e11068 Background: HER2 gene status is a key determinant for anti-HER2 therapy with Herceptin. The common methods used for HER2 test in breast cancer patients are IHC and FISH, which are time-consuming and often generate variable results. Quantitative real-time PCR (qRT-PCR) has emerged as a new gold standard technique for quantifying gene expression in different cells and tissues. In this study, we explored the potential of utilizing this technique to determine HER2 expression status in breast cancer patients. Methods: We collected both freshly-frozen and formalin-fixed paraffin-embedded (FFPE) breast tumor samples at three locations. The total RNA was isolated with a RNA extraction kit and was quantified by RiboGreen method. Each sample was normalized against its total RNA concentration and analyzed by a real-time PCR method with inclusion of the Standards and RNA Control. The HER2 expression level was determined by the copy number, Ct value, as well as the ΔCt against RNA control. The significance of HER2 expression and correlation among the different groups were analyzed by statistical methods. Results: A total of 80 samples have been tested. We noticed that the RT-PCR method generated very consistent results with little intra- and inter-assay variance. We also noticed that HER2 expression in the fresh tumor samples exhibited two distinct populations, although HER2 expression in the normal fresh samples showed the continuous distribution. Similar results were obtained with the FFPE samples. Study of the other breast cancer-related genes, such as EGFR, ER-a, BRCA1, and BRCA2, revealed that their expression, unlike HER2, followed a pattern of continuous distribution. In comparison with FISH test, qRT-PCR method not only produced consistent results, but also able to differentiate breast tumors into two distinct groups, HER2 positive vs. HER2 negative. Conclusions: The qRT-PCR is a more reliable method than the currently used IHC and FISH for HER2 test. Showing two distinct groups of HER2 expression by qRT-PCR further warrants its application in testing HER2 gene status for breast cancer patients.
DEVELOPMENT OF A MULTIPLEX ALLELE-SPECIFIC REAL-TIME PCR METHOD FOR DETECTION OF PIK3CA GENE SOMATIC MUTATIONS AND ITS VALIDATION IN THE TUMORS OF BREAST CANCER PATIENTS
