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. TopoIIa (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 TopoIIa gene amplification status in paraffin-embedded breast cancer samples. To this end, MLPA results were compared with TopoIIa, HER2 chromogenic in situ hybridization (CISH). We also assessed TopoIIa protein expression by immunohistochemistry. Of 353 patients, 9% showed TopoIIa amplification by MLPA and 13% of patients were HER2 amplified. TopoIIa amplification was seen in 42% of HER2-amplified cases and showed no high level amplification without HER2 amplification. Eleven patients displayed TopoIIa loss (3%). Concordance between MLPA and CISH was 91% for TopoIIa and 96% for HER2. Correlation between amplification and overexpression of TopoIIa was significant (P ¼ 0.035), but amplification did not always predict protein overexpression. Loss of the TopoIIa 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 TopoIIa copy number status in paraffin-embedded tissue, and thus an attractive supplement or alternative to CISH.

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Background: Assessment of HER-2/neu status in invasive breast cancer is crucial to establish eligibility for trastuzumab and taxane based chemotherapy. Next to immunohistochemistry (IHC) to evaluate protein overexpression, a second line gene amplification test is required for cases with equivocal protein expression. This study aimed to validate a new PCR based test, called Multiplex Ligation-dependent Probe Amplification (MLPA), as a simple and quick method to assess HER-2/neu gene amplification status in invasive breast cancer.Methods: MPLA results were compared with gene amplification status assessed by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as gold standard, and with protein overexpression by IHC in 518 breast carcinoma patients.Results: About 10% of cases overexpressed HER-2/neu at the protein level (IHC), and 11% of cases showed gene-amplification by MLPA. A high concordance was found between FISH and CISH, MLPA and IHC, and MLPA and CISH. MLPA showed amplification in 7/36 (19%) of the equivocal IHC 2+ cases. However, of the IHC 0/1+ cases, 6/434 (1.4%) were also amplified by MLPA, and amplification was confirmed in all of these cases by FISH/CISH. On the other hand, one of the 48 (2%) IHC 3+ cases was normal by MLPA and lack of amplification was confirmed by FISH/CISH.Conclusion: MLPA is a fast, accurate and cheap method to detect breast cancer HER-2/neu amplification in small quantities of DNA extracted from paraffin blocks, and thereby a reliable alternative to FISH and CISH.