Search tips
Search criteria 


Logo of diagpathBioMed CentralBiomed Central Web Sitesearchsubmit a manuscriptregisterthis articleDiagnostic PathologyJournal Front Page
Diagn Pathol. 2011; 6: 116.
Published online 2011 November 30. doi:  10.1186/1746-1596-6-116
PMCID: PMC3253685

Methylation of cancer related genes in tumor and peripheral blood DNA from the same breast cancer patient as two independent events



Recently it has been suggested that acquisition of methylation of the BRCA1 promoter detectable in peripheral blood (PB) DNA, could give raise to development of breast cancer. In this study, we aimed to investigate a relationship between methylation of three breast cancer related genes in PB DNA, and tumor specific (somatic) methylation of these genes in the same individual.


We have examined methylation status of the BRCA1, APC and RASSF1A promoter regions in a panel of 75 breast tumor and PB DNA samples from the same individual. In our study group, 4.0% of the patients displayed methylation of BRCA1 and APC in both tumor and the corresponding PB DNA. At the same time despite of marked methylation in tumor DNA, no methylation of BRCA1 and APC was seen in PB DNA of 4.3% and 2.7% of the patients respectively. The RASSF1A promoter did not show methylation in PB DNA.


Our results show that for at least a subset of cancer patients methylation of certain cancer related genes in PB DNA does not seem to be directly linked to somatic methylation of the same genes in tumor DNA, and therefore may only be specific to PB DNA.

Keywords: Methylation, cancer predisposition, BRCA1, APC, RASSF1A.

The mechanism of methylation dependent gene deactivation and its significance to cancer pathogenesis is well described, with hypermethylation of tumor suppressor genes, affecting transcriptional activity of the genes, considered to be one of the most important drivers of carcinogenesis. Recently, much attention is paid to the phenomenon of hypermethylation of disease related genes in peripheral blood (PB) DNA and its involvement in the pathology of cancer and other diseases [1-6]. The origins of this phenomenon are unknown. However, it can be hypothesized that aberrant methylation of genes in PB DNA may be a consequence of germ line transmitted methylation changes or somatic aberrations that occurred during early development or through life time under specific environmental conditions. Transmission of methylation changes through germ line is still a problematic notion. There are only limited evidence showing that methylation of certain genes e.g. MLH1 in some cases can be passed through germ line in non-Mendelian fashion [7-11]. Recently, two studies have shown that paternal diet can have an influence on the methylation pattern of the offspring [12,13]. This further supports the significance of germ line transmission of methylation changes, however, these findings have to be more extensively researched in the future. As for environmental pressure on the individuals methylome, the influence of different chemicals on the somatic methylation pattern of the exposed subjects has been demonstrated in animal models, and proven to be especially damaging when the exposure occurred in the early stages of development (as reviewed in [14]). In humans there is mounting epidemiological evidence that environmental exposure can predispose to adult onset diseases. However it is still not clear, how the interactions between individual organisms and the environment occur, and to what extent they involve methylation changes.

Disregarding the origin, the intra individual methylation differences in human PB DNA are being increasingly reported in the literature [4-6]. Furthermore, these changes have been suggested to be a part of a disease predisposition mechanism, which could be based on the theory of constitutional methylation [2].

Constitutional gene methylation was initially defined as abnormal gene methylation observed in all tissues of the body [15]. Constitutional methylation is most likely affecting genes in a mono allelic fashion and if acquired during development, it can be distributed to all tissues of the organism in a mosaic pattern (and therefore seen at very low levels in affected tissues) [2,4]. Drawing analogy from somatic methylation in cancer, constitutional mono allelic methylation changes are likely to render the affected individual prone to development of neoplastic (and other) diseases. This is due to the fact that only one additional hit would be required (according to Knudson's hypothesis of tumor suppressor deactivation [16]) to abolish expression of the constitutionally mono allelic methylated gene and initiate or contribute to carcinogenesis. Moreover, allelic insufficiency could also be a disease-initiating factor.

In the first report, suggesting a link between methylation of the BRCA1 promoter in PB DNA and development of breast cancer with methylated BRCA1, the authors examined methylation status of the BRCA1 promoter in tumors and PB DNA from three breast cancer patients [3]. They showed that the BRCA1 promoter was methylated in both PB DNA samples and matched tumor DNA in all examined individuals [3]. The observed methylation was never seen at a level suggesting monoallelic specificity (50% methylation) but only at low levels (5-14%) reflecting high degree of mosaicism in the screened cell populations. Despite the promising results of this study showing a putative elegant mechanism of direct contribution of constitutional methylation to carcinogenesis, two follow up studies did not result in similar conclusions. In those studies the authors have examined methylation status of the BRCA1 promoter in PB and paired tumor DNA, however only a subset of the tumors, developed by patients with PB BRCA1 methylation, harbored tumor specific methylation of BRCA1 [2,17]. The study by Wong et al. [2] involved 12 breast cancer patients with paired tumor and PB DNA samples. The authors in this study reported generally low levels of methylation detected in PB (for most of the samples less than 5%) and furthermore, three patients with BRCA1 methylation in PB DNA did not display BRCA1 methylation in the paired tumor sample. A subsequent study by Iwamoto et al. [17] showed a more significant lack of direct correlation between methylation of BRCA1 in PB DNA and matched tumor samples.

Based upon the above results we aimed to investigate the presence or absence of a correlation between DNA methylation of cancer related genes in PB and paired tumor DNA from breast cancer patients.

We hypothesized that if the observed methylation of genes in PB DNA reflects constitutional methylation, the same methylation pattern has to be present in tumor DNA (as it originates from healthy tissue harboring constitutional methylation of the specific gene). Consequently if the methylation of those genes cannot be found in tumor DNA, the methylation observed in PB DNA is only specific to PB DNA (not constitutional) and does not directly contribute (in this case) to breast carcinogenesis of the affected individual.

In our study methylation of the BRCA1, APC and RASSF1A promoter regions was analyzed in 75 paired breast tumor and PB DNA samples using the MS-HRM (Methylation Sensitive High Resolution Melting) protocol. All MS-HRM assays were designed according to the guidelines published in [18,19]. The primer sequences used have been published in [20]. The regions targeted by the assays, spanning promoters of the screened genes are shown in Table Table1.1. The experiments were performed as previously described [21]. Briefly, tumor DNA was extracted as described in [22], and a modified salting-out protocol was used for purification of DNA from peripheral blood [23]. 100 ng of genomic DNA was bisulfite modified using EpiTect Bisulfite Kit (Qiagen). The LighCycler® 480 platform (Roche) was used for both PCR amplifications and the subsequent HRM analyses. The PCR mixes consisted of 1× LightCycler® 480 HRM Master mix Roche, 3 mM Mg+2, 0.5 μM of each primer and 4 ng (theoretically) of bisulfite modified DNA template. All reactions were run in triplicates. The methylation status of each sample was scored by comparison of the HRM profile of the sample to the HRM profiles obtained from dilutions of the methylated bisulfite modified template in an unmethylated background (Millipore). The data in this study were analyzed as previously described [21], where MS-HRM and Sanger sequencing were used to confirm that any aberrations of the HRM profile from the profile of the PCR product amplified from 0% methylation template are positive for methylation. Due to the fact that all published studies report the constitutional methylation to occur at very low levels (mosaic fashion) instead of the expected 50% methylation level that could be anticipated for mono allelic methylation, our data analysis was similar to previously published results based on qualitative methylation assessment. Nevertheless, MS-HRM data allow for quantitative methylation measurement and the Figures Figures1,1, ,22 and and33 depict the representative MS-HRM results. It can be argued that for the patients displaying the same methylation pattern in tumor and PB DNA, the detectable in PB DNA methylation, is not PB specific, but derived from circulating tumor cells and/or free circulating tumor DNA. However, it was previously shown that detection of tumor circulating cells is only possible when using enrichment technologies [24], and moreover our peripheral blood sample processing protocol dilutes free circulating tumor DNA and tumor cells under detection limit of MS-HRM (as described in [2]).

Table 1
Frequencies of methylation of analyzed genes in PB and breast tumors.
Figure 1
Representative results for MS-HRM based screening for APC methylation. Panel 1A, show the sensitivity of the assays with MS-HRM profile characteristic for 100% - blue, 10% - green, 1%- pink and 0% - red, mixes of methylated template in unmethylated background. ...
Figure 2
Representative results for MS-HRM based screening for BRCA1 methylation. Panel 1A, show the sensitivity of the assays with MS-HRM profile characteristic for 100% - blue, 10% - green, 1%- pink and 0% - red, mixes of methylated template in unmethylated ...
Figure 3
Representative results for MS-HRM based screening for RASSF1A methylation. Panel 1A, show the sensitivity of the assays with MS-HRM profile characteristic for 100% - blue, 10% - green, 1%- pink and 0% - red mixes of methylated template in unmethylated ...

All three genes analyzed in our study showed methylation in the tumor samples suggesting as previously reported, their involvement in breast cancer pathogenesis Table Table1.1. We observed high frequencies of methylation for all three genes, however the methylation frequencies for APC and RASSF1A were higher than reported in the literature This may be attributed either to an exceptionally high methylation prevalence in our samples or more likely to a high sensitivity of the MS-HRM technology [25].

Two of the examined genes, BRCA1 and APC, showed methylation in both tumor and paired PB DNA at frequencies of 4.4% and 4.1%, respectively (see Table Table11 for details) with one of the paired samples showing methylation of both APC and BRCA1 in tumor and PB DNA. However, at the same time three of the samples in our panel did not show methylation of BRCA1 in tumor DNA despite marked methylation in PB DNA. The same was seen for APC in two of the samples. RASSF1A did not show methylation in any of the PB samples.

Our study shows that a direct link between methylation of cancer related genes in PB DNA and development of cancer is questionable. If a direct link existed and as previously suggested detectable in PB DNA hypermethylation was constitutional [2], all patients with PB DNA methylation should display the same specific methylation pattern in the paired tumor. This is due to the fact that tumor DNA originates from healthy tissue, which (similar to blood tissue) should harbor constitutional methylation that in turn would predispose the affected individual to cancer development. The fact that we have not seen the same methylation pattern for a subset of our paired samples suggests independence of the methylation events in PB DNA and during tumor development. However, at the same time basing on current results, we cannot rule out the presence of a direct link between those two events for the subset of patients displaying methylation in both PB and tumor DNA.

In conclusion, the fact that methylation of the BRCA1 gene in PB DNA correlates with increased risk of breast cancer, allows to anticipate that aberrant methylation of genes in PB and disease predisposition are linked. Especially considering the study by Iwamoto et al. and the study by Wong at al. both indicating a strong correlation between methylation of BRCA1 in PB and breast cancer incidence. However, our present and previously published results do not confirm that the mechanism of that interaction is based solely on constitutional methylation and suggests independence of those two events for at least a subset of cancer patients.

Competing interests

TKW and LLH are listed as inventors on patent pending application on aspects of MS-HRM technology. JO and BBT have no competing interests.

Authors' contributions

TKW performed the experiments, wrote the manuscript, TBB performed experiments, JO and LLH supervised the experiments wrote and the manuscript. All authors approved the manuscript.

Financial support

This study was supported by The Danish Cancer Society, CIRRO - The Lundbeck Foundation Center for Interventional Research in Radiation Oncology and The Danish Council for Strategic Research (TKW, JO, LLH), Aase og Ejnar Danielsens Fond and the Toyota Foundation (TKW, LLH, TBB).


  • Iwamoto T, Yamamoto N, Taguchi T, Tamaki Y, Noguchi S. BRCA1 promoter methylation in peripheral blood cells is associated with increased risk of breast cancer with BRCA1 promoter methylation. Breast Cancer Res Treat. 2011;129:69–77. doi: 10.1007/s10549-010-1188-1. [PubMed] [Cross Ref]
  • Wong EM, Southey MC, Fox SB, Brown MA, Dowty JG, Jenkins MA, Giles GG, Hopper JL, Dobrovic A. Constitutional methylation of the BRCA1 promoter is specifically associated with BRCA1 mutation-associated pathology in early-onset breast cancer. Cancer Prev Res (Phila) pp. 23–33. [PubMed]
  • Snell C, Krypuy M, Wong EM, Loughrey MB, Dobrovic A. BRCA1 promoter methylation in peripheral blood DNA of mutation negative familial breast cancer patients with a BRCA1 tumour phenotype. Breast Cancer Res. 2008;10:R12. doi: 10.1186/bcr1858. [PMC free article] [PubMed] [Cross Ref]
  • Widschwendter M, Apostolidou S, Raum E, Rothenbacher D, Fiegl H, Menon U, Stegmaier C, Jacobs IJ, Brenner H. Epigenotyping in peripheral blood cell DNA and breast cancer risk: a proof of principle study. PLoS ONE. 2008;3:e2656. doi: 10.1371/journal.pone.0002656. [PMC free article] [PubMed] [Cross Ref]
  • Flanagan JM, Munoz-Alegre M, Henderson S, Tang T, Sun P, Johnson N, Fletcher O, Dos Santos Silva I, Peto J, Boshoff C. et al. Gene-body hypermethylation of ATM in peripheral blood DNA of bilateral breast cancer patients. Hum Mol Genet. 2009;18:1332–1342. doi: 10.1093/hmg/ddp033. [PMC free article] [PubMed] [Cross Ref]
  • Woodson K, Mason J, Choi SW, Hartman T, Tangrea J, Virtamo J, Taylor PR, Albanes D. Hypomethylation of p53 in peripheral blood DNA is associated with the development of lung cancer. Cancer Epidemiol Biomarkers Prev. 2001;10:69–74. [PubMed]
  • Ligtenberg MJ, Kuiper RP, Chan TL, Goossens M, Hebeda KM, Voorendt M, Lee TY, Bodmer D, Hoenselaar E, Hendriks-Cornelissen SJ. et al. Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3' exons of TACSTD1. Nat Genet. 2009;41:112–117. doi: 10.1038/ng.283. [PubMed] [Cross Ref]
  • Chan TL, Yuen ST, Kong CK, Chan YW, Chan AS, Ng WF, Tsui WY, Lo MW, Tam WY, Li VS, Leung SY. Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer. Nat Genet. 2006;38:1178–1183. doi: 10.1038/ng1866. [PubMed] [Cross Ref]
  • Morak M, Schackert HK, Rahner N, Betz B, Ebert M, Walldorf C, Royer-Pokora B, Schulmann K, von Knebel-Doeberitz M, Dietmaier W. et al. Further evidence for heritability of an epimutation in one of 12 cases with MLH1 promoter methylation in blood cells clinically displaying HNPCC. Eur J Hum Genet. 2008;16:804–811. doi: 10.1038/ejhg.2008.25. [PubMed] [Cross Ref]
  • Hitchins MP, Wong JJ, Suthers G, Suter CM, Martin DI, Hawkins NJ, Ward RL. Inheritance of a cancer-associated MLH1 germ-line epimutation. N Engl J Med. 2007;356:697–705. doi: 10.1056/NEJMoa064522. [PubMed] [Cross Ref]
  • Hitchins MP, Ward RL. Erasure of MLH1 methylation in spermatozoa-implications for epigenetic inheritance. Nat Genet. 2007;39:1289. doi: 10.1038/ng1107-1289. [PubMed] [Cross Ref]
  • Carone BR, Fauquier L, Habib N, Shea JM, Hart CE, Li R, Bock C, Li C, Gu H, Zamore PD, Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Cell. pp. 1084–1096. [PMC free article] [PubMed]
  • Ng SF, Lin RC, Laybutt DR, Barres R, Owens JA, Morris MJ. Chronic high-fat diet in fathers programs beta-cell dysfunction in female rat offspring. Nature. pp. 963–966. [PubMed]
  • Jirtle RL, Skinner MK. Environmental epigenomics and disease susceptibility. Nat Rev Genet. 2007;8:253–262. doi: 10.1038/nrg2045. [PubMed] [Cross Ref]
  • Dobrovic A, Kristensen LS. DNA methylation, epimutations and cancer predisposition. Int J Biochem Cell Biol. 2009;41:34–39. doi: 10.1016/j.biocel.2008.09.006. [PubMed] [Cross Ref]
  • Knudson AG. Hereditary cancer: two hits revisited. J Cancer Res Clin Oncol. 1996;122:135–140. doi: 10.1007/BF01366952. [PubMed] [Cross Ref]
  • Iwamoto T, Yamamoto N, Taguchi T, Tamaki Y, Noguchi S. BRCA1 promoter methylation in peripheral blood cells is associated with increased risk of breast cancer with BRCA1 promoter methylation. Breast Cancer Res Treat. [PubMed]
  • Wojdacz TK, Dobrovic A, Hansen LL. Methylation-sensitive high-resolution melting. Nature Protocols. 2008;3 [PubMed]
  • Wojdacz TK, Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res. 2007;35:e41. doi: 10.1093/nar/gkm013. [PMC free article] [PubMed] [Cross Ref]
  • Wojdacz TK, Thestrup BB, Cold S, Overgaard J, Hansen LL. No difference in the frequency of locus-specific methylation in the peripheral blood DNA of women diagnosed with breast cancer and age-matched controls. Future Oncology. 2011;12(7):1451–1455. [PubMed]
  • Wojdacz TK, Moller TH, Thestrup BB, Kristensen LS, Hansen LL. Limitations and advantages of MS-HRM and bisulfite sequencing for single locus methylation studies. Expert Rev Mol Diagn. pp. 575–580. [PubMed]
  • Hansen LL, Andersen J, Overgaard J, Kruse TA. Molecular genetic analysis of easily accessible breast tumour DNA, purified from tissue left over from hormone receptor measurement. APMIS. 1998;106:371–377. doi: 10.1111/j.1699-0463.1998.tb01359.x. [PubMed] [Cross Ref]
  • Hansen LL, Yilmaz M, Overgaard J, Andersen J, Kruse TA. Allelic loss of 16q23.2-24.2 is an independent marker of good prognosis in primary breast cancer. Cancer Res. 1998;58:2166–2169. [PubMed]
  • Raynor MP, Stephenson SA, Pittman KB, Walsh DC, Henderson MA, Dobrovic A. Identification of circulating tumour cells in early stage breast cancer patients using multi marker immunobead RT-PCR. J Hematol Oncol. 2009;2:24. doi: 10.1186/1756-8722-2-24. [PMC free article] [PubMed] [Cross Ref]
  • Wojdacz TK, Borgbo T, Hansen LL. Primer design versus PCR bias in methylation independent PCR amplifications. Epigenetics. 2009;4:231–234. [PubMed]

Articles from Diagnostic Pathology are provided here courtesy of BioMed Central