The INFORM HER2 Dual ISH assay compared with FISH to determine HER2 gene status in invasive breast carcinoma.

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25 Background: The HER2 gene, located on chromosome 17 (Chr17), is amplified in 15-25% of patients with invasive breast carcinoma. Amplification and/or HER2 overexpression is associated with poor clinical outcome for these patients; however prognosis is improved if HER2 status indicates eligibility of patients for trastuzumab therapy. Thus, accurate diagnosis of HER2 status through a companion diagnostic is essential. Here we validated the INFORM HER2 Dual ISH Probe Cocktail (Dual ISH) assay as an alternative to FISH, the current gold standard for HER2 testing. The Dual ISH assay is fully automated and is scored using light microscopy. Methods: A multi-site method comparison and inter-laboratory reproducibility study were performed. 5 sites were used to compare Dual ISH results with FISH (Vysis PathVysion). 510 invasive breast carcinoma cases were stained at 3 clinical sites, FISH staining was performed at a fourth site (central laboratory). IHC status was determined at a fifth site (second central laboratory). In addition, 6 cases were evaluated for inter-site (3 sites), inter-reader (6 readers), inter-run (15 runs) and intra-run (duplicate slides) reproducibility. All assay steps were fully automated on a VENTANA BenchMark XT automated slide stainer, using a HER2 repeat-reduced, dinitrophenyl-labeled probe targeting the HER2 gene, detected with silver metallographic detection, and a digoxigenin-labeled Chr 17 probe, detected by an alkaline phosphatase-driven red chromogenic detection. HER2 and CHR17 signals were enumerated using conventional light microscopy allowing interpretation within the morphological context of the specimen. HER2 status was determined as the ratio of HER2/Chr17, where a ratio <2 is non-amplified and a ratio ≥2 is amplified. Results: The positive and negative agreement rates with FISH (95% CI) were 96% (92.6-97.9) and 92.3% (88.6-94.8), respectively. The HER2 Dual ISH assay also was highly reproducible in determining HER2/Chr17 ratio across sites, days, readers and runs. Conclusions: The fully automated INFORM HER2 Dual ISH assay is reproducible and concordant with the manual FISH assay in determining HER2 status in invasive breast carcinoma.