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Home    Συγκριτική μελέτη του mRNA της κυτταροκερατίνης-19 , της MASPIN και του CEA στο αίμα ασθενών με καρκίνο του μαστού και διερεύνηση των μεταβολών τους κατά τη διάρκεια της χημειοθεραπείας ή και ορμονοθεραπείας.  

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Identifier uch.med.phd//2002DIS0725
Title Συγκριτική μελέτη του mRNA της κυτταροκερατίνης-19 , της MASPIN και του CEA στο αίμα ασθενών με καρκίνο του μαστού και διερεύνηση των μεταβολών τους κατά τη διάρκεια της χημειοθεραπείας ή και ορμονοθεραπείας.
Author Σταθοπούλου, Αλίκη Ε
Abstract The purpose of this study was to investigate and compare the diagnostic value of the detection of cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA) and maspin mRNA by nested RT-PCR in the peripheral blood of women with breast cancer and to develop a highly sensitive quantitative RT-PCR hybridization assay for the determination of CK-19 mRNA in peripheral blood. The tumor cell lines MCF-7 and LOVO were used in an experimental tumor cell dilution model to determine the sensivity of the nested RT-PCR for the 3 detection markers. RT-PCR for mRNA of CK-19, CEA and maspin was performed in the peripheral blood of 54 female healthy blood donors, 28 patients with hematologic malignancies, 31 with metastatic colorectal cancer, 75 with operable and 50 with metastatic breast cancer before receiving any cytotoxic chemotherapy. Nested RT-PCR for CK-19 mRNA presented the highest sensivity by detecting 1 tumor cell amongst 10^6 PBMC in 4 out of 5 experiments. CK-19 mRNA was detected in the peripheral blood of 3.7% of healthy blood donors, 14.3% of hematologic malignancies, 33.3% of operable and 42% of metastatic breast cancer patients. CEA mRNA was undetectable in female blood donors and was detected in blood samples of 3.5% of hematologic maglinancies, 19.3% of colorectal cancer and 10% of breast cancer patients. Maspin mRNA was detected in 10.6% of operable and 14% of metastatic breast cancer patients. Maspin mRNA positivity correlated with tumor size in patients with early breast cancer (p=0.027). Subsewuently, peripheral blood from 148 patient with operable (stage I and II) breast cancer, obtained before the initiation of any adjuvant therapy, was tested for the presence of CK-19 mRNA-positive cells. For patients with stage I and II breast cancer, the detection of CK-19 -positive cells in the peripheral blood before adjuvant therapy was assosiated with a 6-fold increased risk relapse (p=0.001). In multivariate analysis, only the presence of CK-19 mRNA-positive cells in the peripheral bloodand the number of involved axillary lymph nodes (>=4 lymph nodes)were independent prognostic factrs assosiated with an increased risk of disease relapse. Conversely, the COx proportional hazard analysis indicated that the detection ocf CK-19 mRNA-positive cells in the peripheral blood , the number involved axillary lymph nodes and the statusof ER expression by the tumor were only important factors affecting the duration of DFI. Our results demonstrate that RT-PCR positivity for Ck-19 mRNA is the most sensitive detection marker for tumor cells in operable and metastatic breast cancer and appropriate for use in clinical practice, although nested RT-PCR for maspin mRNA appearsto be more specific. The molecular detection of CK-19 mRNA - positive cella by RT-PCR in the peripheral blood of patients with stage I and II breast cancer before the initiation of adjuvant therapy has an independent prognostic value as a marker of poor clinical outcome. We have also developed a highly sensitive quantitative RT-PCR hybridization assay for the determination of CK-19 mRNA in the peripheral blood.Quantitation is based on the coamplification of CK-19 mRNA with a recombinant CK-19 RNA internal standard (CK-19 RNA-IS) through RT-PCR. The biotinylated amplification products are immobilized on steptavidin coated wells, hybridized with digoxigenin labeled probes ang determined through an anti-digoxigenin antibody conjugated to alkaline phosphatase by luminometric detection. The hybridization assay was validated with samples containing total RNA from known amounts of CK-19 expressing cells (MCF-7) in the presence of total RNA isolated from peripheral blood mononuclear cells (PBMN) of healthy controls and a constant amount of CK-19 mRNA-IS. Luminescence ratios for CK-19 mRNA and CK-19 mRNA-IS were linearly related to the primary number of MCF-7 we had used. The overall reproducibility of the assay (between-run) varied between 7.4 and 24%. The method can clearly distinguish 10 MCF-7 cells (protocol A) and 1 MCF-7 cell (protocol B) in the presence of 10^6 normal PBMN and is highly specific as none of the healthy controls tested (n=26) has detected CK-19 mRNA levels, while 6 out of 14 breast cancer patients, with verified metastatic and under chemotheraspy, were found [positive.
Language Greek
Issue date 2002-07-01
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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