Abstract
BACKGROUND
Evidence suggests that global blood DNA methylation levels may be associated with the risk of various cancers, but no studies have evaluated this relationship for prostate cancer.
METHODS
We used pyrosequencing to quantify DNA methylation levels at the long interspersed nuclear element 1 (LINE-1) and Alu repetitive elements in pre-diagnostic blood samples from 694 prostate cancer cases and 703 controls from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. We evaluated prostate cancer risk associated with the mean methylation level for each element using logistic regression, adjusting for potential confounders.
RESULTS
We did not observe a significant association with prostate cancer for LINE-1 [Odds Ratio (OR) for the highest compared to the lowest quartile=1.01, 95% Confidence Interval (CI): 0.73-1.39, ptrend=0.99] or Alu (OR=0.94, 95% CI: 0.68-1.29, ptrend=0.69) methylation levels overall. However, for Alu, we observed that higher DNA methylation levels were associated with a significant increased risk for those diagnosed 4 or more years after blood draw (OR=2.26, 95% CI: 1.27-4.00, ptrend=4.4×10−3). In contrast, there was no association for those diagnosed 2 (OR=1.13, 95% CI: 0.67-1.90, ptrend=0.64) or 3 years after draw (OR=1.22, 95% CI: 0.71-2.07, ptrend=0.32), and a decreased risk for those diagnosed less than 2 years after draw (OR=0.40, 95% CI: 0.25-0.65, ptrend=3.8×10−5; pheterogeneity=5.3×10−6).
CONCLUSIONS
While LINE-1 DNA methylation levels were not associated with prostate cancer, we observed an association for Alu that varied by time from blood draw to diagnosis. Our study suggests that elevated Alu blood DNA methylation levels several years before diagnosis may be associated with an increased prostate cancer risk.
Keywords: global DNA methylation, pre-diagnostic, prostate cancer, repetitive element
INTRODUCTION
Global DNA hypomethylation has been observed in tumor relative to histologically normal tissue for a number of cancers [1], including prostate cancer [2]. Although usually thought to occur early in the development of cancer, some data have suggested that this epigenetic alteration may occur late in prostate cancer progression [3]. Some studies have also suggested that global DNA methylation in peripheral blood may be associated with the risk of various cancers, including bladder, breast, renal, gastric, hepatocellular, and head and neck cancer [4-11]; however, the evidence is mixed and not all studies have observed an association [12]. Few of the studies have been prospective and, to date, no previous study has evaluated global DNA methylation levels in peripheral blood in relation to the risk of prostate cancer.
Global DNA methylation can be measured in a variety of different ways [13], which may contribute to the mixed findings with cancer risk in the literature [12]. Long interspersed nuclear element 1 (LINE-1) and Alu elements are abundant transposable DNA elements located within repetitive elements in the genome. As more than one-third of DNA methylation throughout the genome is estimated to occur at repetitive elements, DNA methylation at CpG sites in these elements has been used as a proxy of global cytosine methylation [14]. In this prospective study, we aimed to evaluate the relationship between CpG site DNA methylation at LINE-1 and Alu in peripheral blood DNA and prostate cancer risk.
MATERIALS AND METHODS
Study population
We conducted a nested case-control study of prostate cancer in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial using pre-diagnostic peripheral blood samples. The PLCO Cancer Screening Trial is a randomized trial of more than 150,000 men and women ages 55 to 74 who were enrolled from 10 centers across the United States between 1993 and 2001 to evaluate the impact of specific cancer screening regimens on the risk of mortality from prostate, lung, colorectal, and ovarian cancers [15]. As described previously [15,16], men randomized to the screening arm were offered prostate-specific antigen (PSA) testing at baseline and annually for 5 years and digital rectal exam (DRE) at baseline and annually for 3 years. Men with a PSA test result >4 ng/ml or a DRE exam suspicious for prostate cancer were referred to their personal physician for follow-up. Medical and pathologic records were obtained for participants suspected to have prostate cancer based on screening examination results or self-report on annual follow-up questionnaires. The study was approved by the institutional review boards at the ten centers and the National Cancer Institute.
For the present study, only men randomized to the screening arm of the trial who provided a blood specimen, consented to participate in etiologic studies of cancer, completed a baseline questionnaire, and had no history of cancer prior to randomization were eligible. Cases were preferentially selected from among the non-Hispanic Caucasian men that participated in the Cancer Genetic Markers of Susceptibility (CGEMS) initiative [17], where men with aggressive disease were oversampled. Only pathologically confirmed cases were included, and all cases selected were diagnosed at least one year after providing the blood sample. Controls were free of prostate cancer prior to the censor date for case diagnosis, December 31, 2007. Controls were frequency-matched to cases on age at randomization (five year intervals), year of randomization, year of blood draw, and study year of diagnosis/selection. A total of 707 cases and 707 controls were selected for the study. Excluding participants who had insufficient pre-diagnostic DNA resulted in a final sample size of 694 cases and 703 controls.
Pyrosequencing assays
DNA samples were extracted using Qiagen QIAamp DNA Blood Midi/Maxi kits and shipped to EpigenDx, Inc. (Hopkinton, MA) for analysis. The DNA was bisulfite-converted using a Zymo Research EZ DNA Methylation kit and DNA methylation levels were quantified at 4 CpG sites in LINE-1 and 4 CpG sites in Alu, respectively, using pyrosequencing with assays in triplicate as described elsewhere [7]. Four artificial control samples were included on each plate [one negative control sample (no DNA added), as well as three positive control samples with known global DNA methylation levels: low (0%), partial (50%), and highly methylated (100%)]. In addition, we included 58 blind replicate samples for four of the study subjects (about 15 replicates per subject) interspersed within and between plates for quality control. Sequencing was performed using the Pyrosequencing PSQ96 HS System (Pyrosequencing Qiagen). The methylation status at each CpG site was analyzed as an artificial C/T SNP using QCpG software (Pyrosequencing Qiagen), and the percent of methylation was calculated for each CpG site as methylated cytosine divided by the sum of methylated and unmethylated cytosines. We calculated mean DNA methylation levels across the 4 CpG sites for LINE-1 and the 4 CpG sites for Alu, respectively, to estimate the overall percent methylated cytosine within each element. We excluded results that failed bisulfite control or that had poor bisulfite conversion (based on percent unconverted bisulfite>7%), as well as results that had no signal or a low signal. In addition, we excluded individual triplicate results that had a coefficient of variation (CV) greater than 10 for LINE-1 measures or a CV greater than 15 for Alu measures. The average CV and Intraclass Correlation Coefficient were 4.0 and 0.18 for the triplicate LINE-1 measures and 5.9 and 0.47 for the Alu measures, respectively. After QC exclusions, mean LINE-1 results were available for 691 cases and 700 controls and mean Alu results for 690 cases and 699 controls. The average CVs for QC replicate samples within and between plates were 2.1 and 3.5 for mean LINE-1 and 3.9 and 6.2 for mean Alu, respectively.
Statistical analysis
Using the controls to create cutpoints, we categorized the individual and mean LINE-1 and Alu DNA methylation measures into quartiles and used unconditional logistic regression models to evaluate prostate cancer risk associated with each CpG site individually and the mean across the sites, adjusting for age at blood draw (continuous), year of blood draw, family history of prostate cancer (yes/possible, no), smoking status (never, former, current, pipe/cigar), total folate intake (quartiles defined among controls), and body mass index (BMI) (<25, 25-29, 30 or higher). We selected covariates for adjustment based on an observed association with prostate cancer in the literature or in the present study. We computed p-values for trend using the median value among controls for each quartile entered as a continuous variable in the model.
We also evaluated the relationships between the DNA methylation measures and prostate cancer risk separately for aggressive (defined as Stage III/IV or Gleason score>8) and non-aggressive (Stage I/II and Gleason score<8) cases using polytomous regression. We also used polytomous regression to evaluate these relationships by time from blood draw to diagnosis for the cases (< 2 years, 2 to < 3 years, 3 to < 4 years, and 4 or more years). We computed p-values for heterogeneity across strata using Wald Chi-square tests. In addition, we evaluated potential effect modification of the associations for the mean DNA methylation measures with prostate cancer risk by age at diagnosis/selection (< 65 or ≥ 65), family history of prostate cancer, smoking status, total folate intake, and BMI, using likelihood ratio tests comparing nested models with and without the interaction terms with the DNA methylation measures.
RESULTS
In our study population, about 25% of the prostate cancer cases were aggressive. Cases were more likely to have a family history of prostate cancer than controls and tended to consume higher folate than controls and were less likely than controls to have smoked (Table I). We did not observe differences between cases and controls with respect to age at blood draw or year of blood draw (study matching factors) or BMI (Table I).
Table I.
Study population characteristics for the prostate cancer cases and controls
| Characteristic [mean±sd or n (%)] | Cases (n=694) | Controls (n=703) |
|---|---|---|
| Age at diagnosis/selectiona (years) | 68.6 ± 5.6 | 68.2 ± 5.6 |
| Age at blood drawa | 65.5 ± 5.3 | 65.4 ± 5.2 |
| Year of blood drawa | 1997.6 ± 2.2 | 1997.7 ± 2.4 |
| Family history of prostate cancer | ||
| Yes/possible | 93 (13.4%) | 43 (6.1%) |
| No | 595 (85.7%) | 654 (93.0%) |
| Missing | 6 (0.9%) | 6 (0.9%) |
| Smoking | ||
| Never | 247 (35.6%) | 171 (24.3%) |
| Former | 333 (48.0%) | 371 (52.8%) |
| Current | 52 (7.5%) | 90 (12.8%) |
| Pipe/cigar | 62 (8.9%) | 71 (10.1%) |
| Folate | ||
| Q1 | 124 (17.9%) | 168 (23.9%) |
| Q2 | 192 (27.7%) | 167 (23.8%) |
| Q3 | 186 (26.8%) | 167 (23.8%) |
| Q4 | 164 (23.6%) | 167 (23.8%) |
| Missing | 28 (4.0%) | 34 (4.8%) |
| BMI | ||
| <25 | 177 (25.5%) | 175 (24.9%) |
| 25-29 | 367 (52.9%) | 361 (51.4%) |
| ≥30 | 139 (20.0%) | 161 (22.9%) |
| Missing | 11 (1.6%) | 6 (0.9%) |
| Aggressive prostate cancerb | ||
| Yes | 172 (24.8%) | --- |
| No | 516 (74.4%) | |
| Missing | 6 (0.9%) | |
Study matching factor or combination of study matching factors
Aggressive defined as Stage III/IV or Gleason score≥8; non-aggressive defined as Stage I/II and Gleason score<8
Mean LINE-1 and Alu methylation levels, which were moderately correlated with each other (among controls, Spearman rho=0.26, p-value=1.7×10−12), were similar between cases and controls. The median value for mean LINE-1 was 74.1% for both cases and controls (overall range: 63.3%-82.9%), and the median for mean Alu was 25.7% for both groups (overall range: 20.5%-29.1%). Overall, we did not observe a significant association with prostate cancer risk for mean LINE-1 and Alu (Table II) or any of the individual CpG sites (Supplementary Table SI). For mean LINE-1, the Odds Ratio (OR) for the highest compared to the lowest quartile was 1.01, 95% Confidence Interval (CI): 0.73-1.39, ptrend=0.99, and for mean Alu, the OR was 0.94, 95% CI: 0.68-1.29, ptrend=0.69 (Table II).
Table II.
Mean LINE-1 and Alu DNA methylation levels and prostate cancer risk
| DNA methylation marker quartilesa | LINE-1 b | DNA methylation marker quartilesa | Alu b | |||
|---|---|---|---|---|---|---|
| Quartile | Range | Case/Control | OR (95% CI)c | Range | Case/Control | OR (95% CI)c |
| Q1 | 63.3-73.0 | 171/182 | REF | 20.5-24.6 | 178/175 | REF |
| Q2 | >73.0-74.1 | 188/178 | 1.16 (0.85-1.57) | >24.6-25.7 | 192/199 | 1.01 (0.74-1.37) |
| Q3 | >74.1-75.3 | 185/177 | 1.10 (0.81-1.50) | >25.7-26.3 | 169/162 | 0.96 (0.70-1.32) |
| Q4 | >75.3-82.9 | 147/163 | 1.01 (0.73-1.39) | >26.3-29.1 | 151/163 | 0.94 (0.68-1.29) |
| ptrendd | 0.99 | ptrendd | 0.69 | |||
Defined among controls
Average across positions 1-4
Adjusted for age at blood draw, year of blood draw, family history of prostate cancer (yes/possible, no), smoking status (never, former, current, pipe/cigar), total folate intake (quartiles), and BMI (<25, 25-29, 30 or higher)
Based on a variable assigned the median value (among controls) for each quartile entered as a continuous variable in the model
There was no difference by disease aggressiveness. For mean LINE-1, the OR for the highest compared to the lowest quartile was 0.95 (95% CI: 0.56-1.60, ptrend=0.95) for aggressive disease and 1.04 (95% CI: 0.73-1.49, ptrend=0.85) for non-aggressive disease. For mean Alu, the OR was 0.99 (95% CI: 0.60-1.66, ptrend=0.89) for aggressive disease and 0.93 (95% CI: 0.66-1.32, ptrend=0.75) for non-aggressive disease.
When we evaluated differences by time from blood draw to diagnosis, we observed that higher mean Alu DNA methylation levels were associated with an increased risk of prostate cancer for those diagnosed 4 or more years after blood draw (OR for the highest compared to the lowest quartile=2.26, 95% CI: 1.27-4.00, ptrend=4.4×10−3) (Table III). This association became stronger for those diagnosed 5 or more years after draw (case n=103), with an OR for the highest compared to the lowest quartile of 4.91 (95% CI: 2.25-10.69, ptrend=5.1×10−5). In contrast, no association was observed for those diagnosed 2 (OR=1.13, 95% CI: 0.67-1.90, ptrend=0.64) or 3 years after draw (OR=1.22, 95% CI: 0.71-2.07, ptrend=0.32), and a decreased risk was observed for those diagnosed less than 2 years after draw (OR=0.40, 95% CI: 0.25-0.65, ptrend=3.8×10−5; pheterogeneity=5.3×10−6) (Table III). No association was observed with mean LINE-1 regardless of the time from blood draw to diagnosis (Table III). Results for the individual LINE-1 and Alu positions by time from blood draw to diagnosis (Supplementary Tables SII and SIII) were similar to the results observed for mean LINE-1 and Alu.
Table III.
Mean LINE-1 and Alu DNA methylation levels and prostate cancer risk by time from blood draw to diagnosis for the cases
| Controlsa | Cases | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| DNA methylation marker quartilesb | < 2 yearsc | 2 to < 3 years | 3 to <4 years | ≥ 4 years | pheterogeneityd,e | ||||||
| LINE-1 f | |||||||||||
| Quartile | Range | n | n | OR (95% CI)e | n | OR (95% CI)e | n | OR (95% CI)e | n | OR (95% CI)e | |
| Q1 | 63.3-73.0 | 182 | 53 | REF | 41 | REF | 38 | REF | 39 | REF | |
| Q2 | >73.0-74.1 | 178 | 39 | 0.75 (0.46-1.20) | 50 | 1.22 (0.75-1.98) | 60 | 1.83 (1.13-2.98) | 39 | 1.07 (0.64-1.79) | |
| Q3 | >74.1-75.3 | 177 | 72 | 1.30 (0.84-2.00) | 41 | 0.98 (0.59-1.63) | 37 | 1.06 (0.62-1.80) | 35 | 1.00 (0.59-1.68) | |
| Q4 | >75.3-82.9 | 163 | 54 | 1.04 (0.66-1.64) | 32 | 0.91 (0.53-1.55) | 26 | 0.88 (0.49-1.57) | 35 | 1.18 (0.70-1.99) | 0.83 |
| ptrendg | 0.43 | 0.60 | 0.40 | 0.62 | |||||||
| Alu f | |||||||||||
| Q1 | 20.5-24.6 | 175 | 82 | REF | 40 | REF | 35 | REF | 21 | REF | |
| Q2 | >24.6-25.7 | 199 | 65 | 0.71 (0.47-1.06) | 46 | 1.13 (0.69-1.86) | 42 | 1.15 (0.68-1.94) | 39 | 1.80 (1.01-3.21) | |
| Q3 | >25.7-26.3 | 162 | 41 | 0.50 (0.32-0.78) | 39 | 1.08 (0.65-1.81) | 48 | 1.39 (0.83-2.35) | 41 | 1.90 (1.06-3.41) | |
| Q4 | >26.3-29.1 | 163 | 31 | 0.40 (0.25-0.65) | 39 | 1.13 (0.67-1.90) | 37 | 1.22 (0.71-2.07) | 44 | 2.26 (1.27-4.00) | 5.3×10−6 |
| ptrendg | 3.8×10−5 | 0.64 | 0.32 | 4.4×10−3 | |||||||
Comparison group
Defined among controls
Prostate cancer cases diagnosed less than one year after blood draw were excluded in the study design
P-value for heterogeneity from Wald Chi-square test comparing effects of Q4 (vs. Q1) on the four case groups relative to controls
Adjusted for age at blood draw, year of blood draw, family history of prostate cancer (yes/possible, no), smoking status (never, former, current, pipe/cigar), total folate intake (quartiles), and BMI (<25, 25-29, 30 or higher)
Average across positions 1-4
Based on a variable assigned the median value (among controls) for each quartile entered as a continuous variable in the model
Following up on our findings for Alu, we further evaluated the role of timing of DNA methylation levels relative to diagnosis by conducting a case-only analysis. Among the prostate cancer cases in our study, we observed a significant positive relationship between time from blood draw to diagnosis and mean Alu DNA methylation levels (Spearman rho=0.23, p-value=9.6×10−10), corresponding to higher DNA methylation levels for measurements taken further in time before diagnosis (or, equivalently, lower DNA methylation levels approaching the time of diagnosis). To investigate whether age could have contributed to our findings for Alu by time from blood draw to diagnosis, we also computed the correlation between mean Alu DNA methylation levels and age at blood draw. We did not observe a significant association among either cases or controls (Spearman rho=−0.03 and −0.05, p=0.47 and 0.21, respectively).
We did not observe significant differences (p-interaction<0.05) in the association between mean LINE-1 or Alu DNA methylation levels and prostate cancer risk by age at diagnosis/selection, family history of prostate cancer, smoking status, folate intake, or BMI (data not shown).
DISCUSSION
This is the first study to evaluate the relationship between DNA methylation at LINE-1 and Alu in peripheral blood and prostate cancer risk. We did not observe a significant association with prostate cancer risk for either element overall. However, for Alu, we observed an increased risk of prostate cancer associated with increasing methylation levels (i.e. hypermethylation among cases) for those diagnosed 4 or more years after draw. Notably, the association for Alu tended to further increase with increasing time between blood draw and diagnosis, although estimates were less precise due to smaller case numbers. The lack of association to protective association (i.e. hypomethylation among cases) for Alu methylation levels with blood draws closer to diagnosis and our finding of an association between time from blood draw to diagnosis and Alu methylation levels among the cases suggest that DNA methylation patterns may change over time as the cancer begins to develop.
It is widely held that global DNA hypomethylation may be associated with an increased cancer risk (and thus global hypermethylation associated with a decreased risk) [1] and we recognize that our findings for Alu for those diagnosed 4 or more years after draw are contrary to this hypothesis. However, previous studies that have observed increased cancer risk associated with global hypomethylation in blood (i.e. decreased risk associated with global hypermethylation) have largely used post-diagnostic samples [12] as opposed to pre-diagnostic samples as in our study. The few prospective studies of global DNA methylation that have been conducted to date (i.e. for bladder and renal cancers), although Alu was not evaluated specifically, have tended to observe an increased risk of cancer associated with global hypermethylation [18,19]. The mechanism by which global DNA hypermethylation may increase cancer risk is unclear; however, it has been hypothesized that cells with higher methylation levels may be longer-lived and thus, combined with carcinogen exposure, this methylation pattern may favor clonal expansion of damaged cells [4].
Interestingly, similar contrasting associations to those we observed in the present study by time from blood draw to diagnosis have been reported for pre-diagnostic and post-diagnostic blood samples in studies of global DNA methylation and bladder cancer [4,10,18,20]. In these studies, hypermethylation was observed among cases with pre-diagnostic samples (relative to controls) as opposed to hypomethylation among cases with post-diagnostic samples. Although we excluded prostate cancer cases diagnosed within one year of blood draw, it is likely that those diagnosed less than 2 years after blood draw in our study may have already had prostate cancer at the time of blood draw. Thus, the observed association for Alu for this group in our study may reflect changes in DNA methylation due to the disease process. Previous studies have shown that DNA methylation may also change with age [21], which could potentially contribute to findings by time from draw to diagnosis; however, Alu DNA methylation levels were not associated with age in our study and therefore we do not expect that our findings were driven by an age effect.
While some previous studies evaluating cancer risk in relation to DNA methylation at repetitive elements in peripheral blood have reported similar results for both LINE-1 and Alu [22,24,25], others have found different associations for the two elements [5,23]. Zhu et al reported an association with the risk of all cancers combined for LINE-1 DNA methylation levels, but not for Alu [23]. Similar to our study, a prospective study of gastric cancer by Gao et al observed an association for Alu DNA methylation, but not for LINE-1 DNA methylation [5]. Although both measures have been used as proxies for global DNA methylation levels in the literature, the two measures were not highly correlated in our study, as has also been reported previously in blood [22]. Previous research has also observed different transcription patterns for LINE-1 and Alu in response to cellular stressors [26], suggesting that these elements may have different functional roles.
As we limited our study population to non-Hispanic Caucasians, our study may not be generalizable to other populations. We also acknowledge that DNA methylation levels in blood may not reflect levels in tissue; however, there is growing evidence that blood DNA methylation levels may serve as independent predictors of cancer risk for a variety of solid tumors [12]. We had limited numbers for our stratified analyses by disease aggressiveness and time from blood draw to diagnosis, necessitating replication in future studies. Our ability to evaluate time from blood draw to diagnosis was also limited by the diagnosis of most cases in our study within several years of blood draw (75th percentile=3.7 years), although there was a large range (1-13.9 years). In addition, while it is possible that our findings for Alu may have been driven by changes in DNA methylation levels among the cases over time, we did not evaluate within-individual temporal variability using serial blood samples to directly address this question.
Strengths of our study included the use of pyrosequencing to measure DNA methylation levels as this is a high-throughput, quantitative method that is considered highly sensitive to detect differences in DNA methylation between individuals. In addition, the large, prospective design of the study and our ability to separately evaluate the association between blood DNA methylation measures and prostate cancer risk among those diagnosed several years after providing the blood sample was a further strength.
CONCLUSIONS
While LINE-1 DNA methylation levels in peripheral blood were not associated with prostate cancer risk in our study, we observed an association for Alu DNA methylation levels that varied by time from blood draw to diagnosis. Although replication is needed, our study suggests that elevated blood DNA methylation levels at Alu several years prior to diagnosis may be associated with an increased risk of prostate cancer.
Supplementary Material
SYNOPSIS.
There is growing evidence for a number of cancers that global DNA methylation levels in peripheral blood may be associated with risk. However, few studies have been prospective and, to date, no previous study has evaluated global DNA methylation levels in peripheral blood in relation to the risk of prostate cancer. Using a large, prospective design, we conducted the first study to evaluate blood DNA methylation levels at the LINE-1 and Alu repetitive elements, which are commonly used as proxies of global DNA methylation, in relation to the risk of prostate cancer. While LINE-1 DNA methylation levels were not associated with prostate cancer risk, we observed an association for Alu DNA methylation levels that varied by time from blood draw to diagnosis. Although requiring replication, our findings suggest that elevated Alu DNA methylation levels in peripheral blood DNA several years before diagnosis may be associated with an increased risk of prostate cancer.
ACKNOWLEDGMENTS
This research was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health. The authors thank Drs. Christine Berg and Philip Prorok, Division of Cancer Prevention at the National Cancer Institute, the screening center investigators and staff of the PLCO Cancer Screening Trial, Mr. Thomas Riley, Mr. Craig Williams, Mr. Matthew Moore, and Mr. Michael Furr at Information Management Services, Inc., and Ms. Barbara O'Brien and staff at Westat, Inc. for their contributions to the PLCO Cancer Screening Trial. We also thank the PLCO study participants for their contributions to making this study possible.
Footnotes
Disclosure Statement: M. Poulin is employed by EpigenDx, Inc. The other authors do not have any potential conflicts of interest to disclose.
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