Background Accurate determination of human epidermal growth factor receptor 2 (HER2) status is essential for optimal patient management with trastuzumab (Herceptin(R)). However, standard guidelines do not specify the use of a particular commercial kit, antibody or probe for testing, and discrepancies arise because of variability between kits. The aim of this study was to compare the accuracy of four commercially available fluorescence/chromogenic in situ hybridization (FISH/CISH) kits and validate one for the resolution of borderline immunohistochemistry (IHC) cases. The interpretation pitfalls, optimal threshold values, assay duration and complexity of each kit were also considered.
Methods The Food and Drug Administration (FDA)-approved dual-probe FISH assay PathVysion(R) was chosen as the ‘gold standard’ against which pharmDxTM (dual-probe) and INFORM(R) (mono-probe) FDA-approved FISH kits and the SPoT-Light(R) CISH kit were compared. Tumours were also evaluated by IHC using the FDA-approved HercepTestTM kit and a validated in-house IHC protocol. Fifty-five patients with invasive breast carcinoma were selected to achieve a representative proportion of HER2 IHC 2+ cases.
Results HER2 amplification was observed in 31% of tumours by PathVysion(R) compared to 33% using pharmDxTM. The number of amplified tumours detected by INFORM(R) and CISH differed according to the threshold applied. Agreement was excellent between PathVysion(R) and pharmDxTM (100%), good with SPoT-Light(R) (89%; cut-off of [greater than or equal to]5 signals/nucleus) and moderate with INFORM(R) (76%; cut-off of >4 signals/nucleus). Agreement with INFORM(R) improved to 98% when using a cut-off of [greater than or equal to]6 signals/nucleus.
Conclusions We have shown that with an appropriate cut-off, the INFORM(R) kit was comparable to dual-probe FISH kits for evaluating HER2 status. We also validate and recommend CISH as an appropriate assay for HER2 scoring that is easy to interpret and requires equipment readily found in, or that can be adapted to, all pathology laboratories. For borderline IHC cases, dual-probe FISH analysis remains the most useful protocol to apply.