Biomedical Engineering Reference
In-Depth Information
criteria of overexpression (reviewed in [54]). For example, in some
studies testing was performed using IHC with an antibody to HER-2
developed by the investigators known as the clinical trials assay (CTA).
The CTA assay was composed of two monoclonal antibodies, CB11
and 4D5. The latter antibody is the mouse monoclonal antibody that
was humanized to create trastuzumab [55]. Scoring was IHC 0-1+
(negative), IHC 2+ (weakly positive membrane), or IHC 3+ (strongly
positive membrane staining) (e.g., Fig. 4.2A). Other studies using the
A085 polyclonal antibody from DAKO scored tumors as IHC 0-1+
(negative), IHC 2+ (weakly positive membrane staining on more than
10% of the cells), or IHC 3+ (strongly positive membrane staining on
more than 10% of the cells). The A085 polyclonal antibody (DAKO)
and the CB11 monoclonal antibody (Ventana) are approved by the
FDA for IHC measurement of HER-2 overexpression [54]. Several
studies have shown that the different antibodies vary in terms of
specificity, sensitivity and accuracy [56, 57]. In addition, all of the
IHC based definitions of HER-2 overexpression are subjective.
Fluorescent
hybridization (FISH) was developed to measure
gene amplification in formalin fixed, paraffin embedded tissues by
quantifying the number of copies of the HER-2 gene in interphase
nuclei of cells (Fig. 4.2B) [58]. This method uses fluorescently labeled
HER-2-specific DNA probes or biotin-labeled HER-2-specific DNA
probes, which are then detected by fluorescently labeled avidin [54,
58]. Amplification can be determined by two scoring criteria. In one,
more than four copies of HER-2/cell was considered gene amplification
[58]. The second method normalized the number of copies of HER-2/
cell to the number of copies of chromosome 17 measured by a probe
to the centromere for chromosome 17 (CEP17). In this method, a
HER-2/CEP17 ratio ≥ 2.0 was considered gene amplification [58]. A
number of studies have found that overexpression based on IHC is
rarely seen in the absence of gene amplification [58-60]. Currently
two commercial kits, the PathVysion HER-2 DNA probe kit (Abbott),
which measures the HER-2/CEP17 ratio, and the Inform HER2/
in situ
neu
probe (Ventana), which measures the number of HER-2 copies/cell,
are FDA approved for the measurement of HER-2 amplification [54].
When tested on 117 tumor samples for which HER-2 amplification
status had been previously determined by DNA blotting, these FISH
assays were found to have high sensitivity, specificity, and accuracy
[57]. Importantly, in comparisons of outside laboratory assessments
of HER-2 amplification/overexpression by IHC and FISH with central
