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首页> 外文期刊>The Journal of molecular diagnostics: JMD >PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method.
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PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method.

机译：PIK3CA热点突变扫描，采用新颖且高度灵敏的高分辨率小扩增子熔解分析方法。

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摘要

Somatic mutations in the PIK3CA gene have been discovered in many human cancers, and their presence correlates to therapy response. Three "hotspot" mutations within the PIK3CA gene are localized in exons 9 and 20. High-resolution melting analysis (HRMA) is a highly sensitive, robust, rapid, and cost-effective mutation analysis technique. We developed a novel methodology for the detection of hotspot mutations in exons 9 and 20 of the PIK3CA gene that is based on a combination of PCR and HRMA. The PIK3CA HRMA assay was evaluated by performing repeatability, sensitivity, and comparison with DNA sequencing studies and was further validated in 129 formalin-fixed paraffin-embedded breast tissue samples: 99 tumors, 20 noncancerous, and 10 fibroadenomas. The developed methodology was further applied in a selected group of 75 breast cancer patients who underwent Trastuzumab treatment. In sensitivity studies, the assay presented a capability to detect as low as 1% of mutated dsDNA in the presence of wtDNA for both exons. In the 99 tumor samples (validation group), 12/99 (12.1%) exon 9 mutations and 20/99 (20.2%) exon 20 mutations were found. No mutations were found in noncancerous tissues. In fibroadenomas, we report one PIK3CA mutation for the first time. In the selected group, 30/75 (40%) samples were detected as mutants. The PIK3CA HRMA assay is highly sensitive, reliable, cost-effective, and easy-to-perform, and therefore can be used as a screening test in a high-throughput pharmacodiagnostic setting.
机译：PIK3CA基因中的体细胞突变已在许多人类癌症中发现，它们的存在与治疗反应相关。 PIK3CA基因内的三个“热点”突变位于外显子9和20中。高分辨率熔解分析（HRMA）是一种高度灵敏，稳定，快速且具有成本效益的突变分析技术。我们开发了一种新颖的方法，用于检测基于PCR和HRMA的PIK3CA基因外显子9和20的热点突变。 PIK3CA HRMA分析通过重复性，敏感性和与DNA测序研究的比较进行了评估，并在129个福尔马林固定石蜡包埋的乳腺组织样本中得到了进一步验证：99个肿瘤，20个非癌性和10个纤维腺瘤。所开发的方法被进一步应用到75名接受曲妥珠单抗治疗的乳腺癌患者中。在敏感性研究中，该方法具有在两种外显子均存在wtDNA的情况下检测低至1％突变dsDNA的能力。在99个肿瘤样本（验证组）中，发现12/99（12.1％）外显子9突变和20/99（20.2％）外显子20突变。在非癌性组织中未发现突变。在纤维腺瘤中，我们首次报告了一个PIK3CA突变。在选定的组中，检测到30/75（40％）样品为突变体。 PIK3CA HRMA分析具有高度灵敏性，可靠性，成本效益和易于执行的性能，因此可以在高通量药物诊断环境中用作筛选测试。

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