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Although targeted therapy has been extensively investigated for breast cancers, a molecular target with broad application is currently unavailable due to the high heterogeneity of these cancers. Mammaglobin-A (Mam-A), which is overexpressed in most breast carcinomas, has been proposed as a promising target. However, the lack of specific targeting moieties due to uncertain binding epitopes hampers further translational study. Here, seven potential epitopes of Mam-A were disclosed, and a unique epitope was then identified in most types of breast cancers, despite the genotypic heterogeneity. With phage display technology, the epitope was determined to be N-terminal amino acids 42–51 of Mam-A (N42–51). Then, the N42–51 epitope-specific monoclonal antibody, mAb785, was conjugated to poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with therapeutic agents, thereby enhancing the drug uptake and therapeutic efficacy in different genotypes of breast cancers. The computer simulation of the N42–51 epitope and the mAb785 structures, as well as their interactions, further revealed the specific targeting mechanism of the mAb785-conjugated nanoparticles to breast cancers.

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.

The aim of the current study was to assess the prognostic value of circulating tumor cells (CTCs) and their related markers at different points of chemotherapy regimens in metastatic breast cancer (MBC) patients. The impact of CTCs on progression free survival (PFS) and overall survival (OS) rates were also assessed. Peripheral blood samples were obtained from 66 female patients with MBC at different time intervals for evaluation of CTCs by flow cytometry (FC). cytokeratin 19 (CK19), mammaglobin, prolactin inducible peptide (PIP), aldehyde dehydrogenase 1 (ALDH1) and human chorionic gonadotropin (hCG) were also assessed by qRT-PCR. Analysis of different CTC levels (at 4, 5, and 6 cells/7 ml), showed statistically significant values at 4 cells/7 ml blood. The presence of baseline CTCs

Background Mammaglobin A (MGA), mainly expressed in the breast epithelium, is overexpressed in breast cancer, and has been established as a tumor and promissory marker for the early detection of metastasis. Aim The main aim of this study was to evaluate the association between the presence of the MGA transcript in the peripheral blood of Brazilian breast cancer patients and healthy women and the development of breast cancer and tumor progression. Material and methods The expression of the MGA transcript in peripheral blood of 102 breast cancer patients and 102 healthy women was assessed by RT-PCR. Results MGA mRNA was expressed in the peripheral blood of 39 breast cancer patients and in none of the women from the control group. The presence of MGA was significantly associated with presence of metastasis and age at onset after 60 years. The presence of MGA mRNA in peripheral blood displayed a sensitivity of 38.2%, specificity of 100.0%, positive predictive value (PPV) of 100.0%, and negative predictive value (NPV) of 61.8% as a breast cancer marker. Conclusion This study provides additional evidence of the presence of MGA in the peripheral blood of breast cancer patients, and its applicability as an efficient biomarker for breast cancer (High specificity and PPV). To our knowledge, this is the first study to assess the expression of MGA mRNA in peripheral blood obtained from the Brazilian population.

Mammaglobin B (MGB2) and mammaglobin A (MGB1) are proteins expressed in metastatic breast cancers. The early detection of circulating tumor cells (CTCs) in breast cancer patients is crucial to decrease mortality rate. Herein, novel aptamers were successfully selected and characterized against MGB2 and MGB1 proteins using a hybrid SELEX approach. The potential use of the selected aptamers in breast CTC detection was studied using spiked breast cancer cells in whole blood lysate. The results obtained from this study showed that the selected aptamers (MAMB1 and MAMA2) bind to their target breast cancer cell lines with high affinity (low nanomolar Kd values) and specificity. They also bind to their free recombinant target proteins and show minimal non-specific binding to normal and other cancer cell lines. Additionally, they were able to distinguish a low number of breast cancer cells spiked in whole blood lysate containing normal blood cells. The results obtained in this study indicate the great potential for the use of aptamers to detect MGB1 and MGB2 protein biomarkers, expressed on the surface of breast CTCs.

Sentinel lymph node (SLN) micrometstasis detection improves outcome for breast cancer follow up procedure. The aim of the present study was to identify gene profiles that accurately predicted the outcome of breast cancer patients. Fifty tumor sample from breast cancer patients were analyzed for the expression of 3 genes using quantitative-PCR. Also clinical verification for recurrence to distant organs was performed. Three gene signature were confirmed based on tumor's stage, grade, ER status, using conditional logistic regression. Based on this findings, the negative reported lymph nodes for metastasis, had micro metastasis in significant values. There was a significant difference between normal and cancer samples in 3 gene expression marker and also there was meaningful relationship between three gene expression with tumor's grade, stage according to progression of tumor. A novel gene expression signature predictive of micro metastatic patients was evaluated. In this assessment, relationship between this gene with tumor's features that finding clear role for these genes with tumor's outcome, needs to be established.

It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs) in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19). B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy.

GATA-3 is a transcription factor that has recently been identified by immunohistochemistry to be highly expressed in urothelial and breast carcinomas (CAs). We sought to determine the potential utility of GATA-3 in identifying metastatic breast CA, and to compare its utility with the standard breast markers, GCDFP-15, and mammaglobin A. We identified an archival series of 338 formalin-fixed paraffin-embedded whole-tissue sections of various CAs. Using standard immunohistochemical (IHC) techniques we used mouse monoclonal antibodies to GATA-3 (clones L50-823, HG3-31), GCDFP-15 (23A3), and mammaglobin A (31A5). Both clones of GATA-3 showed positivity in 96% of non-triple-negative breast carcinomas (TNBCs), L50-823 and HG3-31, demonstrating expression in 87% and 63% of TNBCs, respectively; GCDFP-15 and mammaglobin A were expressed in 69% and 61% of non-TNBCs, respectively, and 10% and 17%, of TNBCs, respectively. The L50-823 clone manifested a lower specificity in identifying breast CAs (84%) than did the HG3-31 clone (97%). Both monoclonal antibodies to GATA-3 are very sensitive reagents for the identification of breast CA, surpassing antibodies to GCDFP-15 and mammaglobin A, and offer a significant improvement in identifying TNBCs. However, the L50-823 clone showed a lower level of specificity, which may qualify its utility in the setting of CAs of unknown primary.

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.

UNLABELLED Mammaglobin may be a potential serum biomarker for the differential diagnosis of breast cancer. 260 serum samples were collected from 127 untreated breast cancer patients and 133 healthy volunteers to analyze the sera expression of mammaglobin and its implications for both. The expression vector of pGEX-4T-2-Mammaglobin and pBVIL1-Mammaglobin were constructed and transformed into E.coli.HB101 for expression. The mice were immunized with the purified recombinant protein to prepare monoclonal antibody and to detect by ELISA the serum of normal people and breast cancer patients. Recombinant mammaglobin antigen was effectively expressed in E.coli. Two hybridoma cell lines were obtained after the mice were immunized by pGEX-4T-2-mammaglobin. 133 cases of normal serum and 127 cases of breast cancer serum were analyzed by ELISA. The sera expression level of mammaglobin in breast cancer group (average OD value 0.645±0.223) was significantly (p KEYWORDS mammaglobin; cloning expression; monoclonal antibody; serologic study; breast cancer.

To investigate the prognostic relevance of disseminated tumor cells (DTCs) in bone marrow (BM) assessed by a multimarker mRNA panel consisting of TWIST1, cytokeratin 19 (CK19) and human mammaglobin A (hMAM) mRNA, in patients with early breast cancer.TWIST1 (gene name: TWIST1), CK19 (gene name: KRT19), and hMAM (gene name: SCGB2A2) mRNA was quantitated in BM samples from 191 operable breast cancer patients by real-time reverse transcriptase polymerase chain reaction (RT-PCR).Using the highest relative mRNA concentration of TWIST1 in the control population as a cut-off, 5 of the 191 breast cancer patients showed elevated TWIST1 mRNA levels in their BM by real-time RT-PCR. Two of these patients experienced a systemic relapse during a median follow-up of 98 months. Combining these results with previous hMAM and CK19 mRNA quantifications in the same BM samples, 12 (40%) of the 30 patients with BM positive for at least 1 marker (multimarker positive) experienced a systemic relapse as compared with 18 (11%) of the 161 patients with multimarker-negative BMs. The patients with multimarker-positive BM had significantly shorter systemic recurrence-free survival (P.001, log-rank test), breast cancer-specific survival (P.001), and overall survival (P = .03). The prognostic relevance of BM multimarker detection appeared to be independent of adjuvant treatment, although the difference was not statistically significant in the subgroup of patients who received adjuvant chemotherapy. Multivariate analysis demonstrated the BM multimarker panel status to be a strong independent predictor of clinical outcome.Our results demonstrated the prognostic relevance of BM DTCs assessed by a multimarker mRNA panel consisting of TWIST1, CK19, and hMAM in operable breast cancer patients.

Purpose: To investigate the prognostic value of the molecular detection of circulating tumor cells (CTCs) using three markers [cytokeratin 19 (CK19), mammaglobin A (MGB1), and HER2] in early breast cancer. Experimental Design: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs. Results: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/HER2mRNA− (n = 1), CK19mRNA+/MGB1mRNA−/HER2mRNA+ (n = 42), CK19mRNA+/MGB1mRNA−/HER2mRNA− (n = 21), CK19mRNA−/MGB1mRNA+/HER2mRNA− (n = 5), and CK19mRNA−/MGB1mRNA−/HER2mRNA− (n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGB1+, CK+/MGB1−, CK−/MGB1+, CK+/HER2+, CK+/HER2−, MGB1+/HER2−, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter disease-free survival (DFS; P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor–negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS. Conclusion: The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.

Background The causes of nasal polyposis remain unclear. Mammaglobins have been implicated in its pathogenesis. However, their association with the occurrence of nasal polyps in the presence of allergic rhinitis (AR) has not been explored. The aim of this study was to compare the expression levels of mammaglobins A and B with the nasal polyps of patients with and without AR. Methods Thirty-one patients with bilateral nasal polyposis underwent skin-prick tests to specific aeroallergens. Nasal polyp tissues were obtained from all patients and divided into two groups as nasal polyps with and without AR depending on clinical history and the skin-prick test results. All polyp tissues were analyzed for the levels of mammaglobin A and mammaglobin B by using real-time quantitative polymerase chain reaction technique. Results Of the 16 samples from patients having nasal polyps with AR, only 1 sample expressed a detectable level of mammaglobin A (1/16). There was no detectable expression of mammaglobin A in tissues from the group of nasal polyps without AR (0/15). Expression of mammaglobin B was detected in all nasal polyp tissues from both groups. The expression of mammaglobin B was not significantly different between nasal polyps with AR (median, 25th-75th percentiles; 0.023, 0.013-0.046) and nasal polyps without AR (0.032, 0.007-0.16). Conclusion Expression levels of mammaglobins A and B in nasal polyps are not different between patients with and without AR. Our findings suggest that mammaglobins’ implication in the pathogenesis of nasal polyps is independent of an underlying AR.

Existing serum markers for breast cancer such as CA 15-3, BR 27.29 and CEA lack sensitivity and specificity. The aim of this study was to evaluate the value of new putative breast-specific markers for differentiating breast cancer from non-breast tissues. Expression of mammaglobin A (MGA), B726P, small breast epithelial mucin (SBEM) and MUC1 was measured by RT-PCR. MGA mRNA was detected in 86/162 (60%) breast cancers but in only 1/32 (3%) non-breast tissues; B726P was detected in 44/108 (41%) breast cancers but in none of 20 non-breast tissues, while SBEM was present in 52/103 (51%) breast cancers but in only 1/26 non-breast cancer tissues. In contrast to these novel markers, the established breast cancer marker MUC1 was detected in 72/99 (73%) breast cancers and in 22/32 (59%) of non-breast tissues. Combining MGA with B726P separated breast cancer from non-breast tissue with a sensitivity of 71% and a specificity of 95% while combining MGA with SBEM differentiated breast cancer from non-breast tissues with a sensitivity of 76% and a specificity of 89%. Genes such as MGA, B726P and SBEM that are expressed relatively exclusively in breast tissue are potential new markers for breast cancer.

Looking inside the human body fascinated mankind for thousands of years. Current diagnostic and therapy methods are often limited by inadequate sensitivity, specificity and spatial resolution. Raman imaging may bring revolution in monitoring of disease and treatment. The main advantage of Raman imaging is that it gives spatial information about various chemical constituents in defined cellular organelles in contrast to conventional methods (liquid chromatography/mass spectrometry, NMR, HPLC) that rely on bulk or fractionated analyses of extracted components. We demonstrated how Raman imaging can drive the progress on breast cancer just unimaginable a few years ago. We looked inside human breast ducts answering fundamental questions about location and distribution of various biochemical components inside the lumen, epithelial cells of the duct and the stroma around the duct during cancer development. We have identified Raman candidates as diagnostic markers for breast cancer prognosis: carotenoids, mammaglobin, palmitic acid and sphingomyelin as key molecular targets in ductal breast cancer in situ, and propose the molecular mechanisms linking oncogenes with lipid programming.

Objectives The distinction between metastatic breast carcinomas (BCs) and primary lung carcinomas (PLCs) can be difficult. This study tested the utility of trefoil factor 1 (TFF1) for this purpose and compared it with mammaglobin and GATA protein binding 3 (GATA-3). Methods Tissue microarrays containing 365 BCs and 338 PLCs were stained with TFF1, mammaglobin, and GATA-3, and an H-score was calculated. Sensitivity, specificity, and accuracy were calculated, and logistical regression analysis was performed. Results Accuracy of correctly classifying the tumor type was 81.9%, 71.3%, and 64.0% for GATA-3, mammaglobin, and TFF1, respectively. Odds ratios for selecting BCs were 25.69, 93.15, and 4.17, respectively, with P values less than .001. With a single exception, the best immunopanel included GATA-3 and mammaglobin in all comparisons. Conclusions TFF1 demonstrated breast specificity but was inferior to mammaglobin and GATA-3. Therefore, its routine clinical use may not be justified. TFF1 showed little benefit when added to an immunopanel.

“…we are convinced that analysis of the immune response in the primary tumor following vaccination is likely to be significantly more informative than studies of the immune response in the peripheral blood.”

Objective. To identify novel molecular biomarkers useful for the early diagnosis and therapy of ovarian cancer by gene expression profiling. To compare the genetic fingerprints of flash-frozen ovarian serous carcinomas to those of matched highly purified primary tumor cell cultures. Methods. Gene expression profiles of 19 flash-frozen ovarian serous papillary carcinoma (OSPC) were analyzed and compared to 15 controls (highly purified human ovarian surface epithelium short-term cultures, HOSE) using oligonucleotide microarrays complementary to >14,500 human genes. In addition, gene expression profiling of 5 highly purified primary OSPC cultured in vitro for less than 2 weeks was compared to flash-frozen ovarian carcinoma biopsies obtained from matched samples. Quantitative RT-PCR and IHC staining techniques were used to validate microarray data at RNA and protein levels for some of the differentially expressed genes. Results. Unsupervised analysis of gene expression data readily distinguished normal tissue from flash-frozen OSPC and identified 901 and 557 genes that exhibited >3-fold up-regulation or down-regulation, respectively, in OSPC when compared to HOSE. Mammaglobin 2 , an ovarian secreted protein, was identified as the top differentially expressed gene in OSPC (19 out 19 OSPC versus 0 out of 15 HOSE) with over 827-fold up-regulation relative to HOSE. The claudin and kallikrein family of proteins including the clostridium perfringens enterotoxin receptors claudin 3 and 4 , kallikreins 6 , 7 , 8 , 10 , 11 and the immunomodulatory molecule B7-H4 were found among the most highly overexpressed genes in OSPC when compared to HOSE. Genetic fingerprints of flash-frozen OSPC were found to have high correlation with those of purified primary OSPC short-term in vitro cultures with only 31 out of 8637 genes (0.35%) differentially expressed between the two groups. Conclusions. Short-term in vitro culture of primary ovarian carcinomas may greatly increase the purity of ovarian tumor RNA available for gene expression profiling without causing major alteration in OSPC fingerprints. Mammaglobin 2 , kallikreins 6 , 7 , 8 , 10 , 11 , claudin 3 and 4 and B7-H4 gene expression products represent candidate biomarkers endowed with great potential for early screening and therapy of OSPC patients.

Existing serum-based markers for breast cancer all lack organ specificity. Mammaglobin A (MGA) is a 93 amino acid protein expressed almost exclusively in breast tissue. The aim of our study was to investigate the different forms of MGA protein in fibroadenomas and breast carcinomas. MGA protein was measured by Western blotting in 132 breast cancers, 29 fibroadenomas and 14 nonbreast tissues. MGA protein in breast tissue was found to exist in 2 main forms. These forms migrated with approximate molecular masses of 18 and 25 kDa. Both forms of MGA were detected more frequently in breast carcinomas compared to fibroadenomas. The high molecular weight form of MGA but not the low molecular weight form was found more frequently in hormone receptor-positive than in receptor-negative cancers. Furthermore, an inverse relationship was found between the high molecular weight form of MGA and both tumour grade and proliferation index. No significant correlation was found between the MGA proteins and either tumor size or nodal status. Our results show that MGA protein exists in 2 main forms in breast tissue. As the high molecular weight form correlated positively with hormone receptors and negatively with tumor grade and proliferation rate, its presence is likely to be associated with a favourable prognosis for breast cancer. As expression of MGA is almost breast specific, it is a promising marker for breast cancer. Its most immediate use is likely to be in detecting micrometastases from breast cancer. © 2004 Wiley-Liss, Inc.

Purpose The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity. The aim of this study was to improve tumor cell detection in effusions by molecular approaches. Materials and Methods A total of 157 effusions from patients with tumors and 72 effusions from patients without a history or evidence of malignancy were included in this study. All effusion specimens were evaluated in parallel by cytology, fluorescence in situ hybridization (FISH) for aneuploidy, and reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of human mammaglobin (hMAM) and mammaglobin B (hMAM-B). Results In effusions from patients with tumors, the sensitivities of tumor cell detection by cytology, FISH, and hMAM and hMAM-B detection were 46.2%, 53.3%, 36.4%, and 57.7%, respectively. The corresponding specificities were 94.4%, 97.0%, 87.1%, and 88.6%. Notably, a high percentage of effusions containing malignant cells were in fact transudates, indicating the necessity for molecular diagnostic work-up of transudates collected from patients with tumors. Dependent on the tumor type, the use of appropriate marker combinations improved tumor cell detection in effusions significantly. By combining all four diagnostic tests, a positive test result indicating the presence of malignancy was achieved in 81.1%, with a fairly good specificity of 70.1%. Conclusion Molecular techniques are definitely useful to detect malignancy in cytologically negative effusions. Tumor cell detection in effusions can be significantly improved by FISH and PCR techniques applying appropriate molecular markers. This finding should help to improve tumor staging, prognostic assessment, and treatment monitoring.

Purpose The tumor mRNA expression levels of mammaglobin, a novel breast-specific and breast cancer-associated marker, were correlated with disease outcome in 280 patients with primary breast cancer. Patients and Methods Mammaglobin expression levels were assessed by quantitative reverse transcriptase polymerase chain reaction in frozen tumor tissue from breast cancer patients with a median age of 60 years (range, 30 to 88 years) and a median follow-up of 85 months (range, 2 to 169 months). Results High expression levels were associated with low-grade tumors (P = .018), with positive estrogen and progesterone receptor status (P < .001), and postmenopausal status (P = .010). In the analysis of all patients, low tumor mammaglobin expression levels predicted an early relapse both in Cox univariate (hazard ratio [HR], 0.52; 95% CI, 0.34 to 0.79; P = .002) and multivariate regression analyses corrected for the traditional prognostic factors (HR, 0.55; 95% CI, 0.35 to 0.88; P = .012). The association of mammaglobin expression with the rate of relapse was particularly favorable in patients who received adjuvant tamoxifen treatment (HR, 0.35; 95% CI, 0.17 to 0.71; P = .004). Conclusion These results demonstrate that the assessment of the tumor mRNA expression level of the breast-specific protein mammaglobin could be useful to stratify patients for individual adjuvant treatment strategies.

Aims We have demonstrated previously that gross cystic disease fluid protein-15 (GCDFP-15) and mammaglobin A (MAM) are of limited utility in triple-negative breast cancer (TNBC). GATA-binding protein 3 (GATA-3) is an emerging breast-associated immunohistochemical (IHC) marker with limited data in TNBC. Here, we examined GATA-3 expression in TNBC in comparison with GCDFP-15 and MAM. Methods and results We studied GATA-3, GCDFP-15 and MAM IHC expression in 62 primary and 68 metastatic TNBCs. In primary TNBCs, GATA-3 staining was observed in 25 cases (40%), including 16 cases that were negative for GCDFP-15 and MAM. In metastatic TNBCs, GATA-3 staining was observed in 30 cases (44%), including 16 cases that were negative for GCDFP-15 and MAM. The expression frequency of any of the markers was 56% in primary and 62% in metastatic TNBCs. However, when focal staining was excluded, the expression frequency of any marker dropped to 31% and 44%, respectively. Conclusion GATA-3 is expressed at a higher frequency by IHC in TNBC compared to GCDFP-15 and MAM, although the tissue specificity of the latter markers may be superior. When evaluating a triple-negative tumour, including GATA-3 in a panel of markers may increase the diagnostic accuracy for tissue origin in the appropriate clinical setting.

Background Mammary analog secretory carcinoma (MASC) of the salivary gland has been recently described according to morphological, immunohistochemical, and molecular (ETV6-NTRK3 translocation) similarities with the mammary secretory carcinoma. The most important differential diagnostic considerations of MASC are low-grade adenocarcinoma not otherwise specified (NOS), cystadenocarcinoma, and acinic cell carcinoma (AciCC). These tumors may share an overlapping morphology with MASC, and additional immunohistochemical studies are required to reinforce the diagnosis. Mammaglobin, GCDFP-15, and p63 staining have been reported in MASC. Our study was designed to check the specificity of these antibodies in MASC compared to other frequent tumors of salivary glands. Methods A series of 62 salivary gland tumors [10 MASCs, 5 adenocarcinomas NOS and 2 cystadenocarcinomas with MASC features and without ETV6 rearrangement, one low-grade cribriform cystadenocarcinoma (LGCCC), 9 AciCCs, 10 MECs, 10 adenoid cystic carcinomas (AdeCCs), 5 polymorphous low-grade adenocarcinomas (PLGAs), and 10 pleomorphic adenomas (PAs)] was analyzed by immunohistochemistry with mammaglobin, GCDFP-15, and p63 antibodies. Results Positivity for mammaglobin was observed in all MASCs, cystadenocarcinomas, LGCCC, and PLGAs, in some adenocarcinomas NOS, PAs, and MECs, rarely in AciCCs and never in AdeCCs. Positivity for GCDFP-15 was observed in most of the tumor types except in AdeCCs. Interestingly, cytoplasmic positivity for p63 was observed in most of MASCs and PLGAs while rarely in adenocarcinomas NOS and PAs, and never in the other tumor types. Conclusion Our study revealed the usefulness of mammaglobin and p63 cytoplasmic staining to define which tumors are worth to be screened for ETV6 rearrangement.

The mammaglobin gene has been shown to be preferentially expressed in breast tissue. Few genes match its specificity. Mammaglobin has generated much interest, and studies are ongoing to develop diagnostic tests for breast cancer based on the detection of mammaglobin. While searching the Incyte Genomics Lifeseq database for tissue-specific markers, we observed a second secretoglobin, BU101, also known as lipophilin B. We report here that mammaglobin, in breast tissue, is found as a complex with BU101. The complex was isolated from breast cancer tissue and was characterized as the biologically relevant form of mammaglobin.

Background: We used a combination of genetic subtraction, silicon DNA microarray analysis, and quantitative PCR to identify tissue- and tumor-specific genes as diagnostic targets for breast cancer. Methods and Results: From a large number of candidate antigens, several specific subsets of genes were identified that showed concordant and complementary expression profiles. Whereas transcriptional profiling of mammaglobin resulted in the detection of 70% of tumors in a panel of 46 primary and metastatic breast cancers, the inclusion of three additional markers resulted in detection of all 46 specimens. Immunomagnetic epithelial cell enrichment of circulating tumor cells from the peripheral blood of patients with metastatic breast cancer, coupled with RT-PCR-based amplification of breast tumor-specific transcripts, resulted in the detection of anchorage-independent tumor cells in the majority of patients with breast cancer with known metastatic disease. Conclusion: Complementation of mammaglobin with three additional genes in RT-PCR increases the detection of breast cancers in tissue and circulating tumor cells.

The Mammaglobin gene, a breast-specific member of the uteroglobin gene family, has been previously identified as being overexpressed in some breast tumours, but the cellular origin and relationship to tumour progression are unknown. Using a subtractive hybridization approach, mammaglobin mRNA has also been found to be overexpressed in the in situ compared to the invasive element within an individual breast tumour. Further study by in situ hybridization performed in 13 breast tumours, selected to include normal, in situ, and invasive primary tumour elements, and in most cases axillary lymph node metastases, revealed that mammaglobin expression occurs in all elements, is restricted to epithelial cells, and is significantly increased in tumour cells compared with normal cells ( p< 0.04). Analysis of mammaglobin expression within 20 independent primary breast tumours and their corresponding axillary lymph nodes revealed that all 13 lymph nodes positive and none of the seven nodes negative for metastatic breast carcinoma by histology were mammaglobin-positive by reverse transcription-polymerase chain reaction (RT-PCR) ( p=0.0001). These results suggest that mammaglobin could be a marker of axillary lymph node breast metastases.

The mammaglobin gene encodes a novel secreted protein whose corresponding mRNA is frequently up-regulated in human breast cancer. In non-malignant tissues, expression is also strictly limited to the mammary epithelium. To better understand the mechanisms controlling these patterns of expression, we have isolated the human mammaglobin gene and performed an initial assessment of its promoter activity. Mammaglobin gene architecture is very similar to that of a family of related genes that includes uteroglobin and rat prostatein subunits C1, C2, and C3. However, the mammaglobin gene itself is not well conserved phylogenetically. The human mammaglobin gene is localized by fluorescent in situ hybridization to chromosome 11 band q13, a genomic region frequently amplified in breast neoplasia. The sequence of proximal 1 kb of mammaglobin promoter contains several potential transcriptional control elements and directs high-level expression of a transfected reporter construct in human breast tumor cell lines. However, comparable levels of reporter gene expression are also seen in non-mammary human cell lines. These data suggest that, unlike related gene family members, the striking breast-specific expression and tumor-associated overexpression of mammaglobin is mediated by complex transcriptional control at more distal sequence elements.

Metastases represent the most common type of intracranial neoplasm. In women, 30% of such tumors derive from breast carcinoma. In neurosurgical cases with ambiguous cellular morphology and/or limited biopsy material, immunohistochemistry (IHC) is often performed to distinguish metastases from primary central nervous system (CNS) neoplasms. IHC for mammaglobin-A (MGA), a protein expressed in a majority of breast carcinomas, is commonly applied in this setting, but its utility for distinguishing primary CNS neoplasms from metastatic breast carcinoma is unknown; the reactivity of MGA in primary and metastatic CNS neoplasms has never been described. Here, we describe the frequency and patterns of IHC reactivity for MGA in metastatic and primary CNS neoplasms from patients with well-documented histories of breast carcinoma. Following a published protocol previously applied to non-CNS neoplasms, MGA staining of moderate to strong intensity within 5% or more of a neoplasm was considered positive. On the basis of these criteria, 3 of 12 (25.0%) glioblastomas, 1 of 10 (10.0%) meningiomas, and 47 of 95 (49.5%) metastases were positive. Importantly, the cytoarchitectural staining characteristics among all 4 MGA-positive primary CNS neoplasms (cytoplasmic and nuclear) differed from those of the metastases (cytoplasmic and membranous). These findings suggest that MGA IHC staining intensity and distribution can distinguish metastases from primary CNS neoplasms (P=0.0086) in women with a history of breast carcinoma but also indicate that cytologic staining patterns must be interpreted for more accurate tumor classification.

Background The aim of the present study was to investigate within ovarian carcinoma and normal ovarian biopsies the gene expression of multiple secretoglobin family members relative to mammaglobin B, which we previously reported as a promising novel ovarian carcinoma prognostic marker. Methods Using quantitative real-time Reverse Transcription PCR we tested 53 ovarian carcinoma and 30 normal ovaries for the expression of 8 genes belonging to the secretoglobin family: mammaglobin A, lipophilin A, lipophilin B, uteroglobin, HIN-1, UGRP-1, RYD5 and IIS. Next, we decided to expand the LipB gene expression analysis to a further 48 ovarian carcinoma samples, for a total of 101 tumor tissues of various histologies and to study its protein expression by immunohistochemistry in formalin-fixed paraffin-embedded tumors and normal ovaries. Finally, we correlated lipophilin B gene and protein expression to conventional patient clinico-pathological features and outcome. Results We found significant mammaglobin A, lipophilin A, lipophilin B and RYD5 gene overexpression in ovarian carcinomas compared to normal ovaries. Lipophilin B mRNA showed a higher presence in tumors (75.4%) compared to normal ovaries (16.6%) and the most significant correlation with mammaglobin B mRNA (rs =0.77, p, This investigation was supported by grants from the Nocivelli Foundation, Brescia, Italy, and by grants from the Istituto Superiore di Sanità (Programma onco-proteomica Italia-USA 527B4/4), Rome, Italy. Dr Elisa Rossi is supported by the JAE-Doc program of CSIC funded by FEDER. Moreover, this work was supported in part by grants from NIH R01 CA122728-01A4 and R01 CA154460-01A1, the Honorable Tina Brozman Foundation and Deborah Bunn Alley Ovarian Cancer Research Foundation to ADS

Background To investigate the prognostic significance of disseminated tumor cells (DTCs) in bone marrow (BM) from non-metastatic breast cancer patients before and after surgery. Methods Patients with non-metastatic breast cancer were consecutively recruited to this project during the years 1998–2000. Real-time RT-PCR quantification of a DTC multimarker panel consisting of cytokeratin 19, mammaglobin A and TWIST1 mRNA was performed in BM samples obtained from 154 patients three weeks (BM2) and/or six months after surgery (BM3). The results were compared to previously published data from pre-operative BM analyses for the same patients. Results DTCs were identified in post-operative BM samples (BM2 and/or BM3) from 23 (15%) of the 154 patients investigated. During a median follow-up of 98 months, 10 (44%) of these patients experienced systemic relapse as compared to 16 (12%) of 131 DTC-negative patients. Kaplan-Meier estimates of systemic recurrence-free- and breast-cancer specific survival demonstrated significantly shorter survival for patients with persistent DTCs in BM after surgery (p≤0.001). By multivariate Cox regression analyses, persistent DTCs after surgery was an independent predictor of both systemic recurrence-free- (HR = 5.4, p p p p Conclusions Detection of persistent DTCs in BM samples obtained after surgery identified non-metastatic breast cancer patients at high risk for systemic relapse, and with reduced breast-cancer specific survival. Furthermore, patients with positive DTC status both before and after surgery had a particularly poor prognosis.

Breast cancer is the most frequent cancer in women in the USA and the second most common cause of death in females who develop cancer. Recently, the detection of circulating tumor cells has emerged as a promising tool for monitoring the progression of clinically occult micrometastases in breast cancer patients. Sensitive molecular techniques, primarily based upon the reverse-transcriptase PCR, using various molecules as markers, have been developed to detect circulating tumor cells. Among those molecules, human mammaglobin mRNA has been found to be the most specific marker for the hematogenous spread of breast cancer cells. In this article, we review the current knowledge regarding the use of reverse-transcriptase PCR for detecting human mammaglobin mRNA as a biomarker for circulating tumor cells in breast cancer patients, and evaluate the clinical implications of human mammaglobin since it was first isolated in 1996.

BACKGROUND Molecular characterization of circulating tumor cells (CTCs) is crucial to identify novel diagnostic and therapeutic targets for individualized therapies. We developed a multiplexed PCR-coupled liquid bead array to detect the expression of multiple genes in CTCs. METHODS mRNA isolated from immunomagnetically enriched CTCs was subjected to multiplex PCR for KRT19 (keratin 19; also known as CK19), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2], SCGB2A2 (secretoglobin, family 2A, member 2; also known as MGB1, mammaglobin A), MAGEA3 (melanoma antigen family A, 3), TWIST-1 [twist homolog 1 (Drosophila)], and HMBS (hydroxymethylbilane synthase; also known as PBGD). Biotinylated amplicons were hybridized against fluorescent microspheres carrying gene-specific capture probes and incubated with streptavidin–phycoerythrin. We quantified the captured labeled amplicons and decoded the beads by Luminex flow cytometry. The assay was validated for limit of detection, specificity, and comparison with reverse-transcription quantitative PCR (RT-qPCR), and its clinical performance was evaluated in 64 patients with operable breast cancer, 20 patients with metastasis, and 17 healthy individuals. RESULTS The assay was specific for each gene in complex multiplexed formats and could detect the expression of each gene at the level of a single SK-BR-3 cell. The assay produced results comparable to those for RT-qPCR for each gene. None of the genes tested was detected in the CTC fraction of healthy donors. We detected KRT19, ERBB2, MAGEA3, SCGB2A2, and TWIST1 in 26.6%, 12.5%, 18.7%, 10.9%, and 31.2% of operable breast cancer patients, respectively, and detected the corresponding genes in 65%, 20%, 30%, 20%, and 20% of patients with verified metastasis, respectively. CONCLUSIONS The expression of 6 genes in CTCs can be measured simultaneously and reliably, thereby saving precious sample and reducing the costs and time of analysis.

Morphologic differentiation of breast carcinoma from nonmammary malignancies in fluid specimens can be a diagnostic challenge. Immunocytochemistry is often employed in the differential diagnosis. In this study, we evaluated the expression of mammoglobin (MGB1) in body-cavity fluid specimens and compared its efficacy as a marker for metastatic breast carcinomas with that of gross cystic disease fluid protein-15 (GCDFP-15). Cell blocks from 40 fluid specimens were immunostained with monoclonal antibodies against MGB1 and GCDFP-15. They included 15 breast carcinomas and 25 nonmammary carcinomas (10 lungs, 10 ovaries, 3 gastrointestinal tracts, 1 kidney, and 1 urinary bladder). Positivity was defined as the presence of cytoplasmic staining in 10% or more carcinoma cells. Thirteen (87%) and seven (47%) breast carcinomas showed positive staining with MGB1 and GCDFP-15, respectively. Three (12%) nonmammary carcinomas (2 ovarian and 1 colonic) showed positive MGB1 staining; one (3%) nonmammary carcinoma demonstrated positive GCDFP-15 staining. The differences of MGB1 and GCDFP-15 staining between breast and nonmammary carcinomas were statistically significant (P < 0.05). Both MGB1 and GCDFP-15 are specific markers for metastatic breast carcinomas in cell block fluid specimens (88 vs. 96%). However, MGB1 is more sensitive than GCDFP-15 as a marker for metastatic breast carcinoma (87 vs. 46%).

Lipophilin B (LPB), which is also known as BU101, is a secretoglobin which exists in vivo as a complex with the mammary-specific protein, mammaglobin A (MGA). The aim of our study was to investigate the expression of LPB in a panel of breast and nonbreast tissues and compare its expression with that of MGA. Using RT-PCR, LPB mRNA was detected in 16/25 (64%) of normal breast specimens, 23/30 (77%) of fibroadenomas, 102/156 (65%) of primary breast cancers and in 8/36 (22%) nonbreast tissues. Levels of expression of LPB mRNA were significantly higher in breast cancers compared to both normal breast tissues (p = 0.02) and nonbreast tissue (p < 0.001). In the primary breast cancers, expression of LPB mRNA was positively correlated with the estrogen receptor (p = 0.045) but inversely related to both tumor grade (p < 0.001) and proliferation rates (p = 0.0345). Compared to MGA, expression of LPB was more sensitive but less specific for breast cancer. Using Western blotting, LPB migrated with an approximate molecular mass of 7-8 kDa, the expected molecular mass of free LPB. Immunohistochemical analysis of breast cancers showed that LPB expression was predominantly confined to the cytoplasm of tumor cells. We conclude that expression of LPB is preferentially but not exclusively restricted to breast tissue. Since LPB was expressed relatively specifically in breast tissue and was significantly upregulated in breast carcinomas, it is a promising candidate biomarker for breast cancer.

There are limited data that compare the usefulness of mammaglobin with gross cystic disease fluid protein-15 (GCDFP-15) in the identification of breast carcinomas. Whole tissue sections of 29 breast carcinomas with matched lymph node metastases and 63 breast carcinomas on tissue microarray were stained with mammaglobin cocktail and GCDFP-15 antibodies. In addition, tissue microarrays (US Biomax, Rockville, MD) containing 544 different human tumors were also stained with the mammaglobin antibody cocktail. Positive staining was seen in 67 (55.4%) of 121 breast carcinomas with mammaglobin and in 28 cases (23.1%) with GCDFP-15. In the majority of cases, the staining intensity and number of cells staining were higher with mammaglobin than with GCDFP-15. Positive mammaglobin staining was also seen in 44 (8.1%) of 544 nonbreast tumors. Mammaglobin is a more sensitive marker than GCDFP-15 for breast carcinoma; however, it lacks the specificity of GCDFP-15.

Background Mammaglobin A (SCGB2A2) and lipophilin B (SCGB1D2), two members of the secretoglobin superfamily, are known to be co-expressed in breast cancer, where their proteins form a covalent complex. Based on the relatively high tissue-specific expression pattern, it has been proposed that the mammaglobin A protein and/or its complex with lipophilin B could be used in breast cancer diagnosis and treatment. In view of these clinical implications, the aim of the present study was to analyze the expression of both genes in a large panel of human solid tumors (n = 309), corresponding normal tissues (n = 309) and cell lines (n = 11), in order to evaluate their tissue specific expression and co-expression pattern. Methods For gene and protein expression analyses, northern blot, dot blot hybridization of matched tumor/normal arrays (cancer profiling arrays), quantitative RT-PCR, non-radioisotopic RNA in situ hybridization and immunohistochemistry were used. Results Cancer profiling array data demonstrated that mammaglobin A and lipophilin B expression is not restricted to normal and malignant breast tissue. Both genes were abundantly expressed in tumors of the female genital tract, i.e. endometrial, ovarian and cervical cancer. In these four tissues the expression pattern of mammaglobin A and lipophilin B was highly concordant, with both genes being down-, up- or not regulated in the same tissue samples. In breast tissue, mammaglobin A expression was down-regulated in 49% and up-regulated in 12% of breast tumor specimens compared with matching normal tissues, while lipophilin B was down-regulated in 59% and up-regulated in 3% of cases. In endometrial tissue, expression of mammaglobin A and lipophilin B was clearly up-regulated in tumors (47% and 49% respectively). Both genes exhibited down-regulation in 22% of endometrial tumors. The only exceptions to this concordance of mammaglobin A/lipophilin B expression were normal and malignant tissues of prostate and kidney, where only lipophilin B was abundantly expressed and mammaglobin A was entirely absent. RNA in situ hybridization and immunohistochemistry confirmed expression of mammaglobin A on a cellular level in endometrial and cervical cancer and their corresponding normal tissues. Conclusion Altogether, these data suggest that expression of mammaglobin A and lipophilin B might be controlled in different tissues by the same regulatory transcriptional mechanisms. Diagnostic assays based on mammaglobin A expression and/or the mammaglobin A/lipophilin B complex appear to be less specific for breast cancer, but with a broader spectrum of potential applications, which includes gynecologic malignancies.

Human secretoglobin (SCGB) 2A1 (or lipophilin C, lacryglobin, mammaglobin B) is a small protein of unknown function that forms heterodimers with secretoglobin 1D1 (lipophilin A) in tears. SCGB 2A1 is homologous to mammaglobin (mammaglobin A) and the C3 component of prostatein, the major secretory protein of the rat ventral prostate. Androgen-dependent expression of SCGB 2A1 has been observed in the prostate. Besides identification of SCGB 2A1 in the tear proteome only its mRNA had been detected in the lacrimal gland. Here, we report expression of SCGB 2A1 in all ocular glands and in the keratinized stratified squamous epithelium of the eyelid as well as in the stratified epithelium of the conjunctiva and in the orbicularis oculi muscle. Almost all of these tissues are also known to express the androgen receptor. Therefore, we conclude that presence of the androgen signalling machinery could be the main general determinant of SCGB 2A1 expression. Implications of the presence in tear fluid of an androgen-regulated secretoglobin, which most likely binds hydrophobic ligands, for tear film lipid layer formation and function is discussed.

Purpose: Early detection of breast cancer has implications for the management and treatment of patients with this disease. Currently, there exist no highly sensitive and specific serologic biomarkers for detection of breast cancer. Mammaglobin is predicted to be a secreted protein, and expression of this gene seems to be highly specific in breast cancer. The present studies were undertaken to develop the mammaglobin protein as a serum biomarker for detection of breast cancer. Experimental Design: We characterized the mammaglobin protein as a secreted, 14- to 21-kDa species, which is likely post-translationally processed based on its predicted 7-kDa size. Immunostaining for mammaglobin was conducted. An ELISA was developed for the detection of the mammaglobin protein in serum, and levels were compared between women with and without breast cancer. A receiver operating characteristic curve was used to show sensitivity and specificity for cut points on the continuous mammaglobin scale. Results: The protein was detectable by immunostaining in 72% of breast tumors and not in other tumor types. The ELISA was highly sensitive and specific for detection of mammaglobin protein in tissue culture fluids of breast cancer cells and sera of breast cancer patients. The ELISA differentiated healthy women from those with breast cancer with accurate, repeatable results across time and under varying storage conditions. Conclusion: Our results indicate that mammaglobin, as measured by the ELISA, holds significant promise for breast cancer screening with the realistic potential to impact management of this disease.

The objective of this study was mainly to develop and evaluate a membrane array-based method simultaneously detecting the expression levels of a multiple mRNA marker panel in the peripheral blood for used in complementary breast cancer diagnosis. The mRNA markers employed included cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA), c-Met, Her2/neu, and mammaglobin (hMAM). The specimens of peripheral blood were collected from 80 healthy women and 102 female patients with breast cancer. The expression levels of molecular markers were evaluated by real-time Q-PCR and membrane array. Data obtained from real-time Q-PCR and membrane array were subjected to linear regression analysis, revealing that there was a high degree of correlation between the results of these two methods (r=0.979, P

The breast cancer-associated gene mammaglobin is a member of the secretoglobin protein family and has demonstrated its utility as a breast cancer marker. However, the transcriptional regulation of mammaglobin has not been well-characterized. In this report, we used luciferase reporter assays to identify the 200 bp directly 5' of the transcriptional start site as the minimal promoter region of mammaglobin. Sequence scanning indicated that two PEA3 transcription sites were possibly involved in mammaglobin transcription. By transfecting a PEA3 expression vector into breast cancer cell lines MDA-MB-415 and MCF-7, we determined that exogenous PEA3 was able to drive transcription. Mutational analysis indicated that each PEA3 site was functional. Our reporter system and electrophorectic mobility shift assays (EMSAs) also identified the involvement of a unique repetitive element in mammaglobin transcription. Finally, AP-1 was determined via luciferase assays to be involved in regulating non-PEA3 dependent transcription. Elucidating these cis-acting elements will impact our understanding of transcription of normal breast and breast cancer-associated genes.

To the Editor: In a recent issue of Clinical Cancer Research , two articles ([1][1], [2][2]) addressed the use of PCR to detect micrometastases from breast cancer either in lymph nodes ([1][1]) or in peripheral blood ([2][2]). Both articles found mammaglobin ( mam ) to be an excellent marker for

Mammaglobin is a gene that is expressed almost exclusively in the normal breast epithelium and human breast cancer. It is a member of the secretoglobin gene family and forms a heterodimer with lipophilin B. We have focused on the tissue-specificity of mammaglobin as a potential mechanism for the specific killing of breast cancer cells. By elucidating the promoter region of mammaglobin, we hope to utilize this site as a method for turning on the apoptosis inducer gene, Bax, in breast cancer cells. The Bax gene will only be expressed at levels necessary to induce apoptosis in mammaglobin positive cells. This would include > 80% of all breast cancer cells and some normal breast epithelium. This type of targeted killing could be conceptualized as a biochemical mastectomy; that is, genetic ablation of breast tumor cells and perhaps non-malignant breast epithelium while preserving the adipose and stromal components of the breast. Work is also being done to address the binding specificity of the secreted mammaglobin protein. There is early evidence that the mammaglobin heterodimer may in fact bind to breast and breast cancer cells. If this finding is validated, this creates the possibility that mammaglobin can be tagged with a radioisotope or a toxin, so that binding of the tagged-mammaglobin complex results in the specific killing of that breast cancer cell. Finally, mammaglobin is being explored as a target for immune-based interventions. In vitro studies have demonstrated that T cellmediated immune responses can be induced against mammaglobin-derived peptides expressed by MHC molecules on tumor cells and antigen-presenting cells. In summary, mammaglobin displays several unique features that make it a promising target for intervention.

Background: The aim of this study was to examine the potential usefulness of a mammaglobin multigene reverse transcription-PCR (RT-PCR) assay and a mammaglobin sandwich ELISA as diagnostic tools in breast cancer. Methods: We studied peripheral blood samples from 147 untreated Senegalese women with biopsy-confirmed breast cancer and gathered patient information regarding demographic, and clinical staging of disease. The samples were tested for mammaglobin and three breast cancer-associated gene transcripts by a multigene real-time RT-PCR assay and for serum mammaglobin protein by a sandwich ELISA assay. Results: In 77% of the breast cancer blood samples, a positive signal was obtained in the multigene RT-PCR assay detecting mammaglobin and three complementary transcribed genes. Fifty samples from healthy female donors tested negative. Significant correlations were found between mammaglobin protein in serum, presence of mammaglobin mRNA-expressing cells in blood, stage of disease, and tumor size. Circulating mammaglobin protein was detected in 68% of the breast cancer sera, and was increased in 38% in comparison with a mixed control population. The RT-PCR assay and the ELISA for mammaglobin produced a combined sensitivity of 84% and specificity of 97%. Conclusion: The ELISA and RT-PCR for mammaglobin and mammaglobin-producing cells could be valuable tools for diagnosis and prognosis of breast cancer.

Background Nasal polyps are a common problem that is difficult to diagnose and treat, in part because the cause of nasal polyposis is unknown. Although information on the pathogenesis of polyposis is lacking, there are reports suggesting that a genetic predisposition underlies this disorder. Objective We sought to better understand the basis of nasal polyposis associated with allergic rhinitis. We hypothesize that the expression of unique genes is associated with the nasal polyposis phenotype. Methods We examined 12,000 human genes transcribed in the nasal mucosa of patients with allergic rhinitis with and without nasal polyps. Biopsy specimens of the mucosa of patients with and without polyps were obtained after the patients refrained from the use of topical or systemic steroid therapy for 2 weeks. Results Thirty-four genes were differentially expressed between the patient groups, including those for inflammatory molecules and putative growth factors. The greatest differential expression identified by the array analysis was for a group of genes associated with neoplasia, including mammaglobin, a gene transcribed 12-fold higher in patients with polyps compared with control patients with rhinitis alone. Quantitative RT-PCR confirmed this differential expression and documented that the number of mammaglobin mRNA copies is actually 64-fold greater in tissues of patients with polyps versus control patients. The specificity of mammaglobin protein expression was evaluated by means of immunohistochemistry, which showed specific staining in nasal polyp mucosal goblet cells only in patients with polyps. Conclusion These data suggest that nasal polyposis involves deregulated cell growth, using gene activation in some ways similar to a neoplasm. In addition, mammaglobin, a gene of unknown function associated with breast neoplasia, might be related to polyp growth.

Mammaglobin, known for its mammary tissue specificity, has been discussed as a promising diagnostic marker in breast cancer for almost 10 years. In particular, the application of mammaglobin RT-PCR to detect disseminated breast cancer cells has been reported. More than 25 publications evaluate the detection of mammaglobin mRNA in lymph node, blood, and bone marrow specimens of breast cancer patients. Recently, structural details about the mammaglobin complex have been discovered, and these findings can be implemented to optimize detection of the secreted protein. This review summarizes the findings of almost 50 published studies and the current knowledge about the diagnostic utility of mammaglobin.

Mammaglobin (SCGB2A2) is a breast-specific member of the secretoglobin (SCGB) gene family. SCGB2A2 has previously been found overexpressed in breast tumors but possible associations between its expression and established prognostic tumor characteristics such as the levels of estrogen and progesterone receptors have not yet been investigated. We evaluated SCGB2A2 expression at the mRNA and at the protein level by reverse-transcription polymerase chain reaction and immunocytochemistry in 52 and 32 breast tumors, respectively. Both SCGB2A2 mRNA and protein expression were significantly higher in estrogen-receptor-positive compared to estrogen-receptor-negative tumors (Mann- Whitney rank sum test, p = 0.04; chi-square test, p = 0.01; respectively). In contrast, SCGB2A2 expression did not correlate with progesterone receptor levels or Nottingham grade. As estrogen and antiestrogen treatment of estrogen-positive breast cancer cell lines does not modify SCGB2A2 expression we suggest that SCGB2A2 may be a new independent breast cancer prognostic marker.

Human mammaglobin (hMAM) mRNA is considered to be a promising candidate for a sensitive molecular marker for breast cancer. In this study, we attempted to relate the presence of hMAM mRNA in the peripheral blood with certain established clinico-pathological features of breast cancer in order to validate its clinical utility. A total of 139 subjects including 79 with localized cancer, 33 with metastatic disease, and a control group of 27 individuals were studied. hMAM mRNA expression was assessed by reverse transcriptase polymerase chain reaction on cells from peripheral blood. The expression of hMAM mRNA was found in 0 of the 27 control subjects, 1 of the 8 stage 0 (12.5%) patients, 4 of the 16 stage I (25%) patients, 13 of the 40 stage II (32.5%) patients, 5 of the 15 stage III (33.3%) patients, and 18 of the 33 (54%) cases of metastatic disease. There was a statistically significant (P=0.045) difference in frequency between patients with localized disease (29%) and those with metastatic disease. Although trends of increasing frequency of hMAM mRNA expression existed in patients with unfavorable prognostic factors, including primary tumor size, T stage, N stage, overall stage, presence of angiolymphatic permeation, negative estrogen receptor, high S-phase fraction (>7%), and aneuploid DNA index, none of the differences was significant. In conclusion, the clinical utility of hMAM mRNA may not be in screening or staging of breast cancer, but in following patients after surgery to detect recurrence. Further evaluation of hMAM mRNA in combination with other molecular markers to follow post-operative breast cancer patient is warranted. (Cancer Sci 2003; 94: 99–102)

Mammaglobin, a promising diagnostic marker for breast cancer, forms a covalent complex with lipophilin B. mRNA levels for each component of the complex were determined for a number of breast tumors and normal tissues, and correlation of message expression was highly significant between mammaglobin and lipophilin B (p0.0001). The complex was purified by both standard biochemical techniques and immunoaffinity chromatography. N-Terminal sequencing revealed that mammaglobin and lipophilin B are processed as predicted by cleavage of their signal sequence after amino acids 19 and 21, respectively. Three molecular masses-representing the fully glycosylated form, the complex without one of the carbohydrate chains, and the deglycosylated proteins-are detected by ProteinChip array SELDI-TOF mass spectrometry after partial enzymatic deglycosylation. This is consistent with the two predicted N-linked glycosylation sites in the primary sequence of mammaglobin and each site having an attached sugar of approximately 3500 Da. Reducing agents release lipophilin B from mammaglobin, and the free peptides are seen at their predicted molecular masses in the deglycosylated complex. Molecular modeling, secondary structure prediction, and circular dichroism indicate that the complex is a small alpha-helical globule that has three disulfide bridges and a carbohydrate chain at each pole. LC-ESI-MS shows that mammaglobin and lipophilin B are bonded in a head to tail orientation. This work describes the biochemistry of the mammaglobin/lipophilin B complex and lays the framework for use of this complex as a novel protein-based serological marker for breast cancer.

It has been suggested that mammaglobin may be a useful marker for breast cancer clinical research. Studies investigating the detection of mRNA by RT-PCR from circulating carcinoma cells in the peripheral blood of breast cancer patients have shown that mammaglobin is a highly specific marker and correlates with several prognostic factors, such as lymph node involvement. We aimed to detect cell-free mammaglobin mRNA in the plasma of breast cancer patients and to investigate whether it can be used as a marker for diagnosis of breast cancer.