Oxnard Geoffrey R, Paweletz Cloud P, Kuang Yanan, Mach Stacy L, O'Connell Allison, Messineo Melissa M, Luke Jason J, Butaney Mohit, Kirschmeier Paul, Jackman David M, Jänne Pasi A
Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
Department of Medicine, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
Clin Cancer Res. 2014 Mar 15;20(6):1698-1705. doi: 10.1158/1078-0432.CCR-13-2482. Epub 2014 Jan 15.
Tumor genotyping using cell-free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype.
ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib.
We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression.
Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression.
使用游离血浆DNA（cfDNA）进行肿瘤基因分型有潜力实现对肿瘤生物学的无创评估，但许多现有检测方法操作繁琐且易出现假阳性结果。我们试图确定cfDNA的液滴数字PCR（ddPCR）是否能实现对肿瘤基因型的高度特异性和定量评估。
使用从已知肿瘤基因型的晚期肺癌或黑色素瘤患者采集的血浆，开发针对EGFR、KRAS和BRAF突变的ddPCR检测方法。以基因型不重叠的癌症作为阳性和阴性对照来确定敏感性和特异性。在一项关于厄洛替尼的前瞻性试验中，对EGFR突变型肺癌患者进行反应和耐药性的系列评估。
我们确定了KRAS突变型肺癌标本中EGFR L858R和外显子19缺失的参考范围，从而能够确定具有高敏感性和100%特异性的候选阈值。对四种检测方法的接受操作特征曲线分析显示曲线下面积在0.80至0.94范围内。在cfDNA浓度最佳的标本中敏感性有所提高。对接受厄洛替尼治疗的EGFR突变型肺癌患者进行系列血浆基因分型显示，可以在治疗前检测到EGFR突变，大多数情况下血浆完全反应，并且在客观进展前EGFR T790M水平升高。
使用ddPCR对cfDNA进行无创基因分型显示出可有效转化为临床诊断的检测特性。血浆基因型的系列定量能够对反应和耐药性进行无创评估，包括在影像学进展前16周检测到耐药突变。
