Plasma cfDNA Levels Research Articles

Quantitative Cell-Free DNA, KRAS, and BRAF Mutations in Plasma from Patients with Metastatic Colorectal Cancer during Treatment with Cetuximab and Irinotecan

The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma. Inclusion criteria were histopathologically verified chemotherapy-resistant mCRC, adequate Eastern Cooperative Oncology Group performance status, and organ function. Treatment consisted of irinotecan being administered at 350 mg/m(2) for 3 weeks and weekly administration of 250 mg/m(2) cetuximab until progression or unacceptable toxicity. A quantitative PCR method was developed to assess the number of cfDNA alleles and KRAS and BRAF mutation alleles in plasma at baseline. The study included 108 patients. Only three patients were positive for BRAF mutations. The majority of KRAS mutations detected in tumors were also found in the plasma [32 of 41 (78%)]. Plasma cfDNA and plasma mutant KRAS levels (pmKRAS) were strongly correlated (r = 0.85, P < 10(-4)). The disease control rate was 77% in patients with low cfDNA (<25% quartile) and 30% in patients with high cfDNA [>75% quartile (P = 0.009)]. Patients with pmKRAS levels higher than 75% had a disease control rate of 0% compared with 42% in patients with lower pmKRAS (P = 0.048). Cox analysis confirmed the prognostic importance of both cfDNA and pmKRAS. High levels were clear indicators of a poor outcome. KRAS analysis in plasma is a viable alternative to tissue analysis. Quantitative levels of cfDNA and pmKRAS are strongly correlated and hold promise of clinical application.

Plasma Cell-Free DNA Levels Are Elevated in Acute Puumala Hantavirus Infection

IntroductionPuumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome called nephropathia epidemica (NE). The aim of the present study was to evaluate plasma cell-free DNA (cf-DNA) levels and urinary cf-DNA excretion in acute NE as well as their associations with the severity of the disease.MethodsTotal plasma cf-DNA was quantified directly in plasma of 61 patients and urine of 20 patients with acute NE. We also carried out a qualitative high-sensitivity lab-on-a-chip DNA assay in 20 patients to elucidate the appearance of cf-DNA in plasma and urine.ResultsThe maximum plasma cf-DNA values taken during acute NE were significantly higher than the control values taken after the hospitalization period (median 1.33 µg/ml, range 0.94–3.29 µg/ml vs. median 0.77 µg/ml, range 0.55–0.99 µg/ml, P<0.001). The maximum plasma cf-DNA levels correlated positively with maximum blood leukocyte count (r = 0.388, P = 0.002) and the length of hospital stay (r = 0.376, P = 0.003), and inversely with minimum blood platelet count (r = −0.297, P = 0.020). Qualitative analysis of plasma cf-DNA revealed that in most of the patients cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA (150–200 bp). The visually graded maximum cf-DNA band intensity correlated positively with the maximum quantity of total plasma cf-DNA (r = 0.513, P = 0.021). Maximum urinary excretion of cf-DNA in turn was not markedly increased during the acute phase of NE and did not correlate with any of the variables reflecting severity of the disease or with the maximum plasma cf-DNA level.ConclusionsThe plasma levels of cf-DNA are elevated during acute PUUV infection and correlate with the apoptotic cf-DNA-band intensity. The plasma cf-DNA concentration correlates with some variables reflecting the severity of the disease. The urinary excretion of cf-DNA does not reflect the degree of inflammation in the kidney.

Quantitative analysis of cell-free DNA in the plasma of gastric cancer patients

In the present study, an accurate and reproducible method for quantifying cell-free DNA (cfDNA) in human blood was established and tested for its ability to predict gastric cancer in patients. Using 'Alu81-qPCR' to amplify 81-bp Alu DNA sequences, we first estimated the amount of cfDNA in the serum or plasma of 130 patients with gastric cancer to identify which source of cfDNA is more suitable for the biomarker screening of these patients. The results of Alu81-qPCR revealed that the amount of cfDNA in the plasma was low compared with that in the serum, but was found at similar levels among the samples, indicating that the plasma may be a more suitable source of cfDNA for biomarker screening. For the 54 patients with gastric cancer and the 59 age-matched healthy controls, the mean levels of plasma cfDNA were 2.4-fold higher in the patient group compared with the control group, indicating that plasma cfDNA levels may be useful for predicting patients with gastric cancer. The results of our study suggest that Alu81-qPCR is a more reliable method than other techniques, such as the PicoGreen assay, for quantifying cfDNA in human blood, demonstrating the potential to complement current diagnostic procedures for the management of gastric cancer patients.

Plasma Cell-Free DNA Levels in Children on Peritoneal Dialysis

Background/Aims: Plasma levels of cell-free DNA (cfDNA) are elevated in various clinical conditions including cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. Previously, increased cfDNA levels were reported during hemodialysis. However, there is limited data regarding cfDNA levels in peritoneal dialysis (PD) patients. The aim of this study was to investigate the levels of cfDNA in children on PD. Methods: Twenty-one children on PD (median age: 12; range: 4–18 years) and 21 healthy children (median age: 10; range: 6–16 years) were enrolled into the study. Plasma cfDNA was measured using a real-time quantitative PCR for the beta-globin gene. Results: The median concentrations of cfDNA in the plasma of PD patients and healthy controls were 2,205 genome-equivalents/ml of plasma (range: 39–5,845) and 1,033 genome-equivalents/ml of plasma (range: 254–5,116), respectively (p = 0.026). A significant positive correlation was observed between C-reactive protein levels and plasma cfDNA levels (r: 0.52, p < 0.0001). Conclusion: Our data have demonstrated for the first time that cfDNA is increased in children on PD treatment. However, the mechanism by which the levels of cfDNA is increased and the clinical significance of this finding in PD patients is unclear. Further studies are warranted to clarify the precise mechanism and clinical significance of elevated cfDNA in children on PD.

Cell-Free Plasma DNA during Hemodialysis

Purpose. Cell-free plasma DNA (cfDNA) levels originating predominantly from apoptotic leukocytes were found to rise during hemodialysis (HD) session, and as such are considered a marker of HD membrane biocompatibility. The purpose of our study was to determine the changes of cfDNA during two consecutive high-flux polysulphone HD sessions after a long (HD-L) and short (HD-S) interdialytic interval. Methods. A total of 17 HD patients were examined. Prior to HD and at 30 and 240 min, cfDNA (using real-time PCR) and leukocyte count were determined. Results. No significant difference was found when comparing pre-HD-S with pre-HD-L cfDNA [4893 (1090–28804) vs. 4589 (691–73796) genomic equivalent/mL]. A significant increase in cfDNA at 240 min was seen in HD-L (p < 0.05) but not in HD-S. Leukocyte count correlated with cfDNA levels before HD-S (r = 0.8; p < 0.01); however, no other correlation was seen between routinely measured biochemical markers and pre-HD cfDNA levels or cfDNA changes during HD. The increase in plasma cfDNA during HD did not correlate with dialysis duration, its efficacy, or ultrafiltration. An association between magnitude of diuresis and cfDNA levels in HD-S was found (r = 0.58; p < 0.05). Conclusions. The behavior of cfDNA during HD after long and short interdialytic interval is inconsistent and cannot be explained by changes in laboratory and clinical parameters. The observed relationship of plasma cfDNA levels with diuresis deserves further investigation.