Chromogenic in situ hybridisation for the assessment of HER2 status in breast cancer: an international validation ring study

Netherlands Cancer Institute, Amsterdam, The Netherlands.
Breast cancer research: BCR (Impact Factor: 5.49). 10/2007; 9(5):R68. DOI: 10.1186/bcr1776
Source: PubMed


Before any new methodology can be introduced into the routine diagnostic setting it must be technically validated against the established standards. To this end, a ring study involving five international pathology laboratories was initiated to validate chromogenic in situ hybridisation (CISH) against fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) as a test for assessing human epidermal growth factor receptor 2 (HER2) status in breast cancer.
Each laboratory performed CISH, FISH and IHC on its own samples. Unstained sections from each case were also sent to another participating laboratory for blinded retesting by CISH ('outside CISH').
A total of 211 invasive breast carcinoma cases were tested. In 76 cases with high amplification (HER2/CEP17 ratio >4.0) by FISH, 73 cases (96%) scored positive (scores >or= 6) by 'outside CISH'. For FISH-negative cases (HER2/CEP17 ratio <2.0), 94 of 100 cases (94%) had CISH scores indicating no amplification (score <or= 5), and only three cases were positive by CISH; in the three remaining cases, no CISH result could be obtained. For cases with low-level amplification using FISH (HER2/CEP17 ratio 2.0-4.0), 20 of 35 had CISH scores indicating gene amplification. Inter-laboratory concordance was also very high: 95% for normal HER2 copy number (1-5 copies); and 92% for cases with HER2 copy numbers >or= 6. CISH intra-laboratory concordance with IHC was 92% for IHC-negative cases (IHC 0/1+) and 91% for IHC 3+ cases. Among IHC 2+ cases, CISH was 100% concordant with samples showing high amplification by FISH, and 94% concordant with FISH-negative samples.
These results show that CISH inter- and intra-laboratory concordance to FISH and IHC is very high, even in equivocal IHC 2+ cases. Therefore, we conclude that CISH is a methodology that is a viable alternative to FISH in the HER2 testing algorithm.

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Available from: Josef Rüschoff
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    • "A SISH assay (INFORM HER2 Dual ISH Assay , Ventana Medical Systems, Tucson, AZ) has also been approved by the FDA [15]. Studies show very high concordance rates between CISH, SISH, and FISH; 96% for CISH/SISH, 81% to 100% for CISH/FISH, and 94% to 98% for SISH/FISH [23] [24] [25] [26] [27] [28]. SISH is fully automated, comparatively quick to perform, and, similar to CISH, does not require the use of a specialized microscope [23]. "
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    ABSTRACT: Accurate determination of human epidermal growth factor receptor 2 (HER2) status is critical for optimizing breast cancer outcomes. In 2007, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) developed guidelines for HER2 testing to reduce inaccuracy. However, current ASCO/CAP criteria may restrict access to HER2-targeted therapy for some patient groups who would derive a clear clinical benefit. ASCO/CAP are currently reviewing their guidelines to further optimize HER2 testing and include emerging techniques. Guidelines are critical for optimizing care, as is ongoing research into techniques that accurately and reproducibly assess HER2 status.
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    • "It also requires expert technicians and access to a fluorescence microscope. Also the signals produced by the FISH assay decay within a few weeks (Van de Vijver et al., 2007). More recently, the CISH (chromogenic in situ hybridization) methodology, approved by FDA, has emerged as a potential alternative to FISH (Rosa et al. 2009). "
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    • "First, we can validate CISH as a promising alternative to FISH for determining gene amplification, because it is easy to interpret and the equipment can be readily found in most pathology departments. The potential of CISH as a HER2 status assay for routine practice has been the subject of an international multicentre ring study, to which we have contributed, to validate CISH against the current 'gold standards' [22]. Second, the automated mono-probe assay INFORM provides the opportunity for a more rapid evaluation of samples compared with CISH and the two dual-probe FISH assays evaluated in this study, provided that an appropriate threshold is applied. "
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    ABSTRACT: Accurate determination of human epidermal growth factor receptor 2 (HER2) status is essential for optimal patient management with trastuzumab (Herceptin). However, standard guidelines do not specify a particular commercial kit, antibody or probe for testing, and discrepancies arise from variability between kits. The aim of this study was to compare the accuracy of four commercially available fluorescence/chromogenic in situ hybridisation (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. The Food and Drug Administration (FDA)-approved dual-probe FISH assay PathVysion was chosen as the 'gold standard' against which pharmDx (dual-probe) and INFORM (mono-probe) FDA-approved FISH kits and the SPoT-Light CISH kit were compared. Tumours were also evaluated by IHC with the FDA-approved HercepTest kit and a validated in-house IHC protocol. Fifty-five patients with invasive breast carcinoma were selected as a representative proportion of HER2 IHC 2+ cases. HER2 amplification was observed in 31% of tumours by PathVysion compared with 33% with pharmDx. The number of amplified tumours detected by INFORM and CISH varied with the threshold applied. Agreement was excellent between PathVysion and pharmDx (100%), good with SPoT-Light (89%; cutoff at least five signals per nucleus) and moderate with INFORM (76%; cutoff more than four signals per nucleus). Agreement with INFORM improved to 98% with a cutoff of at least six signals per nucleus. With an appropriate cutoff, the INFORM kit was comparable to dual-probe FISH kits for evaluating HER2 status. We 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.
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