Although HER2 gene status determines the eligibility of breast cancer patients to trastuzumab therapy, only a fraction of HER2 gene-amplified cancers are actually responsive, indicating that complex genotypical profiles might sustain HER2-targeted therapy responsiveness. To identify genetic factors modulating the response to HER2-targeted therapy, the numerical status of chromosome 17 was evaluated in HER2 gene-amplified tumor specimens from 43 patients who received trastuzumab-based treatment for advanced breast cancer, and in 60 unamplified breast carcinomas. Archival tumor specimens were analyzed by interphase fluorescence in situ hybridization (FISH) using a centromeric probe for chromosome 17 simultaneously with the HER2/neu human gene-specific probe. In the 43 patients who received trastuzumab-based treatment, response rate and time to progression (TTP) were compared between subgroups according to chromosome 17 status. Numerical aberrations of chromosome 17 were found in 65% of HER2-amplified tumors (single centromeric signal, 44%; >3 centromeric signals, 21%) and 60% of unamplified cancers (single centromeric signal, 43%; >3 centromeric, 17%). In the 43 treated metastatic breast cancer patients, trastuzumab was combined with docetaxel (35 patients), paclitaxel (2 patients), or vinorelbine (6 patients). Response rate was 92% in patients with >2 centromeric signals (chromosome 17 eusomy/polysomy), and 53% in patients whose tumor showed a single signal (chromosome 17 monosomy) (p=0.005). Chromosome 17 numerical status was not found to affect TTP. Multivariate logistic regression analysis confirmed that the effect of chromosome 17 monosomy on tumor response was independent of other potential clinical variables. Our findings suggest that 17 monosomy defines a subgroup of HER2 gene-amplified breast cancer characterized by reduced responsiveness to trastuzumab-based treatment. Further studies on the imbalance between the amplified HER2 gene and functionally antagonist gene(s) on chromosome 17 are warranted.