Tumor samples

After approval from the Faculty of Medicine Research Ethics Committee, Minia University, 53 Egyptian patients suffering from breast cancer who attended the Minia University hospital and the Minia Oncology center were chosen and signed written informed consent for participation in the study. The patients were investigated and followed from January 2012 to December 2016. Patients with history of diabetes mellitus (DM), metabolic syndrome, and those suffering from coexisting illnesses such as chronic liver and kidney disease were excluded.

All the participants were evaluated according to a standardized form which included medical history (focusing on menstrual history, reproductive history, menopausal history, history of DM, and family history of breast cancer) and physical examination. Height in meters and weight in kilograms were measured, and the body mass index (BMI) was calculated [weight (kg)/height (m)2]. Paraffin blocks of the malignant tissues, the adjacent non-malignant tissues, and the lymph node (LN) metastasis were obtained from each case. The clinical and pathological data are given in Table 1.

We performed immunohistochemical staining on 4 μm thick sections of the most representative paraffin blocks from each tumor. Slides were heated at 60°C and de-paraffinized in xylene. Subsequently, they were rehydrated by replacing graded alcohol with water. Heat-induced antigen retrieval was achieved by boiling sections in a citrate buffer, pH 6, in a microwave oven at 700 W for 20 minutes (four times for 5 minutes each). After boiling, sections were left to cool at room temperature and rinsed thoroughly with PBS for 5 minutes. Endogenous peroxidase was blocked with peroxidase block solution (provided in the EnVision kit, Dako Denmark A/S, Glostrup, Denmark) for 10 minutes and slides were rinsed with PBS. Sections were incubated for 30 minutes with primary mouse monoclonal anti-human chemerin antibody (Abcam, Cambridge, UK) using a 1:400 dilution. The same sections were processed without primary antibodies as negative control.

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Tissue classification

Diagnoses were made based on paraffin-embedded 4 µm tissue sections after H&E staining. Tissues were classified according to histological subtypes as invasive ductal carcinoma of different grades (n=53). Invasive tumors were evaluated according to the Scarff-Bloom-Richardson grade classification modified by Elston et al.30 Normal tissue adjacent to breast cancer was also analyzed as control (n=53). The staging of breast cancer was determined based on the TNM system based on definitions and recommendations of European Society of Medical Oncology, 2015.31

Interpretation of immunohistochemistry staining

Positive signal for chemerin was located mainly in the cytoplasm and to a lesser extent the nucleus of the ductal cells and the score was semi-quantitatively estimated on the basis of both percentage and intensity of positively stained tumor cells. Five views were examined per slide, and 100 cells were counted per view at ×400 magnification. The intensity of chemerin staining was classified using the 4-point scale as 0 (no signal), 1 (weak staining), 2 (moderate staining), 3 (strong staining). Percentage scores were assigned as 0, no staining; 1, 1%–25%; 2, 26%–50%; 3, 51%75%; and 4, 76%–100%. Their multiplication (intensity score × area score) was calculated in each area. Thus, the score should range from 0 to 12. For statistical analysis, chemerin immunostaining scores of 0–4 were defined as weak or low expression, and scores of 5–12 were defined as high expression.

Statistical analysis

The statistical analyses were performed using SPSS version 24.0 (IBM Corporation, Armonk, NY, USA). Data on normal distribution were expressed as mean ± SD and were compared using independent samples t-test. Categorical variables were expressed as percentage and were compared using the chi-squared test or Fisher’s exact test, as appropriate. Non-parametric variables were analyzed using the Mann–Whitney U test. The Pearson correlation coefficients were used to study the correlation between different parametric variables. Spearman rank correlation was used to quantify the association between continuous or ordered categorical variables. Receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were calculated. Kaplan–Meier survival curves were calculated for disease-free survival. A multivariate analysis was performed using the Cox regression model to study the effects of different variables on survival. P-value <0.05 was considered as statistically significant.