Detection of HER2 amplification in breast carcinomas: Comparison of Multiplex Ligation-dependent Probe Amplification (MLPA) and Fluorescence In Situ Hybridization (FISH) combined with automated spot counting
Affiliations: Laboratory for Pathology, PAMM Laboratories, Eindhoven, The Netherlands
Note: [] Corresponding author: Dr. A.J.C. van den Brule, Laboratory for Pathology, PAMM Laboratories, Michelangelolaan 2, 5623 EJ Eindhoven, The Netherlands. Tel.: +31 40 2396100; Fax: +31 40 2396109; E-mail: a.van.den.brule@pamm.nl.
Abstract: In this study the detection of HER2 gene amplification was evaluated using Fluorescence In Situ Hybridization (FISH; PathVysion) in comparison with Multiplex Ligation-dependent Probe Amplification (MLPA), a PCR based technique. These two methods were evaluated on a series of 46 formalin fixed paraffin embedded breast carcinomas, previously tested for protein overexpression by HercepTest (grouped into Hercep 1+, 2+ and 3+). HER2 gene amplification (ratio ≥2.0) by FISH was found in 9/10, 10/30 and 0/6 in IHC 3+, 2+ and 1+/0 cases, respectively. Digitalized automated spot counting performed with recently developed CW4000 CytoFISH software was 100% concordant with manual FISH scoring. Using MLPA 18/46 samples showed a clear HER2 amplification. Comparing MLPA and IHC showed the same results as for FISH and IHC. All but one FISH positive cases (18/19) were confirmed by MLPA for the presence of the gene amplification. The overall concordance of detection of Her2 gene amplification by FISH and MLPA was 98% (45/46). Furthermore, both the level of amplification and equivocal results correlated well between both methods. In conclusion, MLPA is a reliable and reproducible technique and can be used as an either alternative or additional test to determine HER2 status in breast carcinomas.
