Immunophenotypic features of tumor infiltrating lymphocytes from mammary carcinomas in female dogs associated with prognostic factors and survival rates

Abstract

Background: The immune system plays an important role in the multifactorial biologic system during the development of neoplasias. However, the involvement of the inflammatory response in the promotion/control of malignant cells is still controversial, and the cell subsets and the mechanisms involved are poorly investigated. The goal of this study was to characterize the clinical-pathological status and the immunophenotyping profile of tumor infiltrating lymphocytes and their association with the animal survival rates in canine mammary carcinomas.

Methods: Fifty-one animals with mammary carcinomas, classified as carcinomas in mixed tumors-MC-BMT = 31 and carcinomas-MC = 20 were submitted to systematic clinical-pathological analysis (tumor size; presence of lymph node and pulmonary metastasis; clinical stage; histological grade; inflammatory distribution and intensity as well as the lymphocytic infiltrate intensity) and survival rates. Twenty-four animals (MC-BMT = 16 and MC = 8) were elected to the immunophenotypic study performed by flow cytometry.

Results: Data analysis demonstrated that clinical stage II-IV and histological grade was I more frequent in MC-BMT as compared to MC. Univariate analysis demonstrated that the intensity of inflammation (moderate/intense) and the proportion of CD4+ (> or = 66.7%) or CD8+ T-cells (<33.3%) were not associated with worse survival rate. Multivariate analysis demonstrated that only lymphocytic infiltrate intensity > or = 600 (P = 0.02) remained as independent prognostic factor. Despite the clinical manifestation, the lymphocytes represented the predominant cell type in the tumor infiltrate. The percentage of T-cells was higher in animals with MC-BMT without metastasis, while the percentage of B-lymphocytes was greater in animals with metastasized MC-BMT (P < 0.05). The relative percentage of CD4+ T-cells was significantly greater in metastasized tumors (both MC-BMT and MC), (P < 0.05) while the proportion of CD8+ T-cells was higher in MC-BMT without metastasis. Consequently, the CD4+/CD8+ ratio was significantly increased in both groups with metastasis. Regardless of the tumor type, the animals with high proportions of CD4+ and low CD8+ T-cells had decreased survival rates.

Conclusion: The intensity of lymphocytic infiltrate and probably the relative abundance of the CD4+ and CD8+ T-lymphocytes may represent important survival prognostic biomarkers for canine mammary carcinomas.

Figure 1

Histological diagnosis of canine mammary carcinoma samples. Low-power view of tumor specimen classified as carcinoma in benign mixed tumor (MC-BMT) associated with multifocal lymphocytic infiltrate, 200× (A); Higher power view of infiltrating lymphocytes in MC-BMT 600× (A-Inset); Low-power view of tumor specimen classified as mammary carcinoma (MC) in solid type associated with diffuse lymphocytic infiltrate, 200× (B). Histological sections (4 μm) underwent hematoxylin-eosin (HE) staining.

Figure 2

Composition of inflammatory infiltrate associated with canine mammary carcinoma. Infiltrating leukocyte populations from MC-BMT or MC (A), further subcategorized according to the absence (-) or presence (+) of lymph node metastasis (-) (B). The inflammatory cells were characterized by image analysis as described in Material and Methods. Data were expressed as number of cells (#) per eight "Hot Spots" histological fields. Significant differences at p < 0.05 are highlighted by asterisk.

Figure 3

Immunophenotypic profile of tumor infiltrating lymphocyte in canine mammary carcinomas. Analysis of tumor infiltrating T-cells, B-lymphocytes and T-cell subsets from MC-BMT or MC (A), further subcategorized according to the absence (-) or presence (+) of lymph node metastasis (-) (B). Lymphocyte populations and subsets were identified by flow cytometric immunostaining as described in Material and Methods. Data were expressed as percentage of positive cells within gated lymphocytes and CD4⁺/CD8⁺ T-cell ratio. Significant differences at p < 0.05 are highlighted by asterisk.

Figure 4

Survival rates of animals with canine mammary carcinoma. Kaplan-Meier survival curves for animals from MC-BMT or MC (top panel), further subcategorized according to the absence (-) or presence (+) of lymph node metastasis (-) (bottom panels). Animals were submitted to quarterly follow-ups during twelve months and survival rates (%) expressed in days between the surgical excisions of the end of follow-up as described in Methods. The survival curves were estimated with the Kaplan-Meier method followed by Log-rank test and significant differences at p < 0.05 highlighted by asterisk.

Figure 5

Survival rates of animals with canine mammary carcinoma. Kaplan-Meier survival curves for All (MC-BMT and MC) animals (A) categorized according to the lymphocytic infiltrate intensity (<600 and ≥ 600 lymphocytes) further sub grouped according to the histological diagnosis (MC-BMT or MC) (B) (bottom panels). Animals were submitted to quarterly follow-ups during twelve months and survival rates expressed in days between the surgical excisions of the end of follow-up as described in Methods. The survival curves were estimated with the Kaplan-Meier method followed by Log-rank test and significant differences at p < 0.05 highlighted by asterisk.

Figure 6

Survival rates of animals with canine mammary carcinoma. Kaplan-Meier survival curves for animals for All (MC-BMT and MC) animals categorized according to the relative percentage of CD4⁺ T-cells (<66.7% or ≥ 66.7%) and CD8⁺ T-cells (≤ 33.3% or >33.3%) (A). Animals were submitted to quarterly follow-ups during twelve months and survival rates expressed in days between the surgical excisions of the end of follow-up as described in Methods. The survival curves were estimated with the Kaplan-Meier method followed by Log-rank test. Correlation analysis highlighted the significant association between the percentages of CD4⁺ T-cells and CD8+ T-cells with the animal survival in days (B).