Determining Factors in the Therapeutic Success of Checkpoint Immunotherapies against PD-L1 in Breast Cancer: A Focus on Epithelial-Mesenchymal Transition Activation.

Breast cancer is the most common neoplasm diagnosed in women around the world. Checkpoint inhibitors, targeting the programmed death receptor-1 or ligand-1 (PD-1/PD-L1) axis, have dramatically changed the outcome of cancer treatment. These therapies have been recently considered as alternatives for treatment of breast cancers, in particular those with the triple-negative phenotype (TNBC). A further understanding of the regulatory mechanisms of PD-L1 expression is required to increase the benefit of PD-L1/PD-1 checkpoint immunotherapy in breast cancer patients. In this review, we will compile the most recent studies evaluating PD-1/PD-L1 checkpoint inhibitors in breast cancer. We review factors that determine the therapeutic success of PD-1/PD-L1 immunotherapies in this pathology. In particular, we focus on pathways that interconnect the epithelial-mesenchymal transition (EMT) with regulation of PD-L1 expression. We also discuss the relationship between cellular metabolic pathways and PD-L1 expression that are involved in the promotion of resistance in TNBC.

Breast Tumor Cells Highly Resistant to Drugs Are Controlled Only by the Immune Response Induced in an Immunocompetent Mouse Model.

BACKGROUND: The tumor cells responsible for metastasis are highly resistant to chemotherapy and have characteristics of stem cells, with a high capacity for self-regeneration and the use of detoxifying mechanisms that participate in drug resistance. In vivo models of highly resistant cells allow us to evaluate the real impact of the immune response in the control of cancer. MATERIALS AND METHODS: A tumor population derived from the 4T1 breast cancer cell line that was stable in vitro and highly aggressive in vivo was obtained, characterized, and determined to exhibit cancer stem cell (CSC) phenotypes (CD44+, CD24+, ALDH+, Oct4+, Nanog+, Sox2+, and high self-renewal capacity). Orthotopic transplantation of these cells allowed us to evaluate their in vivo susceptibility to chemo and immune responses induced after vaccination. RESULTS: The immune response induced after vaccination with tumor cells treated with doxorubicin decreased the formation of tumors and macrometastasis in this model, which allowed us to confirm the immune response relevance in the control of highly chemotherapy-resistant ALDH+ CSCs in an aggressive tumor model in immunocompetent animals. CONCLUSIONS: The antitumor immune response was the main element capable of controlling tumor progression as well as metastasis in a highly chemotherapy-resistant aggressive breast cancer model.

IL-1ß induced methylation of the estrogen receptor ERα gene correlates with EMT and chemoresistance in breast cancer cells.

Inflammation has been recently acknowledged as a key participant in the physiopathology of oncogenesis and tumor progression. The inflammatory cytokine IL-1ß has been reported to induce the expression of markers associated with malignancy in breast cancerous cells through Epithelial-Mesenchymal Transition (EMT). Aggressive breast cancer tumors classified as Triple Negative do not respond to hormonal treatment because they lack three crucial receptors, one of which is the estrogen receptor alpha (ERα). Expression of ERα is then considered a good prognostic marker for tamoxifen treatment of this type of cancer, as the binding of this drug to the receptor blocks the transcriptional activity of the latter. Although it has been suggested that inflammatory cytokines in the tumor microenvironment could regulate ERα expression, the mechanism(s) involved in this process have not yet been established. We show here that, in a cell model of breast cancer cells (6D cells), in which the inflammatory cytokine IL-1ß induces EMT by activation of the IL-1ß/IL-1RI/ß-catenin pathway, the up regulation of TWIST1 leads to methylation of the ESR1 gene promoter. This epigenetic modification produced significant decrease of the ERα receptor levels and increased resistance to tamoxifen. The direct participation of IL-1ß in these processes was validated by blockage of the cytokine-induced signaling pathway by wortmannin inactivation of the effectors PI3K/AKT. These results support our previous reports that have suggested direct participation of the inflammatory cytokine IL-1ß in the transition to malignancy of breast cancer cells.

Analysis of CD83 antigen expression in human breast fibroadenoma and adjacent tissue / Análise da expressão do antígeno CD83 no fibroadenoma mamário humano e no tecido adjacente

CONTEXT AND OBJECTIVE: Dendritic cell maturation is considered essential for starting an immune response. The CD83 antigen is an important marker of dendritic cell maturation. The objectives here were to analyze CD83 antigen expression in human breast fibroadenoma and breast tissue adjacent to the lesion and to identify clinical factors that might influence this expression. DESIGN AND SETTING: This was a retrospective study at a public university hospital, in which 29 histopathological samples of breast fibroadenoma and adjacent breast tissue, from 28 women of reproductive age, were analyzed. METHODS: The immunohistochemistry method was used to analyze the cell expression of the antigen. The antigen expression in the cells was evaluated by means of random manual counting using an optical microscope. RESULTS: Positive expression of the CD83 antigen in the epithelial cells of the fibroadenoma (365.52; standard deviation ± 133.13) in relation to the adjacent breast tissue cells (189.59; standard deviation ± 140.75) was statistically larger (P < 0.001). Several clinical features were analyzed, but only parity was shown to influence CD83 antigen expression in the adjacent breast tissue, such that positive expression was more evident in nulliparous women (P = 0.042). CONCLUSIONS: The expression of the CD83 antigen in the fibroadenoma was positive and greater than in the adjacent breast tissue. Positive expression of the antigen in the adjacent breast tissue was influenced by parity, and was significantly more evident in nulliparous women.

CONTEXTO E OBJETIVOS: A maturação da célula dendrítica é considerada essencial para o início da resposta imune. O antígeno CD83 é um importante marcador da maturação da célula dendrítica. Os objetivos são analisar a expressão do antígeno CD83 no fibroadenoma mamário humano e no tecido mamário adjacente à lesão e identificar fatores clínicos que possam influenciar esta expressão. TIPO DE ESTUDO E LOCAL: Este é um estudo retrospectivo, realizado em um hospital público universitário, onde 29 amostras histopatológicas de fibroadenomas de mamas e de tecidos mamários adjacentes, de 28 mulheres em idade reprodutiva, foram analisados. MÉTODOS: O método de imunoistoquímica foi utilizado na análise da expressão celular do antígeno. A expressão do antígeno nas células foi avaliada por contagem aleatória e manual utilizando-se microcópio de luz. RESULTADOS: A expressão positiva do antígeno CD83 nas células epiteliais dos fibroadenomas (365,52; desvio padrão ± 133,13) em relação às células do tecido mamário adjacente (189,59; desvio padrão ± 140,75) foi estatisticamente superior (P < 0,001). Vários aspectos clínicos foram analisados, porém, a paridade se mostrou influente na expressão do antígeno CD83 no tecido mamário adjacente, onde a expressão positiva foi mais evidente nas mulheres nulíparas (P = 0,042). CONCLUSÕES: A expressão do antígeno CD83 foi positiva e mais expressiva no fibroadenoma do que no tecido mamário adjacente. A expressão positiva do antígeno no tecido mamário adjacente foi influenciada pela paridade, sendo significativamente mais evidente nas mulheres nulíparas.

Analysis of CD83 antigen expression in human breast fibroadenoma and adjacent tissue.

CONTEXT AND OBJECTIVE: Dendritic cell maturation is considered essential for starting an immune response. The CD83 antigen is an important marker of dendritic cell maturation. The objectives here were to analyze CD83 antigen expression in human breast fibroadenoma and breast tissue adjacent to the lesion and to identify clinical factors that might influence this expression. DESIGN AND SETTING: This was a retrospective study at a public university hospital, in which 29 histopathological samples of breast fibroadenoma and adjacent breast tissue, from 28 women of reproductive age, were analyzed. METHODS: The immunohistochemistry method was used to analyze the cell expression of the antigen. The antigen expression in the cells was evaluated by means of random manual counting using an optical microscope. RESULTS: Positive expression of the CD83 antigen in the epithelial cells of the fibroadenoma (365.52; standard deviation ± 133.13) in relation to the adjacent breast tissue cells (189.59; standard deviation ± 140.75) was statistically larger (P < 0.001). Several clinical features were analyzed, but only parity was shown to influence CD83 antigen expression in the adjacent breast tissue, such that positive expression was more evident in nulliparous women (P = 0.042). CONCLUSIONS: The expression of the CD83 antigen in the fibroadenoma was positive and greater than in the adjacent breast tissue. Positive expression of the antigen in the adjacent breast tissue was influenced by parity, and was significantly more evident in nulliparous women.

Evaluation of monoclonal antibody MIB-1 in the mammary epithelium adjacent to fibroadenomas in premenopausal women treated with tamoxifen.

The purpose of this study was to study the monoclonal antibody MIB-1 in the normal breast epithelium adjacent to a fibroadenoma in women in the luteal phase of the menstrual cycle who were treated with tamoxifen at doses of 10 and 20 mg for 22 days. The proliferative activity of the mammary epithelium adjacent to the fibroadenoma was studied by immunohistochemistry on the basis of the monoclonal antibody MIB-1 (Immunotech, catalog No. 0505, lot 001). The study was randomized and double blind and was conducted on 44 women with fibroadenomas divided into three groups: A (n=16, placebo), B (n=15, tamoxifen, 10 mg), and C (n=13, tamoxifen, 20 mg). Tamoxifen was administered for 22 days starting on the 2nd day of the menstrual cycle, and a biopsy was taken on the 23rd day. Serum estradiol, progesterone, sex hormone binding globulin, follicle-stimulating hormone, luteinizing hormone, and prolactin were measured before treatment (21st and 24th day of the previous menstrual cycle) and on the day of the biopsy. The mean percentage of stained nuclei per 1,000 cells was 9.2 in group A, 4.5 in group B, and 3.2 in group C. The Fisher's test revealed that tamoxifen significantly reduced MIB-1 at doses of 10 and 20 mg compared with the placebo group (p < 0.0001), with no significant differences between doses in terms of proliferative activity (p=0.21). Groups B and C presented a significant increase in progesterone (p=0.038), estradiol (p < 0.001), and sex hormone binding globulin (p=0.001) levels. Elevation of serum follicle-stimulating hormone concentration (p=0.0045) and a fall in prolactin levels (p=0.0055) were observed. We conclude that tamoxifen significantly reduced the proliferative activity of the mammary epithelium at the doses of 10 and 20 mg/day.

Simple mucin-type carbohydrate antigens (T, sialosyl-T, Tn and sialosyl-Tn) in breast carcinogenesis.

Immunohistochemical analysis of the expression of simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) was performed in a series of 43 cases of intraductal hyperplasia without atypia, 9 cases of intraductal hyperplasia with atypia, 54 cases of ductal carcinoma in situ (DCIS) and 26 cases of invasive breast carcinoma. We also studied 36 cases of isolated breast normal epithelium, 20 cases of "normal" breast epithelium adjacent to neoplasms and 14 cases of apocrine metaplasia. All antigens were detected in different frequencies in normal, hyperplastic, metaplastic and neoplastic breast epithelium. Tn and sialyl-Tn are expressed more frequently in malignant than in benign breast epithelium; while Tn expression increases from normal to invasive carcinomas, sialyl-Tn increases until DCIS and drops in invasive carcinomas, suggesting that either there is a failure of a proportion of DCIS to progress to invasive carcinoma or loss of expression of sialyl-Tn when some carcinomas become invasive. The high frequency of Tn and sialyl-Tn expression in breast intraductal proliferations probably reflects incomplete glycosylation in these lesions, which is a well-known tumour-associated phenomenon and supports the assumption that such lesions are putative precursors of breast cancer. T antigen was expressed in all groups studied, but its prevalence differed significantly between normal and neoplastic epithelium. The expression of these antigens in epithelium adjacent to carcinomas is similar to that found in isolated normal breast epithelium, whereas apocrine metaplasia has a pattern of simple mucin-type glycosylation that is specific and distinct from that of the normal breast epithelium, with a high frequency of marked expression of Tn and sialyl-Tn. The similarity of the pattern of expression of simple mucin-type antigens in metaplasia and malignant neoplasia reduces the usefulness of these markers from a diagnostic standpoint.

Spectrum of carcinoembryonic antigen immunoreactivity from isolated ductal hyperplasias to atypical hyperplasias associated with infiltrating ductal breast cancer.

AIMS: To study the immunohistochemical expression of carcinoembryonic antigen (CEA) in ductal hyperplasia of the breast and to investigate its putative relation with atypia and co-existing infiltrating ductal carcinoma. METHODS: Paraffin wax embedded tissue from 37 cases of isolated ductal hyperplasia (five with atypia and 32 without atypia) and 25 cases of ductal hyperplasia associated infiltrating ductal carcinoma (IDC) (seven with atypia and 18 without atypia) was stained with a monoclonal anti-CEA antibody using a standard avidin biotin immunoperoxidase method. RESULTS: CEA immunoreactivity was observed in eight (12.8%) ductal hyperplasia cases. The percentage of CEA positivity in ductal hyperplasia cases with atypia (33.3%) was substantially higher than that observed in cases of ductal hyperplasia without atypia (8.0%). Six cases of ductal hyperplasia associated IDC reacted with CEA; in these six cases the neoplastic cells of the co-existing carcinoma were also CEA positive. The percentage of CEA immunoreactivity in ductal hyperplasia associated IDC was higher than that observed in isolated ductal hyperplasia (24.0 v 5.4%). The percentage of CEA immunoreactivity in atypical ductal hyperplasia associated IDC was similar to that observed in IDC alone (42.9 v 40.0%). CONCLUSIONS: The presence of CEA immunoreactivity has been confirmed in benign proliferative breast lesions. The prevalence of such immunoreactivity increases from 3.1% in isolated, nonatypical ductal hyperplasia to 42.9% in atypical ductal hyperplasia associated IDC. This finding and the similarity of the frequency of CEA positivity in atypical ductal hyperplasia associated IDC and in IDC alone suggests that there is a pathogenetic link between ductal hyperplasia and some types of breast cancer.

The immune response of the mammary gland and its significance for the neonate.

The immune response of the mammary gland is dominated by local production of secretory IgA antibodies (SIgA). These milk antibodies, amounting to about 0.5-1 g/day throughout lactation, are directed against food proteins and microorganisms often present in the intestine. This is presumably explained by the enteromammaric link: after antigenic exposure in the Peyer's patches of lymphoid cells they home to various exocrine glands, including the mammary gland. Similarly, lymphoid cells from the bronchial mucosa, may contribute to the antibody-producing cell population in the mammary gland. SIgA antibodies against common foods like cow's milk and soy proteins are regularly found in milk if such proteins are part of the mother's diet. It is possible, but unproven, that milk antibodies can decrease the exposure of the infant's intestinal mucosa to foreign food proteins introduced during continued breast-feeding. Milk SIgA antibodies do not prevent intestinal colonization by microorganisms, against which the milk antibodies are directed. The SIgA antibodies are thought to exert protection primarily by preventing contact between the microorganisms and the mucosal membranes. In this manner, human milk blocks attachment of otitis media-causing strains of pneumococci and H. influenzae to retropharyngeal cells, possibly explaining why breast-feeding may prevent otitis media. Milk antibodies have anti-attachment capacity, but there is also low molecular weight material in the milk with this capacity. It probably consists of analogues to the oligosaccharide receptor for pneumococci on the retropharyngeal cells.(ABSTRACT TRUNCATED AT 250 WORDS)