HER-2/neu gene amplification assessment in breast cancer patients in Isfahan province by real time PCR, differential PCR and immunohistochemistry.
BACKGROUND
The amplification status of proto oncogene HER-2/neu is one of the major molecular prognosis markers in breast cancer and recent adjuvant treatment with Trastuzumab has increased a request for the evaluation of HER-2/neu status in breast cancer. The aim of our study was the evaluation of HER-2/neu amplification status in malignant breast cancer by PCR techniques such as differential PCR and real time PCR and comparison of results of two methods together and with IHC results in some specimens.


METHODS
86 malignant breast cancer tissue specimens were analysed initially by differential PCR and then by real time PCR. Sections from paraffin-embedded or fresh tissue samples were homogenized by squash and then DNA extraction was performed on cell suspension. A standard curve was initially plotted using BioEasy SYBR Green I for using 2(-ddct) method. A 98bp fragment of HER-2/neu gene was co-amplified in the same reaction tube with a 150bp fragment of INFγ gene for differential PCR.


RESULTS
The IHC results existed only for 27 of 83 assessed samples by dPCR and for 30 of 86 assessed samples by real time PCR. 29 out of 83 (35%) samples tested by dPCR and 28 out of 86 (32.5%) samples tested by real time PCR have HER-2/neu gene amplification.


CONCLUSIONS
There was concordance between the results of real time PCR and differential PCR in 61 of 83 specimens (73.5%) tested by both method. Furthermore, in comparison of IHC results with these two methods, 70% concordance between IHC and differential PCR, 63% concordance between IHC and real time PCR and 55.5% concordance between three methods were observed.
Biology Department, University of Isfahan, Isfahan, Iran. z.hojati@sci.ui.ac.ir
Zohreh
Z
Hojati
Elham
E
Orangi
D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics*; D005260:Female; D005784:Gene Amplification; D018734:Genes, erbB-2; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007492:Iran; D016612:Paraffin Embedding / Q000379:methods; D000090063:Proto-Oncogene Mas; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics
D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases; C000715470:MAS1 protein, human