The detection of circulating tumor cells expressing E6/E7 HR-HPV oncogenes in peripheral blood in cervical cancer patients after radical hysterectomy.

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.

[Papillomavirus infection of the uterine cervix in women using contraception]. / Papilomavírusová infekcia krcka maternice u zien s antikoncepciou.

OBJECTIVE: The authors analyze the prevalence of the anogenital form of HPV infection as one of the most frequent STD, in the female population using different types of contraception. METHODS: Using the method of DNA hybridization, the authors examined for HPV infection of the uterine cervix 245 users of contraception, incl. 127 women using hormonal contraception and 118 women with IUD. 112 women of this group were examined before they started to use contraception and one year after started to use it. The control group was formed by 143 using no contraception. RESULTS: The authors found a significantly higher incidence of HPV infection of the uterine cervix in the group of women using contraception (24.1%) than in the control group (15.4%). On analysis of the group of women using contraception, as compared with the control group, they found a significantly higher incidence of HPV in women with hormonal contraception (26.0%) while the higher prevalence of HPV (by 6.6%) in women with IUD as compared with the control group was not significant. However the differences in the incidence of HPV (4%) between the two groups of women taking hormonal preparations (26.0%) or having a IUD (22%) were not significant. On examination of women before the onset of contraception and after one year of its use the authors found that candidates of contraception had as compared with the control group an insignificantly (by 3.3%) higher prevalence of HPV already before the beginning of contraception. After one year of contraception it increased by another 3.6%. CONCLUSION: With regard to the higher incidence of HPV in women using contraception and with regard to the oncogenic effect of HPV the authors emphasize the importance of regular detailed gynaecological examinations of these women focused on early diagnosis of precancerous conditions of the uterine cervix.