Objective: DNA methylation is a common molecular alteration in human neoplasia and can be detected easily in the bloodstream of patients. Here, we investigated whether DNA methylation in sera is of prognostic significance in breast cancer patients. Methods: Methylation status of BRCA1, p16 and 14-3-3σ was examined by methylation-specific PCR assay in the sera of sporadic breast cancer patients and healthy serum controls. Results: The panel gene methylation frequencies were 29% of sporadic breast cancers for p16, 32% for BRCA1 and 82% for 14-3-3σ; all were significantly associated with grades and estrogen receptor status. Only p16 methylation was associated with histological type. p16 and BRCA1 methylation were associated with progesterone receptor status, while 14-3-3σ was significantly associated with lymph node metastases. Seventy percent of patients with p16 methylation showed elevated serum CEA levels; of the breast cancer patients with BRCA1 methylation, 75.8% showed elevated serum CEA levels and 69.7% showed elevated serum CA15.3 levels. When analyzing all investigated patients, multivariate analysis showed methylated BRCA1 and/or p16 serum DNA to be independently associated with poor outcome, with a relative risk of death of 6.0. Conclusions: Epigenetic markers in sera, especially BRCA1/p16, may be more promising targets for the diagnosis of sporadic breast cancer than previous prognostic markers.

1.
Goldhirsch A, Glick JH, Gelber RD, Coates AS, Senn HJ: Meeting highlights: international consensus panel on the treatment of primary breast cancer. J Clin Oncol 2001;19:3817–3827.
2.
Polychemotherapy for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet 1998;352:930–942.
3.
Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists’ Collaborative Group. Lancet 1998;351:1451–1467.
4.
Hayes DF, Isaacs C, Stearns V: Prognostic factors in breast cancer: current and new predictors of metastasis. J Mammary Gland Biol Neoplasia 2001;6:375–392.
5.
Couch FJ, Weber BL: Breast cancer; in Scriver CR, Beaudet AL, Slyws D, Valle D (eds): The Metabolic and Molecular Bases of Inherited Disease. New York, McGraw-Hill, 2001, pp 999–1031.
6.
Leitch AM: Breast cancer screening: success amid conflict. Surg Oncol Clin N Am 1999;8:657–672.
7.
Silva JM, Dominguez G, Villanueva MJ, Gonzalez R, Garcia JM, Corbacho C, Provencio M, España P, Bonilla F: Aberrant DNA methylation of the p16INK4a gene in plasma DNA of breast cancer patients. Br J Cancer 1999;80:1262–1264.
8.
Kawakami K, Brabender J, Lord RV, Groshen S, Greenwald BD, Krasna MJ, Yin J, Fleisher AS, Abraham JM, Beer DG, Sidransky D, Huss HT, Demeester TR, Eads C, Laird PW, Ilson DH, Kelsen DP, Harpole D, Moore MB, Danenberg KD, Danenberg PV, Meltzer SJ: Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma. J Natl Cancer Inst 2000;92:1805–1811.
9.
Caceres I, Battagli C, Esteller M, Herman JG, Dulaimi E, Edelson M, Bergman C, Ehya H, Eisenberg BL, Cairns P: Tumor cell-specific BRCA1 and RASSF1A hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients. Cancer Res 2004;64:6476–6481.
10.
Muller HM, Widschwendter A, Fiegl H, Ivarsson L, Goebel G, Perkmann E, Marth C, Widschwendter M: DNA methylation in serum of breast cancer patients: an independent prognostic marker. Cancer Res 2003;63:7641–7645.
11.
Lo YM: Prognostic implication of pretreatment plasma/serum concentration of Epstein-Barr virus DNA in nasopharyngeal carcinoma. Biomed Pharmacother 2001;55:362–365.
12.
Jerónimo C, Costa I, Martins MC, Monteiro P, Lisboa S, Palmeira C, Henrique R, Teixeira MR, Lopes C: Detection of gene promoter hypermethylation in fine needle washings from breast lesions. Clin Cancer Res 2003;9:3413–3417.
13.
Hoque MO, Feng QH, Toure P, Dem A, Critchlow CW, Hawes SE, Wood T, Jeronimo C, Rosenbaum E, Stern J, Yu MJ, Trink B, Kiviat NB, Sidransky D: Detection of aberrant methylation of four genes in plasma DNA for the detection of breast cancer. J Clin Oncol 2006;24:4262–4269.
14.
Silva JM, Dominguez G, Garcia JM, Gonzalez R, Villanueva MJ, Navarro F, Provencio M, Martin SS, España P, Bonilla F: Presence of tumor DNA in plasma of breast cancer patients: clinicopathological correlations. Cancer Res 1999;59:3251–3256.
15.
Chen X, Bonnefoi H, Berger S, Lyautey J, Lederrey C, Traub E, Stroun M, Anker P: Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer. Clin Cancer Res 1999;5:2297–2303.
16.
Shao ZM, Wu J, Shen ZZ, Nguyen M: p53 mutation in plasma DNA and its prognostic value in breast cancer patients. Clin Cancer Res 2001;7:2222–2227.
17.
Silva JM, Garcia JM, Dominguez G, Silva J, Miralles C, Cantos B, Coca C, Provencio M, España P, Bonilla F: Persistence of tumor DNA in plasma of breast cancer patients after mastectomy. Ann Surg Oncol 2002;9:71–76.
18.
Leon SA, Shapiro B, Sklaroff DM, Yaros MJ: Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 1977;37:646–650.
19.
Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 2001.
20.
Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996;93:9821–9826.
21.
Grunau C, Clark SJ, Rosenthal A: Bisulifite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 2001;29:e65.
22.
Ferguson AT, Evron E, Umbricht CB, Pandita TK, Chan TA, Hermeking H, Marks JR, Lambers AR, Futreal PA, Stampfer MR, Sukumar S: High frequency of hypermethylation at the 14-3-3 σ locus leads to gene silencing in breast cancer. Proc Natl Acad Sci USA 2000;97:6049–6054.
23.
Esteller M, Silva JM, Dominguez G, Bonilla F, Matias GX, Lerma E, Bussaglia E, Prat J, Harkes IC, Repasky EA, Gabrielson E, Schutte M, Baylin SB, Herman JG: Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst 2000;92:564–569.
24.
Deng CX, Brodie SG: Role of BRCA1 and its interacting protein. Bioessays 2000;22:728–737.
25.
Miyamoto K, Fukutomi T, Asada K, Wakazono K, Tsuda H, Asahara T, Sugimura T, Ushijima T: Promoter hypermethylation and post-transcriptional mechanisms for reduced BRCA1 immunoreactivity in sporadic human breast cancers. Jpn Clin Oncol 2002;32:79–84.
26.
Wang C, Horiuchi A, Imai T, Ohira S, Itoh K, Nikaido T, Katsuyama Y, Konishi I: Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of BRCA1 gene. J Pathol 2004;202:215–223.
27.
Chan DW, Chang HW, Kurman RJ, Lee SM, Shih IM, Goodman SN, Singer G, Cho SK, Sokoll LJ, Montz FJ, Roden R, Zhang Z: Assessment of plasma DNA levels, allelic imbalance, and CA 125 as diagnostic tests for cancer. J Natl Cancer Inst 2002;94:1697–1703.
28.
Klein CA, Blankenstein TJ, Schmidt KO, Petronio M, Polzer B, Stoecklein NH, Riethmuller G: Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. Lancet 2002;360:683–689.
29.
Samowitz WS, Albertsen H, Herrick J, Levin TR, Sweeney C, Murtaugh MA, Wolff RK, Slattery M: Evaluation of a large, population-based sample supports a CpG island methylator phenotype in colon cancer. Gastroenterology 2005;129:837–845.
30.
Zhang CS, Li ZY, Cheng, Jia FQ, Li R, Wu MC, Li K, Wei LX: CpG island methylator phenotype association with elevated serum α-fetoprotein level in hepatocellular carcinoma. Clin Cancer Res 2007;13:944–952.
31.
Guadagni F, Ferroni P, Carlini S, Mariotti S, Spila A, Aloe S, Alessandro R, Carone M, Cicchetti A, Ricciotti A, Venturo I, Perri P, Filippo F, Cognetti F, Botti C, Roselli M: A re-evaluation of carcinoembryonic antigen (CEA) as a serum marker for breast cancer: a prospective longitudinal study. Clin Cancer Res 2001;7:2357–2362.
32.
Wallner M, Herbst A, Behrens A, Crispin A, Stieber P, Burkhard G, Amerz R, Kolligs F: Methylation of serum DNA is an independent prognostic marker in colorectal cancer. Clin Cancer Res 2006;12:7347–7352.
33.
Anker P, Mulcahy H, Chen XQ, Stroun M: Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients. Cancer Metastasis Rev 1999;18:65–73.
34.
Wei SH, Balch C, Paik HH, Kim YS, Baldwin RL, Liyanarachchi S, Li L, Wang ZL, Wan JC, Davuluri RV, Karlan BY, Gifford G, Brown R, Kim S, Huang T, Nephew KP: Prognostic DNA methylation biomarkers in ovarian cancer. Clin Cancer Res 2006;12:2788–2794.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.