Universally-Primed Quantitative Multiplex PCR Short Fluorescent Fragments (upQMPSF): A New Detection Method for HER2 Amplification in Breast Cancer
S Azrak, L Ping, P Starostik, G Wilding, T Khoury. Roswell Park Cancer Institute, Buffalo
Background: Amplification of HER2 in breast cancer identifies patients eligible for Trastuzumab therapy. HER2 status can be evaluated either by immunohistochemistry (IHC) or by fluorescent in situ hybridization (FISH). Interobserver variability is still a problem. The purpose of the study is to develop a new PCR based technology that is more accurate, reliable, less subjective, cost-effective and easier to interpret.
Design: universally-primed Quantitative Multiplex PCR Short Fluorescent Fragments (upQMPSF) for the detection of HER2 amplification involves a multiplex set consisting of 7 genomic regions. These include 4 exons of HER2, and 3 other regions that include 2 flanking the centromere of chr17 and one outside chr17 as internal controls. This approach identifies chr17 polysomy. Compatible multiplex primers were designed to cover the entire exon regions. Furthermore, the numbers of PCR cycles were empirically optimized to retain the quantitative capacity of the PCR, and the products were analyzed using the ABI capillary sequencer. Sixteen random samples of breast cancer that had tumor and normal frozen tissues were analyzed using FISH and upQMPSF. For upQMPSF method, DNA was extracted and for FISH analysis, PathVysion method was used. HER2 was positive by FISH when HER2: CEP17 ratio was more than 2.2 and negative when this ratio was less than1.8. HER2 was positive by upQMPSF when the ratio of overall average for HER2 peak heights for all exons over the internal control in tumor versus normal was 50%. There was no equivocal result.
Results: HER2 FISH identified 4 (25%) positive cases and 12 (75%) negative. upQMPSF identified 3 (18.8%) positive cases and 13 (71.2%) negative. The agreement between FISH and upQMPSF was in 15 of 16 (93.7%) with confidence interval (0.7-1.0). upQMPSF requires 200 nanogram DNA from tumor and normal tissues to run the test. The test takes 1 hour after DNA extraction to get final result.
Conclusions: upQMPSF method may offer an alternative method for the detection of HER2 amplification and may be particularly useful in improving the accuracy of cases that have equivocal results (FISH 1.8-2.2). Given the small amount of DNA required, the test has advantage over other tests like FISH and IHC when the tested tumor is small. A larger study will be conducted to validate these results.
Tuesday, March 10, 2009 9:30 AM
Poster Session III # 61, Tuesday Morning