Abstract
Purpose:
Evidence suggests that mitochondrial DNA (mtDNA) copy number increases in response to DNA damage. Increased mtDNA copy number has been observed in prostate cancer (PCa) cells, suggesting a role in PCa development, but this association has not yet been investigated prospectively.
Methods:
We conducted a nested case-control study (793 cases and 790 controls) of men randomized to the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) to evaluate the association between pre-diagnosis mtDNA copy number, measured in peripheral blood leukocytes, and the risk of PCa. We used logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) and polytomous logistic regression to analyze differences in associations by non-aggressive (Stage I/II AND Gleason grade <8) or aggressive (Stage III/IV OR Gleason grade ≥8) PCa.
Results:
Although mtDNA copy number was not significantly associated with PCa risk overall (OR = 1.23, 95% CI 0.97–1.55, p = 0.089), increasing mtDNA copy number was associated with an increased risk of non-aggressive PCa (OR = 1.29, 95% CI = 1.01–1.65, p = 0.044) compared to controls. No association was observed with aggressive PCa (OR = 1.02, 95% CI = 0.64–1.63, p = 0.933). Higher mtDNA copy number was also associated with increased PSA levels among controls (p=0.014).
Conclusions:
These results suggest that alterations in mtDNA copy number may reflect disruption of the normal prostate glandular architecture seen in early stage disease, as opposed to reflecting the large number of tumor cells seen with advanced PCa.
Keywords: mitochondrial DNA, prostate cancer, prospective, risk factors, copy number, genetics
Introduction
Mitochondria have been implicated in the development of cancer because of their central roles in energy production and apoptosis. Mitochondrial DNA (mtDNA) consists of 16 kilobases of circular double-stranded DNA, encoding 37 genes, 22 mitochondrial transfer RNAs and two ribosomal RNAs [1]. The number of mtDNA copies in a single cell ranges from several hundred to several thousand [2]. Despite the variability across cell types, the copy number is partially heritable and correlated between cell types [2]. There is substantial inter-individual variation in tissue-specific mtDNA copy number in the general population [3] and copy number in peripheral leukocytes reportedly declines with advanced age [4]. As mtDNA lacks protective histones and has diminished repair capacity compared to nuclear DNA, it is more susceptible to reactive oxygen species and other sources of DNA damage [5]. Evidence suggests that mtDNA copy number increases with exposure to such DNA damage [6–9]. Perturbations in mtDNA copy number may therefore serve as markers of DNA damage and/or disease development.
Epidemiologic studies have observed significant associations between elevated mtDNA copy number and increased risk of glioma [10], lung cancer[11], and breast cancer [12] in retrospective analyses, and lung cancer [13], non-Hodgkin lymphoma [14], kidney cancer [15], and pancreatic cancer [16] in prospective analyses, though other retrospective studies report significant associations between decreased mtDNA copy number and increased renal cancer [2, 17], breast cancer [18, 19], and soft tissue sarcoma [20]. In a small, retrospective, hospital-based case-control study of prostate cancer and peripheral leukocyte mtDNA copy number, Zhou, et al., reported a positive association between mtDNA copy number and risk of prostate cancer (PCa) [21]. Among cases, increased mtDNA copy number was also associated with high Gleason score and advanced tumor stage, though not prostate-specific antigen (PSA) at diagnosis [21]. However, as blood specimens were collected after diagnosis, the study is vulnerable to reverse causation and the associations observed may be the result of the disease process.
To evaluate the risk of PCa in association with mtDNA copy number prospectively, we conducted a nested case-control study within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial.
Methods
Study Population
The current study was nested within the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO), whose enrollment and specimen collection procedures have been described [22, 23]. Briefly, approximate 150,000 individuals between the ages of 55 and 74 were enrolled at ten study centers in the USA between 1993 and 2001. Male participants randomized to the screening arm of the trial received screening for PCa with PSA annually for six years and digital rectal exam annually for four years. Non-fasting blood specimens were collected at each visit, and samples were processed and frozen within two hours of collection and stored at −70C. Participants also completed a risk factor questionnaire at baseline and received annual questionnaires to follow-up for all cancer diagnoses. Medical records were obtained and abstracted to confirm all reported cancers. The trial was approved by institutional review boards at the National Cancer Institute and the ten study centers; all participants provided written informed consent.
Cases (n=800) and controls (n=800) for the current analysis were selected from among non-Hispanic Caucasian men who were previously included in the Prostate cancer Genome-wide Association Study of Uncommon Susceptibility loci (PEGASUS) study [24]. Cases and controls were limited to those randomized to the screening arm of the trial without a previous diagnosis of cancer. Controls were frequency-matched to cases in a ratio of 1:1 on age at randomization, fiscal year of PLCO randomization, and year of screening blood draw. Cases were required to have a pre-diagnostic blood sample available at least one year prior to diagnosis, and those diagnosed with PCa during the first year of the study were excluded as likely prevalent cases.
Mitochondrial DNA Copy Number Measurement
DNA was extracted from peripheral blood leukocytes. Measurements of relative mtDNA copy number were performed in triplicate using a fluorescence-based quantitative PCR assay comparing the ratio of the mitochondrial gene ND1 to the nuclear β-globin gene HBB. The ratio of ND1 to HBB threshold cycle numbers, estimated using a linear regression model, is proportional to the mtDNA copy number in each cell. The average of the triplicate measurements was used to characterize mtDNA copy number for this study, with any single measurement differing by more than 10% of the average of the other two discarded. Quality control was assured by interspersing blinded replicate samples throughout batches. The overall coefficient of variation was 7.54%.
Statistical Analysis
Cases and controls were excluded from further analysis if they had insufficient DNA or missing values for mtDNA copy number (n=14), or if they had mtDNA copy number values that were found to be both outliers and influential points (n = 3), leaving 793 cases and 790 controls for analysis.
Bivariate comparisons between cases and controls were done using the χ2 test for categorical variables and the Wilcoxon rank-sum test for continuous variables. We used logistic regression to estimate odds ratios and 95% confidence intervals for the association between PCa and mtDNA copy number modeled as a natural log-transformed continuous variable and in quartiles determined by the distribution of mtDNA copy number among controls. Models were adjusted for matching variables (age at diagnosis/selection and year of screening blood draw, both continuous). Possible confounders, such as body mass index (BMI, kg/m2), educational attainment (≤ 11 years, 12 years/completed high school, vocational tech/some college, college graduate/postgraduate), cigarette smoking status (never, former, current), family history of PCa (yes or no), study center, fruit consumption (g/day), vegetable consumption (g/day), and multivitamin usage (yes or no) were evaluated, but none changed the beta estimate for mtDNA copy number more than 10% and so were not included in the final model. We also performed a conditional logistic regression and results were similar to the unconditional logistic regression adjusted for matching factors, so we chose to present results from the unconditional logistic regression in order to maximize sample size.
We conducted polytomous logistic regression analyses to examine differences in association by stage (I/II vs. III/IV), Gleason score (Gleason < 8 vs. Gleason ≥8), and disease aggressiveness (stage I/II and Gleason < 8 vs. Stage III/IV or Gleason ≥8). To evaluate effect modification, we conducted analyses with multiplicative interaction terms for risk factors with biological plausibility for affecting mtDNA copy number: smoking status (former, never, or current), family history of prostate cancer (yes vs no), multivitamin consumption (yes vs no), and fruit and vegetable consumption (above vs below median). Interactions were evaluated using a likelihood ratio test of the interaction term.
To examine the impact of elapsed time from mtDNA blood draw to prostate cancer diagnosis, we stratified cases by the number of years between the two events (≤ 2, 2–3, 3–4, and >4) and also by disease aggressiveness. Due to small numbers of aggressive PCa cases, the strata of 2–3 years and 3–4 years between blood draw and diagnosis were combined.
We analyzed the relationship between PSA levels and mtDNA copy number among cases and controls separately using linear regression with robust standard errors. We evaluated PSA level taken concurrently with the blood draw used for the assessment of mtDNA copy number for both cases and controls and PSA level measured closest to diagnosis (but within at least one year) for cases. Quartile cutpoints of mtDNA copy number were calculated separately for each analysis.
Statistical analyses were performed using Stata v13.0 (StataCorp LP, College Station, TX).
Results
Cases and controls were comparable in terms of age, BMI, smoking status, education, and self-reported consumption of fruit, vegetables, and multivitamins (Table 1). Cases were more likely than controls to report a family history of PCa (p = 0.02), and the median PSA at blood draw was higher in cases than in controls (p < 0.0001). Among cases, 15% had aggressive PCa, defined as either Gleason score ≥ 8 or Stage III or IV disease.
Table 1.
Study participant characteristics
| Cases (n = 793) | Controls (n = 790) | p-value | ||||
|---|---|---|---|---|---|---|
| Age at diagnosis/selection (n, %) | 55–59 | 25 | 3.2 | 32 | 4.1 | 0.1990 |
| 60–64 | 129 | 16.3 | 140 | 17.7 | ||
| 65–69 | 262 | 33.0 | 224 | 28.4 | ||
| 70–74 | 377 | 47.5 | 394 | 49.9 | ||
| Year of blood collection (n. %) | 1994–1996 | 221 | 27.9 | 220 | 27.9 | 0.6090 |
| 1997–2000 | 400 | 50.4 | 383 | 48.5 | ||
| 2001–2005 | 172 | 21.7 | 187 | 23.7 | ||
| Study Center (n, %) | University of Colorado | 78 | 9.8 | 86 | 10.9 | 0.5760 |
| Georgetown University | 78 | 9.8 | 62 | 7.9 | ||
| Henry Ford Health System | 58 | 7.3 | 46 | 5.8 | ||
| University of Minnesota | 234 | 29.5 | 240 | 30.4 | ||
| Washington University in St. Louis | 79 | 10.0 | 72 | 9.1 | ||
| University of Pittsburgh | 71 | 9.0 | 78 | 9.9 | ||
| University of Utah | 77 | 9.7 | 68 | 8.6 | ||
| Marshfield Clinic Research Foundation | 104 | 13.1 | 124 | 15.7 | ||
| University of Alabama at Birmingham | 14 | 1.8 | 14 | 1.8 | ||
| BMI, kg/m2 (n, %) | < 25 | 205 | 26.4 | 194 | 24.8 | 0.4480 |
| 25–30 | 408 | 52.4 | 402 | 51.3 | ||
| > 30 | 165 | 21.2 | 187 | 23.9 | ||
| Smoking Status (n, %) | Never | 341 | 43.0 | 307 | 38.9 | 0.2420 |
| Current | 73 | 9.2 | 76 | 9.6 | ||
| Former | 379 | 47.8 | 407 | 51.5 | ||
| Education (n, %) | ≤ 11 years | 60 | 7.6 | 68 | 8.6 | 0.2820 |
| 12 years or completed HS | 135 | 17.1 | 141 | 17.9 | ||
| Voc tech or some college | 235 | 29.7 | 258 | 32.7 | ||
| College grad or postgrad | 361 | 45.6 | 323 | 40.9 | ||
| Took Multi-vitamins | No | 441 | 55.6 | 461 | 38.4 | 0.2700 |
| Yes | 352 | 44.4 | 329 | 41.7 | ||
| Fruit Consumption, g/day (median, IQR) | 175 | 96–272 | 173 | 95–274 | 0.8978 | |
| Vegetable Consumption, g/day (median, IQR) | 435 | 301–587 | 438 | 310–592 | 0.5585 | |
| Family History of Prostate Cancer (n, %) | No | 690 | 88.0 | 722 | 92.2 | 0.0200 |
| Yes | 78 | 10.0 | 50 | 6.4 | ||
| Missing | 16 | 2.0 | 11 | 1.4 | ||
| PSA Level at Blood Draw, ng/mL (median, IQR) | 3.19 | 2.19–4.62 | 1.1 | 0.62–1.89 | < 0.0001 | |
| Time from Blood Draw to Selection/Diagnosis (median, IQR) | < 2yrs | 203 | 25.6 | 180 | 22.8 | |
| 2–3 years | 175 | 22.1 | 178 | 22.5 | ||
| 3–4yrs | 140 | 17.7 | 151 | 19.1 | ||
| > 4yrs | 275 | 34.7 | 281 | 35.6 | 0.5970 | |
| Prostate Cancer | ||||||
| Gleason Score (n, %) | 3 | 5 | 0.6 | |||
| 4 | 28 | 3.5 | ||||
| 5 | 64 | 8.1 | ||||
| 6 | 399 | 50.3 | ||||
| 7 | 215 | 27.1 | ||||
| 8 | 35 | 4.4 | ||||
| 9 | 34 | 4.3 | ||||
| 10 | 4 | 0.5 | ||||
| Missing | 9 | 1.1 | ||||
| Stage of Prostate Cancer (n, %) | I | 4 | 0.5 | |||
| II | 712 | 89.8 | ||||
| III | 57 | 7.2 | ||||
| IV | 20 | 2.5 | ||||
| Aggressive Prostate Cancer (n, %) | No | 669 | 85.0 | |||
| Yes | 118 | 15.0 | ||||
| Missing | 6 | 0.0 | ||||
Aggressive prostate cancer defined as Stage III or IV disease, or Gleason ≥ 8
P-values for categorical variables calculated using chi-squared test, for continuous variables using Wilcoxon Rank-Sum test.
Risk of Prostate Cancer by Mitochondrial DNA Copy Number
Overall, the median mtDNA copy number did not differ significantly between cases and controls (94 vs 93, p = 0.1296). After adjustment for age and year of blood draw, we observed a marginal but nonsignificant association between mtDNA copy number and increased risk of prostate cancer (OR = 1.23, 95% CI 0.97–1.55, p = 0.089) (Table 2). However, when the results were stratified by disease aggressiveness, a positive association was found with increasing mtDNA copy number for non-aggressive disease (OR = 1.29, 95% CI = 1.01–1.65, p = 0.044) but not aggressive PCa (OR = 1.02, 95% CI = 0.64–1.63, p = 0.933), though a Wald test for heterogeneity of the coefficients for mtDNA copy number was not statistically significant (p = 0.334). As men with a Gleason score of 4+3 may be considered to have aggressive disease, but we did not have information on in the individual components of Gleason score, we removed men with a Gleason score of 7 from our definition of nonaggressive disease in a sensitivity analysis. Refining our definition of non-aggressive disease to Stage I/II and Gleason < 7 strengthened the association between mtDNA copy number and nonaggressive PCa risk (OR=1.52, 95% CI = 1.16–2.00, p = 0.003). Similarly, polytomous logistic regression analysis of the risk of PCa by Gleason score (≤6, 7, or ≥8) and by stage (I/II vs III/IV), respectively, showed positive associations between mtDNA number and lower Gleason score PCa (OR for PCa with Gleason ≤6 = 1.51, 95% CI = 1.15–1.98, p = 0.003) but not with higher Gleason score (OR = 0.88, p = 0.483 for Gleason 7, OR =1.00, p = 0.998 for Gleason ≥8) nor higher stage (OR = 0.92, p = 0.755) disease (Table 2).
Table 2.
Risk of prostate cancer by quartiles and continuous measure of mtDNA copy number.
| Quartile 1 | Quartile 2 | Quartile 3 | Quartile 4 | Continuous (log transformed) | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| mtDNA copy number range | < 70 | 70 – 92 | 93 – 113 | > 113 | ||||||
| no. cases/no. controls | OR** | no. cases/no. controls | OR (95% CI) | no. cases/no. controls | OR (95% CI) | no. cases/no. controls | OR (95% CI) | OR (95% CI) | p-value | |
| All cases | 172/190 | 1.0 | 213/205 | 1.15 (0.87–1.53) | 180/189 | 1.06 (0.79–1.41) | 228/206 | 1.23 (0.93–1.62) | 1.23 (0.97–1.55) | 0.089 |
| Non-Aggressive | 150/190 | 1.0 | 171/205 | 1.06 (0.79–1.43) | 153/189 | 1.03 (0.76–1.40) | 201/206 | 1.25 (0.94–1.68) | 1.29 (1.01–1.65) | 0.044 |
| Aggressive | 24/190 | 1.0 | 42/205 | 1.84 (1.05–3.20) | 30/189 | 1.17 (0.64–2.13) | 28/206 | 1.13 (0.62–2.05) | 1.02 (0.64–1.63) | 0.933 |
| Stage | ||||||||||
| I/II | 157/190 | 1.0 | 183/205 | 1.08 (0.81–1.45) | 189/165 | 1.07 (0.79–1.44) | 211/206 | 1.25 (0.94–1.66) | 1.26 (0.99–1.61) | 0.057 |
| III/IV | 15/190 | 1.0 | 30/205 | 1.85 (0.96–3.55) | 15/189 | 0.94 (0.45–1.99) | 17/206 | 1.01 (0.49–2.09) | 0.92 (0.53–1.59) | 0.755 |
| Gleason Score | ||||||||||
| ≤ 6 | 97/190 | 1.0 | 134/205 | 1.25 (0.90–1.74) | 114/189 | 1.21 (0.86–1.70) | 151/206 | 1.45 (1.05–2.01) | 1.51 (1.15–1.98) | 0.003 |
| 7 | 58/190 | 1.0 | 51/205 | 0.83 (0.54–1.27) | 47/189 | 0.79 (0.51–1.22) | 59/206 | 0.93 (0.61–1.40) | 0.88 (0.62–1.26) | 0.483 |
| ≥ 8 | 14/190 | 1.0 | 27/205 | 1.91 (0.97–3.78) | 16/189 | 1.08 (0.51–2.28) | 16/206 | 1.06 (0.50–2.25) | 1.00 (0.56–1.79) | 0.998 |
All models are adjusted for age at diagnosis and year of blood draw. OR for all cases was estimated using logistic regression; ORs for subgroups estimated using polytomous regression.
Aggressive PCa defined as either Stage III or IV disease, or Gleason score ≥ 8 at diagnosis.
Reference category
We explored whether other risk factors, such as family history, smoking status, and fruit and vegetable intake, modified the association between mitochondrial DNA copy number and prostate cancer risk, but we failed to find significant associations that would withstand multiple testing (Supplementary Table 1).
Association Between Mitochondrial DNA Copy Number and PSA
Given the positive association with the risk of non-aggressive disease, we examined the association between mtDNA copy number and PSA levels separately among cases and controls (Table 3). Among controls, higher mitochondrial DNA copy number was associated with an increased PSA level (p = 0.014) when measured cross-sectionally at time of blood draw. No significant associations were observed with either PSA at blood draw or diagnosis among cases; however, modeling mtDNA copy number as quartiles demonstrates that the association between mtDNA copy number and PSA among cases may not be strictly linear.
Table 3.
Linear associations between PSA level and mtDNA copy number
| Quartile 2 vs Quartile 1 | Quartile 3 vs Quartile 1 | Quartile 4 vs Quartile 1 | continuous mtDNA CN | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Among Controls | n | Beta | SE | P | Beta | SE | P | Beta | SE | P | Beta | SE | P |
| PSA level at draw | 790 | 0.11 | 0.08 | 0.177 | 0.14 | 0.08 | 0.088 | 0.18 | 0.09 | 0.039 | 0.17 | 0.07 | 0.014 |
| Among All Cases | |||||||||||||
| PSA level at draw | 793 | 0.02 | 0.06 | 0.720 | 0.08 | 0.06 | 0.223 | 0.07 | 0.06 | 0.276 | 0.08 | 0.06 | 0.239 |
| PSA level at diagnosis | 745 | −0.03 | 0.07 | 0.683 | 0.02 | 0.08 | 0.786 | −0.06 | 0.07 | 0.439 | −0.05 | 0.07 | 0.507 |
| Among Aggressive Cases | |||||||||||||
| PSA level at draw | 117 | −0.17 | 0.18 | 0.347 | 0.03 | 0.18 | 0.878 | 0.21 | 0.19 | 0.273 | 0.10 | 0.21 | 0.635 |
| PSA level at diagnosis | 103 | −0.37 | 0.34 | 0.284 | −0.50 | 0.33 | 0.134 | −0.20 | 0.31 | 0.519 | −0.32 | 0.33 | 0.332 |
| Among Non-Aggressive Cases | |||||||||||||
| PSA level at draw | 666 | 0.06 | 0.06 | 0.315 | 0.11 | 0.06 | 0.069 | 0.07 | 0.07 | 0.272 | 0.06 | 0.05 | 0.247 |
| PSA level at diagnosis | 636 | −0.03 | 0.07 | 0.635 | 0.06 | 0.07 | 0.374 | −0.05 | 0.07 | 0.460 | −0.06 | 0.06 | 0.358 |
Both PSA and mitochondrial DNA copy number natural log-transformed.
Among controls, adjusted for age at mtDNA copy number blood draw and cigarette smoking.
Among cases, adjusted for age at mtDNA copy number blood draw, days from draw to diagnosis, and cigarette smoking.
Aggressive cases models excluded one individual as an influential point
To further explore the association between mitochondrial DNA copy number and PSA and the risk of non-aggressive disease, we stratified the non-aggressive cases by PSA at diagnosis (<4.0ng/mL vs ≥4.0ng/mL). Increasing mitochondrial DNA copy number was associated with an increased risk of non-aggressive prostate cancer with high (≥ 4.0ng/mL) PSA at diagnosis (OR = 1.32, 95% CI = 1.02–1.72, p = 0.037), but not low PSA at diagnosis (OR = 1.16, 95% CI = 0.74–1.81, p = 0.527). There were no associations in either PSA strata for risk of aggressive PCa (Table 4).
Table 4.
Analyses of PCa risk and mtDNA copy number with cases stratified by PSA level at diagnosis.
| Quartile 1 | Quartile 2 | Quartile 3 | Quartile 4 | Continuous | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| mtDNA copy number range | < 70 | 70 – 92 | 93 – 113 | > 113 | ||||||
| Overall | Ref | OR (95% CI) | p-value | OR (95% CI) | p-value | OR (95% CI) | p-value | OR | 95% CI | p-value |
| High PSA at diagnosis (≥ 4.0) | 1.0 | 1.16 (0.86–1.57) | 0.318 | 1.12 (0.83–1.52) | 0.459 | 1.25 (0.93–1.68) | 0.144 | 1.26 | 0.98–1.63 | 0.068 |
| Low PSA at diagnosis (< 4.0) | 1.0 | 1.09 (0.66–1.79) | 0.734 | 0.80 (0.46–1.39) | 0.424 | 1.15 (0.70–1.88) | 0.586 | 1.11 | 0.73–1.67 | 0.632 |
| Non-Aggressive Pca | ||||||||||
| High PSA at diagnosis (≥ 4.0) | 1.0 | 1.07 (0.78–1.46) | 0.692 | 1.07 (0.78–1.48) | 0.672 | 1.27 (0.94–1.73) | 0.121 | 1.32 | 1.02–1.72 | 0.037 |
| Low PSA at diagnosis (< 4.0) | 1.0 | 1.04 (0.60–1.79) | 0.892 | 0.87 (0.48–1.57) | 0.64 | 1.17 (0.68–2.00) | 0.573 | 1.16 | 0.74–1.81 | 0.527 |
| Aggressive Pca | ||||||||||
| High PSA at diagnosis (≥ 4.0) | 1.0 | 1.85 (0.99–3.44) | 0.053 | 1.26 (0.65–2.45) | 0.487 | 1.07 (0.54–2.10) | 0.845 | 0.93 | 0.55–1.58 | 0.787 |
| Low PSA at diagnosis (< 4.0) | 1.0 | 1.51 (0.48–4.72) | 0.476 | 0.67 (0.13–2.42) | 0.444 | 1.08 (0.32–3.60) | 0.903 | 1.18 | 0.42–3.37 | 0.752 |
mtDNA copy number natural log-transformed.
Adjusted for age at diagnosis and year of blood draw.
All controls included in each analysis
No differences were observed in the risk of PCa with mitochondrial DNA copy number when cases were stratified from time between blood draw and diagnosis (Supplementary Table 2), either overall or also stratified by PCa aggression.
Discussion
In this prospective, nested case-control study of mtDNA copy number and PCa, although we did not observe a significant association between increased mtDNA copy number and risk of PCa overall, we did find a positive association with non-aggressive PCa compared to controls, which may be partly driven by PSA. Linear regression analysis of mtDNA copy number and PSA level among controls revealed a significant positive association (β = 0.17, SE = 0.07, p = 0.014).
In a smaller, retrospective case-control study of mtDNA copy number and PCa risk in China, Zhou, et al., also reported a significant positive association between mtDNA copy number and risk of PCa (OR above vs below median = 1.85, 95% CI = 1.21–2.83) and no association with PSA at diagnosis among cases. In contrast to our study, Zhou et al, reported positive associations between mtDNA copy number and high Gleason score (p = 0.002) and advanced tumor stage (p = 0.012)[21]; however, these differences in observed results between the two studies may be due in part to differences in study design and populations. Unlike our prospective study, which was nested within a population-based prostate cancer screening trial and utilized pre-diagnostic peripheral blood specimens, the study by Zhou et al was a retrospective, hospital-based case-control study with blood specimens collected after diagnosis for cases. In addition, more than half the men in the study by Zhou et al. had advanced stage (III/IV) disease, presumably due to the lower rate of PSA screening in China [25]. In contrast, the majority of men in our study had low stage (I/II) disease. It should be noted as well that Zhou et al, restricted the controls to men with PSA <4.0ng/mL and a normal digital rectal exam. When we restricted our controls to those with PSA <4.0ng/mL at the time of mtDNA blood draw and a normal digital rectal exam, both the overall (OR = 1.24, 95% CI = 0.91–1.71, p = 0.164) and non-aggressive (OR = 1.31, 95% CI = 0.95–1.81, p = 0.101) associations remained similar to our main findings, but we still failed to see a strong association with aggressive PCa (OR = 1.06, 95% CI = 0.63–1.78, p = 0.821).
MtDNA copy number in circulating leukocytes is increasingly used as a biomarker of oxidative stress [16, 21, 26–28]. For example, polycyclic aromatic hydrocarbons (PAH) have a greater affinity for binding to mtDNA than for nuclear DNA [29], with PAH exposure resulting in a compensatory increase in mtDNA copy number [30, 31]. Low-level occupational benzene exposure is correlated with increased mtDNA copy number [32], and exfoliated saliva cells from current and former smokers show persistent elevations in mtDNA copy number compared to never smokers [7, 33]. Oxidative stress is thought to contribute to the early development of PCa [34]. In light of our results showing increased mtDNA copy number in cases diagnosed with non-aggressive PCa and no association with aggressive PCa, it is possible that elevated mtDNA copy number, hypothesized to reflect increased oxidative stress, may only play a role in the development of non-aggressive or early stage PCa. Studies have shown that antioxidants can reduce oxidative stress in leukocytes and PCa cells, leading to a reduction in PSA levels [35]. Consistent with this, we observed a positive association between mtDNA copy number and PSA levels in controls. Alternatively, the lack of association with aggressive PCa may be the result of a reduced capacity to respond to the damage caused by oxidative stress via a compensatory increase in mtDNA copy number, leading to an aggressive disease phenotype. A decrease in mtDNA copy number is consistent with a reversion to a de-differentiated state and higher cellular proliferation [36]. Finally, the lack of association with aggressive disease may be due to limited statistical power resulting from the small number of aggressive PCa cases in our U.S. screening cohort.
This study has several strengths and weaknesses. Not only is it the largest case-control study of mtDNA copy number and PCa to date, but it is also the first prospective study using mtDNA collected prior to PCa diagnosis. Although given an estimated latency of prostate cancer in the era of PSA testing of 4–9 years, it is possible that some of our cases, and even some controls, had preclinical PCa at the time of their blood draw, we cannot rule out the possibility of reverse causation. However, we required a minimum of one year between the blood draw and diagnosis to lessen this potential and did not observe any differences in the patterns of association after stratifying by time of blood draw to diagnosis (Supplementary Table 2). An important strength of this study is that by setting it within the screening arm of PLCO, both cases and controls underwent PCa screening with PSA testing and DRE and thus, we were able to limit disease misclassification. As this study was set in within a screening trial of standard-risk men, it was not adequately powered to detect small associations between mtDNA copy number and risk of overall PCa; though it represents, to our knowledge, the largest case-control study of mtDNA and PCa to date. Although our power to detect an association for aggressive PCa was limited in the PLCO setting, we did find evidence for an association with non-aggressive disease. As our study was limited to non-Hispanic Caucasians, our results may not be generalizable to other populations, especially populations in which PSA screening is not common. However, by conducting the study within a PCa screening trial, we were able to examine the association between mtDNA copy number and PCa prospectively. Finally, mtDNA copy number in peripheral blood leukocytes may not reflect mtDNA copy number or oxidative stress in the prostate tissue; however, the positive association with PSA levels among controls suggests some degree of correlation, even if imperfect.
Conclusions
In conclusion, we found associations between increasing mtDNA copy number and risk of non-aggressive PCa, and associations between increasing mtDNA copy number and PSA among controls. However, our results require independent replication in order to exclude the possibility that this association is due to chance alone, and additional exploration of the possible biological mechanisms linking PSA and mtDNA copy number is warranted.
Supplementary Material
Funding:
This study was supported by the Intramural Research Program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health.
Footnotes
Disclosure Statement: The authors have no conflicts of interest to disclose.
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