OBJECTIVE:To evaluate risk factors for development of recurrent disease in borderline ovarian tumors.DESIGN:Retrospective study of 10-years single institution population.SETTING:Dept. of Gynecology and Obstetrics, 3rd Medical Faculty of Charles University in Prague.METHOD:59 consecutive cases of borderline ovarian tumors (BOT) were analyzed for age, histopathological type, DNA ploidy, stage, presence of invasive and non-invasive peritoneal implants, type of surgical procedure, residual disease, adjuvant therapy, recurrence and long-time prognosis of the patients.RESULTS:Median follow-up was 47 months (range 1-144). There were 5 (8.5%) patients with DNA aneuploid tumors in the study group; 4 of them were younger than 50 years, 4 of them were early stage serous BOT; no one recur so far. No death of disease was described in the whole study group; only 2 patients (3.4%) developed recurrent disease - both were young patients after conservative surgery for serous diploid stage I/II BOT. Conservative surgery was the only significant factor for recurrence in univariate analysis (p = 0.0159) in our setting.CONCLUSION:DNA ploidy was not proved to be prognostic factor in borderline ovarian tumors in our study group. The only significant risk factor for development of recurrent disease was conservative surgery, with no influence on overall survival.

e21011 Background: Circulating tumor cells (CTC) are potential precursors of metastasis. They are also of use in diagnosing malignancy and for prognostic purposes. Our laboratory has previously isolated CTC from orthotopic nude mouse models of human prostate cancer cells where the PC-3 cancer cells express green fluorescent protein (GFP). It was found that only orthotopic tumors produced CTC and not subcutaneous tumors, which may explain why orthotopic tumors metastasize and subcutaneous tumors do not. However, in this previous study, CTC were observed only after culture. Methods: The present study used the GFP-expressing PC-3 orthotopic model and immunomagnetic beads coated with anti-EpCAM and anti-PSMA. Results: GFP-expressing CTC were isolated within 15 minutes and were readily visualized by GFP fluorescence. The immunomagnetic-bead-captured GFP-expressing PC-3 CTC could be immediately placed in 3-dimensional sponge cell culture where they proliferated. Conclusions: The combination of GFP-expression and immunomagnetic beads is very powerful method to obtain CTC for either immediate analysis or for biological characterization in vivo or 3-dimensional culture for further use in the drug sensitivity testing.

Abstract BACKGROUND: Breast cancer (BC) patients still harbor a considerable risk of metastatic relapse caused by minimal residual disease (MRD) despite of complete removal of primary tumor. The aim of this study was to identify single disseminated tumor cells (DTC) in the bone marrow and circulating tumor cells (CTC) in the peripheral blood as potential biomarkers for therapy response monitoring and metastatic relapse risk prediction. Finally, the gene expression profiles of CTCs have been analyzed. METHODS: A total of 87 patients with diagnosed BC at stage I to III and 115 metastatic patients were enrolled into a prospective study between years 2008 - 2010. Peripheral blood (5ml) for CTC-detection was collected from primary BC patients before chemotherapy (CHT), after 2 cycles of CHT and after the CHT. Metastatic BC patients have been examined for CTCs before starting a new line of the treatment. Bone marrow aspirates from 16 premenopausal BC patients (mean age 31) with primary tumor were analyzed for the presence of DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 (Epimet™, AS Diagnostik, Germany) before surgery. CTC detection in blood was performed by AdnaTest BreastCancerTM(AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. cDNA from isolated CTCs has been further pre-amplified and used for multimarker qPCR gene expression profiling using Biomark® microfluidic 48x48 GE Dynamic arrays (Fluidigm, USA). qPCR results have been analyzed by GENEX vs. 5.2 software (MultiD Analysis). RESULTS: 286 CTC samples have been analyzed in total. The analysis has shown that the expression profiles of CTCs from primary BC patients have been significantly different comparing them to the CTC-profiles from metastatic BC patients for several of tested genes (e.g., CK19, GA7332, MLFIP1, SATB1, PTEN). Interestingly, before surgery of primary tumor DTCs were found in 5/16 patients (31 %) and CTCs in 7/16 (43 %). Both DTCs and CTCs together were found in 4/16 (25%) patients. In 18% of the primary BC patients no dissemination markers were found CONCLUSIONS: Information based on the CTCs-gene expression profiles could provide an additional support for therapy management. Metastatic potential of enriched CTCs will be further evaluated on the single cell level. This work has been supported by Grant Agency of Ministry of Health, Czech Republic (IGA NS9976) and Grant Agency of Charles University in Prague, Czech Republic no. 7709 and 59410. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-01.

INTRODUCTION:Randomly estimated fasting hyperglycaemia in an asymptomatic individual may represent the first sign of pancreatic beta-cell dysfunction.OBJECTIVE:We aimed at specifying the genetic aetiology of asymptomatic hyperglycaemia in a cohort of children and adolescents.SUBJECTS AND METHODS:We analysed the aetiological diagnosis in 82 non-obese paediatric subjects (38 males) aged 0.2-18.5 years (median: 13.1) who were referred for elucidation of a randomly found blood glucose level above 5.5 mmol/l. In addition to fasting glycaemia and circulating levels of insulin and C-peptide, the subjects were tested by an oral glucose tolerance test and an intravenous glucose tolerance test and screened for mutations in the genes encoding glucokinase (GCK), HNF-1alpha (TCF1), Kir6.2 (KCNJ11) (if aged <2 years) and HNF-4alpha (HNF4A) (those with a positive family history of diabetes).RESULTS AND DISCUSSION:We identified 35 carriers of GCK mutations causing MODY2, two carriers of TCF1 mutations causing MODY3, one carrier of a HNF4A mutation causing MODY1 and one carrier of a KCNJ11 mutation causing permanent neonatal diabetes mellitus. Of the remaining patients, 11 progressed to type 1 diabetes mellitus (T1DM) and 9 had impaired glucose tolerance or diabetes mellitus of unknown origin. In 23 subjects, an impairment of blood glucose levels was not confirmed. We conclude that 39 of 82 paediatric patients (48%) with randomly found fasting hyperglycaemia suffered from single gene defect conditions, MODY2 being the most prevalent. An additional 11 patients (13%) progressed to overt T1DM. The aetiological diagnosis in asymptomatic hyperglycaemic children and adolescents is a clue to introducing an early and effective therapy or, in MODY2, to preventing any future extensive re-investigations.

The aim was to determine the effect of fybrinolytic therapy by streptokinase on chemotherapy and radiation response in human colon cancer cells. The cells were treated with different concentrations of gemcitabine, cis-platine and streptokinase, at a single use or in combinations. Radiation was tested at a dose 0.5, 5 and 15Gy in three different schedules. The chemotherapy showed higher cytotoxic effect in combination with streptokinase. On the other hand, the combination of chemotherapy with streptokinase and radiotherapy provide no improvement in sensitivity of cancer cells to treatment. The data suggest that fybrinolytic therapy could influence the effect of chemotherapy.

To the Editor: Maturity-onset diabetes of the young (MODY; MIM# 606391) is a genetically and clinically heterogeneous form of diabetes mellitus, characterized by an autosomal dominant inheritance, early-onset non-insulin-dependent diabetes mellitus and by a primary defect in the pancreatic beta-cell function (1). Until now, six types of MODY diabetes have been identified, depending on the gene causing the disease (2). Screening for glucokinase (GCK) mutations in subjects with clinical characteristics of MODY allows distinguishing between patients with a benign metabolic condition (GCK mutation positive, clinical diagnosis MODY2) and those with a higher risk of progressive hyperglycemia associated with more prevalent and severe diabetic complications (GCK mutation negative). The first mutation in the GCK gene was reported in 1992 (3). Up to now, 195 mutations in GCK have been described, in 285 families (4). Diabetic complications are rare in GCK–MODY, thus GCK–MODY patients only need to be followed by annual HbA1c examination. Also, screening of GCK for heterozygous inactivating mutations allows to determine the subtype of MODY diabetes and to predict the lifelong prognosis. All 12 exons (exons 1a, 1b, 1c and 2–10), the intron–exon boundaries and promotor region of GCK (GenBank accession number, AF04101222) were screened; in 92 Czech probands fulfilling classical MODY criteria, using denaturing highperformance liquid chromatography as previously described (5). The nature of identified mutations was established by direct nucleotide sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) according to manufacture’s instructions. Mutations were confirmed using a second, independent amplification of the affected part of GCK and re-sequenced the following day. The probands were recruited from pediatricians and endocrinologists from the entire Czech Republic. Fifteen different missense mutations were identified in 27 patients. Of these, six were novel missense mutations R250C (exon 7, c.748C.T), L315H (exon 8, c.944T.A), F316V (exon 8, c.946T.G), F419L (exon 10, c.1255T.C), I436N (exon 10, c.1307T.A) and A454E (exon 10, c.1361C.A). Some of the identified GCK missense mutations are located near putative functional domains: R250C was found in the close vicinity of a putative glucose binding site, while F419L was detected near a putative MgATP binding site and could thus affect binding kinetics (6). Five of these mutations co-segregated with hyperglycemia in the family, suggesting that the variants are new diseasecausing mutations. For the novel R250C variant, family members were not available for cosegregation studies. All codons, which are changed by the six novel mutations, are conserved in the human, mouse, rat and chimpanzee genomes and we found none of these mutations in 50 unrelated healthy Czech Caucasian subjects. Therefore, we assume that the mutations are probably novel disease-causing mutations. We also compared the clinical characteristics of patients with GCK mutations and those without mutation in GCK (data not shown in details). In short – the treatment of hyperglycemia with diet was more frequent (p , 0.001) in the group of probands with mutations in GCK and they had a significantly lower frequency of diabetic complications (p 1⁄4 0.02). None of the patients with mutations in GCK was treated with insulin (p , 0.001). Moreover, GCK mutation carriers had a lower level of glycosylated hemoglobin (p1⁄4 0.02). ThemeanHbA1c (%) inGCK-positive probands vs negative was 5.7 0.2 vs 6.5 0.2. In conclusion, we identified 29% of GCK mutation carriers among Czech MODY probands, confirming that mutations in GCK are a common cause of MODY in the Czech population. The present high relative prevalence of GCK–MODY, compared with some other European studies, might reflect not only a specific genetic background, but also the mode of recruitment, because most of the probands in the present investigation were recruited by

The results in this study suggest that microsatellite polymorphism within the transmembrane region of MIC-A gene is associated with genetic susceptibility to adult-onset of type 1 diabetes mellitus (T1DM), MIC-A5.1 allele, corrected P = 0.001, whereas it is not associated with latent autoimmune diabetes in adults (LADA) in Czech population. According to our findings, we can hypothesize that adult-onset T1DM and LADA may have partly different immunogenetic aetiopathogenesis.

Interleukin-18 (IL-18) gene promoter polymorphism is known as a genetic risk factor for child type 1 diabetes mellitus development. To test the role of IL-18 gene polymorphism in predisposition to adult type 1 diabetes (T1DM) and latent autoimmune diabetes in adults (LADA), we analysed SNPs at position −607 (C/A) and −137 (G/C) in the promoter region of IL-18 gene by sequence-specific PCR in 49 T1DM, 66 LADA patients and 139 healthy controls. We found differences in allele, genotype or haplotype distribution in tested patients when compared to frequencies found in control group but these differences did not reach statistical significance. However, there was a difference in −607 (C/A) allele and genotype distribution found in LADA and T1DM patients that reached statistical significance. These results suggest that the IL-18 gene promoter polymorphisms are not associated with adult type 1 diabetes or LADA susceptibility, and according to our findings genes involved in onset and progression of LADA and T1DM are probably different.

HFE-linked hereditary haemochromatosis is a common autosomal recessive disease among Caucasians. The primary pathogenetic mechanism is excessive absorption of iron, which is deposited in various organs with their subsequent damage. In 1996 the gene responsible for haemochromatosis was detected--the HFE gene and its major mutation C282Y. The discovery of further mutations followed. Two sites of point mutations in the HFE gene, C282Y and H63D, are associated with more than 80% of haemochromatosis cases. Another mutation-- S65C--was detected on 8% of chromosomes of haemochromatosis patients, which were negative for mutations C282Y or H63D. The objective of this study was to identify the allele frequency of S65C and other HFE mutations in the Czech population. DNA extracted from 481 randomly selected newborn screening cards (Guthrie cards) from all over the country was analysed by PCR-RFLP. No (0%) sample was identified as homozygous for S65C or C282Y mutation and 8 (1.67%) were homozygous for H63D mutation. Twelve (2.49%) samples were S65C heterozygous, 33 (6.86%) samples were C282Y heterozygous, and 128 (26.61%) were H63D heterozygous. Of these, 11 (2.29%) carried one copy of each mutation, i.e. were compound heterozygous. Two samples were S65C/H63D compound heterozygous and nine were C282Y/H63D compound heterozygous. Allele frequencies for S65C, C282Y, and H63D were 1.25% (95% CI, +/- 0.70), 3.43% (95% CI, +/- 1.15), and 14.97% (95% CI, +/- 2.25), respectively. The observed genotype frequency for S65C, C282Y, and H63D mutations in the Czech Republic agrees with those reported for other Central European populations.