Prognostic value of tumour cell detection in peripheral blood of breast cancer patients.

To investigate the prognostic value of tumour cells in peripheral blood (pB) of breast cancer (BC) patients, pB samples from 143 patients with benign lesions of the breast and from 467 BC patients were tested via a nested RT-PCR assay for mammaglobin mRNA. No sample from patients with benign lesions of the breast was found to be mammaglobin positive in contrast to 5/310 (2%) BC patients with no evidence of disease (NED) and 46/157 (29%) patients with metastatic disease (MD). Two hundred and eighteen BC patients with NED were followed for at least 12 months. All five mammaglobin-positive BC patients relapsed 1-13 months after first examination of positive pB samples in contrast to 27/213 (13%) patients without detectable tumour cells in pB. Fifty-nine BC patients with MD were tested for mammaglobin expression in pB at the time of first diagnosis of MD; 20 of them (34%) were mammaglobin positive. Patients were followed for a median of 19 months (2-51 months). During this time, 19/59 (32%) died due to tumour progression. In Kaplan-Meier survival analysis, BC patients with mammaglobin-negative pB samples at time of diagnosis of MD lived significantly longer than mammaglobin-positive patients (log-rank test: P = 0.0013). In addition, mammaglobin was an independent prognostic parameter and the difference reached significance in univariate as well as in multivariate analysis (P < 0.01). We conclude that the presence of tumour cells in pB of BC patients is of prognostic value.

Identification and localization of M-CoREST (1A13), a mouse homologue of the human transcriptional co-repressor CoREST, in the developing mouse CNS.

By means of subtractive and differential hybridization techniques we have identified a novel murine gene (1A13) the expression of which is developmentally regulated in the mouse brain. Comparison of the nucleotide and predicted protein sequence revealed closest relationship of 1A13 to human CoREST, a transcriptional co-repressor required for regulation of neural-specific gene expression. Thus, we will refer to 1A13 as M-CoREST. As shown by in situ hybridization and Northern blotting, expression of M-CoREST mRNA is abundant in neural tissue at early embryonic stages but declines significantly towards birth, coincident with the progression of CNS maturation.

Statistical validation of the mammaglobin-nested RT-PCR assay for tumor cell detection in blood of breast cancer patients.

A stochastic model was developed to validate the results obtained with the mammaglobin-nested RT-PCR assay for tumor cell detection in peripheral blood of breast cancer patients. Since the assay consists of four PCR setups per peripheral blood sample, the probabilities for receiving 0, 1, 2, 3, or 4 positive setups were calculated. In this model, samples with just 500 mammaglobin mRNA molecules are highly probable to result in at least three positive setups, whereas lower quantities shift the probabilities towards one or two positive setups. In the clinical trial, samples with one or two mammaglobin positive setups were detected in 6/143 (4%) patients with benign lesions of the breast, in 41/310 (13%) breast cancer patients with no evidence of disease and in 39/157 (25%) breast cancer patients with metastatic disease. On the contrary, no sample from patients with benign lesions of the breast resulted in three or four positive setups, but 5/310 (2%) breast cancer patients with no evidence of disease and 46/157 (29%) with metastatic disease. These results correspond with the model: an increased number of tumor cells in peripheral blood lead to a higher amount of mammaglobin mRNA molecules, and these samples may result in at least three positive setups. Samples with three orfour positive setups were mainly derived from breast cancer patients with metastatic disease and only occasionally from patients with no evidence of disease. On account of these results, samples with at least three positive setups are of prognostic value and regarded as tumor cell positive.

[Detection of mammaglobin mRNA as a marker for circulating tumor cells in breast carcinoma]. / Nachweis von Mammaglobin mRNA als Marker für zirkulierende Tumorzellen bei Mammakarzinom.

Mammaglobin (hMAM) has been shown to be a marker for the detection of circulating tumor cells in the peripheral blood (pB) of breast cancer (BC) patients via a nested RT-PCR assay. 286 samples from BC patients were classified into four defined clinical subgroups: prior to and after surgery (pre, post), no evidence of disease (NED) and metastatic disease (MD). hMAM mRNA expression was detected in 2/46 pre (4%), 2/24 post (8%), 4/135 NED (3%) and 35/81 MD (43%) patients. 68 BC patients with NED and negative for hMAM mRNA in their pB were repeatedly tested for at least 6 months. Fifteen of these patients relapsed. Eight of them were hMAM-positive, 5 at time of relapse, one patient 13 months before and two patients 10 and 17 months after relapse was diagnosed. 7/15 BC patients relapsed within 24 months, 5 of them were hMAM-positive versus 3 of 8 patients with later relapses. On the basis of these preliminary results we conclude that tumor cells can be detected via hMAM nested RT-PCR in the pB of BC patients and that hMAM could be a marker for early relapse.

Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via a nested reverse transcriptase polymerase chain reaction assay for mammaglobin mRNA.

PURPOSE: According to current medical research, mammaglobin (hMAM) is expressed exclusively in the mammary glands of adult women and in mammary tumor cell lines. Therefore, we examined hMAM expression as a marker for the detection of carcinoma cells in the peripheral blood of patients with breast cancer (BC). PATIENTS AND METHODS: Blood samples obtained from 114 BC patients at the various stages of their disease and from 68 individuals without BC were screened for hMAM mRNA by a nested reverse transcriptase polymerase chain reaction (RT-PCR) assay. RESULTS: The assay exhibited a calculated analytical limit of one tumor cell per 10(6) to 10(7) WBCs. None of the samples from peripheral blood of 27 healthy individuals were positive, whereas 29 (25%) of 114 samples from BC patients were positive for hMAM mRNA. hMAM mRNA expression was detected in five (28%) of 18 BC patients at diagnosis, in three (6%) of 53 with no evidence of disease, and in 21 (49%) of 43 with metastatic disease. These results correlate with patients' carcinoembryonic antigen (CEA) plasma level and, to some extent, with estrogen receptor status. Two of 41 samples from patients with malignancies other than BC were also positive. CONCLUSION: In contrast to healthy volunteers, hMAM transcripts were detected in the peripheral blood of BC patients. The percentage of positivity relates to the clinical stages of disease, CEA plasma level, and estrogen receptor status. Aberrant hMAM expression might occur occasionally in malignancies other than BC. The clinical relevance of hMAM RT-PCR-based tumor cell detection in the peripheral blood of BC patients should be further evaluated in prospective studies.

Expression of a chemotactic cytokine (MCP-1) in cerebral capillary endothelial cells in vitro.

A full-length cDNA encoding the porcine monocyte chemoattractant protein-1 (pMCPC-1) was isolated from growth-stimulated porcine cerebral capillary endothelial cells (cEC); the pMCP-1 cDNA showed 89% identity to human MCP-1 and was isolated by use of subtractive hybridization and differential screening of two phenotypically different sub-populations of cloned cEC. pMCP-1 was abundantly expressed in cEC grown in the presence of FCS, ECGF and heparin whereas lower expression was observed in cEC kept in FCS-supplemented medium only. As shown by Northern blot analysis, no pMCP-1 transcripts were present in total RNA derived from freshly isolated brain capillaries, large brain vessels or whole brain homogenate. MCP/JE expression was also demonstrated in ECGF/heparin-treated murine cEC. Astrocytes and smooth muscle cells grown in FCS-supplemented medium did not show MCP-1 expression. Treatment of porcine cEC with TNF-alpha increased pMCP-1 mRNA levels in a dose-dependent manner. These data further support the notion that cerebral capillary endothelial cells actively participate in processes of CNS host defense.

Sequence of the porcine full-length cDNA encoding ribosomal protein rpS12.

The full-length cDNA encoding the porcine ribosomal protein rpS12 was shown to be differentially expressed in endothelial and smooth muscle cells. A cDNA clone containing the entire rpS12 sequence was isolated from growth-stimulated capillary endothelial cells by use of subtractive hybridization and differential screening. The porcine rpS12 cDNA coding region exhibits 94% identity to the rat rpS12 and 92% to the human rpS12 cDNAs.

Transcription-independent activation of ornithine decarboxylase activity by heparin in cloned cerebral endothelial cells.

Heparin, a highly sulfated glycosaminoglycan, is known to be obligatory for long-term endothelial cell cultures; it potentiates the mitogenic activities of endothelial cell growth factors and prolongs the replicative life span of the cells. Here we have shown that besides its growth factor-supportive role, heparin exerts a specific action on cerebral capillary endothelial cells (cECs), unrelated to serum or growth factors, by increasing activity of ornithine decarboxylase (ODC; EC 4.1.1.17) in these cells. For our experiments we have used two different types of cloned cECs: type I cECs, grown in the presence of endothelial cell growth factor and heparin, and type II cECs, usually cultivated without growth factors. Heparin action on ODC activity was shown to be dose dependent within the range of 1-100 micrograms/ml. Increasing concentrations of or depletion of endothelial cell growth factor from type I cultures had no effect on ODC activity. The increase in enzyme activity was highest after 30 min to 1 h of heparin treatment. As evidenced by northern analysis, the heparin-mediated enhancement of ODC activity was not accompanied by changes of ODC mRNA levels. Studies of DNA replication revealed that in the absence of heparin-binding growth factors, heparin did not affect the proliferative activity of cloned cECs.

Comparison of gene expression of extracellular matrix molecules in brain microvascular endothelial cells and astrocytes.

By use of random-primed cDNA probes the expression of extracellular matrix molecules in cerebral microvascular endothelial cells (cEC) and in astrocytes from mouse brain was examined. Two phenotypically different batches of cloned cEC were used. Expression of major adhesive ECM molecules, constituting the endothelial basement membrane (i.e., fibronectin, laminin A, B and collagen IV) and of other attachment factors, such as SPARC (osteonectin), tenascin and thrombospondin 1, was examined. We have demonstrated that cEC of different morphology display variations in the expression of fibronectin (FN), thrombospondin 1 (TSP1) and collagen IV (C IV). Astrocytes were shown to contain FN, TSP1, TN and SPARC mRNA. Unexpectedly, SPARC mRNA could not be detected in any of the capillary endothelial cells examined. Therefore, we suggest that astrocytes are likely to be involved in endothelial differentiation and function in the central nervous system via ECM molecule secretion.