Amplification of the HER2 gene, present in 15-30% of breast carcinomas, correlates with poor outcome and is an indication for treatment with trastuzumab. Standard testing methods for HER2 amplification are fluorescence (FISH) or chromogenic in situ hybridization (CISH). In FISH/CISH scoring, correction for chromosome 17 polysomy is believed to be critical for determination of true HER2 amplification as opposed to increased chromosome 17 copy number. The term "polysomy 17" is widely used and defined as > or =3 copies of the chromosome 17 centromere (probe CEP17, D17Z1). Thus, the centromere is assumed to be representative for the entire chromosome. This study aimed to investigate the frequency of polysomy 17 and its association with HER2 amplification in 111 invasive breast cancer patients by CEP17 CISH and by copy number analysis of a set of 17 genes along chromosome 17 using multiplex ligation-dependent probe amplification (MLPA).Chromosome 17 usually showed a complex pattern of gains and losses by MLPA, unrelated to the copy number status of the centromere. Increase in centromere 17 copy number (denoted "polysomy 17"), as assessed by CEP17 CISH, was found in 19% of the patients. Of these patients, 60% also showed amplification of HER2 measured by MLPA. However, none of the 111 patients showed a true polysomy of chromosome 17 by MLPA. Only two patients (1.8%) had a possible gain of 17q. Amplification of 17p was not found in any of the patients, although a possible loss of 17p was found in one patient. In conclusion, this extensive analysis of amplicons along chromosome 17 shows that true polysomy of chromosome 17, either of the whole chromosome or of the short or the long arm, is very rare in invasive breast cancer. Abnormal CEP17 copy numbers may therefore actually stem from high level gains or amplification of CEP17 regardless of copy number gains of the short and long arms of chromosome 17 and, at least in some cases, correction with CEP17 probes may provide misleading HER2 gene status assessment results.