Study design and setting
We studied women enrolled in the Nigerian Integrative Epidemiology of Breast Cancer (NIBBLE) Study, a case-control study of female breast cancer that recruited participants at six government hospitals in Nigeria, five of whom are located in Abuja (National Hospital, University of Abuja Teaching Hospital Gwagwalada, Asokoro District Hospital, Garki Hospital and Wuse General Hospital) and the sixth hospital, the University of Nigeria Teaching Hospital, in Enugu, between January 2014 and July 2016. The details about the study design and setting have been previously published (2).
Participants
Overall, 831 newly diagnosed patients with primary invasive breast cancer aged 18 years and above were identified at their first visit to the clinical sites. Research nurses informed potential participants about the study and obtained their informed consent. Age-matched hospital-based controls (996) were women who did not have cancer or endocrine diseases and were within ±2.5 year of the age of specific patients enrolled within one month in the same hospital. Predominantly, >94.0% of women approached consented to participate. Research nurses conducted face-to-face interviews in the English language (70.6%) or local Nigerian language (29.4%) according to the patient's preference.
Primary exposure
For the LTPA assessment, we used a modification of the U.S. Nurses’ Health Study (NHS) II physical activity questionnaire. The questionnaire measures the average amount of time spent per week on moderate and vigorous leisure-time activities. Participants reported the average time per week spent on each of the following moderate or vigorous activities, in the past year: walking, hiking, jogging, running, bicycling, dancing, playing tennis, soccer, squash; golf, swimming, aerobics, weight lifting or resistance exercise. We calculated participants’ metabolic equivalents (METs) - hour/week of total LTPA by multiplying the number of hours per week of each activity with its corresponding MET values and then summarized all the MET values (32). We excluded one participant with an extreme value of MET. The final MET score was used to create two categories of participants: ‘Leisure-time physical activity’ -participants who met the WHO PA recommendations of at least 150 minutes of moderate-intensity or 75 minutes of vigorous-intensity aerobic LTPA, or an equivalent combination, and ‘Leisure-time physical inactivity’ – for those who did not meet the WHO PA recommendations (33). In addition, we created categories of LTPA in quartiles of METs (< 2.99, 2.99 - 5.80, 5.81 - 11.25, > 11.25) based on the distribution of the study sample.
Breast cancer and breast cancer subtypes
Needle core biopsies were performed using Bard Magnum Biopsy Gun®. Breast specimens were fixed in 10% neutral buffered formalin and processed within 48 hours of fixation with a minimum fixation time of 8 hours in Leica® automatic tissue processors.
Histology
Sections of Paraffin-embedded blocks were cut at 3-4 μm and stained routinely with hematoxylin and eosin stains. Histological features were classified adopting 2003 WHO classification of breast diseases and graded using the Nottingham modification of the Bloom-Richardson grading (34).
Immunohistochemistry (IHC)
Histologically confirmed invasive breast tumors were stained by immunohistochemical techniques using the Thermo Scientific Lab Vision primary antibodies (clones ER-SP1; PR-SP2; Her2-SP3) and Thermo Scientific™ Ultra Vision™ Quanto HRP DAB detection kit according to manufacturer’s recommended protocol (Petrosyan et al., 2002). In brief, formalin-fixed paraffin-embedded (FFPE) tissues were sectioned serially into 4 μm and were placed in histogrip coated microscope slides and incubated overnight at 60 C, deparaffinized in series of xylene (three changes), graded alcohol (2 changes 100%, 90%, and 70% ethanol) and rehydrated in distilled water. Antigen retrieval was performed using a microwave at a level in 10 mM citrate buffer (pH 6.0) at 95 °C for 45 min. Sections were then washed with Phosphate Buffered Saline (PBS) and blocked with hydrogen peroxide for 10 minutes. Then Ultra V was applied to block nonspecific backgrounds staining for 5 minutes. At this stage, sections were washed with PBS, and primary antibodies (ER-SP1; PR-SP2; Her2-SP3) were applied on the sections and incubated at room temperature for 1 hour followed by application of primary antibody enhancer and HRP polymer. Staining was visualized using Diaminobenzidine (DAB) and counterstained with haematoxylin. Sections were dehydrated and cover slipped.
IHC interpretation
We obtained results of estrogen (ER), progesterone (PR) and human epidermal growth factor-2 (HER2) status in about 40% of participants. ER and PR were considered positive if ≥ 1% nuclei of the tumor cells were stained as per the American Society College Oncology/College American Pathology (ASCO/CAP) guidelines (35). HER2 staining was scored as 0, 1+, 2+, or 3+ a positive HER2 result is IHC staining of 3+ (uniform, intense membrane staining of > 30% of invasive tumor cells, (36).
Breast cancer subtypes
Overall, 36% of the cases (301/831) had immunohistochemistry tests that included results of estrogen, progesterone and human epidermal growth factor 2. We classified breast cancer subtypes using combinations of the IHC markers as follows (a) hormone receptor positive (estrogen and progesterone) /HER2 negative (HP), and triple-negative tumors which lacked all 3 markers (TNBC) (37). Therefore, among those cases: 34.0% (n=103/301) were classified as HP, and 47.8% (n=144/301) as TNBC to further analysis subtypes of breast cancer.
Covariates
We collected information on age in years, levels of education (elementary, completed high school, post-high school with no university degree, completed university), marital status (married, single, separated/divorced/widowed), smoking experience (yes vs. no), alcohol use (yes vs. no), age at menarche, number of pregnancies (0, 1 - 3, 4 - 6, ≥ 7), ever use of oral contraceptive (yes vs. no), menopausal status (premenopausal vs. postmenopausal), age at menopause in years and breastfeeding experience of more than one month (yes vs. no). Research nurses measured participant's height, weight, waist, and hip circumferences and we derived body mass index (BMI kg/m2) and waist-hip ratio (WHR) from these measurements. Participants with extreme values of WHR of less than 0.7 or higher than 1.6 and with a BMI less than 10 kg/m2 or greater than 50 kg/m2 were excluded from the analyses (38). To compute socio-economic status, we calculated the ‘wealth index’ using the following variables - house ownership and type of house owned (e.g. home, apartment, house or duplex); source of drinking water (e.g. from outside, well, borehole, piped or bottled); type of cooking fuel; use of separate room for cooking; type of toilet; and ownership of household goods including car and refrigerator. We used Principal Component Analysis (PCA) with varimax rotation to compute factor scores based on the sum of responses to these variables weighted by their factor loading. We used the first component in the PCA that explained most of the variations in the data, to generate a wealth index (39). The wealth index variable was used to classify participants to inferior socio-economic status (lowest 40% of the score distribution), middle (middle 40%) and high (highest 20%) socio-economic class.
Statistical Analysis
From the initial study sample (1,827), the following variables included missing values: marital status (1.8%), education (2.0%), menopausal status (2.5%), pregnancy (2.8%) wealth index (2.9%), LTPA (7.8%), BMI (4.1%), WHR (4.2%), smoking (2.5%), alcohol (2.5%) and occupation (4.8%), age at the first menstrual period (6.9%), use of contraceptives (2.7%) and breastfeeding experience (4.1%). We excluded women with missing values, which reduced the sample size to 1,444. Subsequently, of those, 630 cases were matched with 630 controls based on age (±5 year) using propensity score with the optimal matching procedure.
Overall breast cancer
To examine bivariate associations between independent variables, primary exposure (LTPA) by cases and control, we implemented conditional logistic regressions. Then, for the multivariable models, we selected the independent variables that examined differences between cases and controls p-value < 0.20 in the bivariate analysis. Significantly, alcohol use that has been associated with breast cancer in previous publications was included in our multivariable models as well. In the multivariable odds ratios, we conducted conditional logistic regression models employing Wald tests to identify covariates with the significant association (p-value < 0.05) with overall breast cancer risk and to examine our main study hypothesis.
Breast cancer subtypes
To examine associations between LTPA and breast cancer subtypes, we used from the initial sample, subsamples of HP cases (n=103), and of TNBC (n=144) vs. controls (n=996) and applied unconditional logistic regression models to assess age-adjusted and multivariable odds ratios for each subtype separately.
All analyses were performed using Stata SE version 15.1 (College Station, Texas) and R-Studio Version 1.1.447.