Elsevier

The Lancet Oncology

Volume 9, Issue 4, April 2008, Pages 359-366
The Lancet Oncology

Fast track — Articles
Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case–control study

https://doi.org/10.1016/S1470-2045(08)70038-XGet rights and content

Summary

Background

DNA hypomethylation has been suggested to cause genomic instability and increase cancer risk. We aimed to test the hypothesis that DNA hypomethylation is associated with increased risk of bladder cancer.

Methods

We measured cytosine methylation (5-mC) content in genomic DNA from blood cells from patients with bladder cancer enrolled in a large case–control study in Spain between Jan 1, 1998, and Dec 31, 2001. Cases were men and women with newly diagnosed and histologically confirmed urothelial carcinoma of the bladder. Controls were selected from patients admitted to the same hospital for diseases or conditions unrelated to smoking or other known risk factors for bladder cancer. Controls were individually matched to cases on age (within 5 years), sex, race, and area of hospital referral. 5-mC content was measured in leucocyte DNA by use of a combination of high-performance capillary electrophoresis, Hpa II digestion, and densitometry. Data on demographics, 34 polymorphisms in nine folate metabolism genes, and nutritional intake of six B vitamins (including folate), alcohol, and smoking were assessed as potential confounders. Relative 5-mC content was expressed as a percentage (%5-mC) with respect to the total cytosine content (the sum of methylated and non-methylated cytosines). The primary endpoint was median %5-mC DNA content.

Findings

%5-mC was measured in leucocyte DNA from 775 cases and 397 controls. Median %5-mC DNA was significantly lower in cases (3·03% [IQR 2·17–3·56]) than in controls (3·19% [2·46–3·68], p=0·0002). All participants were subsequently categorised into quartiles by %5-mC content in controls. When the highest quartile of %5-mC content was used as the reference category (Q4), the following adjusted odds ratios (OR) and 95% CI were recorded for decreasing methylation quartiles: OR(Q3) 2·05 (95% CI 1·37–3·06); OR(Q2) 1·62 (1·07–2·44); and OR(Q1) 2·67 (1·77–4·03), p for trend <0·0001. The lowest cancer risk was noted in never smokers in the highest methylation quartile (never smokers in Q4). By comparison with never smokers in the highest quartile, current smokers in the lowest methylation quartile had the highest risk of bladder cancer (Q1: OR 25·51 [9·61–67·76], p for interaction 0·06). In analyses stratified by smoking, hypomethylation was a strong risk factor in never smokers (OR 6·39 [2·37–17·22]). Amount of methylation in controls were not associated with baseline characteristics, micronutrients, or selected genotypes in folate metabolism pathways.

Interpretation

For the first time, to our knowledge, we have shown in a large case–control study that leucocyte DNA hypomethylation is associated with increased risk of developing bladder cancer, and this association is independent of smoking and the other assessed risk factors. Amount of global methylation in genomic DNA could provide a useful biomarker of susceptibility to certain cancer types and further research is warranted.

Funding

Intramural Research Program of the National Institutes of Health, National Cancer Institute, Division of Cancer Epidemiology and Genetics, Bethesda, MD, USA, and Fondo de Investigacion Sanitaria, Spain (G03/174).

Introduction

In tumour tissue, changes in patterns of DNA methylation, such as promoter CpG-island hypermethylation and global (genome-wide) hypomethylation, frequently occur. Global hypomethylation of DNA is thought to contribute to carcinogenesis by the induction of genome instability and gene-specific hypomethylation.1, 2 Genomic instability is a common occurrence in many cancers, and chromosomal instability and, to a lesser extent, microsatellite instability, has been noted in urothelial carcinomas of the bladder.3, 4, 5, 6, 7, 8 Studies have also shown that in bladder cancer, chromosomal instability is widespread, occurs early in the carcinogenic process, and is associated with alterations on chromosome 9, the first and most frequently altered chromosome in the development of bladder cancer.9, 10, 11, 12 One study suggested that loss of heterozygosity (LOH) of chromosome 9 was associated with genome-wide hypomethylation.12

Measures of global cytosine methylation in DNA of blood cells and of healthy tissue have been used as a phenotypic marker of genomic instability and potential cancer risk. One study13 of patients with head and neck cancer noted that amounts of global cytosine methylation were lower in the DNA in blood from patients with cancer compared with controls. Smoking and the methylene tetrahydrofolate reductase (MTHFR) variant genotype were associated with less methylation compared with controls, whereas exposure to human papilloma virus 16 was associated with more global methylation compared with controls.13 Another study14 of cancer-cell lines and tumour tissues showed that most long interspersed nuclear element-1 (LINE-1) sequences were hypomethylated compared with those in lymphocytes and healthy colon mucosa. Only one smaller study15 assessed global methylation as a susceptibility factor for bladder cancer, a tobacco-associated tumour for which genomic instability increases with disease progression.16, 17, 18 That study did not assess other covariates that might also affect the amount of methylation in genomic DNA, such as diet and genetic variation.

To ascertain whether the amount of DNA methylation was associated with risk of bladder cancer, we aimed to measure cytosine methylation (5-mC) content in leucocyte DNA in patients with bladder cancer (cases) and controls who were enrolled in a large hospital-based study (the Spanish Bladder Cancer Study). Information on variation in 34 single nucleotide polymorphisms (SNPs) in nine folate-metabolism-pathway genes, on vitamins B1, B2, B3, B6, B12, and folate, total protein, and alcohol intake was assessed to identify factors that contribute to the amount of genomic methylation in controls and to identify potential confounding factors and risk modifiers for bladder cancer.19, 20, 21

Section snippets

Patients

The study population has been previously described.22, 23 This study was a hospital-based case–control study undertaken between Jan 1, 1998, and Dec 31, 2001, in five regions of Spain (Asturias, Barcelona, Vallés Occidental-Bages, Alicante, and Tenerife). Cases were men and women with newly diagnosed and histologically confirmed urothelial carcinoma of the bladder. Controls were selected from patients admitted to the same hospital for diseases or conditions unrelated to smoking and other known

Results

1219 of 1453 (84%) eligible cases and 1271 of 1442 (88%) eligible controls agreed to participate in the study. 1150 of 1219 (94%) cases and 1149 of 1271 (90%) controls provided suitable genomic DNA for molecular analyses of genotypes and amount of global methylation. Methylation analyses were done on DNA from 775 of 1150 (67%) cases and 397 of 1149 (35%) controls who had available leucocyte DNA for analysis. Nutritional data were available for 588 of 775 (76%) cases and 288 of 397 (73%)

Discussion

In this study, median amounts of global methylation, measured as %5-mC in leucocyte DNA, was significantly lower in cases than controls and lower amounts of DNA methylation (hypomethylation) were independently associated with increased cancer risk. This association was also dose-dependent and not modified by nutritional or polymorphic variants in genes known to be involved in DNA methylation or folate metabolism that were assessed in this study. Although smoking was not associated with amount

References (39)

  • F Jiang et al.

    Centrosomal abnormality is common in and a potential biomarker for bladder cancer

    Int J Cancer

    (2003)
  • A Hartmann et al.

    Frequent microsatellite instability in sporadic tumours of the upper urinary tract

    Cancer Res

    (2002)
  • Y Yamamoto et al.

    Biological characteristics in bladder cancer depends on type of genetic instability

    Clin Cancer Res

    (2006)
  • L Santos et al.

    Chromosome instability and progression in urothelial cell carcinoma of the bladder

    Acta Oncol

    (2003)
  • B Jurgens et al.

    Hypomethylation of L1 LINE sequences prevailing in human urothelial carcinoma

    Cancer Res

    (1996)
  • AR Florl et al.

    DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas

    Br J Cancer

    (1999)
  • F Kimura et al.

    Destabilization of chromosome 9 in transitional cell carcinoma of the urinary bladder

    Br J Cancer

    (2001)
  • DT Hsiung et al.

    Global DNA Methylation Level in Whole Blood as a Biomarker in Head and Neck Squamous Cell Carcinoma

    Cancer Epidemiol Biomarkers Prev

    (2007)
  • MRH Estecio et al.

    LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability

    PLoS ONE

    (2007)
  • Cited by (212)

    • As3MT-mediated SAM consumption, which inhibits the methylation of histones and LINE1, is involved in arsenic-induced male reproductive damage

      2022, Environmental Pollution
      Citation Excerpt :

      Hypermethylation of the global DNA methylation surrogate, LINE1, is associated with reduced sperm motility, lowered sperm quality, and decreased sperm morphology (Cassuto et al., 2016; Tian et al., 2014). Decreased methylation of LINE1 sequences in blood leukocyte DNA occurs in various human cancers and other chronic diseases (Moore et al., 2008; Muka et al., 2016; Wilhelm et al., 2010). Chronic exposure to As is associated with global DNA hypomethylation measured in LINE1 repeats (Hossain et al., 2017).

    • Oxidative stress, epigenetics, and bladder cancer

      2021, Cancer: Oxidative Stress and Dietary Antioxidants
    • LHX6 promoter hypermethylation in oncological pediatric patients conceived by IVF

      2023, Journal of Developmental Origins of Health and Disease
    View all citing articles on Scopus
    View full text