The detection of circulating tumor cells expressing E6/E7 HR-HPV oncogenes in peripheral blood in cervical cancer patients after radical hysterectomy
| Autor: | D Keder, M Vizvaryova, Katarina Zavodna, J Benuska, K Mlada, Z Ferancikova, M Konecny, Vanda Repiská, L Masak, Weismann P, J Kausitz, E Weismanova |
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| Rok vydání: | 2009 |
| Předmět: |
Adult
Oncology Cancer Research medicine.medical_specialty Receptor ErbB-2 Papillomavirus E7 Proteins Uterine Cervical Neoplasms Hysterectomy Immunomagnetic separation Sensitivity and Specificity HeLa chemistry.chemical_compound Circulating tumor cell Internal medicine medicine Humans Receptor Papillomaviridae Cervical cancer biology Reverse Transcriptase Polymerase Chain Reaction Chemistry fungi Reproducibility of Results Epithelial cell adhesion molecule Oncogenes Middle Aged Neoplastic Cells Circulating medicine.disease biology.organism_classification Cell culture Disease Progression biology.protein Cancer research Female Antibody HeLa Cells |
| Zdroj: | Neoplasma. 56:230-238 |
| ISSN: | 0028-2685 |
| Popis: | The aim of this study was to establish the sensitive, specific and clinically acceptable method for detection of tumor cells (TCs) circulating in peripheral blood (PB) of cervical cancer patients without the clinically detectable risk of disease progression. The 7.5 ml of PB of healthy donor was spiked with 5 to 100 cells from SiHa or HeLa cell lines. The spiked tumor cells were collected without gradient centrifugation, by standard gradient centrifugation or by modified gradient centrifugation combined with immunomagnetic separation using EpCAM antibody with affinity for epithelial cell adhesion molecule. The number of collected TCs was determined by EpCAM-FITC-staining and their viability was detected by nested RT-PCR amplifying E6/E7 HR-HPV 16 or HR-HPV 18 oncogenes. For the technical validation of this approach the TCs separation and RT-PCRs were repeated several times. The recovery of viable TCs was reproducibly higher using modified gradient centrifugation combined with immunomagnetic separation in comparison with standard approach. The recovery of TCs in low number of spiked TCs (range from 5 - 20 TCs in 7.5 ml of PB) using modified gradient centrifugation was not reproducible. The recovery of TCs in higher number of spiked TCs (25 TCs and more in 7.5 ml of PB) was reproducible with average recovery about 50 %. The sensitivity of nested RT-PCR amplifying E6/E7 oncogenes was decisively influenced by the number of recovered TCs and the amount of cDNA introduced to RT-PCR, as well. Using this approach we were allowed to detect circulating TCs (CTCs) in cervical cancer patients without metastases, thus this procedure might become a tool to early estimation of disease progression. According to our knowledge, this is the first report describing the use of EpCAM antibody for CTCs detection in cervical cancer patients. |
| Databáze: | OpenAIRE |
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