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Promoter hypermethylation of the tumor-suppressor genes ITIH5, DKK3, and RASSF1A as novel biomarkers for blood-based breast cancer screening

Vera Kloten1, Birte Becker1, Kirsten Winner1, Michael G Schrauder2, Peter A Fasching2, Tobias Anzeneder3, Jürgen Veeck1, Arndt Hartmann4, Ruth Knüchel1 and Edgar Dahl1*

Author Affiliations

1 Molecular Oncology Group, Institute of Pathology, Medical Faculty of the RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany

2 Department of Gynecology and Obstetrics, University Hospital Erlangen, Universitätsstrasse 21-23, D-91054 Erlangen, Germany

3 Patients' Tumor Bank of Hope (PATH) Foundation, Schäftlarnstr. 62, D-81337 München, Germany

4 Department of Pathology, University of Erlangen, Krankenhausstrasse 12, D-91054 Erlangen, Germany

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Breast Cancer Research 2013, 15:R4  doi:10.1186/bcr3375

Published: 15 January 2013



For early detection of breast cancer, the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory. We investigated DNA methylation in circulating free DNA (cfDNA) from blood of breast cancer patients and matched controls to establish a biomarker panel potentially useful for early detection of breast cancer.


We examined promoter methylation of seven putative tumor-suppressor genes (SFRP1, SFRP2, SFRP5, ITIH5, WIF1, DKK3, and RASSF1A) in cfDNA extracted from serum. Clinical performance was first determined in a test set (n = 261 sera). In an independent validation set (n = 343 sera), we validated the most promising genes for further use in early breast cancer detection. Sera from 59 benign breast disease and 58 colon cancer patients were included for additional specificity testing.


Based on the test set, we determined ITIH5 and DKK3 promoter methylation as candidate biomarkers with the best sensitivity and specificity. In both the test and validation set combined, ITIH5 and DKK3 methylation achieved 41% sensitivity with a specificity of 93% and 100% in healthy and benign disease controls, respectively. Combination of these genes with RASSF1A methylation increased the sensitivity to 67% with a specificity of 69% and 82% in healthy controls and benign disease controls, respectively.


Tumor-specific methylation of the three-gene panel (ITIH5, DKK3, and RASSF1A) might be a valuable biomarker for the early detection of breast cancer.