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.
Germline mutations in the BRCA1/2 genes account for the majority of hereditary breast ovarian cancer (HBOC).Identification of causal mutations may have significant impact on clinical management of such families. Despite high mutation detection rate, many HBOC cases remain without identified cause. These cases warrant use of several analysis methods, such as those for large genomic rearrangements and DNA copy number changes, or analysis other genes, shown to be associated with increased HBOC risk.We assessed 585 Slovak HBOC for presence of mutations in BRCA genes. Sequencing revealed mutations in 100 families, representing 17.1% (88% and 12% of mutations were located in BRCA1 and BRCA2, respectively).Four of the mutations, c.80+4del4, c.1938_1947del10 and c.1166delG in BRCA1 and c.6589delA in BRCA2 gene have been described only in Slovak population. Using MLPA analysis, we detected two large genomic rearrangements in 3 families, a deletion of exons 21 and 22, and a rare deletion of a whole BRCA1 gene. Twentyseven different variants of uncertain clinical effect (4 novel) and 14 distinct SNP BRCA1 haplotypes were detected. Their potential effect was considered using the prediction software packages Align GVGD, Pmut and Polyphen.We observed that the best clinical criterion for the initiation of BRCA1 analysis is the presence of breast cancer at 40 years of age in the association with the presence of ovarian cancer diagnosed around the age of 50.Conversely, the best clinical criterion for starting with BRCA2 analysis is the presence of breast cancer diagnosed in older age (above 50), or the presence of breast cancer in conjunction with carcinomas at different sites e.g. prostate, colorectum, ovary, uterus. Finally we have seen that the analyses of other HBOC risk gene TP53 and specific mutation in CHEK2*c.1100delC in Slovak HBOC families were not efficient since no mutations were found in these genes. 2
colorectal carcinoma (crc) represents a serious problem worldwide: in the slovak republic are diagnosed about 2600 new crc cases annually and its incidence is increasing. colorectal cancer patients may succumb to the disease because of local recurrence or local formation of metastasis. Therefore, it is necessary to modulate therapeutic algorithm with new methods, leading to early diagnostic of crc or changing the existing therapeutic procedures. recent progresses have been made in understanding of eGFr pathway involved in crc carcinogenesis, especially the role of ras protein. mutations in KRAS oncogene are frequently found in human cancers, particularly colorectal, pancreatic, billiary tract and lung tumors. The presence of the KRAS mutations in metastatic colorectal cancer patients correlates with lack of response to the certain epidemal growth factor receptor (eGFr) inhibitor therapies, such as panitumumab and cetuximab. consequently, screening for KRAS mutations status may be used as a prognostic marker, because the crc patients with KRAS positive tumors have a worse prognosis.The aim of our study was to establish the methods for rapid and sensitive detection of KRAS mutation status in formalin fixed paraffin embedded (FFpe) tissues dNa. We applied real Time pcr analysis (Therascreen Kras mutation Test Kit) and sequencing analysis (optimised for the analysis of FFpe tissues) to detect somatic mutations in codon 12 and 13 of KRAS gene. Both methods were used concurrently in the panel of dNa isolated from 25 colorectal FFpe tissues tumor. The positive or negative results from all 25 samples were identified by both methods independently. The KRAS mutations were presented in 8 of 25 patients (32%). our results demonstrate that the real Time pcr analysis can be used for detection of somatic KRAS mutations in FFpe clinical samples. however, we also recognize that the sequencing analysis of approximately 200bp amplicons may be used for KRAS mutations status screening, but with care of method sensitivity.
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