Authors' Affiliations: Departments of Oncology Biomarker Development, Bioinformatics, Structural Biology, and Pathology, Genentech, Inc.; Fluidigm Inc., South San Francisco, California; and Expression Analysis, Durham, North Carolina.
Clin Cancer Res. 2014 Apr 15;20(8):2080-91. doi: 10.1158/1078-0432.CCR-13-3114. Epub 2014 Feb 26.
Tailoring cancer treatment to tumor molecular characteristics promises to make personalized medicine a reality. However, reliable genetic profiling of archived clinical specimens has been hindered by limited sensitivity and high false-positive rates. Here, we describe a novel methodology, MMP-seq, which enables sensitive and specific high-throughput, high-content genetic profiling in archived clinical samples.
We first validated the technical performance of MMP-seq in 66 cancer cell lines and a Latin square cross-dilution of known somatic mutations. We next characterized the performance of MMP-seq in 17 formalin-fixed paraffin-embedded (FFPE) clinical samples using matched fresh-frozen tissue from the same tumors as benchmarks. To demonstrate the potential clinical utility of our methodology, we profiled FFPE tumor samples from 73 patients with endometrial cancer.
We demonstrated that MMP-seq enabled rapid and simultaneous profiling of a panel of 88 cancer genes in 48 samples, and detected variants at frequencies as low as 0.4%. We identified DNA degradation and deamination as the main error sources and developed practical and robust strategies for mitigating these issues, and dramatically reduced the false-positive rate. Applying MMP-seq to a cohort of endometrial tumor samples identified extensive, potentially actionable alterations in the PI3K (phosphoinositide 3-kinase) and RAS pathways, including novel PIK3R1 hotspot mutations that may disrupt negative regulation of PIK3CA.
MMP-seq provides a robust solution for comprehensive, reliable, and high-throughput genetic profiling of clinical tumor samples, paving the way for the incorporation of genomic-based testing into clinical investigation and practice.
根据肿瘤分子特征定制癌症治疗方案有望使个性化医疗成为现实。然而，对存档临床标本进行可靠的基因分析受到灵敏度有限和高假阳性率的阻碍。在这里，我们描述了一种新的方法 MMP-seq，它可以在存档的临床样本中进行敏感和特异性的高通量、高内涵基因分析。
我们首先在 66 种癌细胞系和已知体细胞突变的拉丁方交叉稀释中验证了 MMP-seq 的技术性能。接下来，我们使用来自同一肿瘤的匹配新鲜冷冻组织作为基准，在 17 个福尔马林固定石蜡包埋（FFPE）临床样本中表征了 MMP-seq 的性能。为了证明我们方法的潜在临床应用价值，我们对 73 例子宫内膜癌患者的 FFPE 肿瘤样本进行了分析。
我们证明 MMP-seq 能够快速且同时对 88 个癌症基因进行面板分析，检测到频率低至 0.4%的变体。我们发现 DNA 降解和脱氨是主要的错误来源，并开发了实用且稳健的策略来减轻这些问题，并大大降低了假阳性率。应用 MMP-seq 对一组子宫内膜肿瘤样本进行分析，确定了 PI3K（磷酸肌醇 3-激酶）和 RAS 途径中广泛存在的、潜在可操作的改变，包括可能破坏 PIK3CA 负调节的新 PIK3R1 热点突变。
MMP-seq 为临床肿瘤样本的全面、可靠和高通量基因分析提供了一种稳健的解决方案，为将基于基因组的检测纳入临床研究和实践铺平了道路。
