Lenka Zejskova

Quantification of extracellular DNA using hypermethylated RASSF1A, SRY, and GLO sequences - evaluation of diagnostic possibilities for predicting placental insufficiency.

This study evaluated quantification of fetal extracellular DNA in maternal plasma for differentiation between cases at risk of onset of placental-insufficiency-related complications and normal pregnancies. Using real-time polymerase chain reaction, fetal (sex-determining region Y [SRY] and hypermethylated RASSF1A sequence) and total (beta-globin [GLO] gene) extracellular DNA was examined in 70 normal pregnancies, 18 at risk of placental-insufficiency-related pregnancy complications, 24 preeclampsia with or without (w or w/o) intrauterine growth retardation (IUGR) (median 34.0 week), and 11 IUGR (median 28.5 week). IUGR was diagnosed when estimated fetal weight was below the 10th percentile for evaluated gestational age. Although increased levels of extracellular DNA were detected in pregnancies with preeclampsia w or w/o IUGR relative to controls (RASSF1A, p < 0.001; SRY, p = 0.009; GLO, p < 0.001), quantities of fetal extracellular DNA in IUGR were not statistically significant (RASSF1A, p = 0.21; SRY, p = 0.2). RASSF1A, SRY, and GLO achieved 93.1%, 93.6%, and 92.1% accuracy for differentiation between normal pregnancy and preeclampsia w or w/o IUGR. Lower sensitivity was observed for pregnancies with onset of IUGR (RASSF1A, 60.0%; SRY, 80.0%; GLO, 72.7%), but did not influence final accuracy (RASSF1A, 91.6%; SRY, 92.5%; GLO, 89.5%). Among 18 patients at risk, 8 pregnancies involving 3 female and 5 male fetuses developed preeclampsia (n = 4), IUGR (n = 3), and chronic placentopathy causing hypoxia (n = 1). Elevation of extracellular DNA was demonstrated in 3/5 (SRY), 1/8 (hypermethylated RASSF1A), and 4/8 (GLO) patients at the earliest 26 weeks and at the latest 2 weeks before the onset of symptoms. These data indicate that fetal and total extracellular DNA concentrations can be significantly elevated in plasma of patients who later developed placental-insufficiency-related pregnancy complications. However, this is strongly individualized, and not a rule for all cases, and probably depends on the actual occurrence of excessive placental trophoblast apoptosis.

Feasibility of fetal-derived hypermethylated RASSF1A sequence quantification in maternal plasma--next step toward reliable non-invasive prenatal diagnostics.

We determined the feasibility of universal fetal marker detection in maternal circulation. Using real-time PCR, we compared the levels of fetal (SRY and hypermethylated RASSF1A) and total (GLO gene and total RASSF1A) extracellular DNA and fractions of extracellular fetal DNA (SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A) in maternal circulation. Sensitivity and specificity reached 100% as the fetal-specific hypermethylated RASSF1A sequence was detected in all 151 examined plasma samples derived from 70 normal pregnancies with a singleton male (n=51) or female (n=19) fetus sampled throughout gestation and absent in non-pregnant individuals (n=29). A strong positive correlation was observed between fetal-derived hypermethylated RASSF1A and SRY (ρ=0.66, P<0.001), total RASSF1A and GLO (ρ=0.65,P<0.001), SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A ratio (ρ=0.62, P<0.001) in maternal plasma. The results indicate that a universal fetal marker could be useful not only for the confirmation of the presence of fetal cell-free DNA in maternal plasma but could enable quantification of cell-free fetal DNA in pregnancy associated disorders, independently of the sex of the fetus.

The Effect of DYS-14 Copy Number Variations on Extracellular Fetal DNA Quantification in Maternal Circulation

The aims of our research involved to investigate DYS-14 copy number variations in healthy males, to quantify extracellular DNA in maternal circulation in normal versus complicated pregnancies, and to study variations in the DYS-14 copy number in extracellular male fetal DNA. Fifty-five healthy males, 43 uncomplicated male singleton pregnancies (23 sampled at the 16th week and 20 sampled at the 36th week), and 15 pregnancies with placental insufficiency (PI)-related complications (mean 34.1 weeks) were analyzed using real-time PCR with DYS-14 sequence, sex determining region Y (SRY), and beta-globin (GLO) genes used as markers. Increased levels of extracellular DNA were detected in PI-related complications relative to gestational age-matched controls (SRY, p < 0.001; DYS-14, p = 0.007; GLO, p < 0.001). When the mean + 2SD (standard deviation) of controls was used as a cutoff, SRY, DYS-14, and GLO achieved 91.7%, 68.8%, and 94.4% accuracy, respectively, for differentiation between normal and complicated pregnancies. Considerable variations in the DYS-14 copy number in healthy males (mean 52.6) and extracellular DNA were found. A lower DYS-14 copy number was observed in PI-related complications (mean 83.5) compared to uncomplicated pregnancies (16th week: mean 114.2, p = 0.02; 36th week: mean 142.8, p = 0.04). The DYS-14 copy number was higher in extracellular DNA throughout gestation relative to healthy males. We concluded that, regarding interindividual copy number variations, the DYS-14 sequence is not an optimal marker for extracellular fetal DNA quantification for differentiation between normal and complicated pregnancies.

A comprehensive study of TP53 mutations in chronic lymphocytic leukemia: Analysis of 1287 diagnostic and 1148 follow-up CLL samples.

TP53 plays a pivotal role in the process of DNA repair and apoptosis. In 10-20% of patients with chronic lymphocytic leukemia (CLL), the TP53 pathway is affected. In this study, we analyzed the TP53 mutation status in 2435 consecutive CLL samples, including 1287 diagnostic samples and 1148 samples during follow-up, using FASAY (Functional Analysis of Separated Alleles in Yeast) and direct sequencing. In a cohort of 1287 diagnostic CLL samples, we identified 237 cases with TP53 variants, including mutations, temperature-sensitive variants, deletions, insertions and aberrant splicing variants (18.4%). In 1148 follow-up samples, no TP53 clonal evolution was observed.

Fetal cells of mesenchymal origin in cultures derived from synovial tissue and skin of patients with rheumatoid arthritis

The transplacental cell transfer naturally takes place during pregnancy and occurs bi-directionally between the mother and fetus. Using real-time polymerase chain reaction (PCR) assay and sex determining region Y (SRY) gene as a marker, we examined the presence of male fetal cells in cell cultures derived from synovial tissues and skin dermis in women with prior pregnancy history suffering from rheumatoid arthritis (RA) who underwent synovectomy. Male DNA was detected in synovial cell samples derived from carpal, hip, metacarpophalangeal and metatarsophalangeal joints in five out of 13 (38.5%) patients with RA in a frequency range of 0.02-62.55 (mean 12.17) male cells per 10,000,000 total cells. SRY gene positivity was found as well in skin fibroblast cultures in four out of 10 (40.0%) RA patients in a frequency range of 3.26-43.47 (mean 15.42) male cells per 10,000,000 total cells, respectively. The difference in a frequency of fetal-derived male cells between both the cohorts did not achieve the statistical difference (p=0.77). We conclude that persisting male fetal cells are able to grow from non-inflamed tissues as well as from those which have many features characteristic of a stressed tissue. We conclude that persisting male fetal cells are also able to proliferate in cell culture since their presence was detected even in consecutive passages.

A method to identify new molecular markers for assessing minimal residual disease in acute leukemia patients

Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include recurrent cytogenetic abnormalities and mutations in important hematological genes; unfortunately well-characterized targets are lacking in many AL patients. Here we demonstrate a technical approach for the identification and mapping of novel clone-specific chromosomal abnormalities down to the nucleotide level. We used molecular cytogenetics, chromosome microdissection, amplification of the microdissected material, and next-generation sequencing to develop PCR-based MRD assays based on unique breakpoint sequences.

OP31.09: Fetal microchimerism in gynecologic malignancies

the patient with cervical carcinoma by using transvaginal ultrasound (TVUS). Methods: Based on the histological diagnosis of cervical cancer (biopsies and cone) and clinical stage of disease FIGO IB, 26 women have been included in the study. All patients underwent colour Doppler evaluation to assess intratumoral blood flow (colour scores), resistance index (RI), peak systolic velocity (PSV) and tumour size. Results: We were able to calculate the volume of tumour for all 26 patients and final results were: mean volume of tumour is 4.57 cm3, mean volume in pathologoanatomic specimen is 4.96 cm3. We found that TVUS measurement is more accurate in endophytic tumours with longest diameter under 2 cm, but there are problems with exophytic tumours. The mean difference in measurements were with tumours more than 3 cm and in exophytic ones. The ultrasound findings in exophytic tumours and those with the longest diameter more than 3 cm confirmed the presence of parametrial extension and node involvement. Conclusions: Our data suggest that TVUS measurement in estimation of the longest diameter and the volume of tumour with the help of colour Doppler examination in cervical carcinoma has to be considered a necessary procedure that give us information for stage of the disease in small tumours where a conservative management is an issue. However, TVUS has an important supportive aspect in women with cervical carcinoma stage IB willing to use ultrasound as assistance in the process of decision making.