nature genetics | volume 40 | number 7 | july 2008 807 ence channel at the ESR1 locus and found no evidence of CNV. Thus, CNV is unlikely to be the explanation for the discrepancy. The key difference between our study and that of Holst et al.1 is the methodology for scoring FISH-TMA (manual vs. automated) and the criteria used to call amplifications. Holst et al.1 scored as amplified not only cases with an ESR1 to centromere 6 ratio ≥2 but also “tumors with tight signal clusters...independent of their ESR1/centromere 6 ratio.” They report using previously the same definition of amplification for CCND1, ERBB2, MDM2 and MYC8. However, review of this publication reveals use of a single amplification criterion: signal ratio ≥2. As the authors state that “most amplified cases showed a clustered arrangement of additional ESR1 copies”1, we interpret this to mean that most of the ESR1-amplified cases were scored using subjective criteria. In contrast, the automated system we used to score FISH signals employs specific measurement algorithms to detect and quantify such clustered signals. We have previously reported a high correlation between manual and automated scoring of FISH signals and have implemented the use of this system for the scoring of gene amplification events2.The system is FDA approved for the automated scoring of ERBB2 gene amplification (Metasystems). Using this objective set up, we found that only one case had a tight cluster of signals. In summary, our data compiled from several institutions and obtained using two different techniques does not validate the findings of Holst et al., and we conclude that ESR1 amplification in breast cancer is a rare event of unknown clinical significance.