To avoid subjecting breast cancer patients to unnecessary financial burden and significant side effects, the selection of those most likely to response to HER2-directed therapy must be accurate. Our original motivation for developing the tricolor HER2 gene-protein assay was to deliver a tissue-based HER2 test that is more accurate than the separate HER2 IHC and HER2 ISH assays. Because HER2 IHC assays are technically easier to perform than HER2 FISH assays, 80% of newly diagnosed breast cancer cases in the US are analyzed for HER2 status using HER2 IHC . However, the technical issues that can complicate HER2 IHC assays, such as unstandardized antigen retrieval protocols and multiple antibody clones, have led to the recommendation of HER2 ISH as the first-line assay for HER2 status assessment . Tissue quality for HER2 ISH assays can be assessed using the ISH signals in normal cells surrounding the tumor cells as internal controls. In contrast, assessment of tissue quality for HER2 IHC assays is difficult; because there is no proven internal control, false negatives can result .
A 2007 report of the American Society of Clinical Oncology-College of American Pathologists (ASCO-CAP) concluded that 20% of HER2 assays performed in the field were not accurate and established guidelines to improve the accuracy of HER2 testing in breast cancer . However, a 2008 follow up study using survey results from 757 laboratories indicated that substantial gaps remained in assay validation . Lee et al. also reported that only 15% (7/46) of reported studies of the concordance between HER2 IHC and HER2 FISH results achieved the ASCO-CAP guideline of 95% or greater concordance.
Breast tumor heterogeneity is a major cause of discordance between HER2 IHC and HER2 FISH assay results [16, 17] and approximately 5-30% of HER2 positive breast cancer cases exhibit intratumoral genetic heterogeneity . Subtle HER2 genetic heterogeneity of tumor cells has been reported among equivocal cases [17, 19]. An alternative method for determining HER2 status from FFPE breast cancer samples based on the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been proposed, but has not been approved by the FDA. Based on a recent publication comparing the performance of HER2 qRT-PCR-based testing with that of the FDA-approved HER2 IHC and HER2 FISH methods , Ignatiadis and Sotirious have raised concerns about the use of HER2 qRT-PCR for clinical diagnostics . The HER2 qRT-PCR method failed to detect equivocal cases and produced false negative results. Therefore, the need for a better assay to assess HER2 status in breast cancer, particularly in equivocal cases and in cases with tumor heterogeneity, remains.
With an incidence of approximately 4%, HER2 false negative (IHC negative and FISH positive) and false positive (IHC positive and FISH negative) results cannot be ignored [1, 17]. In one study, 9.7% (174/1787) of breast cancer patients were HER2 false positive cases, but they still benefited from adjuvant trastuzumab therapy . In another study, lapatinib therapy had significant positive effects in FISH positive breast cancer patients whose IHC tests had been 0, 1+, or 2+ . Thus, the detection of both false negative and false positive HER2 breast cancer cases is important.
HER2 IHC assays are effective methods for detecting tumor heterogeneity and equivocal cases based on HER2 protein staining under a light microscope, but these assays are semi-quantitative and subjective. Thus, additional quantitative gene analysis is required for equivocal cases using a HER2 ISH assay. HER2 false negative cases will be missed if only HER2 IHC is applied while HER2 false positive cases will be missed if only HER2 ISH method is utilized. Thus, the optimum HER2 testing protocol uses both HER2 IHC and HER2 ISH assays .
To overcome the weaknesses of current HER2 tests, we have successfully developed an automated brightfield tricolor gene-protein assay for the detection of HER2 protein, the HER2 gene, and CEN17. The novel aspect of the assay is the use of a blocker to prevent background staining caused by the binding of the DNP hapten of the HER2 probe to tissue sections after they have been processed through a DAB-based IHC assay. Although three research groups have previously reported the technical achievement of combining HER2 IHC and single color brightfield HER2 ISH to co-visualize HER2 protein and the HER2 gene on FFPE breast cancer tissue sections, all of these combined assays required several manual steps for the ISH procedure.
The HER2 gene-protein assay described herein is a significant improvement in the field because: 1) it demonstrates tricolor co-localization of the HER2 protein, HER2 gene, and CEN17 targets on well-preserved breast cancer tissue sections and 2) it automates the entire protocol of a gene-protein assay from deparaffinization to counterstaining. Extensive analyses of the findings of three pathologists with different levels of HER2 test scoring experience for the combination HER2 gene-protein assay relative to those of the single HER2 IHC and HER2 & CEN17 BISH assays revealed excellent concordance. The statistical analysis suggests that the HER2 gene-protein assay is a robust and reliable assay and provides advantages over single HER2 IHC and HER2 & CEN17 BISH assays.
Among the technical challenges we faced in developing the HER2 gene-protein assay was identifying an appropriate multicolor scheme. Previously, Downs-Kelly  and Ni et al. used AP-based fast red staining of HER2 protein followed by HRP-based silver or DAB staining of HER2 gene, respectively. We evaluated AP-based fast blue detection of HER2 IHC followed by the HER2 BISH assay to obtain a tricolor detection scheme in which HER2 protein was blue, the HER2 gene was black, and CEN17 was red (data not shown), but this scheme proved to be less than optimal; the pathologists had difficulty scoring weak HER2 IHC staining because the fast blue AP-based IHC staining was less crisp and because both fast blue and hematoxylin counterstain are blue. Therefore, because most pathologists are accustomed to scoring DAB-based IHC detection for HER2 protein, we investigated a detection scheme using a combination of conventional DAB-based detection of HER2 protein and BISH detection of HER2 and CEN17 targets. The sequence of HER2 IHC and HER2 & CEN17 BISH staining was also evaluated to optimize HER2 protein staining. As Reisenbichler et al. previously noted, we observed weaker HER2 protein staining when the HER2 IHC portion of the assay was performed after the HER2 & CEN17 BISH portion of the assay (data not shown). They compensated for the weaker HER2 IHC staining by increasing the anti-HER2 antibody incubation time from 30 min to 45 min. In contrast, we determined that the HER2 IHC steps should be performed first to maintain HER2 IHC staining quality, particularly in cases with low expressed HER2 protein.
Reisenbichler et al. also reported that they could not obtain HER2 CISH signals when the CISH assay was performed after HER2 IHC using DAB detection. We encountered a similar obstacle during the development of our HER2 gene-protein assay, but primarily for CEN17 BISH detection. We found that a longer protease digestion time or a higher protease concentration was required to obtain a consistent CEN17 BISH signal with difficult tissue samples to stain for CEN17 signals. As we have demonstrated in this report, our optimized HER2 & CEN17 BISH assay provided successful visualization of the HER2 gene and CEN17 targets after DAB-based HER2 IHC.
Another major issue encountered during assay development was a high level of silver background staining from the silver-based HER2 BISH detection. The silver background staining was observed mainly in the nuclei and also some background staining was seen with DAB staining. It did not occur when the DNP-labeled HER2 probe was omitted from the assay (data not shown). Also, omission of the DAB chromogen and hydrogen peroxide from the IHC procedure prevented silver background staining from silver-based HER2 BISH detection (data not shown). Therefore, we hypothesized that there was an interaction between DAB and the DNP hapten and the BISH detection for the DNP hapten was responsible for the high levels of silver background staining. Because extra washing after the HER2 IHC did not eliminate the silver background staining (data not shown), we concluded that the DAB molecules were covalently bound to the nuclear DNA.
It is well established that DAB is a carcinogen and that carcinogenic agents bind to DNA. Oxidative intermediates of the DAB analogue benzidine have been shown to form covalent bonds to DNA, thereby localizing DAB in the cell nucleus [24, 25]. During the development of the HER2 gene-protein assay, the DNP-labeled probes appeared to be binding to the peroxidase deposited DAB. The exact mechanism of this interaction is unknown, but electron-rich aromatic compounds (such as DAB) and electron-deficient aromatic compounds (such as DNP) are known to form aromatic pi-stacks and/or charge transfer complexes . Therefore, we speculated that another aromatic molecule present in excess during hybridization would act as a competitor for this non-covalent attraction, analogous to the use of protein blockers in protein immunodetection to prevent non-specific protein binding. After testing several compounds with various electronic and aqueous solubility properties (data not shown), we identified naphthol phosphate as a suitable blocker for use during hybridization with DNP-labeled probes.
A HER2 gene-protein assay could be developed by combining two darkfield assays, namely HER2 fluorescence IHC and HER2 FISH assays. However, the current brightfield HER2 gene-protein assay offers several advantages over a darkfield gene-protein assay: 1) the ability to simultaneously observe the HER2 protein, HER2, and CEN17 targets in the context of tissue morphology; 2) the use of an established scoring system for DAB-based HER2 IHC assays; 3) the use of a regular light microscope for slide observations, negating the need for a darkroom; 4) full automation, which is optimal for reproducibility; and 5) permanent preservation of both the IHC and ISH signals. A HER2 gene-protein assay could also be developed using DAB-based HER2 IHC and HER2 FISH assays. This assay would have fewer disadvantages than the gene-protein assay using fluorescence IHC, but would still require an expensive fluorescence microscope and a darkroom. In addition, long term preservation of the FISH signal would be difficult and no completely automated protocol would be available for this assay.