Prediagnostic Circulating Concentrations of Vitamin D Binding Protein and Survival among Patients with Colorectal Cancer

Vitamin D is hypothesized to play a role in the development and progression of colorectal cancer. Colon cancer cells express vitamin D receptor (VDR; refs. 1, 2) and 1-α-hydroxylase (3) that converts the main circulating form of vitamin D, 25-hydroxyvitamin D [25(OH)D], into the active metabolite, calcitriol [1,25(OH)₂D]. Binding of 1,25(OH)₂D to VDR leads to several anticancer effects, including increased cell differentiation and apoptosis (4, 5) and decreased proliferation (6), angiogenesis (7, 8), and metastasis (9, 10).

Vitamin D binding protein (VDBP), also known as the group-specific component, is the major vitamin D carrier protein. Approximately 88% of circulating 25(OH)D is bound to VDBP, while 12% of 25(OH)D is loosely bound to albumin, leaving very little in the free form (11, 12). Experimental studies demonstrate that VDBP has important biological functions that may inhibit tumor growth, such as actin scavenging, macrophage activation, and chemotaxis (13). A meta-analysis of 28 studies examined VDBP levels in relation to the overall risk of multiple cancers, including colorectal cancer, and found borderline decreased risk in individuals with higher VDBP levels [OR, 0.75; 95% confidence interval (CI), 0.56–1.00; ref. 14]. Previous studies did not find an association between VDBP levels and colorectal cancer risk (15–17). However, it is unknown whether prediagnostic VDBP levels influence survival outcomes among patients with colorectal cancer.

The “free hormone hypothesis” postulates that the bound fraction of a hormone is not available to target cells for signaling and gene regulation (18), suggesting that free 25(OH)D and albumin-bound 25(OH)D, which can dissociate during tissue perfusion, may be more biologically active than VDBP-bound 25(OH)D. However, more recent studies found that the 25(OH)D–VDBP complex can also be internalized into cells by transportation of megalin, an endocytic receptor that is expressed in epithelial cells of several organs, including colon (19, 20). Although the link between higher total 25(OH)D levels and better colorectal cancer survival has been well-documented (21–27), the association between bioavailable or free 25(OH)D levels and colorectal cancer survival is unknown.

Building upon our prior analyses of total 25(OH)D levels and colorectal cancer survival (21), we further investigated the associations of prediagnostic plasma levels of VDBP, bioavailable 25(OH)D, and free 25(OH)D with survival among participants diagnosed with colorectal cancer from two prospective U.S. cohorts, the Nurses' Health Study (NHS) and the Health Professionals Follow-Up Study (HPFS).

In 1976, NHS was initiated when 121,700 U.S. female registered nurses ages 30–55 years responded to a mailed questionnaire on risk factors for cancer and cardiovascular disease (28). Blood samples were collected from 32,826 NHS participants between 1989 and 1990. In 1986, HPFS was established when 51,529 U.S. male dentists, optometrists, osteopaths, podiatrists, pharmacists, and veterinarians ages 40–75 years completed a mailed questionnaire on health-related behaviors and medical history (29). Blood samples were collected from 18,225 HPFS participants between 1993 and 1995. In both cohorts, participants received biennial questionnaires to update information on lifestyle factors and medical diagnoses. A high follow-up rate of more than 90% was achieved in both cohorts.

When an incident case of colorectal cancer was identified from self-report or during follow-up of participant deaths, we asked permission to obtain hospital records and pathology reports. Physicians who were blinded to exposure data reviewed medical records, death certificates, or cancer registry data to ascertain the diagnosis of colorectal cancer and record information on important tumor characteristics. We have estimated that 96%–97% of patients were captured by using these methods (30, 31).

Patients diagnosed with colorectal cancer after the date of blood collection through December 2011 were eligible for this study. Patients were excluded if they were non-White (due to inability of the monoclonal assay to accurately measure VDBP in non-Whites) or had reported any cancer (other than nonmelanoma skin cancer) prior to colorectal cancer diagnosis. Patients who were diagnosed with colorectal cancer within 2 years after blood collection were also excluded to minimize bias associated with presence of occult cancer. Among 627 eligible patients with total 25(OH)D levels, 603 had available VDBP levels, from which bioavailable and free 25(OH)D levels were calculated.

The study protocol was approved by the Institutional Review Boards of the Brigham and Women's Hospital (Boston, MA) and the Harvard T.H. Chan School of Public Health (Boston, MA), and those of participating registries as required. All participants provided written informed consent for the researchers to access their medical records. The study was conducted in concordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects (Council for International Organizations of Medical Sciences).

Blood samples were shipped by overnight courier in chilled containers. On receipt, blood samples were centrifuged, aliquoted, and stored in continuously monitored liquid nitrogen freezers at −130°C or below. More than 95% of the blood samples arrived in our laboratory within 24 hours of phlebotomy.

Plasma VDBP was measured at Heartland Assays in 2013 by a mAb-based ELISA (R&D Systems). Plasma total 25(OH)D was measured in the laboratory of Dr. Bruce Hollis (Medical University of South Carolina, Charleston, SC) and Heartland Assays by radioimmunoassay (32). Plasma albumin was measured by a Colorimetric Assay (Roche Diagnostics) in the laboratory of Dr. Nader Rifai (Children's Hospital, Boston, MA). Although all samples were assayed at the same laboratory, cases identified from different questionnaires were assayed in different batches, which are detailed in Supplementary Table S1. The mean intraassay coefficients of variation for VDBP, total 25(OH)D, and albumin were ≤14.8%, ≤11.8%, and ≤4.0%, respectively.

Bioavailable and free 25(OH)D were calculated by the following equations:

• (i) Free 25(OH)D = Total 25(OH)D/(1 + Ka_Albumin × Albumin + Ka_VDBP × VDBP);

• (ii) Bioavailable 25(OH)D = 25(OH)D × (1 + Albumin × Ka_Albumin),

where Ka_Albumin is the affinity of albumin for 25(OH)D (6 × 10⁵) and Ka_VDBP is the affinity of VDBP for 25(OH)D (7 × 10⁸; ref. 11), and all concentrations are in mol/L (18).

Using the TaqMan OpenArray SNP Genotyping Platform (Applied Biosystems), we successfully genotyped two common SNPs in VDBP, rs4588 and rs7041, for 548 patients of our study population. The two SNPs give rise to three predominant haplotypes: GC1F, GC1S, and GC2. Regarding whether the affinity of VDBP for 25(OH)D is affected by these haplotypes, one study found the affinity of GC1F to be four times higher than that of GC2 and double than that of GC1S (33), while three studies demonstrated no difference in the affinity (34–36). Therefore, we calculated bioavailable and free 25(OH)D using a constant affinity of VDBP for 25(OH)D, but our conclusions were essentially unchanged by using the genotype-specific affinities.

Patients were observed until date of death or last follow-up (June 2014 for NHS and January 2014 for HPFS), whichever came first. Ascertainment of deaths included reporting by family or postal authorities, and interrogation of names of persistent nonrespondents in the National Death Index, which has been shown to capture approximately 98% of deaths (37). The primary outcome was overall mortality, and the secondary outcome was colorectal cancer–specific mortality. Because deaths from colorectal cancer mostly occur within the first 5 years after diagnosis, we evaluated 5-year overall mortality as an additional outcome by censoring patients who were alive at the end of the first 5 years.

Cancer stage, grade of tumor differentiation, location of primary tumor, and year of diagnosis (as a surrogate for treatment) were extracted from medical records. Body mass index (BMI) and physical activity were obtained from the questionnaire returned before blood collection.

Plasma vitamin D–related markers were categorized into quartiles by batch (Supplementary Table S1) and analyzed (21). Follow-up time was calculated from colorectal cancer diagnosis to death or censoring. Cox proportional hazards regression was used to calculate HRs and 95% CIs for three outcomes: overall mortality, colorectal cancer–specific mortality, and 5-year overall mortality. We tested for a linear trend across quartiles using an ordinal variable. A priori, we included other prognostic markers and potential confounders in multivariate models, including age at diagnosis, sex, BMI, physical activity, cancer stage, grade of tumor differentiation, location of primary tumor, and year of diagnosis. We additionally adjusted for season of blood collection when the exposure was total, bioavailable, or free 25(OH)D. Interaction between VDBP and the potential effect modifier was assessed by entering in the model the cross-product of the quartile of the biomarker and the stratification variable, evaluated by the likelihood ratio test. The Cox models were tested for and met the proportional hazards assumption. All analyses were performed with SAS Software, version 9.4 (SAS Institute). All P values are two-sided.

Plasma samples were collected at a median of 9.3 years [interquartile range (IQR), 6.1–13.3 years) before colorectal cancer diagnosis. The median VDBP level was 250 μg/mL (IQR, 175–311 μg/mL) and the median total 25(OH)D level was 27.3 ng/mL (IQR, 20.4–33.0 ng/mL), and both were modestly correlated (r = 0.12; P < 0.01; Supplementary Table S2). The median bioavailable 25(OH)D level was 3.36 ng/mL (IQR, 2.26–4.66 ng/mL). As expected, bioavailable 25(OH)D levels were positively correlated with total 25(OH)D (r = 0.59; P < 0.0001) and albumin levels (r = 0.23; P < 0.0001) and negatively correlated with VDBP levels (r = −0.67; P < 0.0001). The median free 25(OH)D level was 8.25 pg/mL (IQR, 5.66–11.19 pg/mL), and was nearly perfectly correlated with bioavailable 25(OH)D levels (r = 0.99; P < 0.0001); thus, we focused our analyses on bioavailable 25(OH)D, which was much more abundant in the circulation.

Patient characteristics were well balanced by quartile of VDBP, except that patients with higher VDBP levels had a lower BMI (Table 1). Patients with higher total 25(OH)D levels had a lower BMI and higher physical activity, compared with those with lower levels (Supplementary Table S3). In addition, patients with higher total or bioavailable 25(OH)D levels were more likely to have their blood samples collected in the summer or fall.

The median time of follow-up among patients who were alive at the end of follow-up was 12.4 years (IQR, 8.0–15.3 years). During the follow-up, we documented 328 deaths, 187 (57.0%) of which were due to colorectal cancer. Non-colorectal cancer causes of death included other malignancies (n = 26), cardiovascular disease (n = 33), neurologic disorders (n = 21), cerebrovascular disease (n = 13), respiratory disease (n = 12), and other or unknown reasons (n = 36). A summary of causes of death by follow-up period after diagnosis is presented in Supplementary Table S4. Of the 187 deaths due to colorectal cancer, 163 (87.2%) occurred within the first 5 years after diagnosis.

Higher VDBP levels were significantly associated with improved overall (P_trend = 0.001) and colorectal cancer–specific survival (P_trend = 0.02; Table 2). Compared with patients in the lowest quartile, those in the highest quartile of VDBP had a multivariate HR of 0.58 (95% CI, 0.41–0.80) for overall mortality and 0.58 (95% CI, 0.37–0.91) for colorectal cancer–specific mortality. The HRs were not materially changed after further adjustment for total 25(OH)D levels. In addition, higher VDBP levels were associated with improved 5-year overall survival (P_trend = 0.001), with a multivariate HR of 0.50 (95% CI, 0.32–0.76), comparing extreme quartiles.

To further address concerns about the possible influence of occult cancer on VDBP levels, we performed sensitivity analyses by excluding patients who developed colorectal cancer within 3, 4, and 5 years after blood sample collection, respectively. Although statistical power was diminished, the association between VDBP levels and patient survival remained largely unchanged (Table 3).

We next evaluated the associations between two VDBP polymorphisms and patient survival. Neither rs7041 nor rs4588 was significantly associated with overall or colorectal cancer–specific survival (P ≥ 0.08; Supplementary Table S5). In models additionally adjusted for these polymorphisms, the significant association between VDBP levels and patient survival remained unchanged (P_trend = 0.001 and 0.008 for overall and colorectal cancer–specific mortality, respectively).

The association of VDBP levels with overall and colorectal cancer–specific survival was examined across strata of potential effect modifiers, including age at diagnosis, time from blood collection to diagnosis, sex, BMI, physical activity, cancer stage, grade of tumor differentiation, location of primary tumor, year of diagnosis, and total 25(OH)D levels, and remained largely unchanged in most subgroups (P_interaction ≥ 0.18; Fig. 1).

Total 25(OH)D levels were not significantly associated with overall (P_trend = 0.09) or colorectal cancer–specific survival (P_trend = 0.08; Table 4). However, higher total 25(OH)D levels were associated with improved 5-year overall survival (P_trend = 0.01), with a multivariate HR of 0.48 (95% CI, 0.30–0.78) comparing the highest with the lowest quartile. The association remained significant after further adjustment for VDBP levels (P_trend = 0.02).

Bioavailable 25(OH)D levels were not associated with either overall (P_trend = 0.39) or colorectal cancer–specific survival (P_trend = 0.43), even in the analyses with 5-year overall survival as the outcome (P_trend = 0.66; Table 4). Free 25(OH)D levels were also not associated with any of these outcomes (P_trend = 0.15, 0.32, and 0.31, respectively; Table 4).

We found that patients with colorectal cancer with the highest prediagnostic plasma VDBP levels had a significant improvement in overall and colorectal cancer–specific survival, independent of total 25(OH)D levels. Bioavailable and free 25(OH)D levels were not associated with overall or colorectal cancer–specific mortality.

To date, only one study has examined the association between circulating VDBP levels and colorectal cancer survival. This analysis of 206 Chinese patients with colorectal cancer measured VDBP level at surgery and detected no association with overall survival (38). In addition, a Canadian study found that the C allele at rs2282679 (a perfect proxy for rs4588) in VDBP was significantly associated with worse disease-free survival among 488 patients with colorectal cancer (39). Our observation that higher VDBP levels were associated with increased survival is biologically plausible. As the major carrier protein of circulating 25(OH)D, VDBP may boost the anticancer effects of 25(OH)D by prolonging its half-life. In addition, VDBP has independent biological functions that may inhibit tumor growth. First, VDBP functions as an actin scavenger, binding to circulating actin released from tissue injury, and thereby preventing vascular occlusion and organ dysfunction (40). Second, VDBP plays a role in immune response through the inflammation-primed conversion to VDBP macrophage-activating factor, which has direct antiangiogenic and antiproliferative activities, in addition to its ability to activate tumoricidal macrophages (41–44). Third, VDBP has an anti-inflammatory effect by directing neutrophils to sites of inflammation (neutrophil chemotaxis; refs. 45, 46). In this study, the association between VDBP levels and patient survival remained significant after controlling for total 25(OH)D levels. To minimize bias in the plasma VDBP levels by the presence of occult cancer, we excluded patients diagnosed within 2 years of blood sample collection in the main analyses, and continued to note an association even when extending this restriction to 5 years.

Multiple prospective cohort studies (21–24), as well as a phase II randomized clinical trial (26), have suggested a benefit of higher vitamin D levels on survival among patients with colorectal cancer. In our previous analysis of 304 patients with colorectal cancer from the same cohort studies, higher total 25(OH)D levels were associated with improved overall survival (21). In this study, with a larger sample size and extended follow-up, higher total 25(OH)D levels were associated with improved 5-year overall survival, but not with overall survival during the entire follow-up. One possible explanation is that vitamin D may not reduce excess mortality from causes other than colorectal cancer. In a recent meta-analysis of 52 randomized controlled trials, vitamin D supplementation was found to reduce the risk of cancer-related death by 16% without being associated with all-cause mortality (47). Another potential explanation is that some patients provided blood samples many years before their diagnosis. Whereas the prospective design of our study is advantageous in reducing bias that results from reverse causation, total 25(OH)D levels measured remotely from diagnosis may not accurately reflect the relevant vitamin D status that influences long-term colorectal cancer survival.

In this study, we found no association between bioavailable or free 25(OH)D levels and colorectal cancer survival even in analyses with 5-year overall survival as the outcome. As higher VDBP levels were associated with improved survival, and resulted in lower concentrations of bioavailable and free 25(OH)D, we would not expect that higher levels of bioavailable or free 25(OH)D would also be associated with improved survival. In a previous case–control study including participants from NHS, total 25(OH)D levels, but not bioavailable or free 25(OH)D levels, were inversely associated with colorectal cancer risk (15). Taken together, these data do not support the “free hormone hypothesis” within the context of circulating 25(OH)D and colorectal carcinogenesis, indicating that total 25(OH)D remains the best measure of clinically relevant vitamin D status.

Our study has several strengths, including the prospective design, long follow-up, high follow-up rate, and detailed data on potential confounders. The prospective design reduces reverse causation, as blood samples were collected years before inadequate nutrition and limited performance status that commonly develop at the time of colorectal cancer diagnosis. The comprehensive assessment of VDBP, total 25(OH)D, and bioavailable and free 25(OH)D, along with availability of albumin levels and VDBP genotype, allowed for a better understanding of the roles of vitamin D–related biomarkers in colorectal cancer survival.

Several limitations of our study deserve comment. We used a single measurement of VDBP and total 25(OH)D from plasma samples collected years before diagnosis, so we were unable to assess the influence of the dynamic changes of these markers. We did not directly measure bioavailable and free 25(OH)D; however, calculated and directly measured concentrations of these markers have been found to be well-correlated (48). We used a mAb-based ELISA to measure VDBP, which is inferior to a polyclonal assay and incapable of measuring VDBP levels in Blacks (48, 49). To address this concern, we excluded non-White patients in the main analyses, and the results were similar after further adjustment for VDBP polymorphisms. Finally, information on treatment was not systematically collected in NHS and HPFS. However, treatment programs were unlikely to have varied by VDBP levels years before diagnosis.

In conclusion, higher prediagnostic plasma VDBP levels were associated with improved overall and colorectal cancer–specific survival among patients with colorectal cancer. Bioavailable or free 25(OH)D levels were not associated with colorectal cancer survival. Additional efforts to understand the mechanisms through which the vitamin D pathway influences colorectal carcinogenesis and cancer progression are warranted.

B.M. Wolpin reports grants and personal fees from Celgene, grants from Eli Lilly, and personal fees from GRAIL and BioLineRx outside the submitted work. J.A. Meyerhardt reports personal fees from COTA (advisory board), Taiho Pharmaceutical (grant review for National Comprehensive Cancer Network), and Ignyta (advisory board) outside the submitted work. S. Ogino reports grants from NIH (grant number R35 CA197735) during the conduct of the study. A.T. Chan reports personal fees from Pfizer Inc., grants and personal fees from Bayer Pharma AG, and personal fees from Janssen Pharmaceuticals and Boehringer Ingelheim outside the submitted work. C.S. Fuchs reports personal fees from Agios, Amylin Pharma, Bain Capital, CytomX Therapeutics, Daiichi Sankyo, Eli Lilly, Entrinsic Health, EvolveImmune Therapeutics, Genentech, Merck, Taiho, and Unum Therapeutics outside the submitted work; serves as a director for CytomX Therapeutics and owns unexercised stock options for CytomX Therapeutics and Entrinsic Health; is a cofounder of EvolveImmune Therapeutics and has equity in this private company; and has provided expert testimony for Amylin Pharmaceuticals and Eli Lilly. K. Ng reports grants from NCI, Department of Defense, and Cancer Research UK during the conduct of the study, as well as grants from Revolution Medicines, Genentech, Gilead Sciences, Trovagene, and Tarrex Biopharma; grants and nonfinancial support from Pharmavite; nonfinancial support from Evergrande Group; and personal fees from Bayer, Seattle Genetics, and Array Biopharma outside the submitted work. No potential conflicts of interest were disclosed by the other authors.

C. Yuan: Conceptualization, data curation, formal analysis, funding acquisition, investigation, visualization, methodology, writing–original draft, writing–review and editing. M. Song: Writing–review and editing. Y. Zhang: Writing–review and editing. B.M. Wolpin: Writing–review and editing. J.A. Meyerhardt: Writing–review and editing. S. Ogino: Funding acquisition, writing–review and editing. B.W. Hollis: Data curation, methodology, writing–review and editing. A.T. Chan: Funding acquisition, writing–review and editing. C.S. Fuchs: Conceptualization, funding acquisition, methodology, writing–review and editing. K. Wu: Methodology, writing–review and editing. M. Wang: Methodology, writing–review and editing. S.A. Smith-Warner: Methodology, writing–review and editing. E.L. Giovannucci: Methodology, writing–review and editing. K. Ng: Conceptualization, data curation, formal analysis, supervision, funding acquisition, investigation, visualization, methodology, writing–original draft, writing–review and editing.

The authors thank the participants and staff of the Nurses' Health Study and the Health Professionals Follow-Up Study for their valuable contributions, as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. The authors assume full responsibility for analyses and interpretation of these data. The Nurses' Health Study was supported by the NIH grants UM1 CA186107, P01 CA87969, and R01 CA49449. The Health Professionals Follow-Up Study was supported by the NIH grant U01 CA167552. This work was additionally supported by the Pussycat Foundation Helen Gurley Brown Presidential Initiative (to C. Yuan and K. Ng); by the NIH grant R35 CA197735 (to S. Ogino); by the NIH grants R01 CA137178 and K24 DK098311 and the Damon Runyon Cancer Research Foundation (to A.T. Chan); by the NIH grant P50 CA127003 (to C.S. Fuchs); by the NIH grants K07 CA148894 and R01 CA205406 and the Project P Fund (to K. Ng); and by the Entertainment Industry Foundation's National Colorectal Cancer Research Alliance (NCCRA).

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