Immunohistochemical Analysis of the Expression of Breast Markers in Basal-like Breast Carcinomas Defined as Triple Negative Cancers Expressing Keratin 5.

Estrogen and progesterone receptors are possible markers for suggesting a mammary origin of metastatic carcinoma, but are useless in cases of triple negative breast cancers (TNBC). Five other potential markers of breast origin were investigated on tissue microarrays in a series of TNBCs showing keratin 5 expression, consistent with a basal-like phenotype. GATA-3 staining was observed in 82 of 115 triple negative cases (71.3%) including 23 cases with >5% staining. Mammaglobin staining was detected in 30 cases (26.0%) including 12 with >5% staining. GCDFP-15 was seen in 23 cases (20.0%) including 9 with >5% staining. NY-BR-1 positivity was present in 7 cases (6.0%) including 3 patients with >5% staining. BCA-225 staining was observed in 74 cases (64.3%); however this latter marker lacks also specificity owing to the reported widespread staining in other malignancies. GATA-3, mammaglobin and GCDFP-15 coexpression was seen in one case (0.9%), whereas GATA-3 and mammaglobin or mammaglobin and GCDFP-15 coexpression was present in 2 and 2 cases (1.7%), respectively. Using at least 5% staining as cut-off, the expression of any of the last 4 markers was 34.7%. The expression of GATA-3, mammaglobin, GCDFP-15 and NY-BR-1 is lower in TNBC-s than in breast carcinomas in general, and this may be even lower in basal-like carcinomas. Although these markers are not fully specific, by using them, a subset of basal-like TNBC-s can be identified as of mammary origin. However, a substantial proportion will not show any staining with any of these markers.

Can GATA3 Immunocytochemistry be Utilized as a Reliable Diagnostic Marker for Metastatic Breast Carcinoma in Cytological Materials? A Comparative Study with Mammaglobin and GCDFP-15 Expression.

OBJECTIVE: Cytomorphologic differentiation of metastatic breast carcinoma from non breast metastases in cytological materials can be difficult. Current breast immunocytochemical markers have low sensitivities. Transcription factor GATA 3 is a promising marker for detecting breast differentiation in cytological materials. The aim of the study was to assess the diagnostic value of GATA 3 as a breast differentiation marker in metastatic cytological materials and to compare it with expression of mammaglobin and gross cystic disease fluid protein-15 (GCDFP-15). MATERIAL AND METHOD: We retrospectively retrieved 133 cases of metastatic breast carcinoma from the archive the of Cytology Unit between December 2013 and June 2015. They included 77 fine needle aspiration and 56 serous effusion samples. Forty-five cytological materials from non mammary metastatic tumors were used as a control. Immunostaining was performed on cell blocks for the presence of GATA 3, mammaglobin and GCDFP-15. RESULTS: GATA 3 nuclear staining was detected in 82.7% of metastatic breast carcinomas, and 11.1% of metastatic non mammary adenocarcinomas (p < 0.001). GATA 3 sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 82.7%, 88.9%, 95.7%, 63.5% and 84.3%, respectively. Mammaglobin and GCDFP-15 staining of metastatic breast carcinoma cases was positive in 70.7% and 47.1%, respectively. GATA 3 staining was significantly higher compared with mammaglobin and GCDFP-15 (p < 0.001). CONCLUSION: GATA 3 is more sensitive marker than mammaglobin and GCDFP-15 for diagnosing metastatic breast carcinoma in cytological cell block materials. Adding mammaglobin to GATA 3 resulted in improvement in its sensitivity. GATA 3 was occasionally positive in some metastatic non mammary carcinoma that may cause misdiagnosis.

Preferential expression of NY-BR-1 and GATA-3 in male breast cancer.

BACKGROUND: Male breast cancer is an uncommon disease often discovered in advanced stage; thus, in the setting of metastatic adenocarcinoma, breast origin must be taken to account. Breast markers as NY-BR-1, GATA-3, mammaglobin, and BRST-2 are established tools for labelling primary and metastatic female breast cancer; however, none of them has been sufficiently studied in male breast cancer. The aim of this study was to analyze the expression of these markers in male breast cancer. MATERIALS AND METHODS: Thirty consecutive cases of male breast cancer and eight loco-regional metastases were re-revaluated, assembled in tissue micro array (TMA), and stained with immunohistochemistry (IHC) for NY-BR-1, GATA-3, mammaglobin, and BRST-2. The IHC stains were scored either positive or negative. In addition, concordant expression patterns of primary tumors and matched metastasis were noted. RESULTS: 30 of 30 (100%) primary tumors and 8 of 8 (100%) metastases were positive for NY-BR-1. 30 of 30 (100%) primary tumors and 6 of 8 (75%) metastases were positive for GATA-3. 22 of 30 (73.3%) primary tumors and 6 of 8 (75%) metastases were positive for Mammaglobin. 18 of 30 (60%) primary tumors and 5 of 8 (62.5%) metastases were positive for BRST-2. Differences in staining percentage were not significant with Fisher's exact test. CONCLUSION: We found a high sensitivity for all the markers analyzed. Moreover, the expression of NY-BR-1 and GATA-3 seemed the most effective for labelling male breast cancer in primary and metastatic setting.

Cationic polyaspartamide-based nanocomplexes mediate siRNA entry and down-regulation of the pro-inflammatory mediator high mobility group box 1 in airway epithelial cells.

High-mobility group box 1 (HMGB1) is a nonhistone protein secreted by airway epithelial cells in hyperinflammatory diseases such as asthma. In order to down-regulate HMGB1 expression in airway epithelial cells, siRNA directed against HMGB1 was delivered through nanocomplexes based on a cationic copolymer of poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) by using H441 cells. Two copolymers were used in these experiments bearing respectively spermine side chains (PHEA-Spm) and both spermine and PEG2000 chains (PHEA-PEG-Spm). PHEA-Spm and PHEA-PEG-Spm derivatives complexed dsDNA oligonucleotides with a w/w ratio of 1 and higher as shown by a gel retardation assay. PHEA-Spm and PHEA-PEG-Spm siRNA polyplexes were sized 350-650 nm and 100-400 nm respectively and ranged from negativity/neutrality (at 0.5 ratio) to positivity (at 5 ratio) as ζ potential. Polyplexes formed either at a ratio of 0.5 (partially complexing) or at the ratio of 5 (fully complexing) were tested in subsequent experiments. Epifluorescence revealed that nanocomplexes favored siRNA entry into H441 cells in comparison with naked siRNA. As determined by flow cytometry and a trypan blue assay, PHEA-Spm and PHEA-PEG-Spm allowed siRNA uptake in 42-47% and 30% of cells respectively, however only with PHEA-Spm at w/w ratio of 5 these percentages were significantly higher than those obtained with naked siRNA (20%). Naked siRNA or complexed scrambled siRNA did not exert any effect on HMGB1mRNA levels, whereas PHEA-Spm/siRNA at the w/w ratio of 5 down-regulated HMGB1 mRNA up to 58% of control levels (untransfected cells). PEGylated PHEA-Spm/siRNA nanocomplexes were able to down-regulate HMGB1 mRNA levels up to 61% of control cells. MTT assay revealed excellent biocompatibility of copolymer/siRNA polyplexes with cells. In conclusion, we have found optimal conditions for down-regulation of HMGB1 by siRNA delivery mediated by polyaminoacidic polymers in airway epithelial cells in the absence of cytotoxicity. Functional and in-vivo studies are warranted.

Sensitivity of neoplastic cells to senescence unveiled under standard cell culture conditions.

BACKGROUND: Cancer cells are typically defined as infinitely proliferating, whereas normal cells (except stem cells) are considered as being programmed to become senescent. Our data show that this characterization is misleading. MATERIALS AND METHODS: Multiplex Ligation-dependent Probe Amplification, TP53 sequencing, real-time polymerase chain reaction (PCR) for MUC1 and SCGB2A2 and immunocytochemistry, together with senescence detection assay and real-time microscopic observations were used to analyze primary neoplastic cells isolated from prostate, breast and colorectal tumors, as well as stable cancer cell lines (MCF7, MDA-MB-468, SW962, SK-MEL28, NCI-H1975 and NCI-H469). RESULTS: In all cases of primary cancer cell cultures, in vitro conditions rapidly revealed senescence in the majority of cells. Two out of six stable cancer cell lines did not exhibit any senescence-associated-ß-Galactosidase-positive cells. Interestingly, four cell lines had small sub-populations of senescent cells (single SA-ß-Gal-positive cells). CONCLUSION: Primary neoplastic cells from different types of cancer (prostate, breast, colon cancer) appear to be senescent in vitro. Apparently, cancer cell lines that have been used for many years in drug-testing analyses have constantly been misleading researchers in terms of the general sensitivity of cancer cells to senescence.

Comparative analysis of CD4+ and CD8+ T cells in tumor tissues, lymph nodes and the peripheral blood from patients with breast cancer.

BACKGROUND: CD4+ and CD8+ T cells are the main types of lymphocytes in cell-mediated immunity and play a central role in the induction of efficient immune responses against tumors. The frequencies of T cell subtypes in the peripheral blood and tumor tissues, and draining lymph nodes (dLN) can be considered as useful markers for evaluation of the immune system in cancers. METHODS: In this study, the frequencies of CD4+ and CD8+ T cells in blood, tumor tissues, and dLN samples of breast cancer patients were compared with each other and with similar tissues from normal individuals. Immunophenotyping was carried out by flow cytometry and the expression levels of CXCL10, granzyme B, and mammaglobin were evaluated by real-time PCR. RESULTS: In the peripheral blood, there were no differences in the T cell subsets between the patients and the normal individuals. The frequency of CD8+ T cells was significantly higher in tumor tissue than normal breast tissues while granzyme B expression was similar. Based on mammaglobin expression levels, dLN have been classified into micro- and macro-metastatic dLN. We found significantly lower frequency of CD4+ in macro-metastatic dLN than micro-metastatic dLN. CD8+ frequency was similar in both dLN; however, granzyme B expression was higher in micro-metastatic ones. There was not any significant difference in CXCL10 expression between the two types of dLN. CONCLUSION: Based on our results, although the tumor does not affect the systemic immunity, tumoral cells affect the local immune system in the tumoral tissues and the metastatic dLN.

GATA3 expression in morphologic subtypes of breast carcinoma: a comparison with gross cystic disease fluid protein 15 and mammaglobin.

GATA3 is a transcription factor, which is involved in the growth and differentiation of several human tissues. Immunohistochemical staining for this marker has proven to be useful in recognizing a number of tumors, most notably those in the urinary tract and breasts. To date, no study has specifically assessed the distribution of GATA3 among different histomorphologic subtypes of breast carcinoma. The surgical pathology archive at our institution was searched, to retrieve cases of breast carcinomas of the following microscopic types-ductal, lobular, mucinous, metaplastic, medullary, apocrine, signet-ring cell, and micropapillary. Tissue microarrays were created, with four 0.6-mm punch specimens from each case. The tissue microarrays were cut at a 5-µm thickness and stained with monoclonal antibodies to GATA3 (Biocare Medical Inc, Concord, CA), mammaglobin (Dako, Carpinteria, CA), and gross cystic disease fluid protein 15 (Dako). Tumors were considered to be positive for those markers if more than 5% of the cells were labeled. Of 55 ductal adenocarcinomas, 51 (92.7%) expressed GATA3. All 4 GATA3-negative tumors were Nottingham grade III lesions that were also nonreactive for estrogen receptor protein. GATA3 was present in 28 (96.6%) of 29 lobular adenocarcinomas, 10 (90.9%) of 11 apocrine adenocarcinomas, 10 (83.3%) of 12 medullary carcinomas, 5 (55.5%) of 9 metaplastic carcinomas, and 1 of 2 signet-ring cell carcinomas. Mucinous carcinomas (23 cases) and micropapillary carcinomas (12 cases) uniformly and strongly labeled for GATA3. GATA3 equaled or surpassed the sensitivity of mammaglobin and gross cystic disease fluid protein 15 in all histologic subgroups of breast cancer in the study. Although most ductal adenocarcinomas were labeled for GATA3, it was absent in high-grade tumors that also lacked estrogen receptor protein. Favorable prognosis types of breast carcinoma (eg, mucinous carcinoma) and aggressive variants such as micropapillary carcinoma were equally reactive for this marker. A proportion of medullary and metaplastic carcinomas was GATA3 negative (17% and 44%, respectively). Thus, those pathologic entities cannot be excluded diagnostically by an absence of GATA3 immunoreactivity.

Human mammaglobin in breast cancer: a brief review of its clinical utility.

Human mammaglobin is a member of the uteroglobin proteins family that has recently been tested as a specific marker for breast cancer. While low levels may be seen in normal breast tissue, expression is increased dramatically in breast cancer and is correlated with higher grade. Detection in blood and body fluids is also correlated with cancer metastasis, and its levels with prognosis. This promises to be a useful screen for early detection of breast cancer, especially in high risk individuals. Mammoglobin has also been used for immunotherapeutic targeting of breast cancer cells. However, there are some controversies regarding its diagnostic efficacy and prognostic value, which warrant further study.

The clinical utility of circulating tumor cells in breast cancer patients: detection by a quantitative assay of h-MAM gene expression.

The aim of this study was to evaluate tumor markers of molecular abnormalities that display tissue specificity, as to detect circulating tumor cells (CTCs) in breast cancer patients. Quantitative real-time RT-PCR was used to determine h-MAM, BCSG1, CK19, and c-erbB2 mRNA levels in peripheral blood (PB) of breast cancer patients. Results were compared with other epithelial cancers (lung or esophagus cancer), benign breast disease, and healthy individuals. We found that h-MAM mRNA was only detectable in the PB of patients with breast cancer (49 of 65, 75.4%), but not in patients with other epithelial cancers, benign breast disease, or healthy individuals. No significant differences in the expression level and positive detection rate of BCSG1, CK19, and c-erbB2 mRNA were observed between breast cancer and other epithelial cancers. Furthermore, the expression level and positive detection rate of h-MAM mRNA in PB were significantly correlated to the breast cancer pathologic stage (p=0.012 and p=0.015, respectively). Chemotherapy, radiotherapy, or total tumor resection (after 7 days of treatment) resulted in a significant decrease in the expression level of h-MAM mRNA in PB compared to the levels prior to the treatment (p<0.001). Importantly, an increase in h-MAM mRNA expression was detected in patients immediately after surgery, as well as 3 days post-surgery. These results indicate that the quantitative analysis of h-MAM mRNA is a useful tool for detecting CTCs in breast cancer patients, and can have a potential diagnostic utility in early micrometastasis, clinical evaluation of cancer treatment efficacy, and post-treatment monitoring of breast cancer patients.

Cutaneous and mammary apocrine carcinomas have different immunoprofiles.

Often the distinction of cutaneous apocrine carcinoma from metastatic mammary apocrine carcinoma to the skin can be a diagnostic dilemma because both tumors share similar histologic features and have overlapping immunohistochemical profile. We compared the expression of adipophilin, cytokeratin 5/6, p63, GATA3, mammaglobin, androgen receptor, estrogen receptor, progesterone receptor, and HER2 by immunohistochemistry in 14 cutaneous apocrine carcinomas (11 primary tumors, 3 metastases) and 26 primary apocrine carcinomas of the breast. Whereas focal adipophilin staining was seen in 36% (5/14) of cutaneous apocrine carcinoma, strong and diffuse adipophilin staining was seen in 88% (22/25) of mammary apocrine carcinoma (P = .0013). Differences in estrogen receptor and progesterone receptor expression were also statistically significant (P = .018 and .043). Androgen receptor was strongly positive in all cutaneous and mammary cases. Although there was no significant difference in the frequency of expression of cytokeratin 5/6, p63, HER2, GATA3, and mammaglobin in cutaneous apocrine carcinoma versus mammary apocrine carcinoma, strong and diffuse cytokeratin 5/6 and/or mammaglobin expression were seen only in cutaneous apocrine carcinoma. In conclusion, cutaneous apocrine carcinoma is likely adipophilin- ER+ PR+/- HER2- and can exhibit strong and diffuse cytokeratin 5/6 and/or mammaglobin expression. On the contrary, a mammary apocrine carcinoma is likely adipophilin+ ER- PR- and often exhibit 3+ HER2 with corresponding HER2 gene amplification. A panel of adipophilin, ER, PR, HER2, cytokeratin 5/6, and mammaglobin may be helpful in distinguishing cutaneous apocrine carcinoma from mammary apocrine carcinoma.

Recombinant mammaglobin A adenovirus-infected dendritic cells induce mammaglobin A-specific CD8+ cytotoxic T lymphocytes against breast cancer cells in vitro.

Mammaglobin A (MGBA) is a novel breast cancer-associated antigen almost exclusively over-expressed in primary and metastatic human breast cancers, making it a potential therapeutic target for breast cancer. The development of dendritic cell (DC)-induced tumor antigen specific CD8(+) cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. In this study we constructed recombinant replication-defective adenoviral (Ad) vectors encoding MGBA and evaluated their ability to trigger anti-tumor immunity in vitro. DCs were isolated from the human peripheral blood monocyte cells (PBMCs) of two HLA-A33(+) healthy female volunteers, and infected with adenovirus carrying MGBA cDNA (Ad-MGBA). After that, the Ad-MGBA-infected DCs were used to stimulate CD8(+) CTLs in vitro and the latter was used for co-culture with breast cancer cell lines. The data revealed that infection with Ad-MGBA improved DC maturation and up-regulated the expression of co-stimulatory molecules and the secretion of interleukin-12 (IL-12), but down-regulated interleukin-10 (IL-10) secretion from DCs. Ad-MGBA-infected DC-stimulated CD8(+)CTLs displayed the highest cytotoxicity towards HLA-A33(+)/MGBA(+) breast cancer MDA-MB-415 cells compared with other CD8(+)CTL populations, and compared with the cytotoxicity towards HLA-A33(-)/MGBA(+) breast cancer HBL-100 cells and HLA-A33(-)/MGBA(-) breast cancer MDA-MB 231 cells. In addition, Ad-MGBA-infected DC-stimulated CD8(+) CTLs showed a high level of IFNγ secretion when stimulated with HLA-A33(+)/MGBA(+) breast cancer MDA-MB-415 cells, but not when stimulated with HLA-A33(-)/MGBA(+) HBL-100 and HLA-A33(-)/MGBA(-)MDA-MB-231 cells. In addition, killing of CD8(+)CTLs against breast cancer was in a major histocompability complex (MHC)-limited pattern. Finally, the data also determined the importance of TNF-α in activating DCs and T cells. These data together suggest that MGBA recombinant adenovirus-infected DCs could induce specific anti-tumor immunity against MGBA(+) breast cancers, which could provide a novel strategy in the immunotherapy of breast cancer.

Clinical relevance of human mammaglobin mRNA in pleural effusion from patients undergoing thoracoscopy: a pilot study.

BACKGROUND: human mammaglobin (hMAM) expression has been reported in pleural effusions (PE). The aim of this study was to assess the clinical relevance of hMAM mRNA in PE from patients who underwent thoracoscopy.
 MATERIAL AND METHODS: A total of 288 patients with PE were studied, 155 of which were diagnosed with malignant and 133 with non-malignant diseases by thoracoscopy. Cells from PE were analyzed by nested hMAM RT-PCR. Statistical analyses were performed to evaluate the diagnostic performance parameters (DPP), the association between hMAM expression and benign or malignant status and the relative risk of cancer for patients with negative thoracoscopy showing hMAM positivity.
 RESULTS: hMAM mRNA was found in 68/288 (23.6%) PE samples of which 51 were from the 155 patients diagnosed with malignant diseases and 17 were from the 133 patients diagnosed with non-malignant diseases. A significant correlation between hMAM expression and malignancy was found (OR=3.04) and the DPP were as follows: sensitivity=32.9%, specificity=87.2%, accuracy=58.0%, positive predictive value=75.0% and negative predictive value=52.7%. Among the patients with negative thoracoscopy (n=133), 5/17 (29.4%) hMAM-positive patients had or developed a tumor during the 18-month follow up period, as compared to 10/116 (8.6%) hMAM-negative patients (relative risk of 4.6 for developing a malignancy).
 CONCLUSION: These findings suggest a possible application of hMAM RT-PCR detection in PE as to identify a false-negative thoracoscopy in non-specific pleuritis.