HER-2/neu amplification testing in breast cancer by multiplex ligation-dependent probe amplification in comparison with immunohistochemistry and in situ hybridization.
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.


CONCLUSIONS
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.
Moelans
Cathy B
CB
Department of Pathology, University Medical Centre Utrecht, Heidelberglaan 100, Utrecht, The Netherlands.
de Weger
Roel A
RA
van Blokland
Marja T M
MT
Ezendam
Chantal
C
Elshof
Sabrina
S
Tilanus
Marcel G J
MG
van Diest
Paul J
PJ
D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005784:Gene Amplification; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D022521:Ligase Chain Reaction / Q000191:economics / Q000379:methods*; D015335:Molecular Probes / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism
D015335:Molecular Probes; D018719:Erb-b2 Receptor Tyrosine Kinases