Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Aug 31.
Published in final edited form as: Epigenetics. 2007 Oct 29;2(4):223–226. doi: 10.4161/epi.2.4.5214

Global DNA Hypomethylation in Liver Cancer Cases and Controls

A Phase I Preclinical Biomarker Development Study

Rafael Guerrero-Preston 1,2,4,*, Regina M Santella 2, Adolfo Blanco 5, Manisha Desai 3, Maria Berdasco 6, Mario Fraga 6
PMCID: PMC13526440  NIHMSID: NIHMS2206290  PMID: 18032927

Abstract

Background:

Global genomic DNA hypomethylation is a feature of genomic DNA derived from solid and hematologic tumors in animal models and human carcinogenesis. Global genomic DNA hypomethylation may be the earliest epigenetic change from a normal to a pre-malignant cell.

Objectives:

To test if global hypomethylation is a good marker for early detection of cancer we used a novel quantification method of 2’-deoxynucleosides to evaluate DNA methylation in liver cancer cases and controls.

Methods:

Frozen tissue from liver cancer patients and controls were obtained from the Cooperative Human Tissue Network. DNA was extracted using standard methods. Genomic DNA samples were boiled and treated with nuclease P1 and alkaline phosphatase. Global genomic DNA methylation patterns were obtained using HPLC for fraction separation and mass spectrometry for quantification. A two-sample t-test was performed using Welch’s approximation for samples with unequal variances. A Wilcoxon rank sum test was also performed.

Results:

A global genomic DNA methylation index measuring methylated cytidine relative to global cytidine in the genome was significantly lower (p value = 0.001) for all cases, mean = 2.43 (95% CI, 2.08, 2.78), when compared to controls, mean = 3.55 (95% CI, 3.16, 3.93).

Discussion:

A correlation between global genomic DNA methylation patterns and type of liver tissue was observed. These results add to the accumulating body of evidence suggesting that global DNA hypomethylation may be a useful biomarker to distinguish between liver cancer cases and controls.

Keywords: global genomic hypomethylation, epigenetics of hepatocarcinogenesis, epigenetic biomarkers for early detection, molecular cancer screening technology, epigenetic approaches to cancer epidemiology

INTRODUCTION

The inheritance of information based on gene expression levels that do not entail changes in DNA sequence is known as epigenetics, as opposed to genetics, which refers to information transmitted on the basis of gene sequence. Cancer is an epigenetic disease characterized by the breakdown of DNA methylation and histones modification patterns.1 DNA methylation, the most important epigenetic modification known, plays a dual role in human cancer. Global hypomethylation, together with both hypomethylation2 and hypermethylation of promoter regions, are fundamental aspects of human neoplasia.3,4

Region-specific hypermethylation of CpG islands leads to the suppression of housekeeping and cell cycle control genes, as well as tumor suppressor and DNA repair genes, resulting in tumor growth and progression.5 Global hypomethylation plays a causal role in tumor formation by promoting chromosomal instability, activation of proto-oncogenes, and loss of heterozygosity, all of which are highly correlated with tumorigenesis.6–8

Genetic and epigenetic alterations following global DNA hypomethylation in non-coding regions have been shown to play a significant role in animal models and human hepatocarcinogenesis. DNA hypomethylation is one of the key events in the initiation of the carcinogenic process in animal models.9 DNA hypomethylation largely affects transposons, leading to their activation and promotion of chromosomal rearrangements and other preneoplastic changes.10 Stable DNA hypomethylation in tissue that undergoes carcinogenesis is also related to cancer progression from normal to tumor cell.11 Although it is not yet well understood why all cancer tissue does not undergo hypomethylation in the same manner, human cancers can be classified into two groups: a low (0–3.4%) hypomethylation group; and a moderately high (6.8–9.5%) hypomethylation group.12

Global hypomethylation is a very early event in human and experimental carcinogenesis and a feature of genomic DNA derived from solid and hematologic tumors, which may precede region-specific hypermethylation in neoplastic transformation from normal to premalignant phenotypes.13 The body of evidence that has accumulated suggests that global hypomethylation may be a potential biomarker for early cancer detection, particularly in populations at risk for cancers lacking effective early detection markers, such as hepatocellular carcinoma.14,15

The purpose of this study is to ascertain the usefulness of using global DNA hypomethylation as a biomarker for early detection of cancer. We used a novel method for quantification of 2’-deoxynucleosides to evaluate a global genomic DNA methylation index as an early detection biomarker in a Phase 1 preclinical discovery study.

METHODS

Frozen tissue samples from liver cancer patients and controls were obtained from the Cooperative Human Tissue Network.16 DNA was extracted using standard methods. Five micrograms of genomic DNA samples were boiled and treated with nuclease P1 and alkaline phosphatase. Global genomic DNA methylation patterns were obtained using HPLC for fraction separation and Mass Spectrometry for quantification.

Fifty micrograms of the hydrolyzed-DNA solution were injected onto a reversed phased Atlantis dC18 column (2.1 × 150 mm; 5 μm particle size) protected by an Agilent guard column (2.1 × 20 mm; 5 μm particle size) at a constant flow of 0.220 ml min−1. Two buffers, 0.1% formic acid in water (Solvent A) and 0.1% formic acid in 50% water: 50% methanol (Solvent B), were used, with an initial gradient of 5% solvent B, then an increase of solvent B to 50% within nine minutes and an isocratic gradient (50% of solvent B) during 25 minutes. Identification of 2’-deoxycytidine (dC) and 5-methyl-2’-deoxycytidine (5mdC) was obtained by UV detection at A254 and A280 using a LC-ESI/MS system. Quantification of global genomic DNA methylation was obtained from integration peak areas of 5mdC relative to global cytidine (5mdC + dC). The DNA methylation index was obtained in triplicate for each sample. The LC-ESI/MS system consisted of an Agilent Series 1100 HPLC system coupled to an Agilent LC/MSD VL mass spectrometer equipped with an electrospray ionization source (Agilent Technology, Palo Alto, California).

Electrospray source conditions were as described in Friso17 with minor modifications. A drying gas flow of 10.0 ml min−1 was employed, with auxiliary 35 psi gas to assist with nebulization and a drying temperature of 350°C. The mass spectrophotometer was operated at a capillary voltage of 4,000 V and spectra were collected in positive ion mode. Significance of results was ascertained with a two-sample t test using Welch’s approximation for samples with unequal variances and a Wilcoxon rank sum test. Significance analyses were conducted in Stata 9.0 (Stata Corporation, Texas, 2006). Logistic regression modeling and graphical representation were done in R 2.5 (R-Project, 2007).

RESULTS AND DISCUSSION

The results of this study are shown in Table 1. The median (range) of the global genomic DNA methylation index value was 2.42 (1.94–3.08) for cases and 3.64 (2.86–4.13) for controls. The standard deviation for the global genomic DNA methylation index was 0.42 for cases and 0.46 for controls and the interquartile range was 1.14 for cases and 1.27 for controls.

Table 1.

DNA samples in triplicate (n = 10 pairs)

Cases Controls
Methylation index median (range) 2.42 (1.94–3.08) 3.64 (2.86–4.13)
Methylation index mean (95% CI) 2.43 (2.08–2.78) 3.55 (3.16–3.93)
Standard Deviation 0.42 0.46
Interquartile Range 1.14 1.27
Open in a new tab

Results of global genomic DNA methylation analysis in tissue of liver cancer cases and controls.

The mean global genomic DNA methylation index value, measuring methylated cytidine relative to global cytidine in the genome, was significantly lower (p value = 0.001 for two sample t-test; p value = 0.01 for Wilcoxon rank-sum test) for all cases, mean = 2.43 (95% CI, 2.08, 2.78), when compared to controls, mean = 3.55 (95% CI, 3.16, 3.93). The significant difference in means and the lack of overlap in confidence intervals for cases and controls suggest that the global genomic DNA methylation index is a useful epigenetic biomarker to distinguish between liver cancer cases and controls.

Figure 1 shows a graphical expression of the logistic regression described by the following expression: Pr (liver cancer) = logit−1 (β0 + β1* methylation) with data overlain. The predictor methylation is the global genomic DNA methylation index value for each case (1) and each control (0).

Figure 1.

Figure 1.

Open in a new tab

Graphical expression of the logistic regression, Pr(liver cancer) = logit-1 (b0 + b1* methylation) with data overlain. The predictor methylation is the global genomic DNA methylation index value for each case (1) and each control (0).

The mean results of three measurements of the global genomic DNA methylation index for eight cases and eight controls are plotted. Lack of sufficient DNA quality in the remaining two samples of cases and controls, prevented us from obtaining an accurate reading of their methylation index.

The most important result in this paper is that a global genomic DNA methylation index was successful in distinguishing between tissue samples of liver cancer cases and controls. We have focused on liver cancer in this proof-of-principle study because most of the animal work in carcinogenesis has been done in hepatocarcinogenesis. This proof-of-principle analysis needs to be validated in a larger data set using a classification rule developed with the data generated in this study to create receiver-operator characteristic curves (ROC).

To explain the advantages of a global genomic DNA methylation index as an early detection tool in human cancer we have created a conceptual model of the determinants of DNA hypomethylation (Fig. 2).

Figure 2.

Figure 2.

Open in a new tab

Determinants of global DNA hypomethylation and its consequences in a transformed cell.

In the main pathway proposed, exogenous factors (biological, chemical, physical, social and life-style factors) cause histones modifications that lead to global DNA hypomethylation.18 Endogenous factors acting through three secondary pathways related to decreased DNA methyltransferase expression,19,20 non-coding RNA silencing21,22 and defective DNA repair,23,24 may also have a direct causal role in global DNA hypomethylation. Factors that activate these three endogenous pathways may lead directly to a loss of global DNA methylation, may also cause chromatin modifications leading to hypomethylation, or may only be an intermediate step leading to histones modifications that are linked to global DNA hypomethylation. The model also shows how the global loss of methylation, mediated by the interaction of exogenous and endogenous factors, leads to abnormalities associated with premalignancy and malignancy: chromosomal instability, aberrant gene expression, loss of imprinting, microsatellite instability and retrotransposons activation.

This study has two main limitations: small sample size and missing data due to lack of pure DNA extraction. However the two-sample t-test and the Wilcoxon rank sum test showed a significant difference between the global genomic DNA methylation index in cases and controls. We had missing data for two cases and two controls which the DNA was not clean enough to be resolved by the LC-ESI/MS assay utilized in this report. We attribute this to technician variability, a factor that will be reduced in future studies.

The LC-ESI/MS quantification method used in this study has a limitation for clinical studies, such that it requires 5 μg of DNA per sample. While being the most sensitive and accurate method to quantify global genomic DNA methylation today, we expect new methods to be developed that reduce the required sample amount of DNA without reducing accuracy in the detection of global genomic DNA methylation patterns.

The enzymatic hydrolysis method and the LC-ESI/MS assay utilized in this report allows for the quantification of total methylated cytosines in the genome and the calculation of a relative methylation index, which can be used to effectively compare methylation changes across different tissues. We now plan on developing a classification rule to distinguish between cases and controls using the methylation data presented here and we will validate this rule on an independent data set.

We have been able to successfully distinguish liver cancer cases from controls using a global genomic DNA methylation assay. Since global DNA hypomethylation is tissue specific in cancer, a continuous global genomic DNA methylation index may be a useful early epigenetic biomarker for cancer research. Future research will be done with a larger sample size to have adequate power and correlate the global hypomethylation index in liver tissue with the global hypomethylation index in blood of the same subjects. Once the global genomic DNA methylation index of liver cancer cases and controls is validated in blood samples, correlation studies will be performed in a prospective cohort to compare the sensitivity and specificity of global DNA hypomethylation as an early detection marker of liver cancer against the current diagnostic marker in blood, circulating levels of α-fetoprotein.

Acknowledgements

This research was supported in part by funds from the federal government, NCI grant number 5T32CA009529-20, NIA grant number 2P30AG-15294 and NCMHD grant number 5S21MD008130-02.

We also want to acknowledge the valuable input of Esteban Ballestar from the Cancer Epigenetics Laboratory at the Spanish National Cancer Research Center and Andrew Gelman, Alfred I. Neugut, Paul Brandt-Rauf, Benjamin Tycko and Federica Perera for providing mentoring, insight and recommendations in different stages of the development of this work.

References

  • 1.Jacinto FV, Esteller M. Mutator pathways unleashed by epigenetic silencing in human cancer. Mutagenesis 2007, 22:247–53. [DOI] [PubMed] [Google Scholar]
  • 2.Kaneda A, Tsukamoto T, Takamura-Enya T, Watanabe N, Kaminishi M, Sugimura T, Tatematsu M, Ushijima T. Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation. Cancer Sci 2004; 95:58–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: A fundamental aspect of neoplasia. Adv Cancer Res 1998; 72:141–96. [PubMed] [Google Scholar]
  • 4.Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 1983; 301:89–92. [DOI] [PubMed] [Google Scholar]
  • 5.Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002; 3:415–28. [DOI] [PubMed] [Google Scholar]
  • 6.Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 2003; 300:455. [DOI] [PubMed] [Google Scholar]
  • 7.Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, Leonhardt H, Jaenisch R. Induction of tumors in mice by genomic hypomethylation. Science 2003; 300:489–92. [DOI] [PubMed] [Google Scholar]
  • 8.Matsuzaki K, Deng G, Tanaka H, Kakar S, Miura S, Kim YS. The relationship between global methylation level, loss of heterozygosity, and microsatellite instability in sporadic colorectal cancer. Clin Cancer Res 2005; 11:8564–9. [DOI] [PubMed] [Google Scholar]
  • 9.Fraga MF, Herranz M, Espada J, Ballestar E, Paz MF, Ropero S, Erkek E, Bozdogan O, Peinado H, Niveleau A, Mao JH, Balmain A, Cano A, Esteller M. A mouse skin multistage carcinogenesis model reflects the aberrant DNA methylation patterns of human tumors. Cancer Res 2004; 64:5527–34. [DOI] [PubMed] [Google Scholar]
  • 10.Goll MG, Bestor TH. Eukaryotic cytosine methyltransferases. Annu Rev Biochem 2005; 74:481–514. [DOI] [PubMed] [Google Scholar]
  • 11.Pogribny IP, Ross SA, Wise C, Pogribna M, Jones EA, Tryndyak VP, James SJ, Dragan YP, Poirier LA. Irreversible global DNA hypomethylation as a key step in hepatocarcinogenesis induced by dietary methyl deficiency. Mutat Res 2006; 593:80–7. [DOI] [PubMed] [Google Scholar]
  • 12.Chalitchagorn K, Ross SA, Wise C, Pogribna M, Jones EA, Tryndyak VP, James SJ, Dragan YP. Poirier LA Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene, 2004; 23:8841–6. [DOI] [PubMed] [Google Scholar]
  • 13.Shen L, Fang J, Qiu D, Zhang T, Yang J, Chen S, Xiao S. Correlation between DNA methylation and pathological changes in human hepatocellular carcinoma. Hepatogastroenterology 1998; 45:1753–9. [PubMed] [Google Scholar]
  • 14.Verma M, Srivastava S. Epigenetics in cancer: Implications for early detection and prevention. Lancet Oncol 2002; 3:755–63. [DOI] [PubMed] [Google Scholar]
  • 15.Giannelli G, Antonaci S. New frontiers in biomarkers for hepatocellular carcinoma. Dig Liver Dis 2006; 38:854–9. [DOI] [PubMed] [Google Scholar]
  • 16.Clausen KP, Grizzle WE, Livolsi V, Newton WA Jr, Aamodt R. Special communication: The cooperative human tissue network. Cancer 1989; 63:1452–5. [DOI] [PubMed] [Google Scholar]
  • 17.Friso S, Choi SW, Dolnikowski GG, Selhub J. A method to assess genomic DNA methylation using high-performance liquid chromatography/electrospray ionization mass spectrometry. Anal Chem 2002; 74:4526–31. [DOI] [PubMed] [Google Scholar]
  • 18.Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, Bonaldi T, Haydon C, Ropero S, Petrie K, Iyer NG, Pérez-Rosado A, Calvo E, Lopez JA, Cano A, Calasanz MJ, Colomer D, Piris MA, Ahn N, Imhof A, Caldas C, Jenuwein T, Esteller M. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 2005; 37:391–400. [DOI] [PubMed] [Google Scholar]
  • 19.Dodge JE, Okano M, Dick F, Tsujimoto N, Chen T, Wang S, Ueda Y, Dyson N, Li E. Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem 2005; 280:17986–91. [DOI] [PubMed] [Google Scholar]
  • 20.James SR, Link PA, Karpf AR. Epigenetic regulation of X-linked cancer/germline antigen genes by DNMT1 and DNMT3b. Oncogene 2006; 25:6975–85. [DOI] [PubMed] [Google Scholar]
  • 21.Lujambio A, Ropero S, Ballestar E, Fraga MF, Cerrato C, Setian F, Casado S, Suarez-Gauthier A, Sanchez-Cespedes M, Git A, Spiteri I, Das PP, Caldas C, Miska E, Esteller M. Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. Cancer Res 2007; 67:1424–9. [DOI] [PubMed] [Google Scholar]
  • 22.Costa FF. Non-coding RNAs: New players in eukaryotic biology. Gene 2005; 357:83–94. [DOI] [PubMed] [Google Scholar]
  • 23.Koturbash I, Pogribny I, Kovalchuk O. Stable loss of global DNA methylation in the radiation-target tissue—A possible mechanism contributing to radiation carcinogenesis? Biochem Biophys Res Commun 2005; 337:526–33. [DOI] [PubMed] [Google Scholar]
  • 24.Pogribny I, Koturbash I, Tryndyak V, Hudson D, Stevenson SM, Sedelnikova O, Bonner W, Kovalchuk O. Fractionated low-dose radiation exposure leads to accumulation of DNA damage and profound alterations in DNA and histone methylation in the murine thymus. Mol Cancer Res 2005; 3:553–61. [DOI] [PubMed] [Google Scholar]

RESOURCES