title: 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.
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 > or =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.
authors.affiliation: Laboratory for Pathology, PAMM Laboratories, Eindhoven, The Netherlands.
authors.forename: Elna
authors.initials: E
authors.lastname: Moerland
authors.forename: Rens L H P M
authors.initials: RL
authors.lastname: van Hezik
authors.forename: Toine C J M
authors.initials: TC
authors.lastname: van der Aa
authors.forename: Mike W P M
authors.initials: MW
authors.lastname: van Beek
authors.forename: Adriaan J C
authors.initials: AJ
authors.lastname: van den Brule
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005784:Gene Amplification; D018734:Genes, erbB-2; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity