Circulating Vitamin D Levels and Risk of Colorectal Cancer in Women

Abstract

Observational data on the association between circulating 25(OH)D and colorectal cancer risk are limited in women. To determine whether prediagnostic levels of 25(OH)D were associated with risk of incident colorectal cancer in the Women’s Health Study (WHS), we conducted a nested case-control study using 274 colorectal cases and 274 controls. Each case was matched to a control by age, ethnicity, fasting status at the time of blood collection, time of day when blood was drawn, and month of blood draw. Conditional logistic regression was used to estimate the odds ratio (OR) and 95% confidence interval (CI) for colorectal cancer by 25(OH)D quartiles. Mean plasma 25(OH)D was lower in cases versus controls (21.9 vs 23.9 ng/mL, p=0.01). In multivariable-adjusted logistic regression models, plasma 25(OH)D was significantly and inversely associated with odds of colorectal cancer (quartile 4 [Q4] versus quartile 1 [Q1]: OR (95% CI): 0.45 (0.25–0.81), p_trend 0.02). Additionally, we observed a somewhat lower risk of colorectal cancer mortality after adjustment for matching variables, randomization treatment and other risk factors (Q4:Q1 OR[95%CI]: 0.40(0.17–0.97); p_trend 0.05). In this cohort of healthy women, we found a significant inverse association between prediagnostic 25(OH)D levels and risk of incident colorectal cancer, and a borderline significant inverse association between prediagnostic 25(OH)D levels and colorectal cancer mortality. These results support a possible association between plasma 25(OH)D and risk of colorectal cancer in women.

Introduction

Colorectal cancer (CRC) is the 3rd leading cause of cancer incidence and death in the US. (1) Laboratory studies suggest that vitamin D and its analogues may inhibit colorectal cancer development and growth through regulation of cellular proliferation and differentiation (2–5) and inhibition of angiogenesis. (6) Based mainly on studies of bone health, a recent report from the Institute of Medicine recommended dietary intake to achieve circulating 25-hydroxyvitamin D (25[OH]D) of >20 ng/mL (50 nmol/L). (7) Epidemiology studies of vitamin D intake and predicted vitamin D plasma status have been fairly consistent in suggesting an association between higher vitamin D and reduced colorectal cancer risk,¹⁰,(8) but limited data exist on women. (9) (10) (11) (12) A recent observational study meta-analysis reported a 26% lower risk of colorectal cancer per 10 ng/mL increment in blood 25(OH)D levels. (13) Vitamin D supplementation randomized control trials have not been supportive of an association between vitamin D and colorectal cancer, but this lack of association may be influenced by the dose of vitamin D used or the duration of the intervention. (14, 15) Among the 36,000 postmenopausal women in the Women’s Health Initiative (WHI), daily calcium (1000 mg) plus low-dose vitamin D₃ (400 IU) did not reduce total cancer incidence, but there was a suggestion of a protective effect against total cancer mortality over the 7-yr follow-up. (14, 15) Beyond any influence on cancer incidence, recent studies suggest that vitamin D could represent an important determinant of cancer survival. (16)

Given the strong laboratory data and conflicting findings from epidemiologic studies, we prospectively examined the association between plasma 25(OH)D and colorectal cancer risk in a large cohort of initially healthy women where plasma samples were collected prior to cancer diagnosis. Plasma 25(OH)D level considered the best indicator for reflecting overall vitamin D status. 25(OH)D is the precursor of 1,25(OH)₂D which binds to vitamin D receptor (VDR), to exert its growth inhibitory effects in colon cancer cells through regulation of several genes that are responsible for cell proliferation, differentiation and apoptosis. (17) We hypothesized that higher 25(OH)D levels would be associated with reduced risk of incident colorectal cancer and colorectal cancer mortality in women.

Materials and Methods

Study Population

The Women’s Health Study (WHS) is a completed randomized, placebo-controlled double-blinded trial originally designed to examine the role of aspirin (100mg every other day) and vitamin E (600 IU every other day) in the prevention of cancer and cardiovascular disease (CVD) among 39,876 women free of cancer and CVD. The randomized controlled trial began in 1992. Every 6 months for the first year and every year thereafter, participants received questionnaires that assessed their compliance with study drug, potential side effects, and clinical outcomes of interest. When the trial ended in 2004, 33,682 women (88.6% of those alive) consented to continue with observational follow-up, reporting on their health habits and medical history annually on questionnaires.

For cases of invasive colorectal cancer reported during the trial or post-trial period, subjects granted written consent for medical record review. Medical records were retrieved for all women that reported a diagnosis of CRC. The retrieved medical records were subsequently reviewed by WHS designated physicians to identify any endpoints. The designated physician reviewers were blinded to the treatment assignment. Cases were individually matched to controls by age (+/− 1 year), ethnicity, fasting status at time of blood collection (>8 hours versus not), time of day when blood was drawn (+/− 4 hours), and month of blood draw. Morbidity follow-up rates were complete for 97.2% and mortality follow-up rates for 99.4%. A total of 453 CRC cases have been identified in the WHS cohort as of 2011 during a mean follow-up duration of 17 years; 321 (71%) provided blood and 132 (29%) did not provide blood; such distribution is also similar to that in the entire WHS blood cohort. The design of 1 case matched to 1 control has yielded a total of 274 CRC cases available for the present study.

Baseline blood collection

Prior to randomization in the WHS, blood was collected by mail using a blood-collection kit containing instructions, tubes, blood draw supplies, a gel-filled freezer pack, and a completed overnight courier air bill. Women were asked to freeze the gel-filled freezer pack overnight to serve as a coolant and to return the completed blood kit to us via overnight courier. Of the 39,876 randomized women in the trial, 28,345 (71%) provided a baseline blood sample. Women who did and did not donate blood were similar for a wide range of variables related to cancer. (18)

25(OH)D Plasma Assay

Plasma 25(OH)D was assayed at Heartland Assays, Inc. (Ames, Iowa) and was measured using a Diasoran radioimmunoassay. All samples for plasma 25(OH)D were shipped in a single batch to the reference laboratory, with laboratory personnel blinded to case, control, or quality control status. The mean intra-assay coefficient of variance for blinded, replicate quality control samples was 6.0 %.

Statistical analysis

Using a nested case-control design with 1:1 matching (by age, ethnicity, month of blood draw, fasting more than 8 hours status) we evaluated whether the risk of colorectal cancer is modified by baseline plasma levels of 25(OH)D. The distributions of baseline characteristics by cancer and control groups were compared using standard measures for two-sample testing, such as t-test and McNemar test. 25(OH)D in control subjects was categorized by quartiles. A conditional logistic regression model was used to calculate the odds ratios (ORs) and 95% confidence intervals (CIs). Tests for trend were calculated by using the median values for the quartiles of 25(OH)D. All P-values were two-tailed. The simple model included the following covariates: season of blood draw and randomized treatment assignment to aspirin or vitamin E. For multivariable-adjusted models, we additionally included established risk factors for CRC assessed at baseline including family history of colorectal cancer, red meat intake, dietary calcium intake, alcohol intake, postmenopausal hormone use, body mass index (BMI), physical activity, smoking status, multivitamin use. Missing values for each covariate, if applicable, were coded as an indicator variable to indicate missing. When analyzing rectal cancer risk and colorectal cancer mortality, we utilized unconditional logistic regression with simple and multivariable adjustment as described above in addition to adjustment for matching factors in either models. Cutpoints for the unconditional models were based on the remaining cases and controls available in the analysis. To evaluate whether aspirin assignment may be an effect modifier for the association between 25(OH)D and CRC development, we treated 25(OH)D as a continuous variable and aspirin assignment as a binary variable (yes/no) for the interaction term in the multivariable regression model. To examine whether the association between 25(OH)D and risk of CRC was modified by other risk factors of CRC, we performed preplanned subgroup analyses using unconditional logistic regression adjusted for the matching factors and other relevant covariates. We examined the association in subgroups defined by BMI (BMI< 30 kg/m², BMI ≥30 kg/m²) and physical activity (above median and below median of reported physical activity). Test for interaction of postmenopausal hormone use, BMI, and physical activity and plasma 25(OH)D on the association between plasma 25(OH)D and colorectal cancer were performed by the Wald test of cross-product terms. We used SAS 9.2 for all analyses.

Results

The median follow-up for this study population is 16.3 years. At baseline, characteristics were similar between CRC cases and controls with regards to BMI, current smoking, family history of CRC, physical activity, and intakes of alcohol, red meat, calcium, and multivitamins (Table 1). There was no difference between cases and controls in baseline postmenopausal hormone use and screening exams. Cases and controls were also similar in blood drawn seasons. However, the baseline plasma 25(OH)D was significantly lower in CRC cases than in controls (cases: 21.9 ng/mL; controls: 23.9 ng/mL, p=0.01). CRC colonoscopy screening rates were similar across 25(OH)D quartiles (p=0.09). Randomized assignment to aspirin and vitamin E was similar in cases and controls. The multivariate-adjusted partial correlations of 25(OH)D with BMI, physical activity, and smoking were −0.21, 0.12, and −0.13, respectively (p values <=0.06 (Table 2).

Table 1.

Baseline characteristics (mean or %) among cases of colorectal cancer and matched controls in the Women’s Health Study^*

Characteristics	Cases	Controls	Pvalue
	Cancer (N=274)	(N=274)
Age, year (SD)	58.8 (8.3)	58.8 (8.3)	matched
Race-Caucasian, %	96.4	96.4	matched
Body mass index, kg/m2	26.7	26.3	0.33
Current smoking (%)	9.9	11.4	0.57
Family history of colorectal cancer (%)	12.0	9.9	0.38
Colorectal cancer screening (%)†	9.4	16.7	0.66
Postmenopausal (%)	74.1	76.3	0.38
Current postmenopausal hormone use (%)††	44.5	41.8	0.29
Current use of multivitamins (%)	36.5	38.0	0.72
Blood drawn in the summer (%)	49.8	44.7	0.33
Physical activity (MET-hrs/week)	15.3	16.5	0.48
Alcohol intake (g/day)	5.1	4.8	0.78
Red meat (servings/day)	0.7	0.7	0.76
Total calcium intake (mg/day)	1042	1062	0.62
Plasma 25(OH)D levels, mean (SD)	21.9 (8.3)	23.9 (9.8)	0.005
Randomized to aspirin treatment (%)	23.2	25.2	0.35
Randomized to vitamin E treatment (%)	22.8	25.4	0.23

^*Mean is reported for continuous variables.

^†Information was obtained at the 12-month follow-up questionnaire. Assessment of colorectal cancer screening included only colonoscopy or flexible sigmoidoscopy.

^††Postmenopausal hormone use is for all cases (perimenopausal and postmenopausal)

Table 2.

Partial correlation of 25(OH)D with BMI, physical activity, and smoking*

Characteristic	Correlation coefficient	Pvalue
BMI (kg/m2)	−0.21	0.0007
Physical Activity(Total Met-hrs/week)	0.12	0.06
Current Smoking (yes/no)	−0.13	0.05

Multivariate model adjusted for randomized treatment assignment to aspirin and vitamin E, season for blood collection (summer, yes/no), body mass index (BMI) (weight (kg)/height (m)²), physical activity (MET-hours/week), family history of colon cancer), smoking status (current), alcohol consumption (g/day), multivitamin use (never, past, current), red meat (servings/day), calcium (mg/day), menopausal status and hormone therapy use.

Table 3 shows the association between plasma 25(OH)D levels and CRC risk in both simple and multivariable models. First, a significant association was observed between plasma 25(OH)D level and the risk for CRC and colon cancer in the simple models. A similar association was observed in the multivariable-adjusted conditional logistic regression models (CRC: quartile 4 [Q4] versus quartile 1 [Q1], OR (95% CI): 0.46 (0.24-0.89), p_trend 0.03; colon, Q4 versus Q1 OR (95% CI): 0.44 (0.21–0.95), p_trend 0.04). Because of a small number of cases, risk for rectal cancer was evaluated using unconditional logistic regression; no significant association was observed between plasma 25(OH)D levels (Q4 versus Q1, OR (95% CI): 0.84 (0.34–2.08), p_trend 0.90) (Table 2). When we used clinical cutpoints with 25(OH)D less than 20 ng/mL as the reference group, the multivariable-adjusted OR (95% CI) of CRC risk for 20–30 ng/mL and greater than 30 ng/mL were 0.98 (0.64–1.50) and 0.58 (0.32–1.05), respectively, p_trend 0.10. In multivariate analyses, no reduction of incident CRC until the 4^th quartile suggests a threshold effect while the CRC mortality analyses suggests a threshold effect for the 2^nd to 4^th quartile. In the overall multivariable regression analysis including aspirin assignment as a covariate, aspirin was not associated with CRC development (OR =0.79, 95% CI 0.55–1.15). We also observed no effect modification by aspirin assignment (p value for interaction=0.88). The unconditional logistic regression for the association between 25(OH)D and CRC risk according to aspirin assignment revealed no clear trend in either groups. The ORs in the higher quartile groups as compared with the lowest quartile according to aspirin treatment were: (a) aspirin-treated group, Q2, 1.40 (0.70–2.78), Q3 1.86 (0.91–3.78), Q4 0.60 (0.30–1.23), p_trend 0.22; (b) aspirin-untreated: Q2 0.57 (0.29–1.13), Q3 0.70 (0.35–1.37), Q4 0.60 (0.30–1.21), p_trend 0.22.

Table 3.

Odds ratios (ORs) and 95% confidence interval (CIs) of colorectal cancer incidence and colorectal cancer mortality according to plasma levels of 25(OH)D in the Women’s Health Study

	Quartiles of 25(OH)D ng/mL				Ptrend
	Q1	Q2	Q3	Q4
	Colorectal cancer incidence
Range, ng/mL	3.7–17.4	17.5–22.3	22.4–29.3	29.4–66.0
No. of cases/controls	79/69	70/70	83/68	42/67
Simple OR†	1.0	0.83 (0.52–1.32)	0.99 (0.60–1.63)	0.45 (0.25–0.81)	0.02
Multivariable OR†	1.0	0.84 (0.50–1.42)	0.97 (0.55–1.73)	0.46 (0.24–0.89)	0.03
	Colon cancer incidence
Range, ng/mL	3.7–17.4	17.5–22.1	22.2–29.2	29.3–66
No. of cases/controls	62/52	49/50	58/51	35/51
Simple OR†	1.0	0.78 (0.45–1.34)	0.84 (0.48–1.49)	0.48 (0.25–0.92)	0.03
Multivariable OR†	1.0	0.80(0.43–1.48)	0.81 (0.41–1.59)	0.44 (0.21–0.95)	0.04
	Rectal Cancer Incidence*
Range ng/mL	4.8–17.4	17.5–22.3	22.4–28.3	28.4–66.0
No. of cases/controls	17/69	17/70	24/61	12/74
Simple OR†	1.0	0.95 (0.44–2.05)	1.67 (0.80–3.50)	0.63 (0.27–1.45)	0.45
Multivariable OR†	1.0	1.16 (0.50–2.67)	1.94 (0.86–4.38)	0.84 (0.34–2.08)	0.90
	Colorectal cancer mortality*
Range	4.8–16.9	17.0–22.0	22.1–28.1	28.2–66.0
No. of cases/controls	23/62	15/71	18/66	11/74
Simple OR†	1.0	0.59 (0.28–1.25)	0.74 (0.36–1.52)	0.42 (0.19–0.94)	0.048
Multivariable OR†	1.0	0.55 (0.25–1.28)	0.57 (0.25–1.28)	0.40(0.17–0.97)	0.051

^†Simple models were adjusted for randomized treatment assignment to aspirin and vitamin E and season for blood collection (summer, yes/no). Multivariable-adjusted models were additionally adjusted for body mass index (BMI) (weight (kg)/height (m)²), physical activity (MET-hours/week), family history of colon cancer), smoking status (current), alcohol consumption (g/day), multivitamin use (never, past, current), red meat (servings/day), calcium (mg/day), menopausal status and hormone therapy use.

^*Rectal cancer incidence and colorectal cancer mortality were evaluated using unconditional logistic regression models adjusting for matching variables (age, race, month of blood draw, fasting status at time of blood draw) because of the small number of cases.

A total of 63 CRC deaths were ascertained in this study population, We observed a significant inverse association between plasma 25(OH)D and CRC mortality in a simple model (Q4 versus Q1, OR (95% CI): 0.42 (0.19–0.94), p_trend 0.048) (Table 2). The multivariable-adjusted model showed that the association was only of borderline significance (Q4 versus Q1: OR (95% CI): 0.40 (0.17–0.97), p_trend 0.051) (Table 2).

Subgroup analysis by tumor characteristics including location (proximal, distal, rectal, p_trends respectively: 0.07, 0.11, and 0.66), stage (A, B, C/D, p_trends respectively: 0.08, 0.41, and 0.41) and grade (well and moderately differentiated, p_trend: 0.29) did not reveal significant association with plasma 25(OH)D. The only exception was found for risk of poorly differentiated tumors (Q4 versus Q1: multivariable-adjusted OR (95% CI): 0.16 (0.03–0.95), p_trend 0.03). No interaction was observed between postmenopausal hormone use (PMH), BMI or physical activity and plasma 25(OH)D for the association between plasma 25(OH)D and colorectal cancer (PMH: p for interaction=0.75; BMI: p for interaction= 0.23; physical activity: p for interaction =0.14).

Discussion

In this nested case-control study, we observed a significant inverse association between pre-diagnostic plasma 25(OH)D and subsequent risk of CRC and a borderline significant inverse relationship for CRC mortality. The significant association was mostly seen in risk for colon but not rectal cancer. No effect modification was found by postmenopausal hormone therapy, obesity and physical activity for the association between 25(OH)D and colorectal cancer. The strongest reduction in incident colorectal cancer and CRC mortality was observed for 25(OH)D levels greater than 29 ng/mL.

Our results are similar to other studies reporting an inverse association between 25(OH)D and CRC. (Table 6) ^12,(19) In the British(20) and the Women’s Health Initiative (WHI)(14) trials, vitamin D did not reduce CRC incidence overall, but nonsignificant reductions in CRC mortality were found. Interestingly, the WHI participants who had the highest baseline levels of plasma 25(OH)D experienced a significant 60% reduction in CRC risk. (16, 21) It is plausible that participants who did achieve a 25(OH)D level beyond 35 ng/mL (through means other than assigned vitamin D supplement) did experience a substantial reduction in CRC risk. (22) Further, in the WHI, although the dose of vitamin D was sub-optimal, supplemental vitamin D did confer non-statistically significant reductions for CRC mortality (RR=0.82, 95% CI = 0.52 to 1.29; P = 0.39), total cancer mortality (RR = 0.89, 95% CI = 0.77 to 1.03; P = 0.12), and total mortality (RR=0.93, 95% CI = 0.83 to 1.01; P = 0.07). (22) Recent meta-analyses have, with few exceptions,(23) found that 25(OH)D levels are inversely associated with risk for CRC(13, 24–27) and colorectal adenoma. (28–30) Table 6 shows a summary of studies that evaluated the association of circulating levels of vitamin D and risk of CRC. (24)^,(13)^{,(11, 12, 31)} The pooled relative risk (RR) of CRC for the highest versus lowest categories of 25(OH)D was 0.67 (95% CI, 0.54 to 0.80). (13) A 10 ng/mL increase in 25(OH)D value was associated with a RR of 0.74 (95% CI, 0.63 to 0.89). (13) There was no heterogeneity among studies of blood 25(OH)D values (p=0.96). (13) The other pooled analysis of Nurses’ Health Study (NHS)(10) and Health Professionals Follow-Up Study (HPFS)(32) reported a significant reduced risk (top v. bottom quintile: RR, 0.66 [0.42–1.05]). (32) Another recent meta-analysis review of 5 large prospective studies (including NHS and WHI(14)) has shown that 25(OH)D ≥ 33 ng/mL had a 51% reduction in risk of CRC compared to those with ≤ 12 ng/mL). (33) Prospective data on vitamin D status and total cancer remain limited(8, 34, 35) Yet, prospective observational studies of plasma 25(OH)D levels generally support an inverse association with risk of neoplasia. (32, 33, 36–44)

Table 6.

Prospective cohort studies of circulating levels of 25(OH)D and risk of colorectal cancer(13, 24)

Study	Country (sex)	Age at blood donation (years)a,b	Study dates (follow-up)	25(OH)D (ng/mL), Cases/controlsa,b,c,d	Range (ng/mL)e	No. of cases/controlsi	RRf,i	95% CI
Garland et al. (36)	US (M, W)	35–75	1975–1983	30.5/33.3a	23	34/67	0.73	0.20–2.66
Braun et al. (37)	US (M, W)	55b	1984–1991	23.6/23.2a	12.9	57/114	0.40	0.10–0.40
Tangrea et al. (38)	Finland (M)	60b	1985–1993	12.1/13.8a	9.5	146/290	0.60	0.30–1.10
Feskanich et al. (10)	US (W)	60.0a	1989–2000	23.6/24.3a, lab 1	20.4	193/383	0.53	0.27–1.04
				27.0/30.3a, lab 2
Wu et al. (32)	US (M)	66.1a	1993–2002	28.7/29.4a	21.0	179/356	0.66	0.42–1.05
Wactawski-Wende et al. (22)	US (W)	50–79	1993–2005	N/A	11.0	306/306	0.75	0.39–1.48
Otani et al. (39)	Japan (M)	56.9a	1990–2003	27.3/27.6b	9.2	163/324	0.73	0.35–1.50
	Japan (W)	56.4a	1990–2003	22.5/22.3b	8.3	160/297	1.10	0.50–2.30
Jenab et al. (62)	Europe (M, W)	30–77	1992–2003	20.7/22.9c	8.0	785/785g	0.77	0.56–1.06
				22.0/22.0d		463/463h
Woolcott et al. (63)	US (M, W)	69.2a	2001–2006	23.2/25.0a	16.0	229/434	0.60	0.33–1.07
Lee et al. (24)	US (M)	56.6a	1982–2000	26.6/25.6a	12.2	229/389	1.08	0.62–1.87
Weinstein SJ et al. (11)	US (M, W)	64b	1993–2001	21/22.7b	12	476/476	0.59	0.36–0.95
Wong YY et al. (31)	Australia (M)	77a	1996–2004	27.1/27.4	N/A	102/3614i	1.04i	0.94–1.14
Jung S et al. (12)	US (M, W)	66.3 (M)j 65.0 (W)j	1986–2008	28 (M)k 32 (W)k	7.8 (M) 10.4 (W)	1059/140,418i	0.55i	0.43–0.71

^amean;

^bmedian;

^cgeometric mean in colon cancer;

^dgeometric mean in rectal cancer;

^erange:the difference in the midpoint between the highest and lowest categories of 25-hydroxyvitamin D blood levels;

^fadjusted relative risks of colorectal cancer for the highest versus lowest categories of 25-hydroxyvitamin D blood level;

^gcolon;

^hrectum;

ⁱcompeting risk proportional hazard model; positive;

^jmedian age of highest quintile for men (M) and women (W);

^kpredicted 25(OH)D score, median of highest quintile.

Clinical trials on vitamin D supplementation for the prevention of cancers have been very limited. Four vitamin D trials have assessed total cancer incidence or total cancer mortality as secondary outcomes. In a British trial(20), 2686 older adults were randomized to 100,000 IU of vitamin D₃ or placebo and followed for up to 5 yrs, vitamin D was not associated with reduced total cancer incidence, but a nonsignificant inverse association for total cancer mortality was noted. In the Nebraska trial(45), 1179 postmenopausal women were randomized to one of three groups—calcium plus vitamin D₃; active calcium plus placebo vitamin D; or placebo calcium plus placebo vitamin D. Women in the calcium plus vitamin D group were significantly less likely to develop cancer than women in the placebo group. In contrast, among the 36,000 postmenopausal women in the WHI, daily calcium (1000 mg) plus low-dose vitamin D₃ (400 IU) did not reduce total cancer incidence, although there was a suggestion of a protective effect against total cancer mortality over the 7-yr follow-up. (14, 15) Lastly, in the Randomized Evaluation of Calcium OR vitamin D (RECORD) trial, 5292 adults (85% women), age 70 years or older, were randomized to vitamin D₃ (800 IU/d), calcium (1000 mg/d), both, or placebo for 24–62 months for the secondary prevention of fractures and were then followed observationally for 3 yrs. (46) Pre- and post-intervention 25(OH)D levels were 15.3 ng/mL and 24.9 ng/mL, respectively, in a randomly selected subgroup of 60 participants. Similar to the findings in the British and WHI trials, there was a nonsignificant inverse association between assignment to vitamin D and cancer mortality (RR=0.85 [0.68–1.06]) but not cancer incidence. Thus, the limited data from randomized trials do not clearly support an overall chemopreventive effect for total cancer. (47)

In its 2011 review, the IOM concluded that large randomized trials of vitamin D at higher doses than tested in the WHI are necessary for a definitive assessment of its effect on site-specific cancers. (7) The association between lower regional UV-B intensity and colorectal and other digestive tract cancers is noteworthy given the evidence suggesting that digestive tract malignancies may be particularly sensitive to vitamin D. (48, 49) Laboratory studies reveal that colorectal cancer cells have vitamin D receptors and 25(OH)D-1-α-hydroxylase, which changes 25(OH)D to the active form of 1,25(OH)₂D. (16) These cells also express vitamin D receptors which translocate to the nucleus and bind vitamin D response factors to control gene expression, after engagement by 1,25(OH)₂D. (50) The generally accepted best indicator of vitamin D nutritional status is plasma 25-hydroxyvitamin D₃ (25(OH) D), since it reflects not only skin exposure to UV-B light and total vitamin D intake but also cholecalciferol production in the skin and hydroxylation of all sources of cholecalciferol in the liver. (21) The optimal 25(OH)D level needed to achieve adequate levels of plasma 25(OH)D for cancer chemoprevention is unknown. (48, 51) Some studies have provided promising evidence. (52–54) In a randomized clinical trial of the effects of 6 months of supplemental calcium (2 g/day) and vitamin D3 (800 IU/d) results suggest that calcium and vitamin D may enhance apoptosis in normal colonic mucosa based on changes in molecular markers of apoptosis. (55) Vitamin D receptor expression is associated with PIK3CA and KRAS mutations in colorectal cancer. (50) The PI3K/Akt pathway mediates many extracellular signals and regulates cell growth, proliferation, invasion, and survival. Tumoral mutations in PIK3CA, the catalytic subunit of PI3K, activates AKT and promotes CRC cell growth. (56)

This study has numerous strengths, including the nested case-control design, rigorous collection of covariate information, and detailed information on tumor characteristics. Limitations of the current study include having only a single measure of 25(OH)D. However, a previous study showed a correlation of 0.70 for repeated measures of plasma 25(OH)D within participants over a one year period, suggesting that a single measurement is a reasonable proxy for long-term levels of 25(OH)D. (57) Furthermore, we only measure CRC screening at baseline, and the reported rate of CRC screening is low because we only reported colonoscopy and flexible sigmoidoscopy screening for CRC. We also cannot rule out that our findings may be due to residual confounding. Yet, likely confounders including BMI, physical activity, supplement use (e.g. vitamin D, multivitamin, and calcium) and dietary calcium were adjusted for in the multivariable model based on the validated baseline questionnaire and food frequency questionnaire (58–60) and we did not observe meaningful changes in our odds ratio estimates after control for these confounders. Nevetheless, residual confounding may be present from unmeasured factors such as inflammation evaluated by inflammatory markers. (61) CRC screening information was obtained at the 12-month follow-up questionnaire and was not significantly different between cases and controls. Our participants are primarily White and we did not have adequate power to examine the association of 25(OH)D in non-Whites. Further studies in non-Whites are needed. Finally, we do not have sufficient sample to conduct analysis for rectal cancer risk and for risk of other tumor characteristics.

In this nested-case control study of women, we observed a statistically significant inverse association between plasma 25(OH)D levels and the subsequent risk of CRC. Participants with 25(OH)D greater than 29 ng/mL experienced the greatest reduction in incident CRC and mortality. In light of the high prevalence of vitamin D insufficiency in the population, future studies should examine whether increasing vitamin D levels reduces the incidence of this lethal malignancy.

Table 4.

25(OH)D and colorectal cancer incidence by Body Mass Index (BMI; kg/m2) subgroups

	Quartiles of 25(OH)D ng/mL				Ptrend
	Q1	Q2	Q3	Q4
BMI<25
Range, ng/mL	6.7–18.9	19.3–23.0	23.1–29.2	29.3–57.1
No. of cases/controls	37/28	34/31	29/34	27/37
Simple OR†	1.0	0.84 (0.41–1.72)	0.66 (0.32–1.36)	0.56 (0.27–1.15)	0.095
BMI25-29.9
Range, ng/mL	3.7–16.4	16.5–21.9	22.1–26.4	26.9–66.0
No. of cases/controls	23/25	23/22	25/21	22/24
Simple OR†	1.0	1.19 (0.52–2.76)	1.20 (0.53–2.74)	0.90 (0.39–2.09)	0.84
BMI≥30
Range, ng/mL	4.8–12.5	12.6–18.6	19.0–26.4	26.5–54.1
No. of cases/controls	14/13	14/12	16/11	10/16
Simple OR†	1.0	1.10 (0.36–3.37)	1.23 (0.40–3.79)	0.54 (0.17–1.68)	0.26

Table 5.

25(OH)D and colorectal cancer incidence by physical activity.

	Quartiles of 25(OH)D ng/mL				Ptrend
	Q1	Q2	Q3	Q4
Physical activity (MET-hrs/week) < median
Range, ng/mL	3.7–15.6	15.7–20.6	20.7–26.9	27.1–57.1
No. of cases/controls	42/27	30/38	43/28	35/31
Simple OR†	1.0	0.49 (0.24–0.99)	0.96 (0.48–1.93)	0.70 (0.34–1.43)	0.66
Physical activity (MET-hrs/week) ≥ median
Range, ng/mL	4.8–17.9	18.2–23.0	23.1–29.0	29.2–66.0
No. of cases/controls	31/38	39/30	33/36	21/46
Simple OR†	1.0	1.74 (0.87–3.50)	1.13 (0.57–2.26)	0.60 (0.29–1.25)	0.09