Predicted 25-hydroxyvitamin D in relation to incidence of breast cancer in a large cohort of African American women

The Black Women’s Health Study (BWHS) began in 1995 when 59,000 African American women aged 21–69 years from across the USA completed mailed health questionnaires. Participants have completed follow-up questionnaires every two years. Follow-up was complete through 2013 for over 85 % of person-time since 1995. The Institutional Review Board of Boston University approved the protocol and reviewed the study annually.

At baseline, participants were asked about use of vitamin D supplements, use of multivitamins, weight, height, number of births, timing of each full-term birth, lactation, age at menarche, use of oral contraceptives, breast cancer in first-degree relatives, vigorous physical activity, alcohol consumption, cigarette smoking, menopausal status, age at menopause, use of supplemental female hormones, years of education, and many other factors. The biennial follow-up questionnaires ascertained occurrences of incident breast cancer and updated information on use of vitamin D supplements and multivitamins, and most other variables. A modified version of the NCI-Block food frequency questionnaire was used to ascertain usual diet in 1995 and 2001 [17].

Each BWHS questionnaire asks about new diagnoses of breast cancer and the year of diagnosis. Participants are contacted for permission to obtain pathology reports and other medical records and data are also obtained from state cancer registries in the 24 states in which 95 % of participants live. We were able to obtain medical records, cancer registry records, or both for approximately 95 % of women who reported incident breast cancer, of which 99 % were confirmed. Only cases of confirmed incident breast cancer were included in the present analysis. In the early years of the study, 1995–2000, testing for estrogen receptor (ER) and progesterone receptor (PR) was not universal, and thus we have missing data on ER and PR status for some participants. Among cases with known status, the proportions with ER+/PR+, ER+/PR-, ER-/PR+, and ER-/PR- tumors are 50 %, 14 %, 2 %, and 34 %, respectively, similar to the distributions observed for African American women in the SEER registry and other population-based data [18‐20]. In previous comparisons of cases with data on receptor status to cases with unknown receptor status, the two groups were similar with regard to the prevalence of known breast cancer risk factors [21].

Collection of blood specimens from BWHS participants began in 2012 and will continue through 2017, by which time all living study participants will have had an opportunity to provide a sample. Of participants approached to date, about 25 % have provided samples. Participants are mailed an informed consent, explanatory materials, a pre-printed laboratory requisition form, and instructions for locating a nearby blood collection site. Blood specimens are collected and tested by Quest Diagnostics (Madison, NJ, USA) an accredited national clinical laboratory [22, 23]. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for measurement of 25(OH)D [24, 25], which was carried out at three Quest central laboratories. National Institute of Standards and Technology Standard Reference Material for 25(OH)D in human serum (NIST SRM 972) was used for quality control. Written informed consent to use the blood samples for health-related research for the entirety of the study was obtained from participants who provided samples.

Development of the prediction model was based on 25(OH)D values obtained from assays of plasma samples provided between 2013 and early 2015, the period immediately following completion of the 2013 BWHS questionnaire. During that period, 3539 participants provided blood samples with signed informed consent. We excluded 276 women with prevalent cancer at the time of the blood draw and 407 who had missing data on any of the candidate predictors of 25(OH)D, for an analytic sample of 2856. Variables that were known or suspected to be related to endogenous levels of 25(OH)D and for which data were available from the 2013 questionnaire were considered as possible predictors: use of vitamin D supplements (with or without calcium, at least twice a week), multivitamin use (at least twice a week, not specified whether they included vitamin D or how much), body mass index (BMI, kg/m²) (considered as both a categorical and a continuous variable), vigorous exercise, walking for exercise, current cigarette smoking, current alcohol consumption, use of female hormones, use of oral contraceptives, and menopausal status. Dietary intake of vitamin D derived from responses to a modified version of the NCI-Block food frequency questionnaire completed in 2001 was also considered as a predictor. A solar UV-B flux variable (high, medium, low levels of solar UV-B radiation) was created as a proxy for ambient sun exposure based on state of residence in 2013 and the reported average annual UV-B radiation in each state [26], and considered for the prediction model.

Repeated k-fold cross-validation was used to derive the best 25(OH)D prediction model [27]. The 2856 specimens were divided into five groups of equal size, with 4/5 serving as a training set and the remaining 1/5 serving as the test set. Using the ‘caret’ package in R [28], stepwise selection by Akaike’s information criterion was performed on each training set to identify the optimal predictors in a generalized linear model for continuous 25(OH)D. The best fit model parameters were then used to predict 25(OH)D in the test set, at which point Pearson’s correlation coefficient was computed for the linear association between observed and predicted 25(OH)D values. This procedure was performed another four times with each remaining 1/5 serving as the test set. We repeated this 100 times, with the full sample divided into a different set of five groups each time. Overall model prediction performance was calculated as the average R-squared value across the 100 repetitions and each of the five folds. The generalized linear models were adjusted for season of blood draw, laboratory, and age, for the purpose of controlling variability when estimating beta coefficients for predictors.

Baseline data from the 1995 BWHS questionnaire were then used in combination with beta-coefficients for each of the predictors to compute a predicted 25(OH)D level at baseline in 1995 for the entire BWHS cohort. Participants were excluded from the analyses if they had missing data on any of the predictors at baseline. Predicted 25(OH)D level was then updated every two years using the same beta coefficients and new values of the predictors. If data were missing for a given variable at some point during follow-up, the value from the previous cycle was carried forward. We then computed a cumulative average of predicted 25(OH)D. The cumulative average method [29, 30] has been used previously in vitamin D prediction models and for exposures such as dietary intake and physical activity [2, 31, 32]. For this approach, the predicted 25(OH)D score for a given time point is the average of scores from previous time points up to and including that time point. This method may better represent average long-term vitamin D status over the period of follow-up for each individual [33].

Analyses of the association between predicted vitamin D status and breast cancer risk included all BWHS participants who had not been diagnosed with cancer prior to enrollment in the cohort and had complete information from the baseline questionnaire on each of the variables included in the 25(OH)D prediction model. Each participant contributed person-time from baseline in 1995 until diagnosis of breast cancer, death, loss to follow-up, or end of follow-up in 2013, whichever came first. Predicted 25(OH)D status was analyzed in quartiles, with highest quartile as the reference category. We used Cox proportional hazards regression, stratified by age (year) and questionnaire cycle (two years) to estimate the incidence rate ratio (IRR) and 95 % confidence interval (CI) for quartile of predicted 25(OH)D in relation to breast cancer incidence, with adjustment for number of births (0, 1, 2, ≥3), age at first birth (<20, 20–24, ≥25), age at menarche (<11, 12–13, ≥14 years), age at menopause (<45, 45–49, ≥50 years, or premenopausal), first-degree family history of breast cancer (yes, no), recent oral contraceptive use (within the previous 5 years), long-term oral contraceptive use (≥10 years), duration of use of estrogen with progesterone postmenopausal hormones (≥5 years), and BMI (<25, 25–29, 30–34, ≥35 kg/m²). Covariates that changed over time were treated as time-dependent. In addition to the overall analyses, we conducted analyses separately for ER+ and ER- breast cancer and within strata of age (<45 years and ≥45 years) and current use of vitamin D supplements (yes, no).