Biomedical Engineering Reference
In-Depth Information
laboratory determinations of HER-2 amplification by FISH, FISH
assays have been found to be more accurate and reproducible [61].
This is likely due, at least in part, to the more quantitative nature of
these measurements.
Two additional methods to determine HER-2 gene amplification
have been developed. Chromogenic
in situ
hybridization (CISH)
and silver enhanced
hybridization (SISH) are similar to FISH
except that the HER-2 DNA probes are detected by using a peroxidase
reaction [62, 63]. This allows detection using a light microscope rather
than a fluorescent microscope, and the stained slides can be stored
indefinitely (unlike FISH slides, which rapidly fade). Comparisons of
the CISH and SISH methods with FISH demonstrated a 94% and 96%
concordance, respectively [62, 63]. Thus the methods are reasonable
alternatives to FISH, but they are not available in the United States
for routine use [54].
To standardize HER-2 testing for both clinical practice and future
clinical trials, the American Society for Clinical Oncology and the
College of American Pathologists developed guidelines for HER-2
testing for use in breast cancer patients [54]. A positive HER-2
test using a validated IHC assay (e.g., the Herceptest or Pathway
antibodies) was defined as a tumor expressing uniform intense
membrane staining on >30% of the invasive tumor cells (IHC 3+).
An equivocal HER-2 test by IHC was defined as a tumor expressing
non-uniform or weak membrane staining on at least 10% of the
cells (IHC 2+). Samples with less staining were defined as negative
(IHC 0 or 1+). These guidelines recommend evaluation of samples
with equivocal IHC staining (IHC 2+) by a validated assay for gene
amplification. A positive test for gene amplification by FISH was
defined as either a HER-2/CEP17 ratio of > 2.2 or > 6 copies of
HER-2/nucleus if no chromosome 17 control is done. An equivocal
FISH test was defined as a HER-2/CEP17 ratio from 1.8-2.2. The
HER-2/CEP17 ratio is derived from the average signal number for
each probe from 60 cells. The indeterminate range was derived from
data suggesting that low level amplification could result in variable
results when read by different pathologists (i.e., when a different set
of 60 cells are scored) [64]. The guidelines noted that most clinical
trials defined amplification by FISH as either HER-2/CEP17 >2.0 or ≥
2.0; thus patients with HER-2/CEP17 from 2.0-2.2 were included on
the trials [54]. Few patients (~3%) will have values between 2.0-2.2
and the guidelines note that the data do not support excluding such
in situ
