HER-2/neu gene amplification, found in certain subtypes of (breast-) cancers, is an independent prognostic factor of poor outcome and determines eligibility for systemic treatment with trastuzumab. TopoII alpha (TOP2A) gene amplification seems to be predictive of response to a class of cytostatic agents called TopoII inhibitors, which include the anthracyclines. The observed increased efficacy of anthracyclines in HER2-positive tumors is thought to arise from the close proximity of both genes on chromosome 17, where the TopoII amplification status will determine the anthracycline sensitivity. This study aimed to validate a new polymerase chain reaction-based test, called multiplex ligation-dependent probe amplification (MLPA), as a simple and quick method to simultaneously assess HER-2/neu and TopoII alpha gene amplification status in paraffin-embedded breast cancer samples. To this end, MLPA results were compared with TopoII alpha, HER2 chromogenic in situ hybridization (CISH). We also assessed TopoII alpha protein expression by immunohistochemistry. Of 353 patients, 9% showed TopoII alpha amplification by MLPA and 13% of patients were HER2 amplified. TopoII alpha amplification was seen in 42% of HER2-amplified cases and showed no high level amplification without HER2 amplification. Eleven patients displayed TopoI alpha loss (3%). Concordance between MLPA and CISH was 91% for TopoII alpha and 96% for HER2. Correlation between amplification and overexpression of TopoII alpha was significant (P=0.035), but amplification did not always predict protein overexpression. Loss of the TopoII alpha gene was almost never associated with loss of its protein. In conclusion, MLPA is an easy and accurate method to simultaneously detect breast cancer HER-2/neu and TopoII alpha copy number status in paraffin-embedded tissue, and thus an attractive supplement or alternative to CISH.