Utilization of Dual ISH and RT-PCR Enhances Resolution of IHC and FISH Double Equivocal Testing Results in Breast Carcinoma
Bryce P Portier, Zhen Wang, Eugen Mincae, Chris Lanigan, Erinn Downs-Kelly, Raymond Tubbs. Cleveland Clinic, Cleveland, OH
Background: Cases classified as equivocal by both IHC and FISH testing for HER2 represent a deficiency in laboratory medicine. Currently, per ASCO/CAP guidelines, cases called equivocal by one methodology are reflex tested by a second methodology (FISH/IHC or IHC/FISH). However, reflex testing fails to resolve HER2 status in all cases. Cases that are equivocal by both FISH and IHC are categorized as “Double Equivocal”. In this study, we examined the utility of using the newly FDA approved Dual ISH HER2 detection system and a Quantitative-Real Time-PCR (Q-RT-PCR) assay to determine the amplification status of HER2 in patients that could not be resolved by standard IHC and FISH testing.
Design: Cases were identified from the Cleveland Clinic electronic records from 1/2008 to 12/2010. Q-RT-PCR was performed on FISH/IHC amplified, equivocal, and non-amplified cases following RNA extraction of macro-dissected tissue utilizing a LightCycler 480 II (Roche Applied Biosciences, Penzberg, Germany). Q-RT-PCR results were expressed as the ratio of HER2 to two reference genes (B2B and GAPDH). Dual ISH (HER2 Inform, Ventana, Tucson, AZ) was performed and scored per manufacturer's instructions.
Results: Q-RT-PCR assay was validated utilizing control IHC/FISH amplified and non-amplified cases. ROC curve analysis of Q-RT-PCR validation assays showed 100% sensitivity and specificity with a cut off score of 7.0 and above identifying HER2 mRNA over-expression. In the IHC/FISH double equivocal population, Q-RT-PCR identified 15 (30%) cases as amplified. Dual ISH applied to the double equivocal cohort identified 13 (26%) cases as amplified. Overall agreement between Q-RT-PCR and DISH for all cases was 90%.
Conclusions: Utilization of Dual ISH, a new FDA approved bright-field HER2 detection system, and a molecular based approach using Q-RT-PCR both showed superior resolution of HER2 status compared to standard IHC and FISH testing methods. This study shows the utility and added sensitivity of adding Dual ISH as a first line HER2 test and adding Q-RT-PCR as a downstream assay, in cases that fail primary screening. Utilization of these two techniques would decrease first round equivocal calls (Dual ISH) and would offer a definitive follow up reflex test (Q-RT-PCR). Both methods are morphology based and are readily incorporable into standard laboratory work flows. Dual ISH has the added benefit of being FDA approved, in addition, since HER2 detection is bright-field based, this opens this procedure to practices that currently lack a FISH laboratory.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 48, Wednesday Morning