Ανάπτυξη και κλινικές εφαρμογές μεθόδων μοριακής διαγνωστικής για την ανίχνευση κυκλοφορούντων καρκινικών κυττάρων στον καρκίνο του μαστού μέσω της έκφρασης της μαστοσφαιρίνης α

Mammaglobin A is expressed mainly by breast epithelial cells and has been recently evaluated as a molecular tumor marker for the detection of circulating breast cancer cells. The aim of our study was to develop a sensitive and specific method for the detection of Mammaglobin A -mRNA positive cells in peripheral blood of breast cancer patients. The method involves: a) the use of nested PCR in peripheral blood of early breast cancer patients. All primers were in silico designed and evaluated in order to avoid false positive results due to genomic DNA contamination. The specificity of the primers used was evaluated in 30 healthy individuals, 20 patients with colorectal cancer and 20 patients with non-small cell lung cancer. The method was applied in 101 patients with operable breast cancer before the administration of adjuvant chemotherapy and 39 patients with metastatic breast cancer. The method is highly specific since the developed assay is free from genomic DNA interferences. Mammaglobin A-mRNA positive cells were detected in 14/101 (13.9%) of early breast cancer patients; 9 of them (64.3%) relapsed during the follow up period. Mammaglobin A was detected in 7/39 (17.9%) of metastatic patients. Multivariate analysis revealed the detection of Mammaglobin A-mRNA positive cells, as an independent risk factor for reduced DFI. b) we developed a real-time RT-PCR methodology for the quantification of Mammaglobin A-mRNA by using LightCycler Technology (Roche Applied Science). In this method, we used a Taqman probe labeled with fluorescent that hybridized to the PCR product while being monitored continuously in the LightCycler. The analytical characteristics of the developed method were evaluated by using standard curve with external standards (known number of copies of Mammaglobin A PCR product).So, by using real-time RT-PCR a high sensitivity for Mammaglobin A-mRNA detection is achieved, since as low as 6 copies/ μL can be detected. The specificity of the method was evaluated in 30 healthy individuals, 5 patients with colorectal cancer and 8 patients with non-small cell lung cancer (NSCLC). The method is specific as Mammaglobin A- mRNA positive cells were not detected in the peripheral blood of healthy volunteers and molecular marker the peripheral blood of patients with colorectal and lung cancer. The method was also applied in 320 patients with operable breast cancer before the administration of adjuvant chemotherapy. In order to determine the quality of the cDNA of these samples, real-time RT-PCR was applied for the quantification of PBGD-mRNA. To the samples that were found positive (n=234), was also applied real-time RT-PCR for the quantification of Mammaglobin A-mRNA. Unfortunately, Mammaglobin-mRNA positive cells were detected only in one out of 234 (<1%) breast cancer patients before adjuvant chemotherapy. In conclusion real-time RT-PCR for the quantification of Mammaglobin A-mRNA is not sensitive enough. c) in order to improve the sensitivity of our methodology, we used immunomagnetic separation steps for the isolation of CTCs, as we have previously established in our laboratory and the selective isolation of mRNA from total RNA. To achieve that, we selected the previously established in our laboratory real-time PCR method for the quantification of CK-19 mRNA, due to the absence of CK-19 background expression by the PBMC. The analytical characteristics of the developed methods were evaluated using positive control mammary (MCF-7) tumor cell line. Since the results of the newly developed assay were encouraging, we compared the methodology that we already use in our laboratory with the methodology of the immunomagnetic separation steps, by applying them in the peripheral blood of 6 patients with metastatic breast cancer. By using the classical methodology, we were unable to detect CK-19 mRNA positive cells in these samples, while, by using the developed assay with the two immunomagnetic separation steps in the same 6 samples, we managed to detect CK-19 mRNA positive cells in 2/6 (33.3%) of patients with metastatic breast cancer.

Grantor dc:publisher

National and Kapodistrian University of Athens

Year dc:date

2007

Authors dc:creator

• Ntoulia, Maria
• Ντούλια, Μαρία

• Καρκινικά κύτταρα
• Καρκίνος μαστού
• Μαστοσφαιρίνη Α
• Ανοσομαγνητικά σφαιρίδια
• PCR πραγματικού χρόνου
• PCR real time
• Tumor cells
• Breast cancer
• Mammaglobin A
• Immuncmagnetic beads
• Φυσικές Επιστήμες
• Χημεία
• Ιατρική και Επιστήμες Υγείας
• Ιατρική Βιοτεχνολογία
• Natural Sciences
• Chemical Sciences
• Medical and Health Sciences
• Medical Biotechnology

Language dc:language

gre

Identifier

10.12681/eadd/1058

OAI identifier oai:identifier

oai:10442/1058