We conducted a consecutive case series study, and 177 eligible newly diagnosed BC patients within the age range of 19-73 years old were recruited from May 2011 to November 2016 at Noor-Nejat Private Hospital in Tabriz, northwest of Iran. The inclusion criteria were histopathological confirmation of primary BC, no chronic inflammation, not vegetarian, not pregnant or a breastfed mother, and no longitudinal usage of drugs (e.g., methotrexate, sulfasalazine, anticonvulsants, and contraceptive drugs). The exclusion criteria consisted of a history of confirmed malignancy during life, history of adjuvant therapy, and history of breast benign disease. Also, those who had a body mass index above 45 kg/m2 were excluded from the study. Individuals' pathological data were recorded from their medical history including histological tumor grade, tumor size, and histopathological subtype (ductal and non-ductal) and immunohistochemistry data such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2-neu).
Face-to-face interview with each participant was conducted by well-trained interviewers. Food frequency questionnaire (FFQ) with 136 food items was used to assess the dietary intake of protein. The accuracy of FFQ-based dietary intake measures of cobalamin and its association with a dietary group of protein had been met in previous studies. Food intake of participants was questioned on a timely basis of daily, weekly, monthly, and yearly. FFQ was completed for the previous year. The amount of average daily intake of a nutrient was estimated by multiplying the time-dependent frequency of food intake to the amount of consumption in grams. Portion size was defined based on the common household utensil and then converted to gram using a standard reference value. A set of photographs showing the major food groups was on hand to express better the consumption magnitude for the food items. Nutritionist IV software (ver.3.5.2; 1994, N-squared computing, San Bruno, USA) was used to compute the intake levels of total calories, macronutrients, and fiber. Total calorie intake was adjusted in terms of nutrient density as described by Willett.
Fresh tissues (tumor and adjacent normal tissues) were collected after dissection and carried in liquid nitrogen to store at - 70  C. Based on microscopic examination, tumor cells on average constituted > 85% tissue sections. Total mRNA was extracted from malignant tissues by means of QIAzol (Qiagen, USA), and RNA cleanup was carried out using the RNeasy Mini Kit (Qiagen, USA). The quantity of mRNA was measured using NanoDrop 2000 (Thermo Scientific, Germany). For complementary DNA (cDNA) synthesis, samples were synchronized at the desired concentration and subsequently reverse-transcribed by QuantiTect (Qiagen, USA) reverse transcriptase with the integral removal of genomic DNA contamination. The threshold cycle (Ct) and subsequently relative expression levels were measured using qRT-PCR by means of LightCycler 480II (Roche, Germany). Primer sets were designed for human RhoA (F: 5'AAGCAGGTAGAGTTGGCTTTGTG3'; R: 5'-ATCGGTATCTGGGTAGGAGAGG-3'), VEGF-A (F: 5'-CTACCTCCACCATGCCAAGT-3'; R: 5'-CCACTTCGTGATGATTCTGC-3'), and VEGFR2 (F: 5'-CATAGTTGTCGTTGTAGGGTA-3'; R: 5'-CATTTAGTTCAGTTCTTGCT-3'). The reaction master mix contained cDNA (~ 200 ng/muL), 10 x SYBER Green (Nanohelix, South Korea), and primers (~ 200-600 pg/muL) were used to have a reaction in a total volume of 25 muL. Thermal cycling included a first denaturation step at 95  C for 15 min, followed by 40 cycles consisting of 24 s at 95  C and 35 s at 62  C. Duplicate reactions were carried out for a single sample. Quantification of fold changes was computed using 2-DeltaDeltact formula. The expression of the hypoxanthine-guanine phosphoribosyltransferase gene (HGPRT; F: 5'-TGGACAGGACTGAACGTCTT-3'; R: 5'-CCAGCAGGTCAGCAAAGAAT-3') was used as an internal normalizing control.
Sample size was calculated based on the formula of mean comparisons and reach 100 subjects after considering the level of significance of alpha = 0.05 (two-sided), statistical power (1 - beta) = 80%, and comparing the mean (SD) relative expression levels of intercellular adhesion molecule-1 (ICAM1) gene (high fiber 1.77 +- 0.47 vs. low fiber 1.94 +- 0.38) provided by Hermsdorff et al.. On the other hand, for conducting principal component factor analysis (PCA), Gorsuch and Hair et al. recommended that sample size can be considered at least 100. A ratio of a sample size to observe variables was also highly suggested to be considered at 10. Taken together, to meet the needed sample size for conducting PCA and to generalize the findings from a sample to a wider primary population, the sample size was estimated to be 177 subjects with BC by considering the necessity of keeping potential covariates controlled in the analysis. The normal distribution of quantitative variables was assessed by using the Kolmogorov-Smirnov test and plotting histogram. Outlier data were assigned by the plotting box plot and removed. Linear regression analysis was carried out to evaluate the correlation magnitude between protein intakes from different dietary sources and fold changes of studied genes. Standardized beta coefficients (beta) and adjusted beta (betaadj.) were obtained from crude (unadjusted) and multivariate (adjusted) regression models, respectively. Models detailing the multivariate linear regression analyses consisted of following variables, just in case, when basic covariates were inter-correlated. The covariates were listed as follow: daily intake of energy (kcal/day); fat (g/day); saturated fat (g/day); cholesterol (mg/day); iron (mg/day); folate (mug/day); dietary, crude, soluble, and insoluble fiber (g/day); weight (kg); waist circumference (cm); hip circumference (cm); waist to hip ratio; body mass index (BMI; kg/m2); tumor size (cm); age at diagnosis (years); age at menarche (years); age at first childbirth (years); number of abortion; number of pregnancies; mean duration of breastfeeding (months); and mean duration of oral contraceptive usage (months). The PCA was carried out to derive appropriate dietary protein components (patterns). Accordingly, eight groups from different dietary protein sources were included in PCA. The eigenvalue is a parameter estimated to represent the sum of the variance of all the variables that can be explained by a given principal component and must be greater than 1.00, and the scree plot was applied to simplify making a decision in determining the number of components to be retained. Factor loadings, the correlation between each variable and a certain component, are presented in the component matrix. Since rotation is an important procedure to interpret the retained component, an orthogonal rotation in terms of the varimax procedure with Kaiser's normalization was carried out. Based on the rotated component matrix, a factor score for each subject on each component is computed to calculate individual's scores on each variable involved in a component. Indeed, an individual's score obtained on each variable included in a component was multiplied by the factor loading for the particular variable. The sum of a person's factor score on a component was then calculated. Subsequently, linear regression analysis was carried out to evaluate the correlation magnitude between the factor scores estimated for each component (pattern of protein sources) and fold changes of studied genes. Scatter plot was used to illustrate the correlations between the dietary patterns of protein and studied genes. Logistic regression analysis was performed to measure the odds ratio (OR) and 95% confidence interval (95% CI). Subgroup analyses based on the dichotomous status of the hormonal receptor of breast tumors (ER, PR, and HER-2) were done to control possibly the related potential effects. Further stratification analyses were relevant to the status of clinical outcomes such as tumor grade, vascular invasion (VI), and involvement of axillary lymph node metastasis (ALNM) to show whether an interested nutrigenomic correlation could be assigned in certain clinicopathological features of BC disease. Data analyses were carried out with the SPSS statistical software, version 16.0 (SPSS Inc., Chicago, IL, USA). All p values were two-tailed, and below 0.05 was considered statistically significant.
The data that support the findings of this study are available from TBZMED, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. However, data are available from the authors upon reasonable request and with permission from TBZMED.
The major ethical issues were expressed to every single participant prior to the enrolment. Each patient had signed an informed consent form before the interview. The protocol of this study and questionnaires were reviewed and approved by the Ethical Committee of the Tabriz University of Medical Sciences (TBZMED; Ethic no: IR.TBZMED.REC.1394.401).
Not applicable.