Automated Reports for Monitoring and Improving Data Quality in a Translational Research Network.

Standardised, automated quality reports were generated at three pilot locations of the decentralized translational research network DKTK with separated local data warehouses (LDW), for assessing syntactic conformity against common data element definitions deposited in a central metadata repository (MDR). Deviations in the LDW were categorised, and locally corrected. Comparisons of reports from two time points confirm a major improvement in data quality in terms of syntactic conformity, an essential prerequisite for network-wide data integration.

Differential regulation of MAGE-A1 promoter activity by BORIS and Sp1, both interacting with the TATA binding protein.

BACKGROUND: As cancer-testis MAGE-A antigens are targets for tumor immunotherapy, it is important to study the regulation of their expression in cancers. This regulation appears to be rather complex and at the moment controversial. Although it is generally accepted that MAGE-A expression is controlled by epigenetics, the exact mechanisms of that control remain poorly understood. METHODS: We analyzed the interplay of another cancer-testis gene, BORIS, and the transcription factors Ets-1 and Sp1 in the regulation of MAGE-A1 gene expression performing luciferase assays, quantitative real-time PCR, sodium bisulfite sequencing, chromatin immunoprecipitation assays and pull down experiments. RESULTS: We detected that ectopically expressed BORIS could activate and demethylate both endogenous and methylated reporter MAGE-A1 promoter in MCF-7 and micrometastatic BCM1 cancer cell lines. Overexpression of Ets-1 could not further upregulate the promoter activity mediated by BORIS. Surprisingly, in co-transfection experiments we observed that Sp1 partly repressed the BORIS-mediated stimulation, while addition of Ets-1 expression plasmid abrogated the Sp1 mediated repression of MAGE-A1 promoter. Both BORIS and Sp1 interacted with the TATA binding protein (hTBP) suggesting the possibility of a competitive mechanism of action between BORIS and Sp1. CONCLUSIONS: Our findings show that BORIS and Sp1 have opposite effects on the regulation of MAGE-A1 gene expression. This differential regulation may be explained by direct protein-protein interaction of both factors or by interaction of MAGE-A1 promoter with BORIS alternatively spliced isoforms with different sequence specificity. We also show here that ectopic expression of BORIS can activate transcription from its own locus, inducing all its splice variants.

Increased serum levels of circulating exosomal microRNA-373 in receptor-negative breast cancer patients.

In this study, we compared the blood serum levels of circulating cell-free and exosomal microRNAs, and their involvement in the molecular subtypes of breast cancer patients. Our analyses on cell-free miR-101, miR-372 and miR-373 were performed in preoperative blood serum of 168 patients with invasive breast cancer, 19 patients with benign breast diseases and 28 healthy women. MicroRNAs were additionally quantified in exosomes of 50 cancer patients and 12 healthy women from the same cohort. Relative concentrations were measured by quantitative TaqMan MicroRNA assays and correlated to clinicopathological risk factors. The concentrations of cell-free miR-101 (p=0.013) and miR-373 (p=0.024) were significantly different between patients with breast cancer and benign tumors. A prevalence of miR-101, miR-372 and miR-373 were found in exosomes. The levels of circulating exosomal (but not cell-free) miR-373 were higher in triple negative than luminal carcinomas (p=0.027). Also, estrogen-negative (p=0.021) and progesterone-negative (p=0.01) tumors displayed higher concentrations of exosomal miR-373 than patients with hormone-receptor positive tumors. Overexpression of miR-373 by transfection of MCF-7 cells showed downregulated protein expression of the estrogen receptor, and inhibition of apoptosis induced by camptothecin. Our data indicate that serum levels of exosomal miR-373 are linked to triple negative and more aggressive breast carcinomas.

Deregulated serum concentrations of circulating cell-free microRNAs miR-17, miR-34a, miR-155, and miR-373 in human breast cancer development and progression.

BACKGROUND: MicroRNAs (miRs) are small, noncoding RNAs that target genes involved in tumor development and progression. In the current study, we investigated the use of circulating miR concentrations as biomarkers in the serum of breast cancer patients. METHODS: We analyzed serum samples from 120 patients with primary breast cancer after surgery and before chemotherapy (M0, classified into 3 subgroups of 40 patients with progesterone/estrogen-positive, HER2-positive, and triple-negative cancer), 32 patients with overt metastasis (M1), and 40 healthy women. Using quantitative TaqMan MicroRNA PCR, we measured the relative concentrations of 6 circulating microRNAs (miR-10b, -17, -34a, -93, -155, and -373) known to be relevant for tumor development and progression. The data were correlated with clinicopathologic risk factors, with particular reference to HER2 and hormone receptor status of the primary tumor and the presence of metastases. RESULTS: The relative serum concentrations of circulating miR-34a [P = 0.013, area under the curve (AUC) 0.636], miR-93 (P = 0.001, AUC 0.699), and miR-373 (P = 0.0001, AUC 0.879) were significantly different between M0 breast cancer patients and healthy women, whereas miR-17 (P = 0.002, AUC 0.679) and miR-155 (P = 0.0001, AUC 0.781) were differently expressed between M0 and M1 patients. Increased concentrations of miR-373 were associated with negative HER2 status of the primary tumor (P = 0.0001). Deregulated concentrations of miR-17 (P = 0.019) and miR-34a (P = 0.029) were detected in patients with progesterone/estrogen receptor-positive and -negative status, respectively. CONCLUSIONS: Our findings indicate that serum concentrations of deregulated microRNAs may be linked to a particular biology of breast carcinomas favoring progression and metastatic spread.

Loss of heterozygosity at tumor suppressor genes detectable on fractionated circulating cell-free tumor DNA as indicator of breast cancer progression.

PURPOSE: LOH on circulating DNA may provide tumor-specific information on breast cancer. As identification of LOH on cell-free DNA is impeded by the prevalence of wild type DNA in blood of cancer patients, we fractionated plasma DNA, and determined the diagnostic and prognostic value of both fractions. EXPERIMENTAL DESIGN: Our cohort of 388 patients with primary breast cancer before chemotherapy was selected from a multicenter study (SUCCESS). Postoperative plasma was fractionated in low- and high-molecular weight DNA by two different column systems. In both fractions, LOH was determined by a PCR-based microsatellite analysis using a panel of 8 polymorphic markers. Circulating tumor DNA in plasma from 30 patients after chemotherapy was additionally analyzed. The significance levels were adjusted for multiple comparisons. RESULTS: More patients (38%) had LOH at all markers in the fraction containing short DNA fragments than in the fraction containing the long DNA molecules (28%, P = 0.0001). In both fractions 32.85% of LOH were concordant. LOH at the markers D3S1605, D10S1765, D12S1725, D13S218, and D17S855 significantly correlated with tumor stage, tumor size, and lymph node metastasis, positive progesterone, and HER2 receptor status. Most importantly, LOH at D12S1725 mapping to cyclin D2 correlated with shorter overall survival (P = 0.004). CONCLUSIONS: The improved detection of LOH on cell-free DNA provides important information on DNA losses of tumor suppressor genes TIG1, PTEN, cyclin D2, RB1, and BRCA1 in breast cancer. In particular, loss of the cyclin D2 gene might become an important prognostic marker easily detectable in the peripheral blood.