One-Day FISH Approach for the High-Speed Determination of HER2 Gene Copy Status in Breast Carcinoma

Abstract

Fluorescence in situ hybridization (FISH) is a commonly used method to detect chromosomal aberrations, for example, to assess human epidermal growth factor receptor 2 (HER2) gene status in breast carcinoma. The classical FISH approach requires overnight incubation for proper hybridization result. Tissue morphologic features are varying because of aggressive pretreatment and application of high temperatures. To eliminate some of the methodological problems, a new 1-day FISH method was recently introduced. The aim of our study was to evaluate the utility of the Instant Quality FISH with the conventional FISH kit from the same provider (Dako pharmDx) for determination of HER2 status.We performed in situ hybridization on the same 40 invasive breast carcinoma samples with both probe kits, and HER2/ CEN17 and chromosome 17/cell nucleus ratios were calculated. FISH signal stability was also tested by the reassessment of the slides after 2 months storage. The accordance regarding HER2 gene amplification status between the 2 FISH kits tested was 100%. There was an excellent correlation between HER2/ CEN17 ratios with a concordance correlation coefficient of 0.958 and correlation coefficient (R) of 0.959. The 1-day HER2 Instant Quality FISH diagnostic kit points with fast and stable reaction showing the same result in the diagnostic practice when compared with the conventional overnight FISH method.Key Words: HER2, IQFISH, FISH, breast cancer, high-speed FISH (Appl Immunohistochem Mol Morphol 2013;00:000-000) T he human epidermal growth factor receptor 2 (HER2, c-erbB-2) gene localizes at 17q21 and encodes a transmembrane tyrosine kinase receptor protein that is involved in the process of cell growth. 1 Amplification of HER2 gene AQ4 occurs in 15% to 25% of invasive breast cancers. 2 The gene amplification is associated with overexpression of the HER2 protein, a feature with strong prognostic impact for increased risk of recurrence and short survival. 3 As the HER2 status predicts therapeutic response to cyclosporamide, methotrexate fluorouracil, 4 and trastuzumab 5,6 as well, the accurate determination of the gene status has great clinical significance.In situ hybridization is a powerful molecular genetic technique allowing the assessment of gene copy numbers by microscopic visualization in interphase nuclei in formaldehyde-fixed and paraffin-embedded sections and tissue microarray samples. 7 Fluorescence in situ hybridization (FISH) is a commonly used method for determining HER2 gene amplification status in breast cancer. 8 Unfortunately, the conventional FISH method performed on histologic specimens is rather time consuming as an overnight incubation is required for proper probe annealing and target DNA hybridization. 9 Moreover, tissue integrity is sometimes influenced by aggressive pretreatment and hybridization reagents. To overcome these problems, a modified FISH method was recently introduced, which requires only 1.5 to 2 hours of hybridization time; thanks to the adjustment of the hybridization buffer a...

The FlexISH assay brings flexibility to cytogenetic HER2 testing

“…Another fast FISH application, Instant Quality FISH (IQFISH), was previously developed by Dako, and has been subjected to benchmark testing . A systematic comparison revealed concordance rates of 95–100% with the automated Omnis and the conventional pharmDx HER2 FISH (both Dako products).…”

“…To this end, commercial manufacturers have developed so‐called ‘fast FISH’ reagents and probes, which should enable a cytogenetic analysis to be completed within a couple of hours. Just recently, HER2 IQFISH pharmDx (Dako) was introduced, allowing rapid and high‐quality HER2 FISH analysis, with results being obtained in ~3.5 h …”

“…To this end, commercial manufacturers have developed so-called 'fast FISH' reagents and probes, which should enable a cytogenetic analysis to be completed within a couple of hours. Just recently, HER2 IQFISH pharmDx (Dako) was introduced, allowing rapid and high-quality HER2 FISH analysis, with results being obtained in~3.5 h. 9,10 Unfortunately, fast FISH approaches are not compatible with routine workflows in pathology laboratories, as they require, for example, incubation temperatures that deviate from routinely used protocols. Additionally, such fast FISH assays cannot be used with overnight hybridization, as this would result in overstained nuclei with a decreased signalto-noise ratio and thus decreased interpretability.…”