Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay
Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay
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DOI：
10.1007/s10549-011-1575-2
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发表时间：
2012-02-01
影响因子：
3.8
通讯作者：
Noguchi, Shinzaburo
中科院分区：
文献类型：
--
作者：
Yamamoto, Noriaki;Nakayama, Takahiro;Noguchi, Shinzaburo
基因启动子异常甲基化是乳腺癌中常见的分子事件。因此，DNA甲基化分析有望成为癌症诊断的新工具。在本文中，我们建立了一种新的高性能 DNA 甲基化测定，即一步甲基化特异性聚合酶链反应 (OS-MSP) 测定，该测定针对分析血清 DNA 中的基因甲基化进行了优化。 OS-MSP 测定旨在检测血清 DNA 中 GSTP1、RASSF1A 和 RAR beta 2 基因的异常启动子甲基化。此外，设计了两个质量控制标记来监测亚硫酸氢盐转化效率和测量血清中的DNA含量。血清样本采集自原发性乳腺癌 (n = 101，I-III 期) 和转移性乳腺癌 (n = 58) 以及健康对照 (n = 87) 患者。如果观察到三个基因中至少一个的甲基化，则 OS-MSP 测定被认为是阳性。对于 I 期乳腺癌（24% 对 8%）和 II 期乳腺癌（26% 对 8%），该测定的灵敏度显着高于涉及传统肿瘤标志物（CEA 和/或 CA15-3）的测定，并且与 III 期乳腺癌（18% 对 19%）和转移性乳腺癌（55% 对 59%）涉及常规肿瘤标志物的测定相似。 OS-MSP 测定的结果和涉及 CEA 和/或 CA15-3 的测定的结果似乎相互补偿，因为当组合用于转移性乳腺癌时，这些测定的灵敏度增加到 78%。总之，我们开发了一种新的 OS-MSP 检测方法，具有更高的灵敏度和便利性；因此，该方法更适合检测血清DNA中异常启动子甲基化。此外，OS-MSP测定与涉及CEA和/或CA15-3的测定的组合有望提高转移性乳腺癌诊断的灵敏度。
Aberrant promoter methylation of genes is a common molecular event in breast cancer. Thus, DNA methylation analysis is expected to be a new tool for cancer diagnosis. In this article, we have established a new, high-performance DNA methylation assay, the one-step methylation-specific polymerase chain reaction (OS-MSP) assay, which is optimized for analyzing gene methylation in serum DNA. The OS-MSP assay is designed to detect aberrant promoter methylation of GSTP1, RASSF1A, and RAR beta 2 genes in serum DNA. Moreover, two quality control markers were designed for monitoring the bisulfite conversion efficiency and measuring the DNA content in the serum. Serum samples were collected from patients with primary (n = 101, stages I-III) and metastatic breast cancers (n = 58) as well as from healthy controls (n = 87). If methylation of at least one of the three genes was observed, the OS-MSP assay was considered positive. The sensitivity of this assay was significantly higher than that of the assay involving conventional tumor markers (CEA and/or CA15-3) for stages I (24 vs. 8%) and II (26 vs. 8%) breast cancer and similar to that of the assay involving the conventional tumor markers for stage III (18 vs. 19%) and metastatic breast cancers (55 vs. 59%). The results of the OS-MSP assay and those of the assay involving CEA and/or CA15-3 seemed to compensate for each other because sensitivity of these assays increased to 78% when used in combination for metastatic breast cancer. In conclusion, we have developed a new OS-MSP assay with improved sensitivity and convenience; thus, this assay is more suitable for detecting aberrant promoter methylation in serum DNA. Moreover, the combination of the OS-MSP assay and the assay involving CEA and/or CA15-3 is promising for enhancing the sensitivity of diagnosis of metastatic breast cancer.