was optimized for investigational purposes in a clinical trial setting. Part of its technical verification included testing of patient tumor samples with a documented diagnosis of FL and DLBCL procured from commercial vendors, and the clinical validation used patient samples from the Epizyme clinical study. Both the technical performance verification method correlation study (104 clinical commercially acquired samples) and the clinical validation accuracy study (341 patient samples from the therapeutic study) used next-generation sequencing as a reference method to establish true vs. false results by cobas EZH2 Test. The reproducibility study used a 15-member panel of DNA samples with varying EZH2 mutation status from procured clinical FL and DLBCL patient samples under multiple variables. RESULTS: Single and rare, infrequent double EZH2 mutations were detected in FL and DLBCL samples. Agreements between results from cobas EZH2 and sequencing were >98% from commercial clinical samples and from the therapeutic study clinical samples. The reproducibility study obtained 178 to 180 valid results for each panel member, with an overall invalid rate of 0.37%. The agreement for each per panel member was 100%. CONCLUSION: cobas EZH2 Test data demonstrated that the test is reliable and will perform well in a commercial customer environment.

Correlation of ROS1 Immunohistochemistry With ROS1 Fusion Status Determined by Fluorescence In Situ Hybridization.

CONTEXT.­: The ability to determine ROS1 status has become mandatory for patients with lung adenocarcinoma, as many global authorities have approved crizotinib for patients with ROS1-positive lung adenocarcinoma. OBJECTIVE.­: To present analytical correlation of the VENTANA ROS1 (SP384) Rabbit Monoclonal Primary Antibody (ROS1 [SP384] antibody) with ROS1 fluorescence in situ hybridization (FISH). DESIGN.­: The immunohistochemistry (IHC) and FISH analytical comparison was assessed by using 122 non-small cell lung cancer samples that had both FISH (46 positive and 76 negative cases) and IHC staining results available. In addition, reverse transcription-polymerase chain reaction (RT-PCR) as well as DNA and RNA next-generation sequencing (NGS) were used to further examine the ROS1 status in cases that were discrepant between FISH and IHC, based on staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells considered as IHC positive. Here, we define the consensus status as the most frequent result across the 5 different methods (IHC, FISH, RT-PCR, RNA NGS, and DNA NGS) we used to determine ROS1 status in these cases. RESULTS.­: Of the IHC scoring methods examined, staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells considered as IHC positive had the highest correlation with a FISH-positive status, reaching a positive percentage agreement of 97.8% and negative percentage agreement of 89.5%. A positive percentage agreement (100%) and negative percentage agreement (92.0%) was reached by comparing ROS1 (SP384) using a cutoff for staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells to the consensus status. CONCLUSIONS.­: Herein, we present a standardized staining protocol for ROS1 (SP384) and data that support the high correlation between ROS1 status and ROS1 (SP384) antibody.