Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer.
Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer.
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DOI：
10.1158/0008-5472.can-13-3392
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发表时间：
2014-04-15
期刊：
影响因子：
11.2
通讯作者：
Sukumar S
中科院分区：
文献类型：
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作者：
Fackler MJ;Lopez Bujanda Z;Umbricht C;Teo WW;Cho S;Zhang Z;Visvanathan K;Jeter S;Argani P;Wang C;Lyman JP;de Brot M;Ingle JN;Boughey J;McGuire K;King TA;Carey LA;Cope L;Wolff AC;Sukumar S
持续检测转移性乳腺癌患者外周血中无细胞肿瘤特异性DNA的能力，为检测肿瘤负荷变化和监测治疗反应提供了机会。我们开发了cMethDNA，这是一种针对一组10个基因的定量多重甲基化特异性PCR检测，包括通过挖掘我们先前报道的乳腺组织中DNA甲基化模式的研究（103个癌症，21个在Illumina HumanMethylation 27 Beadchip上正常），然后在TCGA乳腺癌甲基化组数据库中验证10个基因组而鉴定的新的和已知的乳腺癌高甲基化标志物。对于cMethDNA，在纯化DNA之前，将固定生理水平（50个拷贝）的人工构建的、对每个基因具有特异性的标准非人参考DNA引入恒定体积的血清（300 μl）中，以便于对肿瘤DNA进行灵敏、特异、稳健和定量测定，具有宽的动态范围。在训练组（28例正常，24例癌症）和测试组（27例正常，33例癌症）复发性4期患者血清中检测到癌症特异性甲基化DNA，在测试组中灵敏度为91%，特异性为96%。在一项初步研究中，cMethDNA测定忠实地反映了患者对化疗的反应（N = 29）。原发性乳腺癌中存在的核心甲基化标记保留在疾病诊断后2-11年尸检时收集的血清和转移组织中。总之，我们的数据表明，cMethDNA检测可以检测晚期乳腺癌，并监测转移性乳腺癌妇女的肿瘤负荷和治疗反应。
The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. We developed cMethDNA, a quantitative multiplexed methylation-specific PCR assay for a panel of ten genes, consisting of novel and known breast cancer hypermethylated markers identified by mining our previously reported study of DNA methylation patterns in breast tissue (103 cancer, 21 normal on the Illumina HumanMethylation27 Beadchip) and then validating the 10-gene panel in a TCGA breast cancer methylome database. For cMethDNA, a fixed physiological level (50 copies) of artificially constructed, standard non-human reference DNA specific for each gene is introduced into in a constant volume of serum (300 μl) prior to purification of the DNA, facilitating a sensitive, specific, robust and quantitative assay of tumor DNA, with broad dynamic range. Cancer-specific methylated DNA was detected in Training (28 normal, 24 cancer) and Test (27 normal, 33 cancer) sets of recurrent Stage 4 patient sera with a sensitivity of 91% and a specificity of 96% in the test set. In a pilot study, cMethDNA assay faithfully reflected patient response to chemotherapy (N = 29). A core methylation signature present in the primary breast cancer was retained in serum and metastatic tissues collected at autopsy 2–11 years after diagnosis of the disease. Together, our data suggest that the cMethDNA assay can detect advanced breast cancer, and monitor tumor burden and treatment response in women with metastatic breast cancer.