Quantitative Analysis of ErbB1 and ErbB2 Genes Amplification by a High Performance Liquid Chromatography

Genes for human epidermal growth factor receptors B1 (ErbB1) and B2 (ErbB2) were amplified in breast and ovarian cancers. Both of them were associated with aggressive disease and worse prognosis. The ErbB1 or ErbB2 status of a tumor may provide an indication of the response to ErbB1 and ErbB2 -targeted therapies. For accurate and rapid assessment of amplification of ErbB1 and ErbB2 oncogenes, a High Performance Liquid Chromatography (HPLC) method was developed in this study. DNA was extracted from 30 primary breast tumors and 20 blood samples of healthy donors. ErbB1 and ErbB2 genes along with a reference gene were co-amplificated by Polymerase Chain Reaction (PCR). The PCR products were separated and quantified using an anion- exchange column within 30 min and in a single step. Optimum resolution was obtained when a sodium chloride gradient and a column temperature of 35˚C were used. The results of HPLC analysis of ErbB1 and ErbB2 PCR products were compared with real time PCR method as a gold standard test for 7 tumor samples. The proposed HPLC method was confirmed by real time PCR method. Twenty two and ten of the specimens in our breast cancer cohort showed more than a two-fold amplification of ErbB2 and ErbB1 oncogenes, respectively. Our results were confirmed by real time PCR and showed that HPLC method is a specific, cheap and clinically applicable analytical approach for assessment of ErbB1 and ErbB2 statuses in breast tumors.