A New Method for Obtaining Tumor Interstitial Fluid Applied to Cytokine Analysis of Breast Carcinoma Samples.

BACKGROUND/AIM: Tumor interstitial fluid (TIF), a component of the tumor microenvironment, is a valuable source of molecules and substances that help in diagnosis and prognosis of solid tumors. There is still no consensus on the optimal method for collecting TIF. Therefore, this study aimed to evaluate the effectiveness of a new method of collecting TIF in invasive ductal carcinoma (IDC) samples for cytokine interleukin 1ß (IL1ß) quantification. MATERIALS AND METHODS: Forty women allowed the collection of TIF using absorbent paper strips during the performance of the core biopsy. The samples were stored at a temperature of -80°C and then analyzed using an enzyme-linked immunoassay. RESULTS: The mean values for IL1ß and total protein were 11.39 mg/ml and 2.15 mg/ml, respectively. CONCLUSION: it was possible to quantify the cytokine IL1ß and the total protein concentration present in the tumor tissue through TIF collection with the use of absorbent paper filters, demonstrating the effectiveness of this new method in oncology.

A Phase II Study of Pembrolizumab in Combination With Palliative Radiotherapy for Hormone Receptor-positive Metastatic Breast Cancer.

BACKGROUND: The purpose of this study was to investigate whether combining pembrolizumab with palliative radiation therapy (RT) improves outcomes in patients with hormone receptor-positive (HR+) metastatic breast cancer (MBC). PATIENTS AND METHODS: Eligible patients had HR+/human epidermal growth factor receptor 2-negative MBC; were candidates for RT to ≥ 1 bone, soft tissue, or lymph node lesion; and had ≥ 1 lesion outside the RT field. Patients received 200 mg pembrolizumab intravenously 2 to 7 days prior to RT and on day 1 of repeating 21-day cycles. RT was delivered to a previously unirradiated area in 5 treatments each of 4 Gy. The primary endpoint was objective response rate. The study used a 2-stage design: 8 women were enrolled into the first stage, and if at least 1 of 8 patients experienced an objective response, 19 more would be enrolled. Secondary endpoints included progression-free survival, overall survival, and safety. Exploratory endpoints included association of overall response rate with programmed death-ligand 1 status and tumor-infiltrating lymphocytes. RESULTS: Eight patients were enrolled in stage 1. The median age was 59 years, and the median prior lines of chemotherapy for metastatic disease was 2. There were no objective responses, and the study was closed to further accrual. The median progression-free survival was 1.4 months (95% confidence interval, 0.4-2.1 months), and the median overall survival was 2.9 months (95% confidence interval, 0.9-3.6 months). All-cause adverse events occurred in 87.5% of patients, including just 1 grade 3 event (elevation of aspartate aminotransferase). CONCLUSIONS: RT combined with pembrolizumab did not produce an objective response in patients with heavily pre-treated HR+ MBC. Future studies should consider alternative radiation dosing and fractionation in patients with less heavily pre-treated HR+ MBC.

IL17 Promotes Mammary Tumor Progression by Changing the Behavior of Tumor Cells and Eliciting Tumorigenic Neutrophils Recruitment.

The aggressiveness of invasive ductal carcinoma (IDC) of the breast is associated with increased IL17 levels. Studying the role of IL17 in invasive breast tumor pathogenesis, we found that metastatic primary tumor-infiltrating T lymphocytes produced elevated levels of IL17, whereas IL17 neutralization inhibited tumor growth and prevented the migration of neutrophils and tumor cells to secondary disease sites. Tumorigenic neutrophils promote disease progression, producing CXCL1, MMP9, VEGF, and TNFα, and their depletion suppressed tumor growth. IL17A also induced IL6 and CCL20 production in metastatic tumor cells, favoring the recruitment and differentiation of Th17. In addition, IL17A changed the gene-expression profile and the behavior of nonmetastatic tumor cells, causing tumor growth in vivo, confirming the protumor role of IL17. Furthermore, high IL17 expression was associated with lower disease-free survival and worse prognosis in IDC patients. Thus, IL17 blockade represents an attractive approach for the control of invasive breast tumors.

Analysis of HLA-G gene polymorphism and protein expression in invasive breast ductal carcinoma.

The human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule predominantly expressed in trophoblastic placental cells to protect the fetus during pregnancy. However, evidence has shown that this molecule may be implicated in the immune escape mechanism of tumor cells. Thus, the aim of this study was to evaluate the frequency of 14-bp insertion/deletion HLA-G polymorphism, as well as the expression of this molecule in patients with invasive breast ductal carcinoma (IDC). A significant association between the expression of HLA-G and the presence of metastasis in lymph nodes (p=0.01) was observed and the expression of HLA-G was significantly higher in patients with shorter survival time (p=0.03). The analysis suggests that the polymorphism observed in patients with IDC may be inducing a higher expression of the HLA-G molecule, which may possibly contribute to shorter survival time and a worse clinical prognosis for such patients.

Molecular subtype is determinant on inflammatory status and immunological profile from invasive breast cancer patients.

Breast cancer consists in a chronic inflammatory disease with multiple biological and clinical behaviors. Based on high throughput technologies data, this disease is currently classified according to the molecular expression of estrogen (ER), progesterone (PR) and human epidermal growth factor (HER-2) receptors. In this study, we defined the inflammatory profile of the main molecular subtypes of breast cancer patients: luminal (ER and PR positive, HER-2 negative), HER-2 enriched (HER-2 positive) and triple negative (ER, PR and HER-2 negative). Cytokines panel was assessed by measurement of TNF-α, TGF-ß, IL-1, IL-10 and IL-12 plasmatic levels. Oxidative profile was assessed by determination of lipid peroxidation, total antioxidant capacity of plasma, malondialdehyde levels, carbonyl content and nitric oxide (NO). Clinical data were correlated with inflammatory findings. Our findings demonstrated that patients bearing the luminal subtype displayed high TNF-α, TGF-ß and enhanced oxidative stress levels associated with reduced IL-12. HER-2-enriched group exhibited higher levels of TNF-α, IL-12 and TGF-ß associated with enhanced oxidative stress. Triple-negative subtype exhibited the most aggressive profile of disease behavior, with reduction in both TNF-α and TGF-ß, with high levels of lipid peroxidation and NO. The clinical importance of our findings lies in the fact that the inflammatory status varies in distinct ways due to molecular subtype of breast cancer, opening potential therapeutic targets to future therapies.

Inoculated mammary carcinoma-associated fibroblasts: contribution to hormone independent tumor growth.

BACKGROUND: Increasing evidence has underscored the role of carcinoma associated fibroblasts (CAF) in tumor growth. However, there are controversial data regarding the persistence of inoculated CAF within the tumors. We have developed a model in which murine metastatic ductal mammary carcinomas expressing estrogen and progesterone receptors transit through different stages of hormone dependency. Hormone dependent (HD) tumors grow only in the presence of progestins, whereas hormone independent (HI) variants grow without hormone supply. We demonstrated previously that CAF from HI tumors (CAF-HI) express high levels of FGF-2 and that FGF-2 induced HD tumor growth in vivo. Our main goal was to investigate whether inoculated CAF-HI combined with purified epithelial (EPI) HD cells can induce HD tumor growth. METHODS: Purified EPI cells of HD and HI tumors were inoculated alone, or together with CAF-HI, into female BALB/c mice and tumor growth was evaluated. In another set of experiments, purified EPI-HI alone or combined with CAF-HI or CAF-HI-GFP were inoculated into BALB/c or BALB/c-GFP mice. We assessed whether inoculated CAF-HI persisted within the tumors by analyzing inoculated or host CAF in frozen sections of tumors growing in BALB/c or BALB/c-GFP mice. The same model was used to evaluate early stages of tumor development and animals were euthanized at 2, 7, 12 and 17 days after EPI-HI or EPI-HI+CAF-HI inoculation. In angiogenesis studies, tumor vessels were quantified 5 days after intradermal inoculation. RESULTS: We found that admixed CAF-HI failed to induce epithelial HD tumor growth, but instead, enhanced HI tumor growth (p < 0.001). Moreover, inoculated CAF-HI did not persist within the tumors. Immunofluorescence studies showed that inoculated CAF-HI disappeared after 13 days. We studied the mechanisms by which CAF-HI increased HI tumor growth, and found a significant increase in angiogenesis (p < 0.05) in the co-injected mice at early time points. CONCLUSIONS: Inoculated CAF-HI do not persist within the tumor mass although they play a role during the first stages of tumor formation promoting angiogenesis. This angiogenic environment is unable to replace the hormone requirement of HD tumors that still need the hormone to recruit the stroma from the host.

High concordance of SP3 rabbit monoclonal antibody with FISH to evaluate HER2 in breast carcinoma.

HER2 gene amplification or HER2 protein overexpression predicts a more aggressive clinical course in breast cancer, with a worse response to hormonal therapy, and determines eligibility for the use of the anti-HER2 antibody trastuzumab. For these reasons, the diagnostic assays that determine HER2 status in breast carcinoma have become increasingly important. Our goal was to evaluate the concordance, sensitivity, and specificity of a rabbit monoclonal antibody directed to the extracellular domain of the HER2 receptor (SP3) and compare it with fluorescence in situ hybridization and HercepTest in 179 invasive breast carcinomas. We found that SP3 was in agreement with fluorescence in situ hybridization results in 94.6% of cases. HercepTest and fluorescence in situ hybridization results were in agreement in 95.1% of the cases. Only 4.3% (4/93) of the cases that scored 0/1+ by SP3 were amplified by fluorescence in situ hybridization, and 8.3% (3/36) of cases that scored 3+ were not amplified by fluorescence in situ hybridization. Comparing SP3 with HercepTest, we observed that HercepTest demonstrated higher sensitivity (100.0% vs. 89.0%) but SP3 demonstrated higher specificity (97.0% vs. 89.0%). An important advantage of SP3 (in comparison with HercepTest) is its higher discrimination power (72.1% vs. 34.1%). For these reasons, this antibody could be helpful in the determination of HER2 status in a routine basis.

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

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.

Lymphocyte subpopulations in patients with advanced breast cancer submitted to neoadjuvant chemotherapy.

AIMS AND BACKGROUND: There is an enhanced immune response in patients with breast cancer after the use of chemotherapy. The objective of this study was therefore to investigate alterations in the number of peripheral lymphocytes in patients with breast cancer after neoadjuvant chemotherapy (NC) and the relationship with prognosis. METHODS: Thirty women were analyzed. Their UICC staging was IIb (only T3N0 included) and III (N3 not included). Sample analysis was performed using flow cytometry before the first cycle and 18 to 21 days after the last cycle of NC. The lymphocyte subsets studied were: T (CD3, CD4, CD8), B (CD19, CD23), natural killer (NK) (CD56, CD16), and interleukin-2 (CD25). CD3, CD56, CD8, and CD16 lymphocytes were analyzed with double marking. After x = 3.8 +/- 1.3 cycles of 5-fluorouracil, epirubicin and cyclophosphamide (FEC), 16 patients showed a complete or partial response (group 1). After three cycles 14 showed no response or tumor progression (group 2). A control group of healthy women was used for pretreatment analysis. RESULTS: Before NC there was a significant increase in B lymphocytes and NK cells in comparison to the control group. After NC there was a significant percentage increase in CD3, CD4, CD8, CD25 and CD3+CD56+ cells and a decrease in CD19, CD23, CD56, CD16 and CD16+CD8+ cells. There was a significant fall in the absolute number of CD4, CD19, CD23, CD56, CD16 and CD16+CD8+ lymphocytes and an increase in CD3+CD56+ lymphocytes. Before NC the ratio CD4/CD8 in group 1 was 2.25 +/- 0.5 and in group 2 it was 1.79 +/- 0.5 (P <0.05). CONCLUSIONS: Patients with advanced breast cancer showed increases in B and NK lymphocytes. Neoadjuvant chemotherapy (FEC) caused an increase in CD3+CD56+ and a decrease in B lymphocytes. Patients with an increased CD4/CD8 ratio have a better chance of responding to neoadjuvant chemotherapy.

Relationship between immune state and tumor growth rate in rats bearing progressive and non-progressive mammary tumors.

Impaired immune responses occur frequently in cancer patients or in tumor-bearing animals, but the mechanisms of the tumor-induced immune defects remain poorly understood. The aim of the present study was to determine the relevance of the immune system in the control of tumor growth. We have developed a model of progressive and non-progressive mammary tumor, chemically induced in female Wistar rats. In this model we evaluated the development of an immune response after immunization of rats bearing progressive and non-progressive tumors with a non-related antigen, such as sheep red blood cells. We also studied the activation state of peritoneal macrophages from animals bearing tumors by evaluating the production of free radicals. Our findings indicated that the cell-mediated immunity in rats bearing progressive tumors fails to respond to heterologous antigen in vivo, as demonstrated by a negative delayed-type hypersensitivity reaction, and is accompanied by minor nitric oxide production by peritoneal exudate cells as well as a lower capacity for macrophage activation. The study of non-progressive tumor-bearing rats indicated that the cell-mediated immune response was intact and an activated state of macrophages was found in vivo. The results described in this paper should be taken into account when therapies based on cancer vaccines are chosen for the treatment of cancer.

Molecular biology and gynecology: CD 36 and its interactions with tamoxifen

Tamoxifen (TAM) is an antiestrogenic drug widely used in breast cancer treatment. By using the Differential Display technique in normal and malignant breast tissues, before and during TAM therapy, we were able to demonstrate that expression of the CD36 gene is down-regulated by this drug. CD36 is a cell-surface glycoprotein that acts as a receptor for thrombospondin-1, oxidized-LDL and collagens type I and IV. Thrombospondin-1 is involved in invasion, metastasis and angiogenesis and therefore the down-regulation of CD36 induced by TAM, might correspond to an alternative mechanism of action of this drug. CD36 is also one of the receptors for the oxidized-LDL which in turn is involved in pathogenesis of arteriosclerosis; thus the down-regulation of CD36 during TAM might explain the at least in part the lower levels of myocardial infarction during its use.

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.

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.

Experience with CA 15.3 as a tumor marker in breast cancer.

CA 15.3, a new tumor marker, is a glycoprotein antigen produced in greater amounts by breast tumor cells. It can be quantitatively detected, circulating in human serum or plasma, using an immunoradiometric assay with monoclonal antibodies. In order to evaluate the usefulness of the method and to determine the cut-off for metastatic disease, the CA 15.3 levels were determined in 78 patients (5 patients with breast fibroadenoma and 73 patients with breast cancer). The conclusions of the study are that the CA 15.3 is a useful parameter in the management of patients in different stages of the disease: levels above 36 U/ml are suggestive of metastasis, and above 86 U/ml are indicative of them. On the other hand, CA 15.3 does not seem to be helpful in the pre-operative differential diagnosis of breast lumps.

Monoclonal antibodies against human breast tumor-associated antigens: characterization of B21 antigen.

BACKGROUND: Monoclonal antibodies (MAbs) show promise in the early detection and monitoring of cancer and may have therapeutic applications as well. PURPOSE: The purpose of this study was to characterize MAb B21, a novel murine-derived antibody that has strong reactivity with MCF-7 and T47D cell lines from human breast cancer. METHODS: A number of MAbs that react with breast cancer cell lines were obtained from cultured mouse spleen cells, and one, MAb B21, was selected for detailed analysis. MAb B21 was characterized by immunocytochemical, immunofluorescence, immunoprecipitation, and Western blotting procedures. RESULTS: We found a strong reactivity of MAb B21 with cultured breast cancer cells and cells from human breast tumors, although some reactivity was seen sporadically in non-breast or normal tissue. Negligible reactivity was detected in a series of non-breast cell lines and with normal breast epithelial cell line MCF-10A. However, when MCF-10A cells were permeabilized, allowing the antibody to penetrate within the cells, reaction became apparent. MCF-10A cells, when transfected with the oncogene c-Ha-ras (MCF-10T), gave a positive immunostaining similar to that observed with MCF-7 and T47D cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of L-[35S]methionine-labeled MCF-7 and T47D cell extracts showed distinct immunoprecipitated components, with molecular weight values ranging from 150,000 to 20,000 with the addition of MAb B21. Western blot assays using MAb B21 of SDS-PAGE fractionated/electroblotted proteins from breast cancer cell lines and MCF-10A cells showed specific reaction with a 95,000 component. CONCLUSIONS: Our results indicate that B21 antigen is expressed in neoplastic cells of epithelial origin, mainly breast cancer, and to a minor extent in other cell lines. In addition, MAb B21 recognizes an antigen that is differentially localized during cell transformation. IMPLICATIONS: Our future studies will address the full characterization of MAb B21 and explore its capacity as a tool for therapeutic manipulation.