Study design, study settings and participants
This cross-sectional analytical study was carried out at the Department of Surgery at Bangabandhu Sheikh Mujib Medical University (BSMMU), Shahbag, Dhaka, during the period from July 2018 to September 2019. The inclusion criteria of this study were adult women aged 18 years or above who attended the breast clinic, BSMMU and/or were admitted to the surgery ward of BSMMU. Sample size was determined using the following formula.
$$ n=\frac{{\left({z}_{\alpha }+{z}_{\beta}\right)}^2x\ \left({\sigma}_1^2+{\sigma}_2^2\right)}{{\left({\mu}_1-{\mu}_2\right)}^2} $$
Here, n = estimated sample size, μ1 = mean of the quantitative variable in women with breast cancer, μ2 = mean of the same quantitative variable in women without breast cancer, σ1 = standard deviation of the respective quantitative variable in women with breast cancer, σ2 = standard deviation of the respective quantitative variable in women without breast cancer, Zα = Z value of the standard normal deviate at a given level of significance, and Zβ = Z value of standard normal deviate at a given power. Taking the corresponding values from a previous study by Owiredu et al. [17] (μ1 = 202.00, μ2 = 174.50, σ1 = 53.60 and σ2 = 40.50) and taking a 95% level of significance (Zα = 1.96) and 90% power (Zβ = 0.90), the sample size for each group was 48.81. Finally, a total of 50 participants were intended to be recruited. First, a total of 50 women with breast cancer were chosen as cases. Then they were individually matched to two control subjects (1. women who did not develop breast cancer but had benign breast disease, and 2. Women who had no breast disease and were apparently healthy) according to age (within 1 year). Therefore, a total of 150 participants were considered for inclusion. Key exclusion criteria involved patients with a history of other malignancies or patients receiving any kind of lipid lowering agents or any hormonal medications, including oral contraceptive pills. Women who had any diagnosed case of inherited metabolic disease that causes lipid abnormalities such as familial dyslipidemia and congenital defects in lipid metabolism were also excluded from the study.
Diagnosis of patients, staging and grading of tumors
The presence of a breast mass was confirmed on physical examination by the physician or by mammographic study. A digital mammography device (AMULET Innovality, Fujifilm Corporation, Minato City, Tokyo, Japan) was used for mammographic evaluation of participants using mediolateral oblique and craniocaudal projection. The patients were categorized according to the Breast Imaging Reporting and Data System (BIRADS) final assessment categories- 0: need additional imaging or prior examinations: 1, negative; 2, benign; 3, probably benign; 4, suspicious; 5, highly suggestive of malignancy; and 6, known biopsy proven malignancy. Any lesions were further investigated by core needle biopsy. The College of American Pathologists [18] protocols were used for the histopathological diagnosis of breast cancer. Biopsy-proven confirmed cases were enrolled as “cases”, and biopsy-negative breast mass patients were included as the “benign breast disease groups”. Women who attended the clinic due to suspected breast lesions but were negative for breast cancer both clinically and by mammography were included as controls. Before final inclusion of the controls, a brief history was taken from each control about her past and current disease and medication history. A separate consent form was ensured before core-needle biopsy with precounseling of the potential risk, hazards and benefits of the study. Pathologic staging was based on the American Joint Committee on Cancer 8th Edition [19], and a single experienced pathologist reviewed and determined the characteristics of the primary tumor based on size, axillary nodal status, and resection margin according to immunohistochemical (IHC) staining (see Supplementary Tables 1 and 2 for the staging and tumor guidelines used). Blinding was done to reduce bias of the study. Mammography was performed in collaboration with the Department of Radiology and Imaging, BSMMU.
Data collection procedure
Following obtainment of written informed consent, data collection was performed by the lead investigator with the aid of a structured questionnaire. The information collected in this study consisted of the following: 1) demographic information as well as personal history, such as menstrual history, obstetric history, personal habits, and family history of the disease; 2) duration of the mass, presenting symptoms, relevant clinical history, current medications and treatment; 3) staging and grading of the tumor for the case; and 4) investigations, such as complete blood count, erythrocyte sedimentation rate (ESR), and lipid profile for all subjects. All collected data were recorded in separate case record forms. The researchers were fully aware of the privacy and anonymity of the participants and followed the guidelines of the Declaration of Helsinki throughout the study.
BMI assessment and categorization
Participant weight (in kilograms) was measured using a digital bathroom scale (Model: Meb 9370, Miyako Electronics Ltd., Bangladesh) and height (in meters) was measured using a standard measuring tape. Then BMI was calculated using standard formula. The WHO recommended Asian criteria [20] for BMI categorization was used for this study. A BMI ≥23 was considered overweight and/or obese and a BMI between 18.5 and 22.9 was considered normal weight.
Lipid assay
Analysis of blood samples from cases and their matched controls were done in same batches but in random order. Lipid assays were carried out at the Department of Biochemistry and Molecular Biology, BSMMU. Serum lipid measures were standardized with the Centers for Disease Control (CDC, Atlanta, GA) Lipid Standardization Program. Five milliliters of fasting (at least 12 h) venous blood was collected into Vacutainer® plain tubes, and serum was separated by centrifugation and analyzed on the same day by the enzymatic method in an automated analyzer (Beckman Coulter-AU680) for lipid profiles comprising total cholesterol (TC), triglyceride (TG), LDL and HDL. Dyslipidemia was defined according to the reference levels of the National Cholesterol Education Programme (NCEP) Adult Treatment Panel III (ATP III) guidelines, where hypercholesterolemia was marked as TC > 200 mg/dL or hypertriglyceridemia was marked as TG > 150 mg/dL. The LDL value was categorized as high when it exceeds 130 mg/dl, and HDL was considered low if it was below 40 mg/ dL [21]. Subjects were classified as dyslipidemic when any one of the components of the lipid profile except HDL was beyond the upper limit and in cases where the HDL level was below the lower limit.
Ethical consideration
The study protocol was approved by the Institutional Review Board (IRB), BSMMU (reference number: BSMMU/2018/11716). Written informed consent was obtained from the participants before inclusion.
Statistical analyses
Data were statistically analyzed using software SPSS (version 26). Values were expressed as the mean ± standard deviation. Chi-square tests and analysis of variance (ANOVA) were used for statistical comparisons of categorical and continuous data respectively. Post hoc analysis by Bonferroni adjustments was performed where appropriate. Odds ratios (ORs) were derived by multinomial logistic regression, along with 95% confidence intervals (CIs). P values less than 0.05 were considered statistically significant.