Previously published studies have reported high concordance between HER2
FISH and HER2
CISH performed in breast cancer specimens [7
], however, larger studies that validate the use of HER2
CISH have not yet been presented. In the current investigation 365 primary breast cancer specimens were included and an overall HER2 status agreement close to 98% is reported when comparing test results obtained by HER2
CISH pharmDx™ Kit to results obtained by HER2
FISH pharmDx™ and PathVysion HER-2 FISH DNA Probe Kit. The study population was enriched for HercepTest™ IHC 2+ specimens because most testing modalities pass on such equivocal specimens to a genetic test. For overall agreement the lower 95% confidence interval limits were at or above 96% in the two comparisons, further stressing the reliability of the HER2
CISH pharmDx™ in this comparison to the two FISH analysis methods.
The number of HER2
amplified or HER2 positive consecutively collected specimens reported in this study by the three ISH assays (10.8%, 11.4%, 11.0%) and by HercepTest™ IHC (10.5%) seems to be lower than expected based on previously published data [1
] in which the overall HER2 positivity rate was found at 22% with observations ranging from 9%-74%. However, Ross et al. (2009) [1
] indicate that the true HER2 positivity rate probably is in the range 15-20%, with national reference labs and community hospitals reporting lower rates and tertiary hospitals and cancer centers reporting slightly higher rates.
In the current data analysis a significantly higher average CEN-17 signal counts in HER2 CISH compared to HER2 FISH and PathVysion FISH with no significant difference in HER2 signal counts between the three assays were observed. This resulted in significantly lower HER2/CEN-17 ratios observed for HER2 CISH. There was no clinical diagnostic impact of this change as the concordance agreement calculations revealed very fine agreement between HER2 CISH and the two FISH assays. In support of the good agreement the analysis of test results for specimens having a HER2/CEN-17 CISH ratio below 3.0 revealed that identical ratios were obtained for HER2 CISH, HER2 FISH and PathVysion FISH. Surprisingly, average copy numbers for both HER2 and CEN-17 were higher for the HER2 CISH method, whereas, HER2 CISH standard deviations seemed to be lower for the average HER2/CEN-17 ratio, HER2 and CEN-17 copy numbers compared to PathVysion FISH. This could be interpreted as the HER2 CISH assay being the most sensitive assay.
Also, there was a tendency towards differences between HER2/CEN-17 ratios in highly amplified specimens between HER2 CISH and FISH assays (see Figure and ). This is probably due to the variation in cluster estimation and is not a problem in relation to achieving the correct diagnosis.
As a parallel study, the slide evaluation time was compared between HER2 CISH and PathVysion FISH and as could be expected the average evaluation time for a CISH staining was significantly shorter. This is most likely due to the easy access to the morphological information and hence a faster selection of areas for enumeration. In HER2 CISH a higher number of specimens were reported failed in comparison to staining with PathVysion FISH. This is likely to be due to CISH being a new technique implemented in the US reference laboratory and PathVysion FISH being a well established test method at this site. With respect to HER2 CISH the normal cells in the tissue surrounding the tumor area can be used as internal control securing good staining quality and preventing wrong diagnosis being based on a failed slide.