Breast Cancer Research and Treatment

, Volume 132, Issue 1, pp 165–173 | Cite as

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

  • Noriaki Yamamoto
  • Takahiro Nakayama
  • Masahiro Kajita
  • Tomohiro Miyake
  • Takashi Iwamoto
  • Seung Jin Kim
  • Ayako Sakai
  • Hideki Ishihara
  • Yasuhiro Tamaki
  • Shinzaburo Noguchi
Preclinical Study


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 RARβ2 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.


Breast cancer Methylation DNA Serum 

Supplementary material

10549_2011_1575_MOESM1_ESM.docx (23 kb)
Supplementary material 1 (DOCX 22 kb)


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Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Noriaki Yamamoto
    • 1
    • 2
  • Takahiro Nakayama
    • 1
  • Masahiro Kajita
    • 2
  • Tomohiro Miyake
    • 1
  • Takashi Iwamoto
    • 1
  • Seung Jin Kim
    • 1
  • Ayako Sakai
    • 2
  • Hideki Ishihara
    • 2
  • Yasuhiro Tamaki
    • 1
  • Shinzaburo Noguchi
    • 1
  1. 1.Department of Breast and Endocrine SurgeryOsaka University Graduate School of MedicineSuita-shiJapan
  2. 2.Central Research LaboratoriesSysmex CorporationNishi-kuJapan

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