Population and source of samples
All women over the age of 18 visiting the oncology clinic at Al-Azhar university hospital were invited to volunteer in the study. All new cases of histologically confirmed BC were enrolled. Women that were diagnosed with benign breast lesion, as well as women residing in the same areas and admitted to the hospital, for a wide spectrum of acute, non-neoplastic conditions unrelated to known or likely risk factors for breast cancer were invited to volunteer as controls in the study. Women with gynecological, hormonal or neoplastic diseases were excluded from the study. Peripheral blood and frozen breast tissue samples were collected according to the ethical regulations at Al-Azhar university hospital. Demographic and clinical information was obtained from medical records. The study received approval from the Institutional Review Board at Al-Azhar University. Specimens were collected from 20 treatment-naïve women with primary BC, 15 benign breast lesions (BBL), and 20 healthy volunteers (HV) after providing informed written consent. To avoid a potential influence of major surgical procedures, as well as of neoadjuvant treatment, blood was drawn after core biopsy but before definitive surgery.
Detection and genotyping of high risk HPV
HPV DNA detection and typing were carried out using the “Advanced HPV high-risk Detection and Typing PCR kits (genesig, England), following manufacture protocol. Three high risk HPV types; 16, 18, and 31 were investigated. Each Kit is an in vitro Real -Time amplification test for qualitative detection of HPV specific genotype. It is based on two major processes: isolation of DNA from specimens and Real Time amplification of the E1 – E2 region of HPV. The β-globine gene was used as an internal control. The PCR samples were analyzed by the “One step PlusTM Real Time PCR System with Notebook Computer from Applied Biosystem.
Clinicopathological risk factors were assessed, including age, lymph node metastasis, tumor size, histologic grade, tumor stage, hormonal receptor status including: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Tumor stages were classified according to the American Joint Committee on Cancer (AJCC)-TNM (tumor, node, metastases) classification.
Immunophenotyping of CD4+ Tregs and CD8+ T cells
Blood samples from patients with primary BC, BBL, and HV were procured from treatment-naïve patients prior to any surgery to avoid a potential influence of major surgical procedures and neoadjuvant treatment. Flow Cytometry testing was performed in a single laboratory on fresh blood samples the same day in was collected as previously described [29]. Two ml peripheral blood sample was collected in EDTA tubes. 100 ul of whole blood was incubated for 15 minutes at room temperature with antibodies against CD3, CD4, CD8, CD25, and intracellular transcription factor FOXP3, using immobilization buffer (BD, Biosciences, San Jose, USA). Red cell lysis was performed with FACS Lysing Solution (BD Biosciences). Isotype controls, IgG1a FITC / IgG2 PE and IgG APC were used for detection of non-specific binding background. Compensation settings were established before sample acquisition using color calibrate beads. A minimum of 30,000 CD3+ T cells per sample was acquired using a 4 color FACSCalibur (Beckton Dickenson (BD), USA) and analysis was performed by Cell-Quest Pro software (BD, USA). Gating was performed using the fluorescence-minus-one (FMO) control for each marker. Results were expressed as percent of CD25+ FOXP3+ CD4+ Tregs from the CD4+ lymphocyte gate or CD3+ CD8+ T cells from the lymphocyte gate after subtraction of the isotype control background values (<0.1%).
Statistical analysis
Statistical analysis was done using IBM SPSS® Statistics version 23 (IBM® Corp., Armonk, NY, USA). Numerical data were expressed as mean and standard deviation or median and range as appropriate. Qualitative data were expressed as frequency and percentage. For not normally distributed quantitative data, comparison between two groups was done using Mann-Whitney test (non-parametric t-test). All tests were two-tailed. When more than two groups of data sets were compared, one-way analysis of variance (ANOVA) was performed as described. A p-value < 0.05 was considered significant.