cfDNA Quality Research Articles

8C.07

Objective: Tumor cells undergoing apoptosis or necrosis release cell-free DNA fragments (cf-DNA) of different sizes into the bloodstream. Primary aldosteronism (PA) caused by aldosterone-producing adenoma (APA), but not by bilateral hyperplasia (BAH), has somatic mutations in KCNJ5 gene. Hence, the detection of KCNJ5 mutations in cf-DNA from peripheral blood could allow pinpointing PA patients with APA that must be submitted to Adrenal Vein Sampling (AVS). The aim of the study was to investigate the feasibility of using cf-DNA to detect KCNJ5 mutations in plasma of PA patients. Design and method: Plasma was collected from the right/left adrenal veins from 6 APA patients undergoing AVS. Plasma from 7 patients with stomach cancer and from 6 healthy subjects was used as positive/negative control, respectively, for cf-DNA quality. The integrity index (DII) was calculated as a ratio of 400pb/200pb amplicons. A DII cut off equal or greater than 1.0 was assumed to denote cf-DNA integrity. DNA sequences entailing the region with KCNJ5 gene mutations (G151R, L168R, T158A) were amplified using PCR real time (qPCR) to obtain long (400 bp) and short (200 bp) fragments. Results: The DII for KCNJ5 amplicons on average was consistently > 1.0 in gastric cancer samples, 1.0 in healthy subjects whereas it was < 1.0 in APA patients. The cf-DNA concentration of KCNJ5 amplicons was 10-fold higher in gastric cancer patients than in AVS plasma (4.8 ± 1 vs 0.4 ± 0.1 ng/ul p < 0.01). The latter showed no significant differences between the APA and the contralateral side (0.41 ± 0.1 vs 0.36 ± 0.1). Conclusions: These results confirm the feasibility of isolating cf-DNA not only from patients with malignancies, but also with PA. With current technology the cf-DNA amount and integrity that were obtained suggest the feasibility of using this strategy to detect malignancies. However, at present the results obtained in this study do not support the use of this approach to pinpoint PA with APA based on identification of KCNJ5 mutations. Therefore, further work is needed to develop this innovative and non-invasive strategy that could be useful to pinpoint the patients with APA before the AVS.

Circulating cf-DNA: A promising, noninvasive tool for assessment of early cardio-metabolic risk

In 1948, Mandel and colleagues were the first group to report the presence of circulating nucleic acids in the human bloodstream [1]. The origin of cell-free DNA (cf-DNA) is not fully understood, but apoptosis, necrotic death, cell lysis and active release are possible mechanisms of cf-DNA release into the blood circulation [2]. Circulating cf-DNA is a recent area of increased research interest, with nearly 9000 related papers in the PubMed library. This research has explored a wide range of topics, including the uses of circulating cf-DNA as a cancer prognostic factor, an indicator for intensive care unit admission, and a diagnostic marker of carcinogenesis, injury, sepsis and autoimmune disease [2e5]. The pathophysiological significance of circulating cf-DNA in cardiovascular disease has not received much attention until recently. However, scattered experimental and clinical studies have shed light upon this interesting issue [6e8]. Ten years ago, a clinical study by Chang et al. showed elevated circulating cf-DNA in patients with myocardial infarction [6]. Recently, an observational cohort study clearly demonstrated that plasma cf-DNA levels are a useful biomarker for predicting the outcome after out-of-hospital

Fatal Outcome in Bacteremia is Characterized by High Plasma Cell Free DNA Concentration and Apoptotic DNA Fragmentation: A Prospective Cohort Study

IntroductionRecent studies have shown that apoptosis plays a critical role in the pathogenesis of sepsis. High plasma cell free DNA (cf-DNA) concentrations have been shown to be associated with sepsis outcome. The origin of cf-DNA is unclear.MethodsTotal plasma cf-DNA was quantified directly in plasma and the amplifiable cf-DNA assessed using quantitative PCR in 132 patients with bacteremia caused by Staphylococcus aureus, Streptococcus pneumoniae, ß-hemolytic streptococcae or Escherichia coli. The quality of cf-DNA was analyzed with a DNA Chip assay performed on 8 survivors and 8 nonsurvivors. Values were measured on days 1–4 after positive blood culture, on day 5–17 and on recovery.ResultsThe maximum cf-DNA values on days 1–4 (n = 132) were markedly higher in nonsurvivors compared to survivors (2.03 vs 1.26 ug/ml, p<0.001) and the AUCROC in the prediction of case fatality was 0.81 (95% CI 0.69–0.94). cf-DNA at a cut-off level of 1.52 ug/ml showed 83% sensitivity and 79% specificity for fatal disease. High cf-DNA (>1.52 ug/ml) remained an independent risk factor for case fatality in a logistic regression model. Qualitative analysis of cf-DNA showed that cf-DNA displayed a predominating low-molecular-weight cf-DNA band (150–200 bp) in nonsurvivors, corresponding to the size of the apoptotic nucleosomal DNA. cf-DNA concentration showed a significant positive correlation with visually graded apoptotic band intensity (R = 0.822, p<0.001).ConclusionsPlasma cf-DNA concentration proved to be a specific independent prognostic biomarker in bacteremia. cf-DNA displayed a predominating low-molecular-weight cf-DNA band in nonsurvivors corresponding to the size of apoptotic nucleosomal DNA.

Aging is associated with quantitative and qualitative changes in circulating cell-free DNA: The Vitality 90+ study

As a marker of cellular death, cell-free DNA (cf-DNA) has a utility in diagnosis and prognosis of various disorders. Since aging accompanies increased cellular senescence and death, we aimed to characterize potential age-related alterations in cf-DNA. The study population consisted of 12 nonagenarian women (participants in the Vitality 90+ Study) and 11 healthy control women (aged 22–37 years). Some of the nonagenarians ( n = 8) were also recruited for follow-up after one year. cf-DNA was extracted using two different methods. Total cf-DNA was quantified directly in plasma and the amplifiable cf-DNA was assessed using quantitative PCR. Quality of cf-DNA was analysed with a DNA Chip assay. For all the quantification methods, the concentration of cf-DNA was significantly higher ( p < 0.05) in nonagenarians as compared to controls. The quality of the cf-DNA also displayed a marked difference between nonagenarians and controls; a fragmented pattern or appearance of low molecular weight cf-DNA was observed in the majority of the nonagenarians, whereas in controls, cf-DNA was intact and had a quasi-genomic, high molecular weight appearance. In nonagenarians, the quality of cf-DNA appeared similar in the original and follow-up samples. We propose that some, as yet uncharacterized, aspects of aging are reflected in the appearance of cf-DNA.