The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, United Kingdom.
Clin Cancer Res. 2013 Jun 15;19(12):3276-84. doi: 10.1158/1078-0432.CCR-12-3768. Epub 2013 May 1.
Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number.
The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort.
In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) = 0.92 [95% confidence interval (CI), 0.86-0.99, P = 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2-positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2-negative, with a positive and negative predictive value of 70% and 92%, respectively.
Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer.
数字 PCR 是一种高度精确的 DNA 浓度测定方法。我们通过对循环游离血浆 DNA 的非侵入性分析，将数字 PCR 方法用于检测致癌扩增，并举例说明了该方法在 HER2 拷贝数检测方面的应用。
我们通过实验和生物信息学评估了拷贝数评估的参考基因。染色体 17 着丝粒探针显示效果不佳，选择位于 17q21.31 染色体位置的 EFTUD2 进行分析。在 65 例患者的开发队列血浆样本中确定数字 PCR 检测的参数，并在 58 例转移性乳腺癌患者的独立验证队列血浆样本中进行评估。采用序贯概率比检验将血浆 DNA 数字 PCR 检测结果分配为验证队列中的 HER2 阳性或阴性。
在开发队列中，HER2:EFTUD2 血浆 DNA 拷贝数比值的受试者工作特征曲线下面积（AUC）为 0.92（95%置信区间 [CI]，0.86-0.99，P=0.0003）。在独立的验证队列中，64%（11 例中有 7 例）HER2 扩增型癌症患者被归类为血浆数字 PCR HER2 阳性，94%（47 例中有 44 例）HER2 非扩增型癌症患者被归类为数字 PCR HER2 阴性，阳性预测值和阴性预测值分别为 70%和 92%。
利用数字 PCR 分析血浆 DNA 有可能筛选出转移性乳腺癌中 HER2 扩增的获得。这种方法有可能适用于任何癌症中扩增基因座的分析。
