The source population of this study is from a multicenter hospital-based case-control study which was conducted at two large tertiary care hospitals of Karachi as previously described (17). 477 women with complete records of MBD were extracted from the main study. The inclusion criteria of 178 breast cancer cases were newly diagnosed primary histologically confirmed cases of breast cancer from Aug 2015 to July 2018. Women who were extremely sick, unable to complete the interview, been living outside Pakistan for more than a year, receiving adjuvant or neoadjuvant chemotherapy or radiation, and diagnosis of breast cancer for greater than 6 months before study enrolment were excluded. A total of 299 controls were enrolled from those attending in- and out-patient services for general medical, and surgical departments of the two participating hospitals and who had no previous diagnosis of breast cancer or any other cancer. There is no organized breast cancer screening program in Pakistan and most of the mammography done is either diagnostic or opportunistic screening (18). Therefore, study participants were women undergoing both diagnostic and screening (annual and biennial) mammograms. All mammograms were taken before breast cancer diagnosis among cases. All subjects completed an interview-based questionnaire that included information on age, education, socioeconomic status, parity, age of mother at first birth, breastfeeding, age at menarche and menopause, age of mother at first birth, history of any comorbid or benign breast disease, family history of breast cancer. Menopausal status was either premenopausal or postmenopausal. Participants also reported the average number of hours per week, engaged in physical activity of different intensities for at least ten minutes, like vigorous exercise or moderate exercise of household activities like mopping, etc., and walking. Body mass index BMI (kg/m2) and tumor characteristics were recorded from medical files and reports. Women with missing information of BI-RADS (Breast Imaging-Reporting and Data System) density were excluded. All consenting participants were interviewed, after informed consent, in a separate room to ensure privacy using a structured questionnaire
Mammography measurement details
Two view mammography was performed for all patients comprising of medio-lateral oblique (MLO) and cranio-caudal (CC) views on a computed radiography (CR) system. In the CR system, the X- Rays passing through the breast, grid, and cassette cover are absorbed by the plate reader system that comprises photostimulable storage phosphor (PSP). An electronic latent image is produced on the PSP due to the local absorption of X-ray energy that varies with the anatomical variation of breast parenchyma. The cassette is subsequently placed in the reader that captures the information and converts it to a digital signal that is finally displayed at the workstation (19). The soft copies of the mammographic images were downloaded and reviewed at picture archiving and communication system (PACS) by breast imagers, and qualitative assessment of mammographic density was done by dedicated radiologists with years of experience in interpreting mammography and breast density.
Assessment of Mammographic Breast Density (MBD)
Using the American College of Radiology Breast Imaging Reporting and Data System (BI-RADS 5th edition), the mammographic breast density on mammograms was categorized as; Category 1, Predominantly fatty ( less than 25% glandular); category 2 for scattered fibroglandular (25 to 50% glandular ); category 3 for heterogeneously dense (51 to 75% glandular) and category 4 for dense breast parenchyma ( more than 75% glandular) (20). Categories 3& 4 both are high MBD. Analyses were restricted to patients with an available BI-RADS measurement. The density measurements of the breast contralateral to the tumor were used to avoid a distortion of measurements due to the tumor itself.
Dietary intake assessment with a Food Frequency Questionnaire (FFQ):
Dietary intake assessment was done by using the validated food frequency questionnaire FFQ (21). Intake frequency was categorized into 7 groups and category for each food item was converted to daily intake. Each participant was also asked about their average portion size/ common serving size of the food. The intake frequencies were multiplied by standard portion size to calculate servings per day of all food items. All the food items were grouped into 6 components including fruits, vegetables, dairy, grains, white meat, red meat, and plant proteins and total servings/day was calculated for each food category.
Measurement of serum 25 (OH)D level
After the interview, blood samples were collected from the study participants and serum 25 (OH)D level was measured using ELISA.
Histopathology and estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2/ neu) status of breast cancer cases were retrieved from medical records (22).
The ethical approval was obtained by the Human Research Ethics Committee of the University of Adelaide and the Ethical Review Committees of two hospitals in Karachi Pakistan: Aga Khan University Hospital AKUH & Karachi Institute of Radiation and Nuclear Medicine Hospital KIRAN. Patients who were literate read and signed the informed consent form and informed consent was obtained verbally from those who could not read or write.
Multinomial logistic regression models were applied to compute odds ratios (ORs) and 95% confidence intervals (CIs) for the MBD categories. The fatty and scattered fibroglandular tissue categories were merged and used as a reference. Multivariate models were adjusted for variables found to be significantly associated with breast density and known risk factors for breast cancer such as age, body mass index, age at menarche & menopause in the postmenopausal group, parity, and family history of breast cancer among first-degree relatives, BMI, and the food categories.