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have a ratio more than 2 [ 47 ]. Recent studies have documented
experience of false-positive IHC results due to interpretation
difficulties. Therefore, it is safe to recommend confirmation of
IHC = “3+” cases by gene amplification before commencing
anti-HER2 therapy to avoid toxic and costly treatment to
potential ineligible patents.
When analyzing in situ hybridization samples, the entire
specimen should be screened for amplified regions at a low
power magnification, correlating with positive areas as detected
by immunohistochemistry. This is particularly important in flu-
orescence in situ hybridization samples where a bright field is
not available. A strong correlation has been demonstrated with
IHC 3+ score and in situ hybridization results [ 47 – 49 ]. Areas
with overlapping nuclei, high nonspecific background staining
(dark field methods only), or where there is a weak signal or the
presence of artifacts (e.g., dust grains) should not be included in
the interpretation. Using a 40× magnification, it is recom-
mended that at least 20 evaluable and non-overlapping cells be
counted initially, including cells from different tumor areas to
provide a more uniform score interpretation. Although FISH
has been regarded to be the gold standard in the setting of
HER2 testing for breast cancer bright field techniques have
been found to be superior in gastric/esophagogastric junction
cancer setting [ 47 ].- Recommendation for HER2 testing
In the ToGA subgroup analysis, greatest survival benefit was
demonstrated in those with a high level of protein expression
(IHC = “2+” or IHC = “3+” and FISH positive), compared to
Fig. 6 SISH: A small cluster (arrow) counted as six signals, and larger clusters as
12 signals (arrow head)Duminda Subasinghe et al.