Abstract
Prior studies have suggested that vitamin D may reduce ovarian cancer risk. Thus, we examined whether three single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene (Fok1, Bsm1, Cdx2) were associated with risk of epithelial ovarian cancer in a retrospective case-control study (New England Case-Control study, NECC) and a nested-case control study of three prospective cohort studies: the Nurses’ Health Study (NHS), NHSII, and the Women’s Health Study (WHS). Data from the cohort studies were combined and analyzed using conditional logistic regression and pooled with the results from the NECC, which were analyzed using unconditional logistic regression, using a random effects model. We obtained genotype data for 1,473 cases and 2,006 controls. We observed a significant positive association between the number of Fok1 f alleles and ovarian cancer risk in the pooled analysis (p-trend=0.03). The odds ratio (OR) for the ff versus FF genotype was 1.26 (95% confidence interval (CI)=1.01-1.57). Neither the Bsm1 (p-trend=0.96) or Cdx2 (p-trend=0.13) SNPs were significantly associated with ovarian cancer risk. Among the prospective studies, the risk of ovarian cancer by plasma vitamin D levels did not clearly vary by any of the genotypes. For example among women with the Fok1 FF genotype, the OR comparing plasma 25-hydroxyvitamin D ≥32 ng/mL versus <32 ng/mL was 0.66 (95%CI=0.34-1.28), and among women with the Ff or ff genotype the OR was 0.71 (95%CI=0.43-1.18). Our results of an association with the Fok1 VDR polymorphism further support a role of the vitamin D pathway in ovarian carcinogenesis.
Keywords: Vitamin D, ovarian cancer, prospective
Introduction
Experimental and epidemiologic studies have suggested that vitamin D may be involved in the etiology of ovarian cancer. The vitamin D receptor (VDR) is weakly to moderately expressed in normal ovarian cells, but is more strongly expressed in ovarian cancer cell lines and tumor tissue (1-5). In vitro studies have reported that vitamin D administration inhibited cell growth and induced apoptosis in a dose-dependent manner in both animal (6) and human ovarian cancer cell lines (2, 3, 7-12).
UV-B exposure, which initiates vitamin D production in the skin, has been inversely associated with ovarian cancer mortality in ecologic studies (13-17). Recently, we reported that plasma concentrations of 25-hydroxyvitamin D (a measure of overall vitamin D status) and 1,25-dihydroxyvitamin D (the biologically active but more tightly regulated form) were not associated with risk of epithelial ovarian cancer overall in a prospective study (18). However, 25-hydroxyvitamin D levels were significantly inversely associated with ovarian cancer risk among overweight and obese women, possibly because vitamin D is fat soluble. Further, women with adequate (≥32 ng/mL) versus inadequate (<32 ng/mL) 25-hydroxyvitamin D levels (19) had a 36% decreased risk of serous ovarian cancer (18).
The VDR is a critical component of the vitamin D pathway and a number of common single nucleotide polymorphisms (SNPs) have been identified in this gene (20). We focused on three SNPs that either have been associated with ovarian cancer risk in prior studies (21, 22) or have some known or hypothesized functional effect (20). Thus we examined whether the Fok1 (rs10735810/rs2228570), Bsm1 (rs1544410), and Cdx2 (rs11568820) VDR SNPs were associated with risk of epithelial ovarian cancer in a retrospective case-control study (New England Case-Control study, NECC) and a nested-case control study using data from three prospective cohort studies: the Nurses’ Health Study (NHS), NHSII, and the Women’s Health Study (WHS).
Methods
Study population
Four studies were included in the analysis, including a nested case-control study from the NHS, NHSII, and WHS, and a retrospective case-control study (NECC). The study populations and case-control selections are described below for each study.
New England Case-Control Study (NECC)
The NECC includes 1,231 population-based epithelial ovarian cancer cases and 1,244 controls from Massachusetts and New Hampshire. Participants were enrolled in the study in two phases, from 1992-97 (563 cases, 523 controls) and from 1998-2003 (668 cases, 721 controls). Recruitment methods and eligibility criteria are described elsewhere (23). Briefly, trained interviewers asked participants about potential ovarian cancer risk factors that occurred more than one year prior to the date of diagnosis for cases or the interview date for controls.
Of the 2,347 incident cases of ovarian cancer identified, 1,845 (79%) were eligible and 71% of the eligible cases were enrolled. Controls were identified using random digit dialing, license records, and town resident lists and were frequency-matched to cases by age and state. Additional details of the control selection have been published previously (23). Over 95% of study participants provided a blood specimen at enrollment. DNA was available for 1,173 cases and 1,201 controls for this analysis. The institutional review boards of Brigham and Women’s Hospital and Dartmouth Medical School approved both phases of the study, and all participants provided written informed consent.
Cohort studies (NHS, NHSII, WHS)
The NHS cohort was established in 1976 among 121,700 U.S. female registered nurses, aged 30-55 years, and the NHSII was established in 1989 among 116,609 female registered nurses, aged 25-42 years. Women in both cohorts completed an initial questionnaire and have been followed biennially by questionnaire to update exposure status and disease diagnoses.
In 1989-1990, 32,826 NHS participants provided a blood sample and completed a short questionnaire (24). Briefly, women arranged to have their blood drawn and shipped with an icepack, via overnight courier, to our laboratory where it was processed. In 2001-04, 33,040 additional women provided a buccal cell specimen using a mouthwash protocol. We extracted DNA from each specimen within one week of receipt. Between 1996-1999, 29,611 NHSII participants provided blood samples and completed a short questionnaire (25). Collection methods were similar to those in the NHS. Cohort follow-up was 98% for the NHS blood study in 2004, 99% for the NHS cheek study in 2004, and 98% for the NHSII blood study in 2003.
The WHS is a completed randomized trial examining low-dose aspirin and vitamin E supplementation for the primary prevention of cancer and cardiovascular disease that was initiated in 1992 (26-28). Citrate and EDTA blood samples were collected from 28,345 women prior to randomization. We included women from the treatment and placebo groups. Morbidity and mortality follow-up through 2004 were 97% and 99% complete, respectively.
Cases had no previous history of cancer, except non-melanoma skin cancer, before specimen collection and were diagnosed with ovarian cancer before June 1, 2004 (NHS), June 1, 2003 (NHSII), or December 1, 2004 (WHS). We included incident cases after sample collection from each study plus prevalent cases from the NHS/NHSII who submitted a specimen within four years after diagnosis. Prevalent and incident cases were similar on stage, histology, and survival time (median survival, incident=58 months, prevalent=80 months) (29). Overall, 300 cases (235 incident and 65 prevalent) with DNA were confirmed by medical record review (210 from NHS, 28 from NHSII, and 62 from WHS). Cases were matched to two (WHS) or three (NHS/NHSII) controls, who had intact ovaries at the time of the case diagnosis and no prior history of cancer (except non-melanoma skin cancer), on menopausal status at diagnosis, age (±1 year), and type of sample collection (cheek, blood). Additional details on the control selection have been published previously (18). All three cohort studies were approved by the Committee on the Use of Human Subjects in Research at the Brigham and Women’s Hospital.
Laboratory assays
DNA was extracted from the buffy coat or cheek cells using Qiagen DNA extraction kits (Qiagen Inc., Valencia, CA). Genotyping for samples for all four studies was performed at the Dana Farber/Harvard Cancer Center High Throughput Genotyping Core. All the samples were genotyped for the Fok1, Bsm1, and Cdx2 SNPs in the VDR gene. Whole genome amplified DNA was genotyped using the 5′ nuclease assay (TaqMan) on the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA), in 384-well format. TaqMan primers and probes were designed using the Primer Express Oligo Design software v2.0 (ABI PRISM). Laboratory personnel were blinded to case-control status, and each plate included blinded replicate samples for quality control purposes. Over 94% of the samples were successfully genotyped for each polymorphism, except for the BSM1 polymorphism in the cohort studies (success=92%) due to a lack of DNA for some women in the WHS study. Genotyping failures were considered missing data. The quality control replicate samples (cohort studies: 244 replicates from 43 individuals; NECC: 203 replicates from 52 individuals) were 100% concordant for all genotypes.
Vitamin D analytes were assayed by RIA, as described previously (30), in the prospectively collected heparin (NHS/NHSII) or citrate (WHS) plasma samples (see (18) for details). Case-control sets and samples from the same study were assayed together and labeled to mask case-control status. The intra-assay coefficient of variation, based on blinded quality control replicates, was 8-10% for 25-hydroxyvitamin D and 9-14% for 1,25-dihydroxyvitamin D.
Statistical analysis
We evaluated whether each genotype was in Hardy-Weinberg equilibrium using a chi-square test. This distribution of the alleles was assessed separately in each study (NHS, NHSII, WHS, and NECC), and by sample type for the NHS (cheek versus white blood cell). Unconditional (NECC) and conditional logistic regression (NHS/NHSII/WHS) were used to estimate the odds ratios (OR) and 95% confidence intervals (CI) associated with the main effects of each gene variant. Data analyses were initially conducted separately for the cohort studies, but were combined into one dataset since the p-heterogeneity by study was >0.05 for all analyses. Since the NECC is a case-control study, these analyses were conducted separately from the cohort studies. The two resulting estimates were then combined using a random effects model to obtain pooled effect estimates (31).
We considered multiple a priori potential confounders and included those that changed the risk estimates or were very strong ovarian cancer risk factors in the final model for genetic analyses: number of pregnancies (continuous), postmenopausal hormone use before diagnosis (never, past, current), oral contraceptive use duration (never, <3yr, 3-<5yr, 5+yr), and age at menarche (<12, 12, 13, 14, >14yr). Other potential confounders such as BMI, tubal ligation, and smoking did not substantially change risk estimates and therefore were not included in the final model. We additionally adjusted for age and study center in the NECC, as these were frequency matching variables. We calculated the P for trend for each unit increase in the number of minor alleles (log-additive model) using the Wald test. For analyses including plasma vitamin D levels we additionally adjusted for body mass index at blood draw (continuous), season of blood draw (winter to early spring [January, February, March, April], summer to early fall [July, August, September, October], late-spring/late fall [May, June, November, December]) and interaction terms of study with duration of oral contraceptive use and body mass index; this mimics the statistical model used in our prior vitamin D analysis (18).
Our primary analysis included both invasive and borderline cases. However, in secondary analyses, we evaluated genetic associations among histologic subtypes of cases (all invasive, serous invasive, serous borderline, endometrioid, and mucinous). We also stratified by season of diagnosis (summer, other), age at diagnosis (<55, ≥55 yr), menopausal status at diagnosis (premenopausal, postmenopausal), body mass index (BMI, <25, ≥25 kg/m2), and oral contraceptive use (never, ever). Multiplicative interaction terms between the above strata and genotypes (homozygous wildtype vs. heterozygous and homozygous variant) were used to determine the p-heterogeneity. We also examined statistical interactions between the SNPs. These analyses used unconditional logistic regression for both the cohort and case-control studies, additionally adjusting the cohort analysis for the matching factors and study.
Further, we examined the relationship between plasma vitamin D levels and ovarian cancer risk in prospectively collected cases and their matched controls in the three cohort studies. Outliers (32) were identified separately by sample type and set to missing using methods described previously (18). We used a cutpoint of 32 ng/mL for 25-hydroxyvitamin D, which reflects vitamin D adequacy (19)) and cohort-specific medians for 1,25-dihydroxyvitamin D. We used unconditional logistic regression, adjusting for matching factors and potential confounders, to estimate ORs and 95% CIs.
All tests of statistical significance were two-sided and considered significant if p≤0.05. SAS version 9.1 (SAS Institute, Inc., Cary, NC) was used for the analyses.
Results
Women ranged in age from 39 to 79 years old (mean=62 yr) in the NHS, from 31 to 52 years old (mean=42 yr) in the NHSII, and from 45 to 73 years old (mean=56 yr) in the WHS at blood collection (Table 1) (18). Women in the NECC ranged in age from 16 to 77 years old (mean=51 yr) at study entry. The characteristics of the NECC population have been described previously (29). Overall, the risk factor distributions were as expected, within each study; NHSII women were on average younger and had a longer duration of oral contraceptive use than women in the NHS. We obtained genotype data for 1,473 cases (NECC: 1,173 and NHS/NHSII/WHS: 300) and 2,006 controls (NECC: 1,201 and NHS/NHSII/WHS: 805).
Table 1.
Characteristics of women in the Nurses’ Health Studies (NHS and NHSII), the Women’s Health Study (WHS), and the New England Case-Control Study (NECC).
| NHS/NHSII | WHS | NECC | ||||
|---|---|---|---|---|---|---|
| Cases | Controls | Cases | Controls | Cases | Controls | |
| n | 238 | 684 | 62 | 121 | 1173 | 1201 |
| Age at diagnosis (yr)* | 59.3 (10.0) | 59.2 (10.1) | 55.8 (7.2) | 55.6 (7.1) | 51.3 (12.8) | 50.8 (13.0) |
| Duration of OC use (mo)* | 24.1 (37.8) | 27.5 (43.9) | 21.7 (19.3) | 24.6 (19.0) | 25.1 (45.7) | 36.7 (52.2) |
| Parity* | 2.7 (1.5) | 3.0 (1.7) | 2.1 (1.6) | 2.6 (1.6) | 1.7 (1.6) | 2.2 (1.7) |
| BMI (kg/m2)* | 26.0 (5.5) | 25.7 (4.7) | 24.6 (3.9) | 25.0 (4.4) | 26.3 (6.3) | 25.7 (5.5) |
| Tubal ligation† | 36 (15.1) | 145 (21.2) | 12 (19.4) | 32 (26.5) | 166 (14.2) | 220 (18.3) |
| Menopause† | ||||||
| Premenopausal | 48 (20.2) | 157 (23.0) | 15 (24.2) | 36 (29.8) | 552 (47.1) | 585 (48.7) |
| Postmenopausal | 175 (73.5) | 487 (71.2) | 35 (56.5) | 69 (57.0) | 563 (48.0) | 562 (46.8) |
| Unknown | 15 (6.3) | 40 (5.9) | 12 (19.4) | 16 (13.2) | 58 (4.9) | 54 (4.5) |
| PMH use† | ||||||
| Never | 49 (28.0) | 157 (32.2) | 6 (17.1) | 12 (17.4) | 387 (68.7) | 362 (64.4) |
| Past | 27 (15.4) | 101 (20.7) | 5 (14.3) | 5 (7.3) | 53 (9.4) | 61 (10.9) |
| Current | 93 (53.1) | 197 (40.5) | 24 (68.6) | 52 (75.4) | 121 (21.5) | 137 (24.4) |
| Unknown | 6 (3.4) | 32 (6.6) | 0 (0.0) | 0 (0.0) | 2 (0.4) | 2 (0.4) |
| Family history of ovarian cancer† |
18 (7.6) | 23 (3.4) | 2 (3.2) | 7 (5.8) | 60 (5.1) | 34 (2.8) |
| Morphology†,‡ | ||||||
| Invasive | 199 (83.6) | NA | 62 (100) | NA | 907 (77.3) | NA |
| Borderline | 36 (15.1) | 0 (0.0) | 266 (22.7) | |||
| Histology†,‡ | ||||||
| Serous invasive | 115 (48.3) | NA | 50 (80.7) | NA | 479 (40.8) | NA |
| Serous borderline | 20 (8.4) | 0 (0.0) | 168 (14.3) | |||
| Mucinous | 30 (12.6) | 4 (6.5) | 153 (13.0) | |||
| Endometrioid | 31 (13.0) | 4 (6.5) | 171 (14.6) | |||
| Clear cell | 7 (2.9) | 4 (6.5) | 143 (12.2) | |||
The mean (standard deviation) are presented. OC = oral contraceptives; BMI = body mass index
The n (%) are presented; for PMH use, among postmenopausal women only. PMH = postmenopausal hormones
May not add up to total number of cases due to unknown status and other histologic subtypes. Not applicable for controls.
We evaluated the genotype frequencies of the Fok1, Bsm1, and Cdx2 VDR SNPs in each study population separately. There were no statistically significant differences in the genotype distributions of these SNPs between the cases and controls within each study and genotype distributions were in Hardy-Weinberg Equilibrium among the controls, except for Bsm1 in the NECC controls (p=0.03, data not shown). Review of the screen shots for these plates revealed appropriate clustering, thus this finding is likely due to chance. The minor allele frequencies across the four studies were 0.39 for Fok1 f allele, 0.40 for the Bsm1 B allele, and 0.21 for the Cdx2 A allele.
We observed a non-statistically significant increased risk of ovarian cancer with increasing numbers of the Fok1 variant f allele in the NECC and the combined cohort studies separately that became statistically significant (p-trend=0.03) in the pooled analysis (Table 1). The pooled OR comparing women with the ff versus FF genotype was 1.26 (95% CI=1.01-1.56), with an intermediate risk for the Ff genotype (pooled OR=1.13, 95%CI=0.96-1.56). Results were somewhat attenuated when only including invasive cases likely due to the smaller sample size (pooled OR, Ff+ff vs. FF=1.21, 95%CI=0.96-1.53). The association between Fok1 and ovarian cancer risk appeared stronger for the serous borderline (pooled OR, Ff+ff vs. FF=1.66, 95%CI=1.17-2.36) and endometrioid (pooled OR, ff vs. FF=1.61, 95%CI=1.02-2.51) histologic subtypes. The corresponding, pooled OR for serous invasive cases was 1.05 (95%CI=0.87-1.28) and for mucinous tumors was 1.11 (95%CI=0.79-1.55). The Fok1 association was similar by age, menopausal status, season at diagnosis, BMI, and oral contraceptive use history as well as when excluding prevalent cases from the NHS and NHSII (data not shown).
Neither the Bsm1 or Cdx2 polymorphisms were statistically significantly associated with overall ovarian cancer risk in study-specific or pooled analyses (Table 1). For example the pooled OR comparing the Bsm1 BB to the bb genotype was 0.95 (95%CI=0.76-1.18) and for the Cdx2 GG versus AA genotype was 1.00 (95%CI=0.71-1.42). Results were similar among invasive cases only and by histologic subtype for the Bsm1 polymorphism (data not shown). However the association between the Cdx2 polymorphism was statistically significant for invasive ovarian cancer overall (pooled OR, GA+AA vs. GG=1.20, 95%CI=1.02-1.41, p-trend=0.05) and for serous invasive tumors (comparable OR=1.37, 95%CI=1.13-1.67, p-trend=0.004), but not for the other subtypes (p-trend>0.56). In general, associations were similar by strata of BMI, age, menopausal status, and season at diagnosis (data not shown); however there was a statistically significant interaction between the Bsm1 genotype and oral contraceptive use history (p-heterogeneity=0.02). There was a modest positive association between the number of B alleles and risk among never users (OR, BB vs. bb=1.31; 95%CI=0.94-1.82; p-trend=0.08) and a suggestive inverse association among ever users (OR=0.77; 95%CI=0.54-1.10; p-trend=0.13).
We further examined whether there was a combined effect of multiple SNPs on ovarian cancer risk. The Bsm1 SNP association was similar across strata of the Fok1 and Cdx2 genotypes. However there was a nearly statistically significant interaction (p=0.07) between the Fok1 and Cdx2 SNPs. Compared to the reference group of FF and GG, respectively, women with any other genotype were at a statistically significantly increased risk of ovarian cancer in the pooled analysis. Specifically, the OR for the women with the Ff+ff and GG genotype was 1.32 (OR=1.37 for invasive cases) and for women with the GA+AA genotype regardless of Fok1 genotype was 1.36 (ORs=1.28 for invasive cases).
There were 210 incident cases and 522 matched controls with both genotype and plasma data in the three prospective cohort studies. We examined whether the association between plasma vitamin D concentrations differed by VDR genotype (Table 2). Overall, the risk of ovarian cancer comparing plasma 25-hydroxyvitamin D <32 ng/mL versus ≥32 ng/mL or 1,25-dihydroxyvitamin D above versus below study-specific medians was similar by Fok1, Bsm1, and Cdx2 genotype. For example, among women with the Fok1 FF genotype, the OR comparing ≥32 versus <32 ng/mL of 25-hydroxyvitamin D was 0.66 (95%CI=0.34-1.28) and among those with Ff or ff genotype the OR was 0.71 (95%CI=0.43-1.18). Plasma vitamin D levels were not clearly associated with the VDR SNPs among controls (data not shown).
Table 2.
Odds ratios (OR) and 95% confidence intervals (CI) for the association between vitamin D receptor SNPs and ovarian cancer risk in the New England Case-Control Study (NECC), the Nurses’ Health Study (NHS), NHSII, and the Women’s Health Study (WHS)
| NECC* | NHS / NHSII / WHS*,† | Pooled‡ | |||
|---|---|---|---|---|---|
| Genotype | N, case/control |
OR (95% CI) | N, case/control |
OR (95% CI) | OR (95% CI) |
| Fok1 | |||||
| FF | 409 / 450 | 1.00 (ref.) | 98 / 304 | 1.00 (ref.) | 1.00 (ref.) |
| Ff | 502 / 511 | 1.08 (0.89, 1.30) | 141 / 340 | 1.28 (0.93, 1.76) | 1.13 (0.96, 1.33) |
| ff | 193 / 175 | 1.23 (0.95, 1.58) | 49 / 113 | 1.37 (0.89, 2.10) | 1.26 (1.01, 1.57) |
| P-trend | 0.12 | 0.10 | 0.03 | ||
| Ff + ff | 1.12 (0.94-1.33) | 1.30 (0.97, 1.75) | 1.16 (1.00, 1.35) | ||
| Bsm1 | |||||
| bb | 409 / 430 | 1.00 (ref.) | 94 / 267 | 1.00 (ref.) | 1.00 (ref.) |
| bB | 521 / 518 | 1.07 (0.89, 1.29) | 143 / 353 | 1.20 (0.87, 1.66) | 1.10 (0.94, 1.30) |
| BB | 183 / 203 | 0.93 (0.73, 1.20) | 41 / 114 | 1.00 (0.64, 1.58) | 0.95 (0.76, 1.18) |
| P-trend | 0.80 | 0.72 | 0.96 | ||
| bB + BB | 1.03 (0.87, 1.23) | 1.15 (0.85, 1.57) | 1.06 (0.91, 1.23) | ||
| Cdx2 | |||||
| GG | 670 / 746 | 1.00 (ref.) | 179 / 496 | 1.00 (ref.) | 1.00 (ref.) |
| GA | 399 / 356 | 1.23 (1.02, 1.48) | 92 / 220 | 1.09 (0.79, 1.49) | 1.19 (1.02, 1.39) |
| AA | 51 /56 | 1.01 (0.67, 1.51) | 14 / 36 | 0.99 (0.51, 1.92) | 1.00 (0.71, 1.42) |
| P-trend | 0.11 | 0.74 | 0.13 | ||
| GA + AA | 1.20 (1.01, 1.43) | 1.07 (0.79, 1.45) | 1.16 (1.00, 1.36) | ||
NECC: unconditional logistic regression; NHS/NHSII/WHS: conditional logistic regression. Adjusted for number of pregnancies, postmenopausal hormone use, oral contraceptive use duration, and age at menarche. Additionally adjusted for age and study center for the NECC analysis.
P-heterogeneity between NHS/NHSII and WHS was 0. 70 for Fok1, 0.36 for BSM1, and 0.72 for CDX2.
P-values for tests for heterogeneity comparing the NECC and cohort results were all >0.36
Discussion
To our knowledge, this is the largest ovarian cancer study to examine the association between SNPs in the VDR gene and risk, and it is the first study to examine whether these SNPs modify the association of plasma 25-hydroxy and 1,25-dihydroxyvitamin D levels with risk. Overall our results suggest that the Fok1, but not the Bsm1 and Cdx2, polymorphism is associated with ovarian cancer risk. Further, the combined Fok1 and Cdx2 genotype may be important as well, although additional studies are needed to confirm a potential interrelationship. In general, the association with plasma vitamin D levels did not vary by VDR genotype, suggesting that these two factors may act independently.
Our results are consistent with those observed in a case-control study of ovarian cancer from Hawaii (n=72 Caucasian cases), which observed more than a 2-fold increase in risk for either the Ff or ff genotype versus the wildtype FF, but no association for the Bsm1 or Cdx2 SNPs (22). Interestingly, no association with the Fok1 genotype was observed for Japanese women (n=94 cases), suggesting that this relationship may differ by race/ethnicity. A second study (21), using a nested case-control design among two cohort studies, did not observe any associations between the Fok1 (RR, ff vs. FF=1.23, 95% CI=0.61–2.51) or Bsm1 (RR, AA vs. GG=1.08, 95% CI=0.54-2.17) SNPs and ovarian cancer risk; however, the sample size was relatively small (n=170 cases), limiting power to detect a modest association. In a meta-analysis of the current cohort and case-control studies plus the two prior studies, the summary OR comparing women with the Fok1 ff versus FF allele was 1.28 (95%CI=1.05-1.58; Figure 1), suggesting an overall positive association for this SNP.
Figure 1.
Forest plot of the odds ratios and 95% confidence intervals comparing risk of ovarian cancer for women with the Fok1 ff versus FF allele in the vitamin D receptor for the cohort studies (NHS, NHSII, and WHS) and the NECC study in the current analysis and two prior published reports. The summary odds ratio is 1.29 (95%CI=1.05-1.58, p=0.01).
We observed that the association of the Bsm1 VDR SNP with risk differed by oral contraceptive use history; however the log-additive Bsm1 associations were not statistically significant in either never or ever users. The two prior studies did not examine interactions by oral contraceptive use (21, 22). There are some data supporting a potential biologic relationship between the vitamin D pathway and oral contraceptive use. Among premenopausal women, current oral contraceptive use is associated with higher vitamin D levels in Caucasians and African-Americans, and levels decline after women stop using oral contraceptives (33, 34). In addition, a small randomized trial of oral contraceptive use in premenopausal women observed changes in bone metabolism after three months only among the intervention group who had the Bsm1 BB and Bb genotypes (35). However, further research is needed to replicate this association and elucidate the underlying biologic mechanisms.
We also observed a nearly statistically significant interaction between the Fok1 and Cdx2 genotypes, such that women with the FF and GG genotypes, respectively, had the lowest risk of cancer. Women with any other genotype combination had over a 30% increased risk. Prior studies have not examined this combination of genotypes (21); thus it will be important to examine this potential interaction in larger studies.
Our finding that the Fok1 f allele is associated with an increased risk of ovarian cancer is consistent with functional data on the Fok1 SNP. The variant f allele has an earlier start codon, leading to a protein with three extra amino acids that is less transcriptionally active and has a lower transactivation of VDR target genes than the F allele (20, 36, 37). Further, the variant protein may have a decreased capacity to inhibit cellular growth after administration of vitamin D (38). This suggests that women with the f allele have a less active VDR, which may increase their ovarian cancer risk. The Bsm1 SNP is in the 3-prime untranslated region of the VDR gene and may alter mRNA stability; the B allele has been associated with increased osteopontin, calcitrol, and 1,25-dihydroxyvitamin D in serum (20). However, this potential functional effect may not be important in ovarian cancer carcinogenesis, given that we did not observe an association between this SNP and either risk of ovarian cancer or circulating vitamin D levels. Finally the Cdx2 G->A SNP alters the binding site of a CDX transcription factor, with the G allele having a lower binding affinity (20, 39, 40). These studies also observed that the A allele was associated with increased VDR expression in intestinal cells and enhanced calcium absorption. To our knowledge, no functional studies have examined a potential biologic interplay between the Fok1 and Cdx2 SNPs; however such data could lend support to the interaction we observed with ovarian cancer risk.
Experimental data also support a role of the vitamin D pathway specifically in ovarian carcinogenesis. A number of studies have observed that 1,25-dihydroxyvitamin D inhibits ovarian cancer cell growth (2, 3, 7, 9-12) and increases apoptosis (8). High 1,25-dihydroxyvitamin D levels also can increase vitamin D receptor expression in ovarian cancer cell lines (8). One study reported that 25-hydroxyvitamin D slightly increased cell growth of ovarian cancer cells; however this effect was reduced at higher concentrations of 25-hydroxyvitamin D exposure (11). Recent data suggest that ovarian cancer cells and tissue contain measurable levels of 1α-hydroxylase and 24-hydroxlase (1, 4, 11), the former of which can convert 25-hydroxyvitamin D to the more active 1,25-dihydroxyvitamin D (3, 4). It is possible that 1,25-dihydroxyvitamin D formed through this process acts intracellularly or as an autocrine/paracrine factor (41). Since ovarian tumor tissue expresses the VDR (1-5), these data suggest in total that some ovarian tumors may have a functional vitamin D pathway that could potentially be a target for prevention or treatment.
Interestingly we did not observe that the association of plasma vitamin D levels with ovarian cancer risk differed by VDR genotype; however the sample size precluded detecting small to modest effects or comparing extreme ends of the vitamin D spectrum. Examining a potential interrelationship between VDR genotype and plasma vitamin D levels could be important, as it is possible that women with genotypes associated with a higher risk of ovarian cancer may benefit more from high plasma vitamin D levels. Biologic data support this hypothesis. Colin, et al. (38) reported that the Fok1 polymorphism in cultured peripheral blood mononuclear cells from postmenopausal women was associated with growth inhibition only at low, physiologic doses. They suggested that the polymorphism may be clinically relevant only among those with insufficient vitamin D levels. Further experimental and epidemiologic research is needed to elucidate these relationships.
Our study has several limitations and strengths. One limitation is that the NHS/NHSII and WHS collected different sample types (heparin and citrate plasma, respectively) for measuring plasma vitamin D levels. Citrate plasma can dilute specimens, thus lowering the measured concentrations (42), although the levels were similar between the two studies when adjusting for the dilution factor (18). Further the study population was primarily of European ancestry, thus we were not able to examine these associations in other races/ethnicities. The strength of this study was the ability to combine four studies, one retrospective case-control study and three prospective cohort studies. The similar associations across studies and the statistically significant results in both the pooled analysis and meta-analysis with prior studies, lend support that our findings are not spurious; however given that we made a number of comparisons the results could be due to chance. The ability to pool the results also substantially increased our sample size to approximately 1,500 cases and 2,000 controls.
In conclusion, we observed that increasing copies of the Fok1 f allele in the VDR gene were positively associated with ovarian cancer risk. We did not observe a strong interrelationship between 25-hydroxyvitamin D and Fok1 genotype. Additional research is needed to further evaluate possible relationships between multiple vitamin D pathway genes and circulating levels of vitamin D. Overall, these results provide further support that the vitamin D pathway may play a role in the etiology of ovarian cancer.
Table 3.
Relative risks* (RRs) and 95% confidence intervals (CIs) for the association between pre-diagnostic 25-hydroxy and 1,25-dihydroxy vitamin D levels and ovarian cancer risk in the Nurses’ Health Study (NHS), NHSII, and Women’s Health Study, stratified by polymorphisms in the vitamin D Receptor gene.
| 25-hydroxyvitamin D† | 1,25-dihydroxyvitamin D† | |||
|---|---|---|---|---|
| Polymorhpism | <32ng/mL | ≥32 ng/mL | <median | ≥median |
| Fok1 | ||||
| n, cases/controls | 166/385 | 44/137 | 101/264 | 108/257 |
| FF | 1.0 | 0.66 (0.34, 1.28) | 1.0 | 1.03 (0.59, 1.81) |
| Ff | 1.0 | 0.80 (0.45, 1.43) | 1.0 | 1.35 (0.83, 2.19) |
| ff | 1.0 | 0.49 (0.17, 1.38) | 1.0 | 0.57 (0.24, 1.32) |
| Ff + ff | 1.0 | 0.71 (0.43, 1.18) | 1.0 | 1.08 (0.71, 1.64) |
| Bsm1 | ||||
| n, cases/controls | 158/369 | 41/125 | 94/250 | 104/243 |
| bb | 1.0 | 0.61 (0.30, 1.27) | 1.0 | 0.89 (0.50, 1.58) |
| bB | 1.0 | 0.75 (0.41, 1.35) | 1.0 | 1.27 (0.77, 2.08) |
| BB | 1.0 | 0.70 (0.26, 1.92) | 1.0 | 1.14 (0.47, 2.77) |
| bB+BB | 1.0 | 0.73 (0.43, 1.22) | 1.0 | 1.23 (0.80, 1.89) |
| Cdx2 | ||||
| n, cases/controls | 160/371 | 43/132 | 97/254 | 105/248 |
| GG | 1.0 | 0.59 (0.36, 0.98) | 1.0 | 1.15 (0.75, 1.74) |
| GA+AA | 1.0 | 0.91 (0.46, 1.81) | 1.0 | 0.97 (0.55, 1.74) |
Used unconditional logistic regression adjusting for number of pregnancies, postmenopausal hormone use, oral contraceptive use duration, age at menarche, age, menopausal status at diagnosis, cohort, season of blood collection, body mass index at blood collection, and the interaction of study with oral contraceptive use duration and body mass index.
p-heterogeneity across Fok1, Bsm1, and Cdx2 genotypes for 25-hydroxyvitam in D are 0.81, 0.81, and 0.32, respectively and for 1,25-dihydroxyvitam in D are 0.47, 0.53, and 0.65, respectively.
Acknowledgements
We would like to thank the women in the NHS, NHSII, WHS, and NECC studies for their valuable participation. We also thank Marilyn Chown, Allison Vitonis, and Jeanne Sparrow for data management support.
Financial support: Support for this project was from NIH grants P01 CA87969, CA49449, CA67262, CA50385, P50 CA105009, HL-43851, HL-080467, and CA-47988. Dr. Gates was supported by the NCI training grant R25 CA098566.
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