Abstract
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.
Similar content being viewed by others
References
Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72:141–196
Bird A (1992) The essentials of DNA methylation. Cell 70:5–8
Brock MV, Hooker CM, Ota-Machida E, Han Y, Guo M, Ames S, Glockner S, Piantadosi S, Gabrielson E, Pridham G, Pelosky K, Belinsky SA, Yang SC, Baylin SB, Herman JG (2008) DNA methylation markers and early recurrence in stage I lung cancer. N Engl J Med 358:1118–1128. doi:10.1056/NEJMoa0706550
Das PM, Singal R (2004) DNA methylation and cancer. J Clin Oncol 22:4632–4642. doi:10.1200/JCO.2004.07.151
Dulaimi E, Hillinck J, Ibanez de Caceres I, Al-Saleem T, Cairns P (2004) Tumor suppressor gene promoter hypermethylation in serum of breast cancer patients. Clin Cancer Res 10:6189–6193. doi:10.1158/1078-0432.CCR-04-0597
Egger G, Liang G, Aparicio A, Jones PA (2004) Epigenetics in human disease and prospects for epigenetic therapy. Nature 429:457–463. doi:10.1038/nature02625
Esteller M (2007) Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 8:286–298. doi:10.1038/nrg2005
Esteller M (2008) Epigenetics in cancer. N Engl J Med 358:1148–1159
Evron E, Dooley WC, Umbricht CB, Rosenthal D, Sacchi N, Gabrielson E, Soito AB, Hung DT, Ljung B, Davidson NE, Sukumar S (2001) Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR. Lancet 357:1335–1336
Fackler MJ, McVeigh M, Mehrotra J, Blum MA, Lange J, Lapides A, Garrett E, Argani P, Sukumar S (2004) Quantitative multiplex methylation-specific PCR assay for the detection of promoter hypermethylation in multiple genes in breast cancer. Cancer Res 64:4442–4452. doi:10.1158/0008-5472.CAN-03-3341
Fiegl H, Millinger S, Mueller-Holzner E, Marth C, Ensinger C, Berger A, Klocker H, Goebel G, Widschwendter M (2005) Circulating tumor-specific DNA: a marker for monitoring efficacy of adjuvant therapy in cancer patients. Cancer Res 65:1141–1145. doi:10.1158/0008-5472.CAN-04-2438
Grunau C, Clark SJ, Rosenthal A (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29:E65
Hayatsu H, Shiraishi M, Negishi K (2008) Bisulfite modification for analysis of DNA methylation. Curr Protoc Nucleic Acid Chem Chapter 6, Unit 6.10. doi:10.1002/0471142700.nc0610s33
Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054. doi:10.1056/NEJMra023075
Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93:9821–9826
Hoque MO, Feng Q, Toure P, Dem A, Critchlow CW, Hawes SE, Wood T, Jeronimo C, Rosenbaum E, Stern J, Yu M, Trink B, Kiviat NB, Sidransky D (2006) Detection of aberrant methylation of four genes in plasma DNA for the detection of breast cancer. J Clin Oncol 24:4262–4269. doi:10.1200/JCO.2005.01.3516
Jones PA, Baylin SB (2007) The epigenomics of cancer. Cell 128:683–692. doi:10.1016/j.cell.2007.01.029
Kim MS, Lee J, Sidransky D (2010) DNA methylation markers in colorectal cancer. Cancer Metastasis Rev 29:181–206. doi:10.1007/s10555-010-9207-6
Lo PK, Sukumar S (2008) Epigenomics and breast cancer. Pharmacogenomics 9:1879–1902. doi:10.2217/14622416.9.12.1879
Lofton-Day C, Model F, Devos T, Tetzner R, Distler J, Schuster M, Song X, Lesche R, Liebenberg V, Ebert M, Molnar B, Grutzmann R, Pilarsky C, Sledziewski A (2008) DNA methylation biomarkers for blood-based colorectal cancer screening. Clin Chem 54:414–423. doi:10.1373/clinchem.2007.095992
Ludwig JA, Weinstein JN (2005) Biomarkers in cancer staging, prognosis and treatment selection. Nat Rev Cancer 5:845–856. doi:10.1038/nrc1739
Martens JW, Margossian AL, Schmitt M, Foekens J, Harbeck N (2009) DNA methylation as a biomarker in breast cancer. Future Oncol 5:1245–1256. doi:10.2217/fon.09.89
Mehrotra J, Ganpat MM, Kanaan Y, Fackler MJ, McVeigh M, Lahti-Domenici J, Polyak K, Argani P, Naab T, Garrett E, Parmigiani G, Broome C, Sukumar S (2004) Estrogen receptor/progesterone receptor-negative breast cancers of young African-American women have a higher frequency of methylation of multiple genes than those of Caucasian women. Clin Cancer Res 10:2052–2057
Mulero-Navarro S, Esteller M (2008) Epigenetic biomarkers for human cancer: the time is now. Crit Rev Oncol Hematol 68:1–11. doi:10.1016/j.critrevonc.2008.03.001
Muller HM, Widschwendter A, Fiegl H, Ivarsson L, Goebel G, Perkmann E, Marth C, Widschwendter M (2003) DNA methylation in serum of breast cancer patients: an independent prognostic marker. Cancer Res 63:7641–7645
Robbins P, Pinder S, de Klerk N, Dawkins H, Harvey J, Sterrett G, Ellis I, Elston C (1995) Histological grading of breast carcinomas: a study of interobserver agreement. Hum Pathol 26:873–879
Stearns V, Yamauchi H, Hayes DF (1998) Circulating tumor markers in breast cancer: accepted utilities and novel prospects. Breast Cancer Res Treat 52:239–259
Umbricht CB, Evron E, Gabrielson E, Ferguson A, Marks J, Sukumar S (2001) Hypermethylation of 14–3-3 sigma (stratifin) is an early event in breast cancer. Oncogene 20:3348–3353. doi:10.1038/sj.onc.1204438
Acknowledgments
This research was supported by Promotion of Cancer Research (Japan) for the 3rd Term Comprehensive 10-Year Strategy for Cancer Control. The authors thank Noriko Oka and Kaya Tai for their assistance during the assay development.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yamamoto, N., Nakayama, T., Kajita, M. et al. 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. Breast Cancer Res Treat 132, 165–173 (2012). https://doi.org/10.1007/s10549-011-1575-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10549-011-1575-2