Abstract 4217: A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC)
| Autor: | Victoria H. Brophy, Anona Bristol, Sung Lee, Ramani Ravirala, Namneet Kaur, Peter Schlag, Joyce Kramer, Stephen Quashnick, Stephen Soviero, Corey Hoeppner, Minet Negash |
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| Rok vydání: | 2013 |
| Předmět: | |
| Zdroj: | Cancer Research. 73:4217-4217 |
| ISSN: | 1538-7445 0008-5472 |
| Popis: | Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Somatic mutations in the PIK3CA gene encoding the p110α catalytic subunit of the PI3K protein have been linked to human cancer and have been found to occur at a high frequency in BC (25-40%). Therapeutics that target key nodes in the PI3K signaling pathway are currently being developed and stratifying patients based on their PIK3CA mutation status is becoming more important. We describe the analytical performance of the cobas® PIK3CA Mutation Test (For Research Use Only), a multiplex allele-specific PCR assay designed to detect 17 mutations in exons 1, 4, 7, 9 and 20 in the PIK3CA gene in 3 PCR wells. The test detects ∼94% of known PIK3CA mutations found in BC. The amount of DNA required for the assay (150 ng) can typically be isolated from a single 5 μm FFPET section with automated test results generated within 8 hours. The analytical sensitivity of the test, defined as ≥95% mutation detection rate, was found to be ∼5% mutant allele in BC FFPET tumor sections representing 4 prevalent PIK3CA mutations (H1047R, E545K, E542K, and Q546K). Additionally, the test was able to detect all 17 mutations at ≤ 5% mutant allele in plasmid/gDNA blends. The test was also compared to 2X bi-directional Sanger sequencing using 110 BC FFPET specimens. The PCR test detected mutations in 50 specimens that were Sanger (+) (100% positive agreement). Moreover, the test detected mutations in 4 Sanger (-) specimens and detected a second mutation in 1 Sanger (+) specimen that was not detected by Sanger. All 5 of the PCR (+)/Sanger (-) were confirmed mutant by 454 sequencing. | | | Sanger Sequencing | | |:--------------------------- | --------------------- | ----------------- | --------------------- | ----- | | | | Mutation Detected | Mutation Not Detected | Total | | cobas® PIK3CA Mutation Test | Mutation Detected | 50* | 4 | 54 | | | Mutation Not Detected | | 56 | 56 | | | Total | 50 | 60 | 110 | * *One specimen had an additional mutation. When a panel of BC FFPET specimens was repeatedly tested over multiple days by 2 operators using 2 instruments and 2 reagent lots, a correct call was obtained in 99.5% (191/192) of tests. The results demonstrate that the cobas® PIK3CA Mutation Test is a fast, accurate and reproducible assay that is more sensitive than Sanger sequencing for the detection of PIK3CA mutations in BC tumors. Citation Format: Peter Schlag, Corey Hoeppner, Anona Bristol, Namneet Kaur, Stephen Quashnick, Ramani Ravirala, Joyce Kramer, Minet Negash, Victoria Brophy, Sung Lee, Stephen Soviero. A real-time PCR assay for the detection of PIK3CA mutations in formalin-fixed paraffin embedded tissue (FFPET) specimens of breast cancer (BC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4217. doi:10.1158/1538-7445.AM2013-4217 |
| Databáze: | OpenAIRE |
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