Total Cell-free DNA Research Articles

Quantification of the placental epigenetic signature of the SERPINB5 gene in maternal plasma of pregnancies complicated by small for gestational age

IntroductionTo investigate the association between pregnancies with small for gestational age (SGA) neonates and the concentration of cell-free fetal DNA or cell-free total DNA in maternal plasma during the first and second trimesters using tissue-specific epigenetic characteristics of the SERPINB5 gene. MethodsA nested case–control study was conducted with maternal plasma collected at 11 to 26 gestational weeks from 51 women with SGA neonates and 102 controls. We performed a real-time quantitative methylation-specific PCR to quantify concentrations of unmethylated-SERPINB5 (U-SERPINB5) as a cell-free fetal DNA marker and methylated-SERPINB5 (M-SERPINB5) as a cell-free total DNA marker. ResultsA positive correlation was observed between U-SERPINB5 and M-SERPINB5 concentrations in both control (r = 0.363, p < 0.001) and SGA groups (r = 0.548, p < 0.001). Moreover, the concentration of U-SERPINB5 or M-SERPINB5 was significantly positive correlated with gestational age at sampling in both controls (U-SERPINB5: r = 0.397, p < 0.001; M-SERPINB5: r = 0.275, p = 0.005) and SGA (U-SERPINB5: r = 0.274, p = 0.052; M-SERPINB5: r = 0.439, p = 0.001). However, the concentration of U-SERPINB5 or M-SERPINB5 was not correlated with birthweight. At 11–14 weeks, U-SERPINB5 and M-SERPINB5 concentrations in SGA did not differ significantly from those of controls. There were also no statistically significant differences in the concentrations of U-SERPINB5 and M-SERPINB5 between SGA and controls at 15–26 weeks of gestation. DiscussionOur findings suggest that U-SERPINB5 and M-SERPINB5 concentrations in maternal plasma during early pregnancy are not associated with pregnancies who delivered SGA neonates.

Maternal plasma cell‐free DNA in the prediction of pre‐eclampsia

To examine whether maternal plasma concentrations of total cell-free (cf)DNA and fetal fraction at 11-13 and 20-24 weeks' gestation in pregnancies that subsequently develop pre-eclampsia (PE) are different from those without this complication. Total cfDNA and fetal fraction were measured in 20 cases of early PE requiring delivery at < 34 weeks, in 20 cases of late PE with delivery at ≥ 34 weeks and in 200 normotensive controls, at 11-13 and 20-24 weeks' gestation. Total cfDNA and fetal fraction measured at 11-13 weeks were converted to multiples of the median (MoM), corrected for maternal characteristics and gestational age. The distributions of total cfDNA and fetal fraction at 20-24 weeks were expressed as MoM of values at 11-13 weeks. The Mann-Whitney U-test was used to determine the significance of differences in the median values in each outcome group relative to that in the controls. In the early-PE group at 11-13 weeks, compared with controls, there was a significant increase in median total cfDNA (2104 genome equivalents (GE)/mL vs 1590 GE/mL) and a decrease in median fetal fraction (6.8% vs 8.7%). In the late-PE group at 20-24 weeks, compared with controls, there was a significant decrease in median fetal fraction (8.2% vs 9.6%). These significant differences between groups were not observed when the values were converted to MoM. Measurements of total cfDNA and fetal fraction in maternal plasma at 11-13 and 20-24 weeks are not predictive of PE.

Circulating muramyl dipeptide is negatively associated with interleukin-10 in the frail elderly.

Elevated levels of serum cytokines, a marker of immune activation and chronic inflammation, are commonly associated with age and are a significant risk factor for all-cause mortality in the elderly. This phenomenon is very similar to that exhibited by individuals with diseases of inflammatory etiology and chronic viral infections such as human immunodeficiency virus (HIV). Although the origin of chronically elevated cytokines with age is unknown, for chronic diseases and viral infections, a role for circulating bacterial products and other pattern recognition receptor (PRR) ligands has been suggested. Given this, we sought to examine whether the levels of circulating cytokines (tumour necrosis factor (TNF), interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12) in the advanced-age, frail elderly (n = 135) correlated with plasma levels of lipopolysaccharide (LPS), muramyl dipeptide (MDP), 16S ribosomal DNA, total cell-free DNA and host-derived mitochondrial DNA. After adjusting for multiple testing, no associations between circulating products and donor age, sex or comorbidities were observed. However, a significant negative correlation between MDP and IL-10 was identified. Given the anti-inflammatory nature of IL-10, a negative relationship with a potent inflammatory agonist such as MDP is not surprising and suggests a potential role for circulating MDP in the propagation of age-related immune activation.

The effect of exercise and metformin treatment on circulating free DNA in pregnancy

IntroductionSome pregnancy complications are characterized by increased levels of cell-free fetal (cffDNA) and maternal DNA (cfmDNA), the latter may also be elevated during physical strain. This study aims at assessing the impact of exercise and metformin intervention in pregnancy, and to compare the levels of cell free DNA in pregnant women with or without PCOS diagnosis. MethodsConsecutive women from two previous randomized controlled trials in pregnancy were included. Women came from a trial with organized exercise vs. standard antenatal care in pregnancy and a trial of metformin vs. placebo in PCOS women. Levels of cffDNA, cfmDNA and cell-free total DNA (cftDNA) were measured by qPCR. ResultsTraining in pregnancy did not affect the levels of cffDNA, cfmDNA or cftDNA.PCOS-women treated with metformin had lower levels of cfmDNA and cftDNA at week 32 (mean ± SD: 301 ± 162 versus 570 ± 337, p = 0.012, 345 ± 173 versus 635 ± 370, p = 0.019); otherwise the levels were comparable to PCOS-controls. Metformin-treated PCOS-women had higher cffDNA at inclusion, in the 1st trimester; later on in pregnancy the levels in the metformin and placebo groups were equal.A comparison of pregnant women in the exercise study (TRIP) to placebo-treated pregnant PCOS-women, showed the levels of cffDNA, cfmDNA or cftDNA during mid-pregnancy (weeks 18–36) to be equal. DiscussionTraining during pregnancy was not associated with altered levels of cffDNA cfmDNA or cftDNA, but metformin treatment may reduce cfmDNA and cftDNA in pregnant PCOS women.

Cell-free DNA in healthy individuals, noncancerous disease and strong prognostic value in colorectal cancer.

The purpose was to investigate total cell-free DNA (cfDNA) in colorectal cancer (CRC) patients during treatment with second-line chemotherapy and in healthy controls and patients with different comorbidities. Patient treated with second-line irinotecan for metastatic CRC (n = 100), a cohort of healthy controls with and without comorbidity (n = 70 and 100, respectively) were included. cfDNA was quantified by an in-house developed quantitative polymerase chain reaction from plasma samples drawn prior to the first cycle of chemotherapy and at time of progression. cfDNA levels were significantly higher in CRC compared to controls, with a clear capability for discriminating between the groups (receiver operation curve analysis; area under the curve 0.82, p < 0.0001). Patients with high levels had a shorter survival from irinotecan compared to those with lover levels. The cohort independent upper normal limit divided patients into high and low risk groups. The progression-free survival (PFS) was 2.1 months [95% confidence interval (CI) 2.0-3.4] and 6.5 (95% CI 4.2-7.2) months [hazard ratio (HR) 2.53; 95% CI 1.57-4.06, p < 0.0001] and overall survival (OS) 7.4 months (95% CI 4.3-8.7) and 13.8 months (95% CI 11.9-18.9; HR 2.52; 95% CI 1.54-4.13, p < 0.0000), respectively. Cox regression multivariate analysis showed a PFS HR of 1.4 (95% CI 1.1-1.7) for each increase in cfDNA quartile, p = 0.03 and 1.6 (1.3-2.0) for OS, p < 0.0001, respectively. A combined marker analysis with plasma KRAS mutations added further prognostic impact, which was consistent when performed on the samples drawn at time of progression. In conclusion, cfDNA measurement holds important clinical information and could become a useful tool for prediction of outcome from chemotherapy in mCRC.

Circulating cell-free total and Epstein–Barr virus DNA in pediatric lymphomas.

e21004 Background: Quantification of serum EBV DNA and/or cell-free total DNA (cf-DNA) may become valuable sources for diagnosis and prognosis evaluation in pediatric lymphomas, and also for diseas...

Cell-free DNA levels in colorectal cancer patients treated with irinotecan, healthy controls, and non-cancer patients with comorbidity.

3559 Background: The present study investigated the clinical value of total cell free DNA (cfDNA) measurement in metastatic colorectal cancer patients treated with second-line irinotecan monotherap...

MC13-0088 Controls for quantification of total cell free DNA qPCR analyses in plasma

MC13-0088 Controls for quantification of total cell free DNA qPCR analyses in plasma

Non-Invasive Prenatal Testing of Trisomy 18 by an Epigenetic Marker in First Trimester Maternal Plasma

BackgroundQuantification of cell-free fetal DNA by methylation-based DNA discrimination has been used in non-invasive prenatal testing of fetal chromosomal aneuploidy. The maspin (Serpin peptidase inhibitor, clade B (ovalbumin), member 5; SERPINB5) gene, located on chromosome 18q21.33, is hypomethylated in the placenta and completely methylated in maternal blood cells. The objective of this study was to evaluate the accuracy of non-invasive detection of fetal trisomy 18 using the unmethylated-maspin (U-maspin) gene as a cell-free fetal DNA marker and the methylated-maspin (M-maspin) gene as a cell-free total DNA marker in the first trimester of pregnancy.Methodology/Principal FindingsA nested case-control study was conducted using maternal plasma collected from 66 pregnant women, 11 carrying fetuses with trisomy 18 and 55 carrying normal fetuses. Median U-maspin concentrations were significantly elevated in women with trisomy 18 fetuses compared with controls (27.2 vs. 6.7 copies/mL; P<0.001). Median M-maspin concentrations were also significantly higher in women with trisomy 18 fetuses than in controls (96.9 vs. 19.5 copies/mL, P<0.001). The specificities of U-maspin and M-maspin concentrations for non-invasive fetal trisomy 18 detection were 96.4% and 74.5%, respectively, with a sensitivity of 90.9%.ConclusionsOur results suggest that U-maspin and M-maspin concentrations may be useful as potential biomarkers for non-invasive detection of fetal trisomy 18 in the first trimester of pregnancy, irrespective of the sex and genetic variations of the fetus.

A repertoire of biomarkers helps in detection and assessment of therapeutic response in epithelial ovarian cancer

In epithelial ovarian cancer (EOC), the cancer antigen 125 (CA-125) has been conventionally used to help in diagnosis and assessment of response to treatment. Currently, YKL-40 (Tyrosine-Lysine-Leucine-40) and circulating cell-free DNA are being evaluated for possession of similar ability. In this study, we aimed to assess the ability of a repertoire of potential biomarkers in detecting and assessing therapeutic response, in advanced EOC. Blood levels of CA-125, YKL-40, total cell-free DNA (CFDNA), cell-free nuclear DNA (CFnDNA), and cell-free mitochondrial DNA (CFmDNA) levels were measured in 100 untreated patients of advanced EOC from November 2009 to June 2011, and again on treatment completion from the 20 patients who appeared for follow-up analysis. Significantly, higher proportion of untreated patients had serum CA-125 >3 times upper limit of normal (ULN) (90.0%; P < 0.0001) and plasma YKL-40 >ULN (77.0%; P < 0.0001), both of which significantly decreased, Posttherapy. posttherapy, CFDNA (P < 0.0001), and CFnDNA (P < 0.0001) levels significantly decreased as compared to pretreatment levels. Positive and significant correlations existed between pretherapy CFDNA and CFnDNA [Spearman rho (ρ) = 1.000; P < 0.0001], and also with CFmDNA (ρ = 0.301; P = 0.002), separately between CFnDNA and CFmDNA (ρ = 0.303; P = 0.002), as well as between plasma YKL-40 and patient age (ρ = 0.353; (P < 0.0001). On treatment completion, CFDNA and CFnDNA levels showed positive and significant correlation (ρ = 1.000; P < 0.0001). Therefore serum CA-125 and plasma YKL-40 aid detection and assessment of therapeutic response, in advanced EOC. CFDNA and CFnDNA help in estimating extent of therapeutic response in advanced EOC.

Noninvasive prenatal diagnosis experience in the Çukurova Region of Southern Turkey: detecting paternal mutations of sickle cell anemia and β‐thalassemia in cell‐free fetal DNA using high‐resolution melting analysis

This study used a high-resolution melting (HRM) technique to detect paternal mutations for the noninvasive prenatal diagnosis (NIPD) of β-thalassemia and sickle cell anemia (HbS). We also determined the levels of cell-free fetal DNA and total cell-free DNA. We used the HRM technique for fetal genotyping of paternal mutations in maternal plasma from 32 pregnancies at risk of β-thalassemia and 57 pregnancies at risk of HbS. The DNA levels in maternal plasma were measured using real-time quantitative PCR. Multiples of the median (MoM) values were calculated in women at risk for β-thalassemia or HbS. Twenty-two paternal mutations were detected in 89 pregnant women. Although we were successfully able to detect the paternal β-thalassemia mutations, the mutant HbS fetuses could not be distinguished from maternal background in the early weeks of pregnancy. The detection of DYS14 in male fetuses was 100%. The MoM values of women at high risk of having HbS-affected fetuses were higher than those for the other groups. High-resolution melting is a useful method for NIPD of β-thalassemias by detecting paternal mutations in the maternal plasma. Cell-free fetal DNA quantification and MoM values were not informative for HbS or β-thalassemias in early pregnancy.

Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24 h after venipuncture

ObjectivesCirculating cell-free fetal DNA (ccffDNA) in maternal plasma is an attractive source for noninvasive prenatal testing (NIPT). The amount of total cell-free DNA significantly increases 24h after venipuncture, leading to a relative decrease of the ccffDNA fraction in the blood sample. In this study, we evaluated the downstream effects of extended processing times on the reliability of aneuploidy detection by massively parallel sequencing (MPS). Design and methodsWhole blood from pregnant women carrying normal and trisomy 21 (T21) fetuses was collected in regular EDTA anti-coagulated tubes and processed within 6h, 24 and 48h after venipuncture. Samples of all three different time points were further analyzed by MPS using Z-score calculation and the percentage of ccffDNA based on X-chromosome reads. ResultsBoth T21 samples were correctly identified as such at all time-points. However, after 48h, a higher deviation in Z-scores was noticed. Even though the percentage of ccffDNA in a plasma sample has been shown previously to significantly decrease 24h after venipuncture, the percentages based on MPS results did not show a significant decrease after 6, 24 or 48h. ConclusionsThe quality and quantity of ccffDNA extracted from plasma samples processed up to 24h after venipuncture are sufficiently high for reliable downstream NIPT analysis by MPS. Furthermore, we show that it is important to determine the percentage of ccffDNA in the fraction of the sample that is actually used for NIPT, as downstream procedures might influence the fetal or maternal fraction.

Plasma circulating cell-free DNA and uteroplacental blood flow in pre-eclamptic patients

Objective The aim of the present study is to evaluate the plasma total cell-free DNA and cell-free fetal DNA level in maternal venous samples obtained from pre-eclamptic pregnant women during the third trimester (gestational age>28 weeks) and whether it shows a correlation with uteroplacental blood flow. Materials and methods This was a case–control study in which plasma total cell-free DNA, cell-free fetal DNA levels, and uteroplacental blood flow including umbilical artery resistance index, uterine artery resistance index and pulsitility index, and middle cerebral artery/umbilical artery pulsitility index were measured in 60 women diagnosed with pre-eclampsia, representing the study group, and another 60 normotensives were taken as controls. Both total cell-free DNA and cell-free fetal DNA were quantified by real-time PCR using the RASSF1A gene before and after treatment by methylation-sensitive restriction enzyme, respectively, irrespective of the sex of the fetus. Results Our results showed that there was a positive correlation between plasma cell-free fetal DNA, total cell-free DNA, and uteroplacental blood flow in pre-eclamptic women (P<0.05). To detect pre-eclampsia, the best cut-off value for plasma cell-free fetal DNA level was 80 copies/ml and that for plasma total cell-free DNA level was 137 copies/ml. Conclusion Our results found that there was a positive correlation between plasma cell-free fetal DNA level and the severity of pre-eclampsia. In addition, we found a correlation between plasma cell-free fetal, total cell-free DNA level, and uteroplacental blood flow in pre-eclamptic women during the third trimester.

Ethical considerations when offering noninvasive prenatal testing

Technology in the prenatal setting is advancing at an exceptional rate, and these advancements will likely result in major changes to current pregnancy screening and testing paradigms. In Australia, prenatal testing is increasingly becoming a routine part of antenatal care and pregnant women are offered an assortment of screening and diagnostic tests, which give them information about their fetus, and can identify potential anomalies before it is born. In Victoria (Australia), each year approximately 4% of babies will be born with a birth defect or fetal abnormality many of which are diagnosed in uterus. Greater than 97% of women in Victoria have at least one or more ultrasounds during pregnancy and greater than 80% of pregnant women take up screening for Down syndrome, with the majority having First Trimester Combined Screening (FTCS). FTCS combines maternal serum analytes — pregnancy associated plasma protein-A (PAAP-A) and beta human chorionic gonadotrophin (free β-hCG) — with results from the nuchal translucency ultrasound to give a risk figure for Down syndrome and Trisomy 18. Approximately 5% of women will receive a false positive result from FTCS, meaning they screen positive for Down syndrome or Trisomy 18 and do not have an affected pregnancy. Women who are considered to be “increased risk” either because of a screening result or ultrasound finding are offered invasive diagnostic testing whereby a fetal sample is obtained via chorionic villus sampling (CVS) or amniocentesis and sent for conventional or molecular karyotyping (microarray). These tests carry a miscarriage risk of up to 1% above the background rate of miscarriage. There has been increasing demand for a safe and reliable alternative to invasive diagnostic testing and very recently, noninvasive prenatal testing (NIPT) has become commercially available to women in America (2012), Australia and many other countries, including Brazil (2013). NIPT is an advanced screening test, which relies on the fact that small fragments of cell-free fetal DNA and RNA circulate in maternal serum. In the first and second trimesters of pregnancy, approximately 6–10% of total cell free DNA (cfDNA) circulating in maternal serum is thought to be fetal in origin; this fetal fraction rises to 10–20% in the third trimester. Using massively parallel sequencing technology, scientists can sequence cfDNA fragments in maternal plasma and detect specific chromosome aneuploidies such as trisomies 21, 13 and 18, much earlier in pregnancy than has previously been possible,

Improvement of detection of paternally inherited fetal mutant genes for β-globin in maternal plasma by PNA clamp

To study improvement of detection of paternally herited fetal mutant genes for β-globin in maternal plasma by PNA clamp to seek a noninvasive prenatal diagnostic method for β-thalassemia. A total of 38 maternal blood samples were collected at 7 to 20 weeks of gestation, samples in which the father carried CD41-42 mutation and mother carried normal gene or the other point mutation for β-thalassemia were examined. The results of fetal DNA in amniotic fluid, cord blood or peripheral blood of newborns were used as the gold standard for comparison. In the study group, the total cell-free DNA was extracted from maternal plasma using QIAamp DNA Blood Mini Kit. After extraction, the total cell-free DNA was separated by agarose gel (1%) electrophoresis, and the cell-free DNA with a size of 100-300 bp was retrieved from the gel slice. Then, the retrieved DNA-free cell underwent PCR amplified with a PNA clamp. The genotype was confirmed by the conventional method (reverse dot blot hybridization), and the results were compared to gold standard. Simultaneously, two control groups with different PCR procedures were set up. The PCR procedure of control group A was amplified with the extracted total cell-free DNA and PNA clamp, and the PCR procedure of control group B was amplified with the retrieved size-fractionated DNA-free cell without PNA clamp. Plasma samples from 38 pregnant women were detected using PCR products for hybridization, the results were compared with the gold standard. Regarding the 21 samples confirmed by gold standard with fetal genotype 41-42M/N, 19, 8, 12 cases were detected as fetal genotype 41-42M in study group, control group A and control group B respectively, the sensitivity was 90.5% (19/21), 38.1% (8/21) and 57.1% (12/21) respectively;Concerning the 17 samples confirmed by gold standard with fetal normal genotype, the amount of false positive cases were 1, 2 and 1 respectively. The respective specificity was 94.1% (16/17), 94.1% (16/17) and 88.2% (15/17) respectively. The respective accuracies were 92.1% (35/38), 63.2% (24/38) and 71.1% (27/38) respectively. The difference in sensitivity and specificity was pairwise compared by means of McNemar's test. There was significant difference between new study group and control group A or control group B (all P﹤0.05). The method of detection of paternally inherited fetal mutation genes for β-thalassemia using small size of fetal DNA-free cell in maternal plasma with PNA clamp had several advantages of reliable sensitivity, specificity and accuracy, indicating its potential of clinical practicality.

Cell-Free Fetal DNA and Cell-Free Total DNA Levels in Spontaneous Abortion with Fetal Chromosomal Aneuploidy

BackgroundCell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA) with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy.Methodology/Principal FindingsA nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC) curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both). The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001) than in SA women with normal karyotype (r = 0.465, P = 0.002) and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037). The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852–0.945) and 0.939 (95% CI, 0.903–0.975), respectively.ConclusionsSignificantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA with fetal chromosomal aneuploidy, regardless of fetal gender.

A multifactorial relationship exists between total circulating cell‐free DNA levels and maternal BMI

A multifactorial relationship exists between total circulating cell‐free DNA levels and maternal BMI

Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield

ObjectiveCell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used.MethodsMicrofluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples.ResultsTotal cell-free DNA quantity increased significantly with time in samples stored in K3EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4°C did not prevent these changes.ConclusionWhen samples can be processed within eight hours of blood draw, K3EDTA tubes can be used. Prolonged transfer times in K3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.

Serum cell‐free DNA in renal cell carcinoma

Currently, there are no established diagnostic and prognostic serum markers for renal cell carcinoma (RCC). The objective of this study was to evaluate the putative significance of serum cell-free DNA. Preoperative serum samples from 200 consecutive patients with sporadic, solid renal tumors were analyzed (157 patients with RCC and 43 patients with benign renal tumors). Quantitative real-time polymerase chain reaction was used to assess total cell-free DNA (ring finger protein 185 [RNF185]) and CpG island methylation of Ras association domain family member 1A (RASSF1A) von Hippel-Lindau (VHL), prostaglandin-endoperoxidase synthase 2 (PTGS2), and P16 (cyclin-dependent kinase inhibitor 2A). Associations with RCC, pathologic variables, and disease-specific survival were evaluated. Total cell-free DNA levels and CpG island methylation of RASSF1A and VHL were highly diagnostic for RCC with an area under the receiver operating characteristic curve of 0.755, 0.705, and 0.694, respectively. VHL methylation was detected more frequently in patients with clear cell RCC than in those with other subtypes (P = .007). Total cell-free DNA levels were higher in patients with metastatic RCC (P < .001) and necrotic RCC (P = .003) and were associated with poorer disease-specific survival (P < .001). In multivariate analysis, the tumor stage, size, grade, and necrosis (SSIGN) score (P < .001) and categorized total cell-free DNA levels (P = .028) were retained as independent prognostic factors. The current results indicated that cell-free DNA represents a novel serum-based diagnostic and prognostic biomarker for RCC. Total serum cell-free DNA levels and CpG island methylation of RASSF1A and VHL may be useful diagnostic biomarkers for RCC. VHL methylation of cell-free DNA is suggestive of clear cell RCC. Total serum cell-free DNA may be a useful prognostic biomarker that may assist in tailoring postoperative surveillance and therapy. External prospective validation of these data will be required.

Cell-free Nucleic Acids as Potential Markers for Preeclampsia

Preeclampsia is one of the leading causes of maternal and fetal/neonatal mortality and morbidity worldwide. Therefore, widely applicable and affordable tests are needed to make an early diagnosis before the occurrence of the clinical symptoms. Circulating cell-free nucleic acids in plasma and serum are novel biomarkers with promising clinical applications in different medical fields, including prenatal diagnosis. Quantitative changes of cell-free fetal (cff)DNA in maternal plasma as an indicator for impending preeclampsia have been reported in different studies, using real-time quantitative PCR for the male-specific SRY or DYS 14 loci. In case of early onset preeclampsia, elevated levels may be already seen in the first trimester. The increased levels of cffDNA before the onset of symptoms may be due to hypoxia/reoxygenation within the intervillous space leading to tissue oxidative stress and increased placental apoptosis and necrosis. In addition to the evidence for increased shedding of cffDNA into the maternal circulation, there is also evidence for reduced renal clearance of cffDNA in preeclampsia. As the amount of fetal DNA is currently determined by quantifying Y-chromosome specific sequences, alternative approaches such as the measurement of total cell-free DNA or the use of gender-independent fetal epigenetic markers, such as DNA methylation, offer a promising alternative. Cell-free RNA of placental origin might be another potentially useful biomarker for screening and diagnosis of preeclampsia in clinical practice. Fetal RNA is associated with subcellular placental particles that protect it from degradation. Its levels are ten-fold higher in pregnant women with preeclampsia compared to controls. In conclusion, through the use of gender-independent sequences, the universal incorporation of fetal nucleic acids into routine obstetric care and into screening or diagnostic settings using combined markers may soon become a reality. Effort has now to be put into the establishment of standardized and simplified protocols for the analysis of these biomarkers in a clinical setting.