Development of automated brightfield HER2 and chromosome 17 centromere (CEN17) double in situ hybridization application for breast carcinomas and its performance comparison to manual HER2 fluorescence in situ hybridization (FISH)

Abstract

11080 Background: HER2 dual color FISH is a ‘gold standard’ quantitative assay for selecting breast cancer patients for trastuzumab therapy. Current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists. Also, a specialized fluorescence microscope with oil immersion objectives in a dark room is necessary for the analysis and FISH slides are difficult to archive. Our objectives were: 1) to develop an automated BDISH application for HER2 and CEN17 that produces permanent slides and 2) to compare the assay performance with HER2 FISH tested breast carcinomas cases. Methods: BDISH assay was developed with a nick translated HER2 DNA probe and a CEN17 oligoprobe on an automated slide stainer. Detection of HER2 and CEN17 signals was accomplished with the silver, hydroquinone, and H2O2 reaction and the fast red and naphthol phosphate reaction, respectively. BDISH specificity was optimized with xenograft tumors [MCF7 (non-amplified HER2) and BT-474 (amplified HER2)]. BDISH performance was evaluated with 237 routinely processed breast carcinoma cases. Interpretation was conducted with a conventional brightfield microscope without oil immersion objectives. Data were analyzed for the concordance between BDISH and FISH scores for HER2 gene status assessment. Results: Specific signals for single HER2 copies were visualized as discrete black dots while single CEN17 copies were detected as slightly larger red dots in the same cells of xenograft tumor sections. Overall concordance was 93% between BDISH and FISH of breast carcinoma cases. Interobserver concordance was 97%. Investigation for the discordant cases with BDISH slides revealed that the heterogeneity of tumor cell populations within the same tissue section was a significant reason for the discordance. Conclusions: We successfully developed an automated BDISH assay that is specific for HER2 and CEN17 targets. Our study demonstrated a high concordance between observers interpreting BDISH results of clinical breast carcinoma cases. The majority of BDISH discordance with FISH was due to the tumor cell heterogeneity in the tumor sample. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. Ventana Medical Systems, Inc.