collin 10 cebp 19 1632

collin 10 cebp 19 1632
Published OnlineFirst on May 25, 2010 as 10.1158/1055-9965.EPI-10-0180
& Prevention
Research Article
Circulating Folate, Vitamin B12, Homocysteine, Vitamin B12
Transport Proteins, and Risk of Prostate Cancer: a CaseControl Study, Systematic Review, and Meta-analysis
Simon M. Collin1, Chris Metcalfe1, Helga Refsum3,5, Sarah J. Lewis1, Luisa Zuccolo1, George Davey Smith1,2,
Lina Chen1, Ross Harris6, Michael Davis1, Gemma Marsden4, Carole Johnston3, J. Athene Lane1, Marta Ebbing7,
Kaare Harald Bønaa9, Ottar Nygård7,8, Per Magne Ueland7,8, Maria V. Grau10, John A. Baron10,
Jenny L. Donovan1, David E. Neal11, Freddie C. Hamdy4, A. David Smith3, and Richard M. Martin1,2
Background: Disturbed folate metabolism is associated with an increased risk of some cancers. Our objective was to determine whether blood levels of folate, vitamin B12, and related metabolites were associated
with prostate cancer risk.
Methods: Matched case-control study nested within the U.K. population–based Prostate testing for cancer
and Treatment (ProtecT) study of prostate-specific antigen–detected prostate cancer in men ages 50 to 69
years. Plasma concentrations of folate, B12 (cobalamin), holo-haptocorrin, holo-transcobalamin total transcobalamin, and total homocysteine (tHcy) were measured in 1,461 cases and 1,507 controls. ProtecT study
estimates for associations of folate, B12, and tHcy with prostate cancer risk were included in a meta-analysis,
based on a systematic review.
Results: In the ProtecT study, increased B12 and holo-haptocorrin concentrations showed positive associations with prostate cancer risk [highest versus lowest quartile of B12 odds ratio (OR) = 1.17 (95% confidence
interval, 0.95-1.43); Ptrend = 0.06; highest versus lowest quartile of holo-haptocorrin OR = 1.27 (1.04-1.56);
Ptrend = 0.01]; folate, holo-transcobalamin, and tHcy were not associated with prostate cancer risk. In the
meta-analysis, circulating B12 levels were associated with an increased prostate cancer risk [pooled OR =
1.10 (1.01-1.19) per 100 pmol/L increase in B12; P = 0.002]; the pooled OR for the association of folate with
prostate cancer was positive [OR = 1.11 (0.96-1.28) per 10 nmol/L; P = 0.2) and conventionally statistically
significant if ProtecT (the only case-control study) was excluded [OR = 1.18 (1.00-1.40) per 10 nmol/L;
P = 0.02].
Conclusion: Vitamin B12 and (in cohort studies) folate were associated with increased prostate cancer risk.
Impact: Given current controversies over mandatory fortification, further research is needed to determine
whether these are causal associations. Cancer Epidemiol Biomarkers Prev; 19(6); 1632–42. ©2010 AACR.
The folate-mediated one-carbon metabolic pathway is
fundamental to DNA synthesis, repair, and methylation
(1). The role of folate antagonists in treating hematological (2) and trophoblastic (3) malignancies is well known,
and genetic studies have suggested that folate pathway
gene polymorphisms may be associated with colorectal
Authors' Affiliations: 1 Department of Social Medicine and 2 Medical
Research Council Centre for Causal Analysis in Translational
Epidemiology, Department of Social Medicine, University of Bristol,
Bristol, United Kingdom; 3 Department of Physiology, Anatomy and
Genetics and 4 Nuffield Department of Surgery, University of Oxford,
John Radcliffe Hospital, Oxford, United Kingdom; 5 Department of
Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine,
University of Oslo, Oslo, Norway; 6Health Protection Agency, London,
United Kingdom; 7Department of Heart Disease, Haukeland University
Hospital and 8 Institute of Medicine, University of Bergen, Bergen,
Norway; 9 Department of Heart Disease, University Hospital of North
Norway, Tromsø, Norway; 1 0 Departments of Medicine and of
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
and gastric cancers (4). Several epigenetic mechanisms related to folate metabolism, including CpG island and histone methylation, DNA uracil misincorporation, and
chromosomal rearrangements, have been observed in
prostate tumor cells (5, 6).
Studies of dietary intake and blood levels of folate, vitamin B6, methionine, and homocysteine have generally
found no associations with risk of prostate cancer (7-17),
Community and Family Medicine, Dartmouth Medical School, Lebanon,
New Hampshire 03766; and 11Department of Oncology, University of
Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom
Note: Supplementary data for this article are available at Cancer Epidemiology, Biomarkers & Prevention Online (
Corresponding Author: Simon M. Collin, University of Bristol, Canynge
Hall, 39 Whatley Road, Bristol BS8 2PS, United Kingdom. Phone: 1173313934; Fax: 117-9287292. E-mail:
doi: 10.1158/1055-9965.EPI-10-0180
©2010 American Association for Cancer Research.
Folate, B12, and Prostate Cancer
although there is some evidence that high dietary intake
and blood levels of vitamin B12 are associated with increased risk (12-15). One recent study reported a positive
association between folic acid supplementation and prostate cancer risk (18). However, results from the same
study suggested inverse associations with baseline dietary and plasma folate, as did three other studies (8, 19,
20), and the main trial finding was not replicated in a
larger trial (21). Although differences in study design
may partly explain these contradictory findings (22),
any role of folate metabolism is likely to be complex, possibly involving a dual effect in which low-folate concentrations are associated with increased risk of cancer
initiation, whereas high concentrations, or folic acid supplementation, are associated with more rapid progression following disease onset (23). Answers to these
research questions are urgently needed to inform the debate over mandatory fortification of food with folic acid
and vitamin B12.
We used data from a cross-sectional case-control study
nested within the U.K. population–based Prostate testing
for cancer and Treatment (ProtecT) study to investigate
whether plasma concentrations of folate, vitamin B12,
and total homocysteine (tHcy) were associated with the
risk of localized and/or advanced prostate cancer detected by means of prostate-specific antigen (PSA) testing.
We included our results in a meta-analysis of data from
studies identified by a systematic review of the literature.
In the ProtecT case-control study, we also measured
the concentrations of total transcobalamin and holotranscobalamin, and calculated the concentration of
holo-haptocorrin. Haptocorrin and transcobalamin are
B12 transport proteins to which total circulating B12 is
bound, as holo-haptocorrin and holo-transcobalamin, in
an approximate 80:20 ratio (24). Holo-transcobalamin is
an alternative marker of impaired B12 absorption (25), and
decreased levels have been associated more strongly than
total B12 with conditions related to impaired folate and
B12 metabolism (26, 27). Raised levels of holo-haptocorrin
have been reported in some cancers (28, 29), possibly as a
result of upregulated haptocorrin production by tumor
cells (30).
Materials and Methods
Study population
The ProtecT study is a randomized controlled trial of
treatments for localized prostate cancer. Between 2001
and 2009, all (∼227,300) men ages 50 to 69 years in 300
general practices located around nine U.K. cities (centers)
were invited to have a PSA test at a prostate check clinic
appointment. Participants with a PSA level between 3.0
and 19.9 ng/mL (∼10% of men tested) were invited to
attend the center's urology department for digital rectal
examination and 10-core trans-rectal ultrasound-guided
biopsy. Men with a PSA level of ≥20 ng/mL were referred as a matter of urgency to a urologist and were eligible to participate in the treatment trial only if localized
cancer was confirmed. A diagnosis of localized prostate
cancer was defined as a positive biopsy, clinical stage T1
to T2, NX, M0; advanced prostate cancer was defined as
positive biopsy, clinical stage T3 to T4, or N1 or M1. All
men provided written informed consent. Trent Multicentre Research Ethics Committee approved the ProtecT
study and allied prostate cancer research under the auspices of Prostate Mechanisms of Prostate cancer and
Selection of cases and controls
The study size (1,500 cases and 1,500 controls) was determined a priori to detect an effect estimate [odds ratios
(OR)] of 1.26 (exposure odds in cases, 0.42) comparing
the highest versus lowest three quartiles of vitamin and
metabolite concentration at 5% significance and 80%
power. Cases were selected at random from among all
men diagnosed (by July 2008) with localized or advanced
cancer who had consented to a blood sample for research. Eligible controls were men who had a PSA level
of <3 ng/mL, or who had a PSA level of ≥3 ng/mL and a
negative biopsy result, and who had consented to provide a research blood sample. Controls were stratum
matched to cases by 5-year age group and by the primary
care practice from which they were recruited, thereby
matching for calendar time as prostate check clinics were
completed sequentially. For the assays investigated in the
current analysis, one control per case was selected at random from the pool of eligible controls in each stratum.
Blood sample handling
A standardized blood collection and storage protocol
was in place across all collecting centers. Blood was
drawn from nonfasting participants at the time of their
initial PSA test. Plasma samples were collected using
the BD Vacutainer PPT 8.5-mL polymer gel and spraydried K2EDTA separator tube (Becton, Dickinson and
Co), centrifuged at 2,200 relative centrifugal force within
10 minutes of blood draw, and transported upright at 4°C
to the local processing laboratory. The plasma was transferred to intermediate cryo-vials for medium-term storage and was frozen at −80°C within 36 hours of draw.
Samples were transferred to the central biorepository
hub on dry ice. Plasma samples were thawed at 4°C in a
shaking water bath, mixed thoroughly using the Stuart
SB3 Blood Rotator Mixer (Bibby Scientific Ltd) for
10 minutes, centrifuged for 10 minutes at 4°C at 4,500
rpm in the Beckman 25R Allegra centrifuge (Beckman
Coulter Ltd.), and aliquotted into Starlab 1.5 mL cryotubes (STARLAB Ltd). The plasma was then stored at
−80°C and transferred on dry ice to the Department
of Physiology, Anatomy and Genetics, University of
Oxford, for assay.
Biochemical analyses
Plasma concentrations of folate, B12, holo-transcobalamin, and total transcobalamin were measured by automated (Perkin-Elmer MultiProbe 11 liquid handling
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
Collin et al.
system, Perkin-Elmer Life and Analytical Sciences) microbiological assay using Lactobacillus casei for folate (31)
and Lactobacillus leichmannii for B12 (32), holo-transcobalamin,
and total transcobalamin (33). Plasma tHcy was measured
by automated (Abbott IMx system, Abbott Laboratories)
fluorescence polarization immunoassay (34). Between-batch
coefficients of variation were, respectively, 7.4% for folate,
7.1% for B12, 8.2% for holo-transcobalamin and total transcobalamin, 10% for holo-haptocorrin, and 3.3% for tHcy. Each
batch contained an approximate 1:1 mix of case and control
samples, and laboratory staff were blind to case-control
status. Assays that gave out-of-range results were repeated
with diluted (if too high) or larger (if too low) samples. Full
results were obtained for all bar one sample (insufficient
volume for tHcy assay). Folate was measured as
<2 nmol/L in four participants and holo-transcobalamin
as <9 pmol/L in 13 participants; values of 1.8 nmol/L
and 8 pmol/L, respectively, were substituted for these
results. Holo-haptocorrin concentration was calculated by
subtracting holo-transcobalamin concentration from B12
concentration; hence, it does not include cobalamin
Other covariates
Self-reported data on ethnicity, smoking, alcohol,
medications and dietary supplements, family history
of prostate cancer (father and brother), height, and
weight were collected from Diet, Health and Lifestyle
questionnaires, which are completed before the receipt
of the initial PSA test result. Self-reported height was
used to calculate body mass index (BMI, kg/m2), along
with nurse-measured weight where available (92.5% of
cases and 92.8% of controls) and self-reported weight
Systematic review and data extraction
Eligible studies of the association of prostate cancer
risk with serum or plasma folate, B12, or tHcy levels were
identified by searching the Medline and Embase online
databases using text search terms for “folate/folic,”
“B12/cobalamin,” and “tHcy,” each in conjunction with
the MeSH heading “Prostatic Neoplasms” and text terms
“prostate cancer” and “prostatic carcinoma.” No language or publication date restrictions were imposed.
All databases were last searched on September 26,
2009. References of retrieved articles were screened.
Case-control and cohort studies that reported associations of blood (serum or plasma) levels of folate, B12,
and tHcy with prostate cancer risk were included. We
also included data from the placebo arms of randomized
controlled trials of folic acid and B12 supplementation.
Studies reported their results in several different ways
and presented various models with different adjustments. We selected the age-adjusted estimate or a more
fully adjusted estimate where available, except where
the model was deemed by us to be overadjusted (e.g., adjusted for vegetable intake). Data were extracted independently by two investigators (SMC and RH).
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
Statistical methods
Vitamins and metabolites. Circulating vitamin and metabolite concentrations were categorized into quartiles
(with cut-points based on the their distributions among
controls), and ORs as a measure of relative risk of prostate
cancer per quartile of vitamin and metabolite were estimated by conditional logistic regression to account for
the matching variables (5-y age group and recruiting general practice), further adjusted for exact age as a continuous variable. Linear trends across quartiles were tested in
these models using the mean value for each quartile. ORs
for associations with advanced and localized cancer versus controls were compared using a multinomial logistic
regression model. This model provides a statistical test for
heterogeneity in ORs comparing associations of the vitamins and metabolites of interest with localized versus advanced prostate cancers. It is an unconditional model;
hence, it was adjusted for exact age and the study center
where the recruiting general practice was based (ninelevel variable). Pairwise correlations between circulating
vitamin and metabolite concentrations (and with PSA
level) in controls were measured by Spearman's rank correlation coefficient. Given previous suggestions of possible
U-shaped relationships (23), we used fractional polynomials to investigate possible departures from linearity in
the relationships between vitamin and metabolite levels
(as continuous measures) and prostate cancer risk (35). Circulating vitamin and metabolite concentrations were natural log transformed (all had nonnormal distributions) for
inclusion in multivariable linear regression models. These
models were used to assess potential confounders (see
“other covariates”), to assess the effects of mutually adjusting vitamin and metabolite levels for each other, and to test
for interaction between folate and B12, and between folate
and alcohol on prostate cancer risk. We investigated by linear regression whether log-transformed vitamin and metabolite concentrations were associated with PSA level
among controls because any such association could bias
the PSA-based detection of prostate cancer.
Meta-analysis. To compare across studies, we calculated the log OR or hazard ratio per unit increase in vitamin
and metabolite concentration. For studies presenting their
results within categories of exposure (e.g., quantiles), we
used the mean or median exposure in each category when
they were reported and calculated the log OR per unit
increase in exposure using the method of Greenland
and Longnecker (36). When the mean or median in each
group was not reported and a range of exposure in each
group was given, we estimated the mean exposure in
each group using the method of Chêne and Thompson
(37). Having fitted means to each group, the data were analyzed using the Greenland and Longnecker method (36).
We used Stata's metainf command to investigate whether
the exclusion of any one study would significantly change
the pooled estimate, that is, whether the pooled point
estimate with one study excluded would lie outside the
95% confidence interval (95% CI) of the pooled estimate
with all studies included (38).
Cancer Epidemiology, Biomarkers & Prevention
Folate, B12, and Prostate Cancer
Software. All statistical analyses were done using Stata
Release 11 (StataCorp.).
and with B12 (correlation coefficient = 0.22). None of the
vitamin or metabolite concentrations were associated
with PSA levels among controls.
Baseline characteristics
Of the 3,019 cases and controls for whom plasma concentrations were measured, 51 were in unmatched strata.
The remaining 2,968 men were in 587 strata. There was a
small surplus of controls in 66 strata and a small deficit in
29 strata; hence, the final analysis was based on 1,461 cases
[1,298 (89%) localized, 163 (11%) advanced] and 1,507 controls. There were no differences in the baseline characteristics of cases and controls (Table 1), but anthropometric
(height and weight) and life-style data (smoking, alcohol
consumption, and vitamin supplement) were missing
(mainly due to nonreturn of questionnaires) for a higher
proportion of controls (26-27%) than cases (18-19%).
The majority of pairwise combinations of folate, B12,
holo-haptocorrin, holo-transcobalamin and total transcobalamin, and tHcy were correlated (Supplementary
Table S1). Folate was most strongly correlated with tHcy
(correlation coefficient = −0.51), but was also correlated
with holo-transcobalamin (correlation coefficient = 0.31)
Plasma vitamin and metabolite levels, and prostate
cancer risk
Of the six vitamins and metabolites, circulating concentrations of B12, holo-haptocorrin, and total transcobalamin
were associated with prostate cancer risk in the basic conditional logistic regression models (Table 2). Higher quartiles of B12 showed a trend (Ptrend = 0.06) toward increased
risk, although this positive association was evident only
weakly for the highest versus lowest quartile (OR = 1.17;
95% CI, 0.95-1.43; P = 0.1). Holo- haptocorrin concentration was positively associated (Ptrend = 0.006) with prostate cancer risk (OR = 1.27; 95% CI, 1.04-1.56; P = 0.02
comparing highest versus lowest quartiles). Total transcobalamin had an inverse association (Ptrend = 0.04) with
risk of localized prostate cancer, evident only weakly for
the highest versus lowest quartile (OR = 0.80; 95% CI,
0.64-1.00; P = 0.1).
For B12, total transcobalamin, and holo-haptocorrin,
the lowest (or highest) ORs appeared in the second
quartile, but models incorporating fractional polynomial
Table 1. Baseline characteristics of participants stratified by case-control status
Ethnicity (white)
BMI (kg/m2)†
Tobacco use†
Alcohol consumption in
the past 12 mo†
Mean (SD)
Mean (SD)
Current smoker
Ever smoked
Almost daily or more often
Once or twice per week
Once or twice per month
Special occasions or never
Vitamin supplement use in
past 12 mo (yes/no)†
Circulating vitamin and metabolite
Folate (nmol/L)
Vitamin B12 (pmol/L)
Holo-haptocorrin (pmol/L)
Holo-transcobalamin (pmol/L)
Total transcobalamin (pmol/L)
tHcy (μmol/L)
Median (5th-95th percentile)
(n = 1,461)
(n = 1,507)
62.5 (5.1)
27.9 (3.7)
62.3 (5.1)
28.2 (4.1)
Median (5th-95th percentile)
*χ2 test (proportions), Student's t test (means), or two-sample Wilcoxon rank-sum (Mann-Whitney) test (medians).
Numbers of cases and controls for whom these data were available: BMI (1,183 cases and 1,105 controls), current smoker (846
cases and 808 controls), ever smoked (1,208 cases and 1,117 controls), alcohol consumption (1,204 cases and 1,116 controls), and
vitamin supplementation (875 cases and 831 controls).
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
Collin et al.
Table 2. ORs for prostate cancer by categorical (quartiles) and continuous (loge‐transformed) measures
of plasma vitamin and metabolite concentrations
Quartiles in controls
(n = 1,507)
All cases
(n = 1,461) vs
Localized cases
(n = 1,298) vs
Advanced cases
(n = 163) vs
OR* (95% CI)
OR* (95% CI)
OR* (95% CI)
Folate (nmol/L)
Per loge
0.94 (0.84-1.06)
Vitamin B12 (pmol/L)
Per loge
1.19 (0.98-1.43)
Holo-haptocorrin (pmol/L)
Per loge
1.21 (1.01-1.44)
Holo-transcobalamin (pmol/L)
Per loge
0.99 (0.86-1.14)
Total transcobalamin (pmol/L)
Per loge
0.76 (0.55-1.05)
Per loge
0.90 (0.69-1.19)
1.00 (Reference)
0.67 (0.39-1.18)
1.47 (0.89-2.45)
0.71 (0.42-1.22)
0.96 (0.86-1.08)
0.90 (0.67-1.21)
Pheterogeneity‡ = 0.5
1.00 (Reference)
0.70 (0.40-1.22)
0.93 (0.55-1.57)
1.00 (0.61-1.65)
1.21 (0.99-1.48)
1.04 (0.64-1.68)
Pheterogeneity‡ = 0.4
1.00 (Reference)
0.85 (0.50-1.44)
0.68 (0.39-1.19)
1.24 (0.75-2.04)
1.23 (1.02-1.48)
1.05 (0.67-1.64)
Pheterogeneity‡ = 0.3
1.00 (Reference)
1.02 (0.60-1.75)
1.00 (0.61-1.65)
0.85 (0.50-1.44)
1.01 (0.87-1.18)
0.93 (0.66-1.32)
Pheterogeneity‡ = 0.7
0.71 (0.50-0.99)
1.45 (0.65-3.25)
= 0.06
1.00 (Reference)
0.93 (0.54-1.59)
1.16 (0.68-1.95)
1.05 (0.61-1.79)
0.88 (0.66-1.17)
0.94 (0.47-1.91)
Pheterogeneity‡ = 0.3
*From conditional logistic regression matching on 5-y age group and recruiting center, further adjusted for exact age (continuous).
From conditional logistic regression matching on 5-y age group and recruiting center, further adjusted for exact age (continuous),
using quartile means as a linear variable.
From multinomial logistic regression, adjusted for age (continuous) and study center, using log-transformed concentration.
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
Cancer Epidemiology, Biomarkers & Prevention
Folate, B12, and Prostate Cancer
terms for each vitamin or metabolite as continuous variables gave no indication of departure from linearity (all
P > 0.1). There were no differences in the associations of
vitamin or metabolite concentrations with prostate cancer according to whether the cancer was localized or advanced with the exception of total transcobalamin
concentration, which was inversely associated with the
risk of localized cancer but not associated with advanced cancer (Pheterogeneity = 0.06).
Table 2 also shows age-adjusted estimates for each vitamin and metabolite as continuous log-transformed variables (OR per log e approximates to a doubling in
concentration) that were included in multivariable conditional logistic regression models. We found little or no
change in the estimates for each vitamin and metabolite
when mutually adjusted for each other, and there was no
interaction between levels of folate and B12. In multivariable conditional logistic regression based on men for
whom anthropometric (BMI) and life-style (smoking,
alcohol consumption, and vitamin supplementation)
data were available (811 cases and 779 controls), these
covariates did not confound the associations of vitamin
and metabolite concentrations with prostate cancer risk,
and there was no interaction between levels of folate
and alcohol intake.
Systematic review and meta-analysis
Our literature search identified 414 studies, of which
20 were eligible. Of these, six, five, and three studies
provided data on blood concentrations of folate (7, 13,
15, 18, 19, 21), B12 (7, 13, 15, 18, 21), and tHcy (7, 13, 21),
respectively (Supplementary Table S2). The excluded
studies reported data on dietary folate or B12 intake only, or concentrations of other folate pathway vitamins
and metabolites, or outcomes other than prostate cancer
risk. The results of the Hultdin et al. study (13) were
replicated in a subgroup analysis by Johansson et al.
(15) using the same samples but a different assay
(Lactobacillus leichmannii microbiological assay instead
of Quantaphase II radioassay) and adjusted for BMI,
smoking, and concentrations of folate and tHcy. We
used the Hultdin et al. data (13) although the Johansson
Figure 1. Meta-analyses of associations of circulating folate, vitamin B12, and tHcy concentrations with prostate cancer risk.
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
Collin et al.
Figure 2. Meta-analyses of associations of circulating folate, vitamin B12, and tHcy concentrations with prostate cancer risk in prospective cohort studies.
et al. (15) data gave very similar results. We used
unpublished data12 from the placebo arms of two randomized controlled trials: one of folic acid and aspirin
supplementation for the chemoprevention of colorectal
adenomas (18) and one of folic acid, vitamin B12, and
vitamin B6 supplementation for the lowering of tHcy
among patients with ischaemic heart disease (21).
We conducted dose-response meta-analyses, combining our results for serum concentrations of folate, B12,
and tHcy with those extracted from the literature and
those obtained directly from authors (Fig. 1). The pooled
(random effects) estimates were as follows: OR of 1.11
(95% CI, 0.96-1.28) per 10 nmol/L folate (P = 0.2), OR
of 1.10 (95% CI, 1.01-1.19) per 100 pmol/L B12 (P = 0.002),
and OR of 0.91 (95% CI, 0.69-1.19) per 10 μmol/L tHcy
(P = 0.5). Hence, there was no strong statistical evidence
for an association of folate or tHcy with prostate cancer
risk, but there was evidence to support a 10% higher
Unpublished data.
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
odds per 100 pmol/L increase in circulating B12. There
was some heterogeneity in the associations of folate
(I2 = 40%) and B12 (I2 = 47%) with prostate cancer risk.
There were too few published studies to assess smallstudy bias. Influence analysis using Stata's metainf command showed that excluding any one study from the
meta-analysis of associations of B12 and tHcy with prostate cancer risk did not significantly change either the
fixed or random effects pooled estimates (Fig. 2). However, exclusion of the ProtecT case-control study from
the meta-analysis of the associations of folate with prostate cancer risk changed the fixed-effects pooled estimate
to an OR of 1.19 (95% CI, 1.03-1.37) per 10 nmol/L folate
(P = 0.02) and left little heterogeneity between the remaining studies (I2 = 13%), all of which were prospective
cohort studies (Fig. 2). The median baseline concentrations of B12 and tHcy were similar in all studies, but baseline concentrations of folate were higher in the Figueiredo
(USA) and ProtecT studies (Fig. 2), and among nonSwedish subjects in the Johansson (pan-European) study.
The number of studies was too small to support metaregression analysis to test whether the measures of effect
Cancer Epidemiology, Biomarkers & Prevention
Folate, B12, and Prostate Cancer
in each study were related to these baseline values. However, examination of effect estimates against the median
baseline values of folate, vitamin B12, and tHcy gave no
indication that heterogeneity between studies was attributable to differences in baseline concentrations
Data from the ProtecT study, when combined with results from all other studies, suggest that high circulating
concentrations of vitamin B12 may be associated with an
increased risk of prostate cancer (Fig. 1). The ProtecT data
also showed that high circulating concentrations of holohaptocorrin were associated with increased risk, and high
circulating concentrations of total transcobalamin with
decreased risk of prostate cancer. We found no association of folate with prostate cancer risk in the ProtecT data.
This result strongly influenced the meta-analysis, which
would otherwise have shown a clear positive association
of circulating folate with increased prostate cancer risk
(Fig. 2). It is possible that folate is positively associated
with the rate of progression of localized prostate cancer;
hence, the ProtecT study (based on PSA-detected prevalent cases) would not detect an effect observed in European cohort studies based mainly on clinically detected
cases. We found no associations of tHcy with prostate
cancer risk, either in our own data or in the meta-analysis.
Although there was considerable variation in baseline folate levels between studies, this did not seem to explain
between-study differences in the measures of association
of folate with prostate cancer risk. This is consistent with
Johansson et al. (15) who found no difference of effect between Swedish versus non-Swedish subjects in their panEuropean study, despite baseline levels of folate being
much lower in Sweden.
The ProtecT study is by far the largest study to date of
associations of circulating folate pathway vitamin and
metabolite concentrations with risk of prostate cancer,
contributing 81%, 48%, and 62% by (inverse variance)
weight to the fixed effects meta-analytic results for folate,
B12, and tHcy, respectively (Fig. 1). The coefficients of
variation for our assay results were low, and any measurement error would attenuate effect estimates to the
null rather than generate the observed associations. The
study's main limitation is that blood samples were drawn
after the occurrence of disease; hence, causality cannot be
directly inferred from our results. However, men were
unaware of their disease status, so they were unlikely
to have changed their behavior or diet in response to
the disease. In addition, our results were similar for advanced and localized disease, which is contrary to what
would be expected if effects were secondary to disease
status. As with all prostate cancer case-control studies
based on PSA testing followed by biopsies, some measurement error would be present due to the imperfect nature of the diagnostic process (39). Non-Caucasian men
were not represented in the ProtecT study, or in any of
the other studies in our meta-analysis [except for a small
proportion (7%) in the Aspirin/Folic Acid Polyp Prevention trial]; therefore, our findings may not be generalizable to all populations. The small number of advanced
cases in ProtecT means we may have been underpowered to detect true differences in associations of vitamin
or metabolite concentrations with prostate cancer according to whether the cancer was localized or advanced.
Fractional polynomial analysis of the ProtecT data did
not indicate departure from linearity for the associations
of B12, holo-haptocorrin, and total transcobalamin with
prostate cancer risk, but these associations were evident
only in the top quartiles when analyzed as categorical
variables. Whether there really is only an effect at extreme elevations could not be discerned from our review
of the literature, although two studies showed a similar
pattern, with associations evident only in the top quartile
of circulating folate (13, 15).
That we found no confounding or effect modification
by BMI, smoking, or alcohol consumption of associations
between vitamin and metabolite concentrations and
prostate cancer is consistent with other studies (7, 13,
14, 20). Among men with prostate cancer in the ProtecT
study, those who reported little or no sexual activity had
higher plasma B 12 concentrations (data not shown).
However, among men with raised PSA levels in the ProtecT study, there was no association between sexual dysfunction and prostate cancer; hence, confounding by
sexual activity is unlikely to explain our findings (40).
The positive association of B12 with prostate cancer risk
could be causal, or due to reverse-causation or coincidental. A causal association would be consistent with the epigenetic mechanisms of prostatic carcinogenesis if these
were triggered by elevated levels of B12 independently
of folate and tHcy. Although some associations of B12
with DNA methylation have been observed in rats
(41, 42) and humans (43, 44), such a process as a cause
of prostate cancer remains speculative. Vitamin B12 is
an essential cofactor of methionine synthase, and we reported in a previous meta-analysis that the A2756G polymorphism of this enzyme is associated with an increased
risk of prostate cancer (45). We proposed a causal “activating polymorphism” mechanism that might be consistent with our finding for B12, but this is speculative.
One possible mechanism of reverse causation could be
prostate tumor cells having an increased demand for B12
due to the increased biosynthesis of polyamines (46),
which in turn upregulates the activity of methionine
synthase (47). Another mechanism could be that elevated
levels of plasma B12 are due to the increased production of
haptocorrin by prostate tumor cells (48), an effect which
may explain high levels of B12 observed in myelogeneous
leukemias and metastatic cancers (30). Whether prostate
carcinomas can raise plasma concentrations of haptocorrin and B12 by such processes remains hypothetical, and
we did not observe stronger positive associations of plasma B12 and holo-haptocorrin with advanced versus localized
prostate cancer. However, the plausibility of reverse causation as an explanation for our findings is perhaps reinforced
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
Collin et al.
by the absence of an association of holo-transcobalamin,
representing the bioavailable fraction of B12, with prostate
cancer risk. We cannot suggest a clear biological explanation
for the inverse association of total transcobalamin with localized prostate cancer. Finally, it is conceivable that folate pathway genes could be affected coincidentally by epigenetic
alterations that play a causal role in prostate cancer. For example, the multidrug resistance protein gene (MRP1), which
is overexpressed in several cancers including prostate cancer
(49), has recently been shown to play a role in cellular efflux
of B12 (50).
That we found similar associations of folate, B12, and
tHcy with advanced and localized prostate cancer is consistent with previous studies (7, 13-15), although Johansson
et al. (15) reported (from a cohort study) borderline
heterogeneity (P = 0.05) between localized and advanced
cases for B12: a doubling in vitamin B12 concentration
was associated with an OR of 1.69 (95% CI, 1.05-2.72;
P = 0.03) of advanced cancer, whereas B12 concentrations were not associated with risk for localized disease
(OR = 0.96; 95% CI, 0.71-1.29; P = 0.8). We found marginal evidence of similar heterogeneity (P = 0.09) for
total transcobalamin [advanced disease OR = 1.46
(95% CI, 0.65-3.26); localized disease OR = 0.70 (95%
CI, 0.50-0.99)]. Levels of B12 and total transcobalamin in
our data were uncorrelated, and our finding may be
due to chance rather than a corroboration of Johansson
et al.'s (15) finding for B12.
The only studies to date of dietary B 12 intake and
prostate cancer risk both reported positive associations:
Vlajinac et al. (12) found 2-fold higher odds (OR = 2.07;
95% CI, 1.08-3.97; Ptrend = 0.02) for the highest versus
lowest tertile, and Weinstein et al. (among smokers; ref.
14) found 36% higher odds in the highest versus lowest
quintile (OR = 1.36; 95% CI, 1.14-1.62; Ptrend = 0.01). Both
studies reported that these results withstood adjustment
for dietary covariates: Vlajinac et al. (12) for intake of
total energy, protein, total fat, saturated fatty acids,
carbohydrate, total sugar, fiber, retinol equivalent,
α-tocopherol, folate, sodium, potassium, calcium, phosphorus, magnesium, and iron, and Weinstein et al. (14)
for total energy, total protein, animal protein, total fat,
animal fat, folate, B6, methionine, iron, and specific
foods that are correlates of B12 intake (fish, organ meats,
sausages, cholesterol, fatty acids, vitamins, and minerals). Hence, there was no confounding by other nutrients that cooccur in foods high in B12 and which may
be associated with prostate cancer risk. Both studies also
adjusted for nondietary covariates. Although these results may suggest a possible causal relationship, the
limitations of studies based on food frequency questionnaires are well known, particularly with regard to dietcancer associations (51). Indeed, the B12 dietary intake
finding by Weinstein et al. (7) was not found in their
earlier, albeit smaller, study of circulating concentrations
of B12.
We conclude that our finding of a positive association
of circulating B12 with increased prostate cancer risk could
Cancer Epidemiol Biomarkers Prev; 19(6) June 2010
be explained by reverse causality. However, given current
controversies over mandatory B12 fortification (52), further research to eliminate a causal role of vitamin B12 in
prostate cancer initiation and/or progression is required,
including Mendelian randomization analyses (53) and
repeat measurements of B12 and holo-haptocorrin levels
during prostate cancer development and/or before and
after treatment. Our meta-analysis did not entirely rule
out a positive association of circulating folate with increased prostate cancer risk. As with B12, even a weak
positive association would be a significant public health
issue, given the high prevalence of prostate cancer, and
a legitimate concern about the potential harms versus
benefits of mandatory folic acid fortification (23).
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Author Contributions: S.M. Collin, C. Metcalfe, and R.M. Martin had
full access to all of the data in the study and take final responsibility for
the integrity of the data, the accuracy of the data analysis, and the
decision to submit for publication. Hypothesis development and acquisition
of funding: R.M. Martin (principal investigator), G.D. Smith, H. Refsum,
A.D. Smith, S.J. Lewis, C. Metcalfe, J. Donovan, D.E. Neal, and F.C.
Hamdy (coinvestigators). Original ProtecT study design: J. Donovan, D.E.
Neal, and F.C. Hamdy. Acquisition of data: S.M. Collin, R.M. Martin,
M. Davis, G. Marsden, C. Johnston, J.A. Lane, M. Ebbing, K.H. Bønaa,
O. Nygård, P.M. Ueland, M.V. Grau, and J.A. Baron. Analysis and
interpretation of data: S.M. Collin, C. Metcalfe, H. Refsum, R. Harris,
M. Ebbing, M.V. Grau, and R.M. Martin. Drafting of the manuscript:
S.M. Collin. Critical revision of the manuscript for important intellectual
content: S.M. Collin, C. Metcalfe, H. Refsum, S.J. Lewis, L. Zuccolo,
G.D. Smith, L. Chen, R. Harris, J.A. Lane, M. Ebbing, K.H. Bønaa,
O. Nygård, P.M. Ueland, M.V. Grau, J.A. Baron, J. Donovan, D.E. Neal,
F.C. Hamdy, A.D. Smith, and R.M. Martin. Statistical analysis:
S.M. Collin, C. Metcalfe, M. Ebbing, and M.V. Grau. Administrative,
technical, or material support: S.M. Collin, M. Davis, G. Marsden,
C. Johnston, and J.A. Lane. Day to day senior study supervision:
R.M. Martin and C. Metcalfe.
Additional Contributions: We thank all members of the ProtecT study
research group for the tremendous contribution; especially the following
who were involved in this research: Prasad Bollina, Sue Bonnington,
Debbie Cooper, Andrew Doble, Alan Doherty, Emma Elliott, David
Gillatt, Pippa Herbert, Peter Holding, Joanne Howson, Liz Down,
Mandy Jones, Roger Kockelbergh, Howard Kynaston, Teresa Lennon,
Norma Lyons, Hilary Moody, Philip Powell, Stephen Prescott, Liz
Salter, and Pauline Thompson; and Cynthia Prendergast (University of
Oxford) for her invaluable help with the biochemical analyses.
Grant Support
World Cancer Research Fund UK (grant number: 2007/07). The National Cancer Research Institute (administered by the Medical Research
Council) provided support for the development of the ProtecT epidemiologic database through the Prostate Mechanisms of Progression and Treatment collaborative. The ProtecT study is supported by the UK NIHR
Health Technology Assessment Programme (projects 96/20/06, 96/20/
99). Support for the ProtecT biorepository in Cambridge is provided by
NIHR through the Biomedical Research Centre.
Role of the Funders: The funders were nonprofit organizations with no
participating role in the study.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received 02/17/2010; revised 03/30/2010; accepted 04/05/2010;
published OnlineFirst 05/25/2010.
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