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The Concordance Between the Clinical Trials Assay (CTA) and Fluorescence in Situ Hybridization (FISH) in the Herceptin Pivotal Trials. Robert D. Mass, Corsee Sanders, Kasian Charlene, Lori Johnson, Tajuana Everett, Steve Anderson, Genentech, Inc, South San Francisco, CA; Lab Corp of America, Research Triangle Park, NC.

Overexpression of HER2 at the 2+ or 3+ level by immunohistochemistry (IHC) was required for enrollment in the pivotal Herceptin metastatic breast cancer trials. The CTA involves two separate IHC assays performed with either monoclonal antibodies 4D5 or CB11. Subjects were eligible if either assay was scored at 2 or 3+. If both were performed, the final score was the higher of the two results. Concordance between the CTA and another IHC, HercepTest (HT), is 79%. This was the basis for FDA approval of HT to aid in the selection of patients for Herceptin therapy. We undertook a similar concordance study, utilizing clinical material submitted for screening for the Herceptin pivotal trials, comparing the CTA to HER2/neu gene amplification measured by the PathVysion FISH assay. In the pivotal trials, 5998 subjects were screened for HER2 expression; 1915 [32%] were positive by the CTA and 4083 [68%] were negative. A random sample of 623 specimens (1:1 ratio of positive:negative) were selected for this analysis, 317 CTA+ and 306 CTA-. Specimens were not freshly cut from blocks. They had been stored between 2 and 4 years as 4-6m sections on glass slides. Each section was assayed for HER2/neu amplification using the protocol specified in the package insert of the PathVysion assay. Amplification was defined as a signal ratio of ³ 2. For the total 623 specimens tested, a FISH signal result was obtained in 529. Assay failure occurred in 19.9% of CTA- and 10.4% CTA+ samples. Amplification in the 0,1+,2+,3+ groups was 4.2%, 6.7%, 23.9%, and 89.3% respectively. The sample concordance was 81.3%, similar to the CTA/HT concordance of 79%. Single copy overexpression was 31%, predominantly in the 2+ group. Amplification was rarely (4.6%) noted in the CTA- group. The higher assay failure rate in the CTA- group may be due to non-assay related factors such as tissue fixation. These may have also resulted in false negative results for IHC. These data suggest that HER2/neu amplification status may provide similar predictive value for Herceptin benefit as compared to HercepTest. A direct analysis of Herceptin benefit, based on FISH score, will be presented.