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1.  Application of a Multigene RT-PCR assay for the detection of Mammaglobin and complementary transcribed genes in breast cancer lymph nodes 
Clinical chemistry  2002;48(8):1225-1231.
Background: Mammaglobin mRNA expression is found in 70-80 % of primary and metastatic breast tumor biopsies. The potential breast tumor markers B305D, B726P and GABAπ complement the expression of mammaglobin. Collectively the expression profile of these four genes could be utilized as a diagnostic and prognostic indicator for breast cancer.
Methods: A multigene RT-PCR assay was established to detect the expression of mammaglobin, GABAπ, B305D and B726P simultaneously. Specific primers and Taqman® probes were used to analyze combined mRNA expression profiles in primary breast tumors and metastatic lymph node specimens.
Results: The multigene RT-PCR assay detected significant expression signals in 27/27 primary and in 50/50 lymph-node-metastatic breast tumor samples. Specificity studies demonstrated no significant expression signal in 27 non-breast cancer lymph nodes, in 22 various normal tissues or in 14 colon tumor samples.
Conclusion: The novel RT-PCR-based assay described here provides a sensitive detection system for disseminated breast tumor cells in lymph nodes. In addition, this multigene assay could also be used to test peripheral blood and bone marrow samples.
PMCID: PMC1482782  PMID: 12142378
2.  Expression analysis of mammaglobin A (SCGB2A2) and lipophilin B (SCGB1D2) in more than 300 human tumors and matching normal tissues reveals their co-expression in gynecologic malignancies 
BMC Cancer  2006;6:88.
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.
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.
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.
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.
PMCID: PMC1513245  PMID: 16603086
3.  Immunohistochemical characterization of neoplastic cells of breast origin 
Diagnostic Pathology  2012;7:73.
After skin cancer, breast cancer is the most common malignancy in women. Tumors of unknown origin account for 5-15% of malignant neoplasms, with 1.5% being breast cancer. An immunohistochemical panel with conventional and newer markers, such as mammaglobin, was selected for the detection of neoplastic cells of breast origin. The specific objectives are: 1) to determine the sensitivity and specificity of the panel, with a special emphasis on the inclusion of the mammaglobin marker, and 2) to compare immunohistochemistry performed on whole tissue sections and on Tissue Micro-Array.
Twenty-nine metastatic breast tumors were included and assumed as tumors of unknown origin. Other 48 biopsies of diverse tissues were selected and assumed as negative controls. Tissue Micro-Array was performed. Immunohistochemistry for mammaglobin, gross cystic disease fluid protein-15, estrogen receptor, progesterone receptor and cytokeratin 7 was done.
Mammaglobin positive staining was observed in 10/29 cases, in 13/29 cases for gross cystic disease fluid protein-15, in 20/29 cases for estrogen receptor, in 9/29 cases for progesterone receptor, and in 25/29 cases for cytokeratin 7. Among the negative controls, mammaglobin was positive in 2/48, and gross cystic disease fluid protein-15 in 4/48.
The inclusion of MAG antibody in the immunohistochemical panel for the detection of tumors of unknown origin contributed to the detection of metastasis of breast cancer. The diagnostic strategy with the highest positive predictive value (88%) included hormone receptors and mammaglobin in serial manner.
Virtual slides
The virtual slide(s) for this article can be found here:
PMCID: PMC3468373  PMID: 22726568
Tumors of unknown origin; Breast; Mammaglobin; Immunohistochemistry
4.  Mammaglobin peptide as a novel biomarker for breast cancer detection 
Cancer Biology & Therapy  2013;14(4):327-332.
Among the different types of tests used for cancer diagnosis, molecular tests have been increrasingly incorporated because of their ability to detect either expression or functional changes in the molecules associated with the disease. Mammaglobin is a protein found in mammary tissue and can be detected in serum. This protein has been proposed as a biomarker to diagnose breast cancer, given that patients exhibit an increased amount of the protein in serum and tumor tissue, in comparison to healthy individuals. The ELISA test was used in the present study to detect mammaglobin in blood samples from 51 breast cancer patients and 51 control individuals. Antibodies against mamaglobin were generated in rabbits by using the following synthetic peptides: A (amino acids 13 to 21), B (amino acids 31 to 39), C (amino acids 56 to 64) and a D peptide, corresponding to the protein isoform without three amino acids (59, 60 and 61 amino acids) from peptide C. All peptides were immunogenic and allowed generation of antibodies that were able to discriminate patients from controls. The best results were obtained for antiserum B, achieving the best sensitivity (86.3%) and specificity (96%).
PMCID: PMC3667872  PMID: 23358476
ELISA; biomarker; breast cancer; diagnosis; human mammaglobin
5.  Real-time RT–PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer 
British Journal of Cancer  2006;94(5):672-680.
This study assessed the ability of real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT–PCR detection method. The mean follow-up time was 786 days (+/− 487). Kaplan–Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results.
PMCID: PMC2361203  PMID: 16495933
breast cancer; disseminated epithelial cells; cytokeratin-19; mammaglobin; RT–PCR
6.  Multimarker RT–PCR assay for the detection of minimal residual disease in sentinel lymph nodes of breast cancer patients 
British Journal of Cancer  2006;94(5):681-685.
The presence of metastases in lymph nodes is the most powerful prognostic factor in breast cancer patients. Routine histological examination of lymph nodes has limited sensitivity for the detection of breast cancer metastases. The aim of the present study was to develop a multimarker reverse transcriptase–polymerase chain reaction (RT—PCR) assay for the detection of minimal residual disease in sentinel nodes of breast cancer patients. RNA was extracted from 30 sentinel lymph nodes (SLN) obtained from 28 patients, three primary breast cancers (positive controls), three lymph nodes from patients with benign diseases, and peripheral blood lymphocytes of 10 healthy volunteers (negative controls). RT–PCR was performed using the following markers; cytokeratin (CK)-19, NY-BR-1 and mammaglobin B. RT–PCR results were compared to enhanced histopathologic examination and immunohistochemistry (IHC). All three positive controls showed strong PCR amplification for all three markers. None of the 13 negative controls was amplified by any of the three markers. Among the 30 SLN analysed, breast cancer metastases were detected in six SLNs by routine histology, in eight by IHC and in 15 by RT–PCR. We conclude that a multimarker RT–PCR assay probing for NY-BR-1, mammaglobin-B, and CK-19 is more sensitive compared to enhanced pathologic examination. This method may prove to be of value in breast cancer staging and prognosis evaluation.
PMCID: PMC2361196  PMID: 16495929
RT–PCR; breast neoplasms; micrometastases; mammaglobin; NY-BR-1
7.  Gene expression of circulating tumour cells in breast cancer patients 
The diagnostic tools to predict the prognosis in patients suffering from breast cancer (BC) need further improvements. New technological achievements like the gene profiling of circulating tumour cells (CTC) could help identify new prognostic markers in the clinical setting. Furthermore, gene expression patterns of CTC might provide important informations on the mechanisms of tumour cell metastasation.
Materials and methods
We performed realtime-PCR and multiplex-PCR analyses following immunomagnetic separation of CTC. Peripheral blood (PB) samples of 63 patients with breast cancer of various stages were analyzed and compared to a control group of 14 healthy individuals. After reverse-transcription, we performed multiplex PCR using primers for the genes ga733.3, muc-1 and c-erbB2. Mammaglobin1, spdef and c-erbB2 were analyzed applying realtime-PCR.
ga733.2 overexpression was found in 12.7% of breast cancer cases, muc-1 in 15.9%, mgb1 in 9.1% and spdef in 12.1%. In this study, c-erbB2 did not show any significant correlation to BC, possibly due to a highly ambient expression. Besides single gene analyses, gene profiles were additionally evaluated. Highly significant correlations to BC were found in single gene analyses of ga733.2 and muc-1 and in gene profile analyses of ga733.3*muc-1 and GA7 ga733.3*muc-1*mgb1*spdef.
Our study reveals that the single genes ga733.3, muc-1 and the gene profiles ga733.3*muc-1 and ga733.3*3muc-1*mgb1*spdef can serve as markers for the detection of CTC in BC. The multigene analyses found highly positive levels in BC patients. Our study indicates that not single gene analyses but subtle patterns of multiple genes lead to rising accuracy and low loss of specificity in detection of breast cancer cases.
PMCID: PMC3352225  PMID: 19748849
Mamma carcinoma; pcr; gene profile
8.  Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT–PCR in patients with metastatic breast cancer 
British Journal of Cancer  2009;102(2):276-284.
The detection, enumeration and isolation of circulating tumour cells (CTCs) have considerable potential to influence the clinical management of patients with breast cancer. There is, however, substantial variability in the rates of positive samples using existing detection techniques. The lack of standardisation of technology hampers the implementation of CTC measurement in clinical routine practice.
This study was designed to directly compare three techniques for detecting CTCs in blood samples taken from 76 patients with metastatic breast cancer (MBC) and from 20 healthy controls: the CellSearch CTC System, the AdnaTest Breast Cancer Select/Detect and a previously developed real-time qRT-PCR assay for the detection of CK-19 and mammaglobin transcripts.
As a result, 36% of patients with MBC were positive by the CellSearch System, 22% by the AdnaTest, 26% using RT–PCR for CK-19 and 54% using RT–PCR for mammaglobin. Samples were significantly more likely to be positive for at least one mRNA marker using RT–PCR than using the CellSearch System (P=0.001) or the AdnaTest (P<0.001).
We observed a substantial variation in the detection rates of CTCs in blood from breast cancer patients using three different techniques. A higher rate of positive samples was observed using a combined qRT-PCR approach for CK-19 and mammaglobin, which suggests that this is currently the most sensitive technique for detecting CTCs.
PMCID: PMC2816650  PMID: 19953098
circulating tumour cells; breast cancer; CellSearch System; RT–PCR; AdnaTest
9.  Molecular Markers in Peripheral Blood of Iranian Women with Breast Cancer 
Cancer Microenvironment  2012;6(1):109-116.
A biomarker is a quantifiable laboratory measure of a disease specific biologically relevant molecule that can act as an indicator of a current or future disease state. The purpose of this study is to detect the expression of RNA biomarkers using Cytokeratin 19 (CK-19), Mammaglobin (MAM), Carcinoembryonic antigen (CEA), Mucin (MUC), C-Myc, erb-B2, a proliferation marker (Ki-67), Epidermal growth factor receptor (Her2/neu) and Estrogen receptor (ER) in Iranian women who were diagnosed with breast cancer. In this study, 90 samples; 60 cancer patients and 30 healthy controls were considered. 73.4 % patients were in stage I/II and 26.6 % were in stage III/IV. Patients were selected prior to the administration of any adjuvant systemic therapy. Total RNA extraction was obtained from peripheral blood of each patient and healthy control. Reverse transcription polymerase chain reaction (RT-PCR) method was used for detection of mRNA of the selected biomarkers of circulating breast cancer cells in blood. Molecular characterization is assessed as a method for early detection of breast cancer. For this purpose, eleven specific primers were selected and RT-PCR was used. The data of RT-PCR revealed that expression of MUC1, CK19, CEA, MAM, erbB-2, Ki67 and C-Myc biomarkers were significantly different between breast cancer patients and healthy controls. On the other hand, ERα, ERβ and Her2 markers were not significantly different between the two mentioned groups. Biomarkers detection of breast cancer patients could be assessed as a diagnostic factor and its potential for conveying as a prognostic factor require further studies, with a larger number of patients.
PMCID: PMC3601212  PMID: 22828927
Breast cancer; CEA; CK19; C-Myc; ER; erbB-2; Her2; Ki67; MAM; MUC1
10.  A Mammaglobin-A Targeting Agent for Non-invasive Detection of Breast Cancer Metastasis in Lymph Nodes 
Cancer research  2010;71(3):1050-1059.
Pathologic axillary lymph node (ALN) status is an important prognostic factor for staging breast cancer. Currently, status is determined by histopathology following surgical excision of sentinel lymph node(s), which is an invasive, time consuming, costly and potentially morbid procedure. This work describes an imaging platform for the non-invasive assessment of ALN status, eliminating the need for operation in patients without nodal involvement. A targeted imaging probe (MamAb-680) was developed by conjugation of a mammaglobin-A specific monoclonal antibody to a near-infrared fluorescent dye. Using DNA and tissue microarray, mammaglobin-A was validated as a cell-surface target that is expressed in axillary lymph node positive patient samples but is not expressed in normal lymph nodes. In vivo selectivity was determined by intravenous injection of MamAb-680 into mice with mammaglobin-A positive and negative mammary fat pad (MFP) tumors; and by peritumoral MFP injection of the targeted imaging probe in mice with spontaneous ALN metastases. Fluorescence imaging showed that probe was only retained in positive tumors and metastases. As few as 1000 cells that endogenously express mammaglobin-A were detected in ALN indicating high sensitivity of this method. Hence, this approach has potential for translation into clinical use for the non-invasive staging of breast cancer.
PMCID: PMC4130564  PMID: 21169406
breast cancer; imaging; lymph node metastasis; mammaglobin-A; targeted agent
11.  Secretoglobin expression in ovarian carcinoma: lipophilin B gene upregulation as an independent marker of better prognosis 
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.
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.
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 < 0.001). By immunohistochemical analysis, we showed higher lipophilin B expression in the cytoplasm of tumor cells compared to normal ovaries (p < 0.001). Moreover, lipophilin B gene overexpression was significantly associated with serous histology (serous vs clear cell p = 0.027; serous vs undifferentiated p = 0.007) and lower tumor grade (p = 0.02). Lower LipB mRNA levels (low versus high tertiles) were associated to a shorter progression-free (p = 0.03, HR = 2.2) and disease-free survival (p = 0.02, HR = 2.5) by univariate survival analysis and, importantly, they remain an independent prognostic marker for decreased disease-free (p = 0.001, HR = 3.9) and progression-free survival (p = 0.004, HR = 2.8) in multivariate Cox regression analysis.
The present study represents the first quantitative evaluation of secretoglobin gene expression in normal and neoplastic ovarian tissues. Our results demonstrate lipophilin B gene and protein upregulation in ovarian carcinoma compared to normal ovary. Moreover, lipophilin B gene overexpression correlates with a less aggressive tumor phenotype and represents a novel ovarian carcinoma prognostic factor.
PMCID: PMC3706350  PMID: 23819652
Ovarian carcinoma; Secretoglobins; Lipophilin B; Gene expression; Prognosis; Biomarker
12.  Comparative Analysis of CD4+ and CD8+ T Cells in Tumor Tissues, Lymph Nodes and the Peripheral Blood from Patients with Breast Cancer 
Iranian Biomedical Journal  2015;19(1):35-44.
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.
PMCID: PMC4322231  PMID: 25605488
Breast neoplasms; CD4+ T lymphocyte; CD8+ T lymphocytes; CXCL10; Granzymes
13.  Elevated mammaglobin (h-MAM) expression in breast cancer is associated with clinical and biological features defining a less aggressive tumour phenotype 
Breast Cancer Research  2003;5(3):R65-R70.
Mammaglobin (h-MAM) is expressed mainly by breast epithelial cells, and this feature has been used to detect circulating breast cancer cells and occult metastases in sentinel axillary lymph nodes of breast cancer patients. However, the biological role of mammaglobin is completely unknown.
We studied 128 fresh-frozen breast cancer specimens by means of reverse transcriptase–polymerase chain reaction and quantified their h-MAM mRNA expression. This was then correlated with histological and nuclear grade, oestrogen and progesterone receptor expression, c-erb-B2 and mutant p53 expression, as well as with cellular proliferation measured by means of the Ki67 labelling index, DNA ploidy and S-phase, and finally with the presence or not of invaded axillary nodes in the mastectomy specimen.
In the univariate analysis, high h-MAM expression (above the median for the whole group) correlated significantly (P < 0.05) with oestrogen and progesterone receptor expression, diploid DNA content, low Ki67 labelling index, low nuclear grade and almost significantly (P = 0.058) with the absence of axillary nodal invasion in the mastectomy specimen. In a final, multivariate model, only progesterone receptor expression, diploid DNA content and absence of nodal invasion were found to be independently associated with high h-MAM expression.
All of the features associated with mammaglobin expression reflect, without exception, a less aggressive tumour phenotype. Further studies are needed to clarify whether this is attributable to h-MAM expression itself, or to another mechanism of which mammaglobin expression forms part.
PMCID: PMC165002  PMID: 12793902
breast; cancer; mammaglobin; reverse transcriptase polymerase chain reaction
14.  Detection of circulating epithelial cells in the blood of patients with breast cancer: comparison of three techniques 
British Journal of Cancer  2005;92(5):906-912.
This study compares the sensitivities and specificities of three techniques for the detection of circulating epithelial cells in the blood of patients with breast cancer. The number of circulating epithelial cells present in the blood of 40 patients with metastatic breast cancer and 20 healthy volunteers was determined by: immunomagnetic separation (IMS) and laser scanning cytometry (LSC), cell filtration and LSC and a multimarker real-time RT–PCR assay. Numbers of cytokeratin-positive cells identified and expression of three PCR markers were significantly higher in the blood of patients with breast cancer than in healthy volunteers. Using the upper 95% confidence interval of cells detected in controls to determine positive patient samples: 30% of patients with metastatic breast cancer were positive following cell filtration, 48% following IMS, and 60, 45 and 35% using real-time RT–PCR for cytokeratin 19, mammaglobin and prolactin-inducible peptide. Samples were significantly more likely to be positive for at least one PCR marker than by cell filtration (83 vs 30%, P<0.001) or IMS (83 vs 48%, P<0.001).The use of a multimarker real-time RT–PCR assay was therefore found to be the most sensitive technique for the detection of circulating epithelial cells in the blood of patients with breast cancer.
PMCID: PMC2361897  PMID: 15714202
breast cancer; micrometastases; circulating tumour cells
15.  Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells 
BMC Cancer  2002;2:14.
Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells.
In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (EGFR), mammaglobin 1 (MGB1), epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA) were tested. Two new epithelial-specific markers ELF3 and EphB4 were also tested.
MUC1 was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR.
ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment.
PMCID: PMC115840  PMID: 12031094
16.  Mammaglobin B is an independent prognostic marker in epithelial ovarian cancer and its expression is associated with reduced risk of disease recurrence 
BMC Cancer  2009;9:253.
Traditional prognostic factors in epithelial ovarian cancer (EOC) are inadequate in predicting recurrence and long-term prognosis, but genome-wide cancer research has recently provided multiple potentially useful biomarkers. The gene codifying for Mammaglobin B (MGB-2) has been selected from our previous microarray analysis performed on 19 serous papillary epithelial ovarian cancers and its expression has been further investigated on multiple histological subtypes, both at mRNA and protein level. Since, to date, there is no information available on the prognostic significance of MGB-2 expression in cancer, the aim of this study was to determine its prognostic potential on survival in a large cohort of well-characterized EOC patients.
MGB-2 expression was evaluated by quantitative real time-PCR in fresh-frozen tissue biopsies and was validated by immunohistochemistry in matched formalin fixed-paraffin embedded tissue samples derived from a total of 106 EOC patients and 27 controls. MGB-2 expression was then associated with the clinicopathologic features of the tumors and was correlated with clinical outcome.
MGB-2 expression was found significantly elevated in EOC compared to normal ovarian controls, both at mRNA and protein level. A good correlation was detected between MGB-2 expression data obtained by the two different techniques. MGB-2 expressing tumors were significantly associated with several clinicopathologic characteristics defining a less aggressive tumor behavior. Univariate survival analysis revealed a decreased risk for cancer-related death, recurrence and disease progression in MGB-2-expressing patients (p < 0.05). Moreover, multivariate analysis indicated that high expression levels of MGB-2 transcript (HR = 0.25, 95%, 0.08–0.75, p = 0.014) as well as positive immunostaining for the protein (HR = 0.41, 95%CI, 0.17–0.99, p = 0.048) had an independent prognostic value for disease-free survival.
This is the first report documenting that MGB-2 expression characterizes less aggressive forms of EOC and is correlated with a favorable outcome. These findings suggest that the determination of MGB-2, especially at molecular level, in EOC tissue obtained after primary surgery can provide additional prognostic information about the risk of recurrence.
PMCID: PMC2724548  PMID: 19635143
17.  Comparison of molecular and immunocytochemical methods for detection of disseminated tumor cells in bone marrow from early breast cancer patients 
BMC Cancer  2014;14:514.
Disseminated tumor cells (DTCs) have potential to predict the effect of adjuvant treatment. The purpose of this study was to compare two methods, reverse transcription quantitative PCR (RT-qPCR) and immunocytochemisty (ICC), for detecting breast cancer DTCs in bone marrow (BM) from early breast cancer patients.
We investigated a subset (n = 313) of BM samples obtained from 271 early breast cancer patients in the “Secondary Adjuvant Taxotere Treatment” (SATT)-trial. All patients in this study had node positive or intermediate/high-risk node negative non-metastatic disease. The DTCs were detected by ICC using AE1-AE3 anti-cytokeratin monoclonal antibodies. Patients with DTCs detected in their BM by ICC after standard adjuvant fluorouracil, cyclophosphamide, epirubicin (FEC) chemotherapy were offered docetaxel treatment. For comparison, 5 × 106 mononuclear cells from the aliquoted BM samples were also analyzed by RT-qPCR using a multimarker (MM) assay based on the tumor cell mRNA markers keratin 19 (KRT19), mammaglobin A (hMAM), and TWIST1. In the MM-assay, a sample was defined as positive for DTCs if at least one of the mRNA markers was positive.
The MM RT-qPCR assay identified DTCs in 124 (40%) of the 313 BM samples compared with 23/313 (7%) of the samples analyzed by ICC. The concordance between the MM RT-qPCR and ICC was 61% (Kappa value = 0.04) and twelve of the BM samples were positive by both methods. By RT-qPCR, 46/313 (15%) samples were positive for KRT19, 97/313 (31%) for TWIST1, and 3/313 (1%) for hMAM mRNA. There were no statistically significant associations between the individual mRNA markers.
The RT-qPCR based method demonstrated more DTC-positive samples than ICC. The relatively low concordance of positive DTC-status between the two different assessment methods suggests that they may be complementary. The clinical relevance of the methods will be evaluated based on future clinical outcome data.
Trial registration NCT00248703.
PMCID: PMC4223548  PMID: 25023626
Disseminated tumor cells; RT-qPCR; Immunocytochemistry; Breast cancer; Bone marrow
18.  Mammaglobin-A cDNA Vaccination of Breast Cancer Patients Induces Antigen-Specific Cytotoxic CD4+ICOShi T Cells 
Mammaglobin-A (Mam-A) is a 10 kD secretory protein that is overexpressed in 80% of primary and metastatic human breast cancers. Previous studies from our laboratory demonstrated that Mam-A cDNA vaccine can induce Mam-A specific CD8 T-cell responses and mediate regression of human breast cancer xenografts in NOD/SCID mice. In this report, we present our results on a phase I clinical trial of a Mam-A cDNA vaccination in breast cancer patients with stage-IV metastatic disease, including the impact of vaccination on the expression of the inducible co-stimulator molecule (ICOS) on CD4 T cells. Specimens from seven patients with stage IV metastatic cancer were available for these analyses. Patients were vaccinated with a Mam-A cDNA vaccine on days 0, 28 and 56, and immune responses were assessed at serial time points following vaccination. At six months following the first vaccination, flow cytometric analysis demonstrated a significant increase in the frequency of CD4+ICOShi T cells from 5 ± 2% pre-vaccination to 23 ± 4% (p<0.001), with a concomitant decrease in the frequency of CD4+Foxp3+ T cells (Treg) from 19 ± 6% to 10 ± 5% (p<0.05). ELISpot analysis of CD4+ICOShi sorted T-cells demonstrated that following vaccination the cytokines produced by Mam-A-specific T cells switched from IL-10 (78 ± 21 spm pre-vaccination to 32 ± 14 spm 5 months post-vaccine p<0.001) to IFN-γ (12 ± 6 spm pre-vaccination to 124 ± 31 spm 5 months post-vaccine p<0.001). The ratio of CD4+ICOShi T cells to CD4+Foxp3+ T cells increased from 0.37 ± 0.12 prior to vaccination to 2.3 ± 0.72 (p=0.021) following vaccination. Further, these activated CD4+ ICOShi T-cells induced preferential lysis of human breast cancer cells expressing Mam-A protein. We conclude that mammaglobin-A cDNA vaccination is associated with specific expansion and activation of CD4+ICOShi T cells, with a concomitant decrease in Treg frequency. These encouraging results strongly suggest that Mam-A cDNA vaccination can induce antitumor immunity in breast cancer patients.
PMCID: PMC3656506  PMID: 22678162
DNA Vaccine; Mammaglobin-A; Breast Cancer; T-cells; ICOS
19.  Density-dependent lineage instability of MDA-MB-435 breast cancer cells 
Oncology Letters  2013;5(4):1370-1374.
The use of cell lines in cancer research is strongly dependent on the avoidance of contaminations, the correct attribution of a cell line to the initial primary tumor and stability. Previous studies have identified expression of melanocytic molecular markers in the widely used breast cancer cell line, MDA-MB-435. In the present study the three breast cancer cell lines, MCF-7, MDA-MB-231 and MDA-MB-435, were systematically analyzed for mRNA and protein expression of major epithelial (cytokeratin isoforms), mammary (mammaglobin) and melanocytic (melan A and S100-protein) markers. Protein expression was identified by immunocytochemistry and quantitative RT-PCR was used to determine mRNA levels. While MCF-7 and MDA-MB-231 cells unambiguously revealed an epithelial/mammary phenotype, MDA-MB-435 cells were found to exhibit epithelial/mammary and melanocytic features dependent on cell density. Subconfluent cells demonstrated epithelial characteristics only, however, densely growing, confluent cells also expressed melanocytic markers. Consistent with gain of melanocytic features, the expression levels of mammaglobin mRNA decreased in these cells. These results indicate that the three cell lines are primarily of epithelial phenotype, however, MDA-MB-435 cells revealed lineage infidelity in dense cultures with a gain in melanocytic phenotype. These characteristics must be taken into consideration when analyzing cancer-relevant genes and their expression profiles in vitro.
PMCID: PMC3629269  PMID: 23599796
in vitro study; breast cancer cell lines; melanocytic phenotype
20.  Detection of mammaglobin mRNA in peripheral blood is associated with high grade breast cancer: Interim results of a prospective cohort study 
BMC Cancer  2008;8:55.
We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis.
Patients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells.
At a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (mam) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN).
This study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the mam gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.
PMCID: PMC2292197  PMID: 18289390
21.  Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer 
British Journal of Cancer  2005;92(10):1948-1952.
In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-α, IL-1β) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells.
PMCID: PMC2361769  PMID: 15841077
mammaglobin; maspin; breast cancer; micrometastasis
22.  Mammary analog secretory carcinoma of the thyroid gland: A primary thyroid adenocarcinoma harboring ETV6–NTRK3 fusion 
ETV6–NTRK3 fusion was identified in several cancers including the recently described mammary analog secretory carcinoma (MASC) of the salivary glands and a minority of papillary thyroid carcinomas. We describe three cases of primary MASC of the thyroid gland and provide a detailed clinical and pathological characterization of the tumor morphology, immunoprofile, and genetic background. Immunohistochemistry for PAX8, TTF-1, thyroglobulin, mammaglobin, GCDFP-15, S-100 protein, and p63 was used to define the tumor immunophenotype. Fluorescence in situ hybridization for ETV6 rearrangement was performed in three, and the next-generation sequencing assay MSK-IMPACT™ (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets) was performed in two cases. Primary MASC of the thyroid occurred in two women and one man, age 47–72 years. All patients presented with high T stage, infiltrative, locally aggressive tumors with extrathyroidal extension. Two cases were associated with well-differentiated papillary thyroid carcinoma. Histologically, they appeared as low-grade tumors, resembling MASC of the salivary glands and labeled positive for mammaglobin, GCDFP-15, S-100 protein, p63, weakly positive for PAX8, and negative for TTF-1 and thyroglobulin. Fluorescence in situ hybridization revealed ETV6 rearrangement in all cases. In two tested cases MSK-IMPACT™ confirmed the presence of ETV6–NTRK3 gene fusion. Two patients had at least two local recurrences, one was alive with disease, and one was alive and free of disease after 14 and 17 years, respectively. The third patient was alive and free of disease after 2 years. MASC of the thyroid is histologically, immunophenotypically, and genetically similar to its salivary gland counterpart. Thyroid MASC can be associated with a well-differentiated papillary thyroid carcinoma component, supporting follicular cell origin. Clinically, these carcinomas may show frequent recurrences but are associated with long-term survival. Patients with MASC of the thyroid may potentially benefit from Trk molecular-targeted therapy.
PMCID: PMC5019170  PMID: 27282352
23.  Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR 
BMC Cancer  2011;11:422.
Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.
We quantified by RT-qPCR CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+, and mammaglobin gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.
RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, CK-19 was detected in 42.4%, HER-2 in 13.6%, MAGE-A3 in 21.2%, hMAM in 13.6%, TWIST-1 in 42.4%, and hTERT α+β+ in 10.2%. In 26 patients with verified metastasis, CK-19 was detected in 53.8%, HER-2 in 19.2%, MAGE-A3 in 15.4%, hMAM in 30.8%, TWIST-1 in 38.5% and hTERT α+β+in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.
Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.
PMCID: PMC3224356  PMID: 21967632
24.  HLA Class II tetramers reveal tissue-specific regulatory T cells that suppress T-cell responses in breast carcinoma patients 
Oncoimmunology  2013;2(6):e24962.
Regulatory T cells (Tregs) play an important role in controlling antitumor T-cell responses and hence represent a considerable obstacle for cancer immunotherapy. The abundance of specific Treg populations in cancer patients has been poorly analyzed so far. Here, we demonstrate that in breast cancer patients, Tregs often control spontaneous effector memory T-cell responses against mammaglobin, a common breast tissue-associated antigen that is overexpressed by breast carcinoma. Using functional assays, we identified a HLA-DRB1*04:01- and HLA-DRB1*07:01-restricted epitope of mammaglobin (mam34–48) that was frequently recognized by Tregs isolated from breast cancer patients. Using mam34–48-labeled HLA Class II tetramers, we quantified mammaglobin-specific Tregs and CD4+ conventional T (Tcon) cells in breast carcinoma patients as well as in healthy individuals. Both mammaglobin-specific Tregs and Tcon cells were expanded in breast cancer patients, each constituting approximately 0.2% of their respective cell subpopulations. Conversely, mammaglobin-specific Tregs and CD4+ Tcon cells were rare in healthy individuals (0.07%). Thus, we provide here for the first time evidence supporting the expansion of breast tissue-specific Tregs and CD4+ Tcon cells in breast cancer patients. In addition, we substantiate the potential implications of breast tissue-specific Tregs in the suppression of antitumor immune responses in breast cancer patients. The HLA Class II tetramers used in this study may constitute a valuable tool to elucidate the role of antigen-specific Tregs in breast cancer immunity and to monitor breast cancer-specific CD4+ T cells.
PMCID: PMC3716760  PMID: 23894725
breast cancer; mammaglobin; HLA Class II; multimer; regulatory T cells; suppression; tetramer; Tregs; tumor-specific T cells
25.  Expression of human mammaglobin as a marker of bone marrow micrometastasis in breast cancer 
The aim of the present study was to detect the expression of human mammaglobin (hMAM) mRNA in the bone marrow (BM) of patients with breast cancer and determine the relationship between micrometastasis and clinicopathological parameters as well as selected molecular markers and breast cancer prognosis. The expression of hMAM mRNA in the BM of patients with breast cancer was determined by RT-PCR. The expression of ER, PR and Cath-D in cancer tissues was detected by immunohistochemistry. A positive expression rate for hMAM of 38.2% in 102 patients with stage I–III breast cancer was found. The expression of hMAM was higher in patients with T2–3 (>2 cm) tumors than in those with T1 tumors (≤2 cm) (χ2=19.20, P=0.001) and in patients with stage II or III tumors than in patients with stage I tumors (χ2=15.101, P=0.001). The expression of hMAM in the BM of breast cancer patients categorized as grade 1 was lower than that in those of grade 2 or 3 (χ2=8.522, P=0.014), and hMAM expression was related to the pathological type of tumor (χ2=6.892, P=0.032) and the degree of axillary lymph node metastasis (χ2=14.050, P=0.001). The expression of hMAM in BM was much higher in patients with ER(−) or ER(+) tumors than in those with ER(++ or +++) (χ2=11.800, P=0.003), and those with PR (χ2=8.759, P=0.013). hMAM expression in BM was also significantly positively correlated with Cath-D expression (χ2=6.623, P=0.036). However, no correlation was found between hMAM expression and patient age (χ2=1.056, P=0.304). There was a strong correlation between patients with positive expression of hMAM in the BM and the presence of distant metastases (P=0.009). In conclusion, micrometastasis in the BM correlates with certain clinical pathological parameters and several tumor markers. Patients with positive expression of hMAM in the BM have a greater chance of distant metastasis and poor prognosis. The detection of micrometastasis may be one of the most advantageous markers for predicting the prognosis of breast cancer.
PMCID: PMC3438616  PMID: 22969928
breast cancer; bone marrow micrometastasis; human mammaglobin; biological factor; RT-PCR; immunohistochemistry; prognosis

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