It is an indirect method of measuring gene amplification and is based on the ability of antibodies to identify HER-2 proteins expressed by fixed cells in frozen or paraffin embedded tissue sections.
The ability to use small punches of tumour identified by the pathologist in paraffin blocks makes this a potentially very valuable technique, as it is also be possible to measure gene amplification or mutation by PCR of DNA markers.
For application of the 2-ΔΔCt method, the efficiencies of all measured gene amplifications were first examined using diluted samples and confirmed to be appropriately equal.
This can be determined by measuring HER2 protein levels using immunohistochemistry (IHC) and/or by measuring HER2 gene amplification by in situ hybridization (ISH).
One recent study applied a standardised methodology used in breast tumour diagnostic assessment to measure both gene amplification and expression of ERBB2 (Lae et al, 2010).
Wt p53 status in cancers is often associated with elevated Mdm proteins (reviewed in Wade et al).. To explore whether gene copy number of Mdm2 and/or Mdmx are abnormal in BrCas, we measured gene copy amplification by FISH in a BrCa tissue micro-array (TMA).
We observed a high amount of FFPE samples with DNA degradation measured with beta-globin gene amplification.
Retrospective studies have shown that HER2/neu gene amplification, measured using FISH, is the best predictive marker of response to trastuzumab-based therapy [ 14].
For HER2, no (0) or weak (1+) membrane protein immunostaining was considered low HER2 expression (HER2–); and moderate (2+) or strong membrane protein immunostaining (3+) was considered HER2 overexpression (HER2+) based on previously validated results from the same pathology laboratory comparing IHC with HER2 gene amplification measured by fluorescent in situ hybridization methods [ 17].
Indeed, our results contrast with those of a smaller series in a neoadjuvant setting, in which response did not correlate with the level of HER-2 gene amplification measured by FISH (Buzdar et al, 2007); this study concerned 45 patients receiving paclitaxel followed by 5-fluorouracile, epirubicin, and cyclophosphamide (FEC) with concurrent trastuzumab.
