Apoptosis and production of TNF-alpha by tumor-associated inflammatory cells in histological grade III breast cancer

Abstract

Tumor necrosis factor alpha (TNF-alpha) is a cytokine that acts as an important mediator of the apoptotic process that also demonstrates selective citotoxicity against malignant breast tumor cells. In the present study, the presence of apoptotic tumor cells and the synthesis of TNF-alpha by inflammatory cells were investigated in tissue samples from grade III invasive breast cancer with long-term follow-up. In situ detection of tumor apoptotic cells was investigated by direct immuno-peroxidase of digoxigenin-labeled genomic DNA. The production of TNF-alpha and tumor cell proliferation were investigated by immunohistochemical procedures. Our data demonstrated that patients with a clinical history of cancer recurrence and metastasis presented a lower number of cancerous apoptotic cells, higher tumor proliferation rates, and lower TNF-alpha expression rates by inflammatory cells than what is observed among patients diagnosed with the same histopathological breast cancer type but in the absence of tumor recurrence and metastasis.

Fig. 1

Histopathological analyses and proliferative index. a Poorly differentiated ductal carcinoma. Classified as grade III. Arrows: note several tumor-associated mononuclear inflammatory cells (hematoxilin and eosin 200× magnification). Tumoral cells area delineated by a blue line. b Proliferative index investigated by Ki67 antibody (grupo II): many Ki 67 positive cells in grade III ductal carcinoma (400× magnification). Arrows: indicates a Ki 67 positive cell. In set: negative control of the immunohistochemical procedure(400× magnification). c Number of Ki67 positive cells. Tumoral cells observed in ten microscopic fields. At least 500 cells, with 400× magnification were counted. Results expressed in %, (Mean+/−SD), N=25, of total tumor cells. Not statistically significant differences (p>0.05) were noted in Group I when compared to Group II

Fig. 2

Positive staining of TNFα in tumor surrounding inflammatory cells in grade III ductal carcinoma. Arrows: indicate positive cells. Tumoral cells area delineated by a blue line. a Group I: patients without metastasis or recurrence (400× magnification). In set: negative control of the immunohistochemical procedure(200× magnification). b Group II: patients with recurrence and metastasis (400× magnification). c Number of TNFα positive cells. Inflammatory cells observed in ten microscopic fields. At least 1,000 cells, with 400× magnification were counted. Results expressed in %, (Mean+/−SD), N=25, of total tumor-associated inflammatory cells.Statistically significant differences (p<0,05) were noted in Group I when compared to Group II

Fig. 3

Apoptotic tumor cells in grade III ductal carcinoma detected by TUNEL method. Arrows: indicate apoptotic cells. a Group I: patients without metastasis or recurrence (400× magnification). In set: negative control of the immunohistochemical procedure(100× magnification). b Group II: patients with recurrence and metastasis (400× magnification). c) Number of apoptotic cells. Tumoral cells observed in ten microscopic fields. At least 500 cells, with 400× magnification were counted. Results expressed in %, (Mean+/−SD), N=25, of total tumor cells. Statistically significant differences (p<0.05) were noted in Group I when compared to Group II