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1.  Breast Cancer Metastasis Suppressor-1 promoter methylation in primary breast tumors and corresponding Circulating Tumor Cells 
Molecular cancer research : MCR  2013;11(10):10.1158/1541-7786.MCR-13-0096.
Breast Cancer Metastasis Suppressor-1 differentially regulates expression of multiple genes, leading to metastasis suppression without affecting orthotopic tumor growth. We evaluated BRMS1 promoter methylation as prognostic biomarker in primary breast tumors and studied BRMS1 promoter methylation in a subset of corresponding Circulating Tumor Cells (CTC) for the first time. We analyzed 118 formalin-fixed paraffin embedded samples: 5 pairs of breast tumors and adjacent non-cancerous tissues, 14 non-cancerous tissues, 10 benign fibroadenomas, and 84 primary breast tumors. Peripheral blood mononuclear cells from 39/84 of these patients were fixed in cytospins. BRMS1 methylation status was investigated in all FFPE and cytospin stained CTC using methylation specific PCR. BRMS1 expression in cytospins was examined by double-immunofluorescence using anti-BRMS1 and pancytokeratin A45-B/B3 antibodies. BRMS1 promoter methylation was not observed in noncancerous breast tissues (0%), and benign fibroadenomas (0%), while it was observed in 36.9% of primary breast tumors. BRMS1 promoter methylation in primary tumors was associated with reduced disease-free interval (P=0.009) while a trend towards a reduced overall survival was also observed (P=0.071). 13/39 cytospin samples (33.3%) were positive for the presence of CTC and the total number of the detected CTC was 41. Most CTC (80.5%) were negative for BRMS1 or maintained low expression, implying that BRMS1 is down regulated in these cells. BRMS1 promoter methylation was observed in 5/39 (12.8%) samples. BRMS1 promoter methylation in primary breast tumors provides prognostic information for DFS. BRMS1 expression in CTC was highly heterogeneous, between patients and even in the same patient.
PMCID: PMC3868947  PMID: 23744981
Breast Cancer Metastasis Suppressor gene-1; BRMS1; circulating tumor cells; CTC; DNA methylation; operable breast cancer
2.  Myeloid-derived suppressor cell measurements in fresh and cryopreserved blood samples 
Journal of Immunological Methods  2012;381(1-2):14-22.
Myeloid-derived suppressor cells (MDSC) present in the human peripheral blood, represent a heterogeneous population of cells with monocytic and granulocytic features. To provide guidelines for reliable assessments of the frequency and function of MDSC, we compared fresh vs. cryopreserved peripheral blood mononuclear cell (PBMC) samples obtained from normal controls and patients with cancer. PBMC were obtained from 4 healthy donors and 21 patients with cancer. They were stained with labeled antibodies, and the frequency of DR−/LIN−/CD11b+, DR−/LIN−/CD15+, DR−/LIN−/CD33+ and DR−/low/CD14+ cells was determined by flow cytometry before and after cryopreservation. CFSE-based suppressor assays were used to test inhibitory functions of MDSC. Arginase I expression and reactive oxygen species (ROS) upregulation in MDSC subsets were evaluated by flow cytometry. The DR−/low/CD14+ and DR−/LIN−/CD11b+ subsets of MDSC were found to be more resistant to the cryopreservation/thawing procedure compared to the DR−/LIN−/CD15+ and DR−/LIN−/CD33+ subsets. The frequency of the latter two MDSC subsets was significantly reduced after cryopreservation. All but DR−/LIN−/CD15+ cells inhibited proliferation of autologous CSFE-labeled CD4+ cells but lost suppressor activity after cryopreservation. Only DR−/LIN−/CD15+ cells were positive for Arginase I, but lost its expression after cryopreservation. Only fresh DR−/LIN−/CD11b+ and DR−/LIN−/CD15+ cells produced ROS after in vitro stimulation.
Studies of human MDSC should be performed in fresh blood samples. If samples have to be cryopreserved, monitoring of CD11b+ and CD14+ MDSC subsets provides the most reliable results. Arginase I expression or stimulated ROS production assessed by flow cytometry are useful markers for MDSC subsets only in fresh samples.
PMCID: PMC3385927  PMID: 22522114
myeloid-derived suppressor cells (MDSC); MDSC subsets; cell cryopreservation; flow cytometry
3.  A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples 
BMC Cancer  2012;12:486.
CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.
We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.
The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.
The developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.
PMCID: PMC3495665  PMID: 23088560
Methylation-sensitive high-resolution melting analysis; Cystatin M; CST6; DNA methylation; Breast cancer; Methylation specific PCR
4.  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
5.  Correlation of breast cancer risk factors with HER-2/neu protein overexpression according to menopausal and estrogen receptor status 
BMC Women's Health  2005;5:1.
Several researchers have claimed that classification of tumours on the basis of HER-2/neu overexpression or amplification may define a subset of breast cancer in which the net effect of a risk factor could be rather more obvious and its impact on breast cancer development more clear. We decided to investigate, in a group of patients from a geographical area with a low incidence of breast cancer, whether HER-2/neu positive tumours are correlated with established or suspected risk factors for breast cancer and thus to identify distinct subgroups of high risk women.
This study analysed data from patients who attended the Breast Unit at the University Hospital of Heraklion, Crete, Greece between 1996 and 2002. 384 women with primary invasive breast cancer were compared with 566 screened women who were referred to the Unit and had not developed breast neoplasm by the time the data were analysed. Risk factor data were obtained from each subject by personal interviews using a structured questionnaire. The detection and scoring of the HER-2/neu protein, estrogen and progesterone receptor expression were performed using immunochemistry. Odds ratios and 95% confidence intervals were determined by chi-square test and logistic regression analysis. Case-case odds ratios were calculated in order to measure the risk heterogeneity between HER-2/neu+ and HER-2/neu-tumours. Separate analyses were performed for premenopausal and postmenopausal women and according to estrogen receptor status.
In multivariate analysis without HER-2/neu stratification, an increased breast cancer risk was associated with only four of the factors examined: use of oral contraceptives (OR = 4.40, 95%C.I: 1.46–13.28), use of HRT (OR = 7.34, 95%C.I: 2.03–26.53), an age at first full pregnancy more than 23 years (OR = 1.91, 95%C.I: 1.29–2.83) and body mass index more than 29 kg/m2 (OR = 3.13, 95%C.I: 2.02–4.84). Additionally, a history of abortion or miscarriage (OR = 0.56, 95%C.I: 0.38–0.82) was correlated with a decreased risk of breast cancer. In the case to case comparison only BMI >29 kg/m2 revealed a relative connection that was stronger with positive than with negative HER-2/neu tumours (ratio of OR's = 2.23, 95%C.I: 1.20–4.15, p = 0.011). This may indicate evidence of heterogeneity of a rather significant degree for this factor. In the ER negative group an age at first full pregnancy >23 years and a BMI >29 kg/m2 were associated with an increased risk in both HER-2/neu groups, but the association was significantly stronger for the latter factor in the positive HER-2/neu tumours (ratio of OR's = 2.46, 95%CI: 0.97–6.21).
Our study did not confirm that the established or putative hormonal breast cancer risk factors differ regarding their relations with HER-2/neu+ versus HER-2/neu-breast tumours, with the exception of increased BMI. Further innovative studies with larger sample sizes are needed to examine how the status of these potentially modifiable breast cancer risk factors interacts with biological markers such as HER-2/neu oncoprotein.
PMCID: PMC549187  PMID: 15694000

Results 1-5 (5)