HER-2/neu amplification testing in breast cancer by Multiplex Ligation-dependent Probe Amplification: Influence of manual- and laser microdissection

Background: Accurate assessment of HER-2/neu status is crucial for proper prognostic information and to offer direct appropriate treatment for breast cancer patients. Next to immunohistochemistry (IHC) to evaluate HER2 protein overexpression, a second line gene amplification test is generally deemed necessary for cases with equivocal protein expression. Recently, a new PCR based test, called Multiplex Ligation-dependent Probe Amplification (MLPA), was introduced as a simple and quick method to assess HER-2/neu gene amplification status in invasive breast cancer. MLPA was previously shown to correlate well with IHC and in situ hybridization (ISH), but a low tumor percentage in the tissue tested could negatively affect the accuracy of MLPA results. Methods: To examine this, MLPA was repeated in 42 patients after serial H&E section guided manual dissection with a scalpel and after laser microdissection of the tumor. Results: Both dissection techniques led to higher HER2 gene copy numberratios and thereby made MLPA more quantitative. Concordance between MLPA and ISH improved from 61% to 84% after manual microdissection and to 90% after laser microdissection. Conclusion: Manual and laser microdissection similarly increase the dynamic range of MLPA copy number ratios which is a technical advantage. As clinically a dichotomization between normal and amplified suffices and MLPA is relatively unsensitive to tumor content, microdissection before MLPA may not be routinely necessary but may be advisable in case of very low tumor content (≤30%), when MLPA results are equivocal, or when extensive ductal carcinoma in situ is present. Since differences between manual and laser microdissection were small, less time consuming manual microdissection appears to be sufficient.