566 Background: Controversy exists regarding the optimal assay to measure HER2 status. We previously reported that breast cancer patients with HER2 amplified tumors benefit from dose intense doxorubicin-based therapy (Muss, NEJM, 1994). In the current study, we directly compare HER2 status, using the FDA-approved PathVysion (PV) FISH kit, and the new Tricolor (TC) FISH system (both from Vysis, Inc), which, in addition to the HER2 and chromosome 17 (CEP17) probe, contains a third probe against the TOPO2 gene, a target of doxorubicin therapy. Because the TC system involves different assay conditions, it was important to determine if this affected HER2 status and predicting outcome. METHODS The PV and TC methods were performed on two sections from the same tumor block from 408 stage II breast cancer patients registered to CALGB 8541 (dose-intensive CAF therapy; cyclophosphamide, doxorubicin and 5FU, Cancer and Leukemia Group B). Three different measures were performed: HER2 amplification status, using the ratio of HER2:CEP17 signals, where a ratio >=2.00 defined a case as amplified, per kit instructions; HER2 amplification status based only on the HER2 signal, where >=4 signals defined a case as amplified; and polyploidy of chromosome 17, where >=3 signals defined polyploidy. RESULTS There was no significant difference between the two kits in assigning a case as HER2 amplified or not amplified using the ratio definition (McNemar's exact test, p=.18; kappa=0.862). Of the 14 discordant cases, most had ratios near the cutpoint (e.g. 1.71 by PV vs 2.03 by TC). Using the HER2 signal alone (not normalized to CEP 17), we found significant differences between the two kits, with TC giving a higher frequency of amplification (McNemar's test, p=.02). The TC system also identified a higher frequency of chromosome 17 polyploidy. Using the ratio definition, both kits predicted outcome similarly (K-M plots nearly superimposable). CONCLUSION Using the chromosome 17 probe to normalize HER2 amplification status yields the most consistent results for HER2 measurement and predicting clinical outcome in a comparison between the PathVysion kit and the new Tricolor probe FISH system. No significant financial relationships to disclose.