Molecular characterisation of the Her2-Top2A amplicon in breast cancer
Date
2010-09-17
Authors
Herd, Olivia Jayne
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Abstract
The HER2 gene is amplified in 20-30% of breast cancers, a common cancer amongst
South African women. HER2 amplification is associated with a poor prognosis and
predicts response to treatments such as Herceptin. The gold standard for HER2 testing
is Fluorescent in situ Hybridisation (FISH) with dual colour probes for the HER2
gene and chromosome 17 centromere (CEP17) internal control. According to
international guidelines, a HER2/CEP17 ratio >2.2 is considered positive. The HER2
FISH test is complicated by the emergence of ambiguous cases with increased CEP17
signals that cannot be accounted for by chromosome 17 polysomy (> 6 copies of
CEP17) and that may hide true HER2 gene amplification.
The aims of this study were to characterise the HER2 amplicon, in particular the copy
number of genes in the vicinity of the HER2 gene, and to design an alternative control
probe that could clarify the HER2 gene status in ambiguous cases. In addition, results
on 1558 breast cancer specimens sent for routine testing were analysed to determine
the trends of HER2 amplification amongst South African women.
The rate of HER2 gene amplification was significantly higher (p < 0.05) in African
patients (52%) than in Caucasian patients (43%). In Caucasian women, the rate of
HER2 amplification in the younger group (68%) was significantly higher (p < 0.05)
than in the general Caucasian group (43%), while the same was not seen in the
African cohort.
Nineteen ambiguous cases with more than 9 copies of CEP17 were further
investigated. FISH assays with four different probe kits (PathVysion HER-2:
Poseidon Repeat free TOP2A, HER2, CEP17: and Vysis PML-RARA respectively)
were performed to determine the copy number of the HER2, TOP2A, RARA genes
and CEP17. An in-house dual colour probe kit was designed using the ACTG1 gene
as a control for HER2. Of the 19 ambiguous cases, 16 had centromeric amplification,
showing that CEP17 is no longer an adequate internal control in FISH HER2 testing.
The TOP2A gene was only amplified in HER2 positive cases and the RARA gene
was only amplified when the TOP2A gene was also amplified. FISH with ACTG1 as
v
a control clearly revealed HER2 amplification in ambiguous cases on image analysis
and gave HER2/ACTG1 ratios significantly higher than HER2/CEP17 ratios.
However, screening of an additional 40 unambiguous cases showed an increased copy
number, although limited ( 8), of the ACTG1 gene in four patients; this warrants
further testing to assess the value of this gene as a control. Interestingly, a trend was
observed for ACTG1 increased copy number in HER2 negative cases, this may point
to the presence of a driver gene whose amplification tends to be mutually exclusive from HER2 amplification.
Description
MSc (Med), Faculty of Health Sciences, University of the Witwatersrand
Keywords
breast cancer, Her2-Top2A amplicon