Patients, samples, and follow-up
This study was approved by the Ethics Committee on Animal Use of the Federal University of Minas Gerais, Brazil (protocol #81–2013). Sixty-six female dogs diagnosed with mammary carcinomas from 2011 to 2015 were used in this study. All patients were submitted to surgical mastectomy as treatment option with subsequent microscopic evaluation of the removed mammary tumors. They were classified according to the World Health Organization’s histologic criteria for canine mammary tumors diagnosis [16] and to updated classification propositions [17]. Subsequently, tumors were graded using the Nottingham grading system [18]. When dogs presented more than one malignant neoplasm, the tumor with the more aggressive clinical and histopathologic features (e.g., larger size, undifferentiated histology) was selected, as recommended by other authors [19]. The WHO’s staging system [20] was used for determination of clinical stage based on animals’ clinical, radiographic, and sonographic findings.
Patients’ data were obtained by phone interviews with owners and by reviewing the clinical data available at the Institution’s Veterinary Teaching Hospital regarding animals that died at the place. Overall survival (OS) was calculated and characterized as the time from surgery/tumor removal to the date of animal death/euthanasia due to tumor-related causes and/or end of the study. When the owners did not answer the or when animals died from other causes data was censored.
Immunohistochemical staining
For immunohistochemistry, 3µm-thick sections were obtained from paraffin blocks and mounted on poly-L-lysine-coated slides. Sections were deparaffinized and rehydrated and subsequently submitted to heat-induced epitope/antigen retrieval for 30 seconds at 125oC using the Trilogy antigen retrieval buffer (Cell Marque Corporation, Rocklin, CA) in a pressure chamber (Pascal Pressure Chamber, Dako, Carpinteria, CA). Proteinase K antigen retrieval was used when suitable, with subsequent incubation of tissue sections with a mouse anti-macrophage antibody (clone MAC387, 1:400, AbD Serotec, Oxford, UK). Canine lymph node tissue was used as positive control for reactions while negative controls were obtained by using isotype-matched primary antibodies. For anti-macrophage immunohistochemistry, the staining was revealed by using a polymer based detection system (Envision G2 System/AP, rabbit/mouse, Dako) with liquid permanent red as chromogen (Permanent red substrate-chromogen, Dako) and visualized under light microscopy.
Immunolabelling and quantification of tumor-associated macrophages
Immunoreaction was considered as positive for anti-macrophage antibody when cytoplasmic diffuse or granular staining (but no nuclear) was observed and simultaneously demonstrating macrophage morphology. Since the antibody also stains granulocytic cells, macrophages were distinguished from other cells, such as neutrophils by nuclear morphologic, a non-segmented nucleus. The number of stained TAMs were counted in each tumor in five hot spot areas first detected at low magnification and then captured at x200 magnification (total area of 1 mm2) without any previous knowledge of patients’ clinical data. TAM hot spots were chosen rather than random fields, because these areas are thought to be biologically the most important [21–22]. For cutoff point analysis, the medians were used to categorize tumors into high and low TAM groups.
In order to distinguish intratumoral (i.e., intratumoral) and stromal TAM, criteria previously applied by other authors [10] and based on differentiation of stromal from intratumoral lymphocytes [23–24] were used. Intratumoral TAM was defined as macrophages within tumor cell nests an in direct contact with tumor cells, and stromal TAM were characterized as macrophages infiltrating tumor stroma (Figure 1). After counting, the number of intratumoral, stromal, and total TAM (sum of both types) was calculated. Median values were calculated for each histologic location and used for categorization of tumors as with low or high TAM infiltration.
Statistical analysis
TAM infiltration was compared using analysis of variance (ANOVA) with TAM value as a continuous variable. Correlation between the number of macrophages and clinicopathologic features was assessed using Spearman rank order correlation or the Mann-Whitney U test, which was used for comparison between different histologic locations. Survival curves were produced using the Kaplan-Meier method and differences in survival time between patients were evaluated using the log-rank test. Results were considered as statistically significant when P ≤ 0.05.