Plasma DNA Levels Research Articles

Plasma T790M and HGF as potential predictive markers for EGFR-TKI re-challenge.

Re-challenge with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) has been suggested to potentially improve survival in certain populations of patients with advanced lung cancer, but predictive markers for the success of EGFR-TKI re-challenge have not been identified. The present study analyzed 16 re-challenges with EGFR-TKI undertaken in 12 patients with lung adenocarcinoma by investigating T790M and hepatocyte growth factor (HGF) in plasma coupled with clinical characteristics. EGFR mutations in plasma DNA were detected using the wild inhibiting PCR and quenched probe system for exon 19 deletions, and T790M and L858R were detected using the mutation-biased PCR and quenched probe system. HGF levels in the plasma were measured by enzyme-linked immunosorbent assay, and the ratio of HGF levels prior to re-challenge to those prior to the previous EGFR-TKI treatment was calculated. Two re-challenges demonstrated partial response, six remained as stable disease and eight had progressive disease (PD). A total of 4 of the 5 patients with a history of T790M positivity based on plasma DNA levels had PD. A total of 7 of the 8 patients who had ≥1.5-fold elevation of HGF prior to re-challenge with EGFR-TKI suffered PD. Elevation of the HGF ratio to ≥1.5 was significantly associated with poor response to EGFR-TKI re-challenge. Having no history of T790M and an HGF ratio <1.5 was significantly associated with a positive response to EGFR-TKI re-challenge. A combination of T790M detection and HGF quantification using plasma is a potentially useful assay system for predicting the effect of EGFR-TKI re-challenge. Future prospective studies are required to confirm the predictive validity of these markers.

The Alphabet Soup of HIV Reservoir Markers.

Despite the success of antiretroviral therapy in suppressing HIV, life-long therapy is required to avoid HIV reactivation from long-lived viral reservoirs. Currently, there is intense interest in searching for therapeutic interventions that can purge the viral reservoir to achieve complete remission in HIV patients off antiretroviral therapy. The evaluation of such interventions relies on our ability to accurately and precisely measure the true size of the viral reservoir. In this review, we assess the most commonly used HIV reservoir assays, as a clear understanding of the strengths and weaknesses of each is vital for the accurate interpretation of results and for the development of improved assays. The quantification of intracellular or plasma HIV RNA or DNA levels remains the most commonly used tests for the characterization of the viral reservoir. While cost-effective and high-throughput, these assays are not able to differentiate between replication-competent or defective fractions or quantify the number of infected cells. Viral outgrowth assays provide a lower bound for the fraction of cells that can produce infectious virus, but these assays are laborious, expensive and substantially underestimate the potential reservoir of replication-competent provirus. Newer assays are now available that seek to overcome some of these problems, including full-length proviral sequencing, inducible HIV RNA assays, ultrasensitive p24 assays and murine adoptive transfer techniques. The development and evaluation of strategies for HIV remission rely upon our ability to accurately and precisely quantify the size of the remaining viral reservoir. At this time, all current HIV reservoir assays have drawbacks such that combinations of assays are generally needed to gain a more comprehensive view of the viral reservoir. The development of novel, rapid, high-throughput assays that can sensitively quantify the levels of the replication-competent HIV reservoir is still needed.

Monitoring tumor specific mutations in plasma during systemic treatment of biliary tract cancer.

318 Background: We have previously reported a rate of 23% RAS/RAF mutations in plasma from patients with KRAS exon 2 and 3 wild-type, non-resectable biliary tract tumors. We wanted to explore the changes in circulating tumor specific DNA (ctDNA) during systemic chemotherapy in these patients. Methods: Patients with non-resectable biliary tract cancer treated within a phase II trial were included if they had KRAS exon 2 and 3 wild-type tumor tissue, had quantifiable levels of tumor specific DNA in plasma and progressive disease on imaging. They received gemcitabine, oxaliplatin and capecitabine with either bevacizumab or panitumumab. Treatment continued for up to six months until progression. Blood sampling and evaluation according to RECIST 1.1 were done every 12 weeks. Droplet Digital PCR was performed on DNA isolated from 4 ml plasma. A pre-amplification step was done and adequate positive and negative controls were included. The extended RAS and BRAF mutation analysis covered 20 mutations in KRAS exons 3/4, NRAS exon 2/3, PIK3CA and BRAF V600E. The percentage of tumor specific DNA relative to total DNA was reported. Results: The inclusion criteria were met by 13 patients, 10 women and three men. The typical pattern was seen in eight cases, where the percentage of tumor specific DNA dropped at least half during therapy and rose at least two-fold at progression. In three patients, a baseline sample was not available or there was not an initial drop, but the ctDNA rose at progression. One patient had an initial drop, but not a rise a progression based on imaging. The last patient progressed rapidly. Conclusions: This exploratory analysis pointed toward changes in percentage of tumor specific mutations in plasma as a marker of effect and progression. Dynamics of liquid biopsies is a promising tool in monitoring biliary tract cancer patients during systemic therapy. Clinical trial information: NCT01206049.

Associations among Cognitive Functions, Plasma DNA, and White Matter Integrity in Patients with Early-Onset Parkinson's Disease.

Early-onset Parkinson's disease (EOPD) patients are symptomatic at a relatively young age, and the impacts of the disease on both the patients and their caregivers are dramatic. Few studies have reported on the cognitive impairments seen in EOPD, and the results of these studies have been diverse. Furthermore, it is still unclear what microstructural white matter (WM) changes are present in EOPD patients. As such, we conducted this study to investigate the microstructural WM changes experienced by EOPD patients and their association with cognitive function and plasma DNA levels. We enrolled 24 EOPD patients and 33 sex- and age-matched healthy volunteers who underwent complete neuro-psychological testing (NPT) to evaluate their cognitive function and diffusion tensor imaging (DTI) scanning to determine their fiber integrity. The plasma DNA measurements included measurements of nuclear and mitochondrial DNA levels. Fractional anisotropy (FA) maps were compared using voxel-based statistics to determine differences between the two groups. The differences in DTI indices and NPT scores were correlated after adjusting for age, sex, and education. Our results demonstrate that patients with EOPD have elevated nuclear DNA levels and wide spectrums of impairments in NPT, especially in the executive function and visuospatial function domains. Exploratory group-wise comparisons of the DTI indices revealed that the patients with EOPD exhibited lower DTI parameters in several brain locations. These poorer DTI parameters were associated with worse cognitive performances and elevated plasma nuclear DNA levels, especially in the anterior thalamic radiation region. Our findings suggest that the thalamus and its adjacent anterior thalamic radiation may be important in the pathogenesis of EOPD, as they appear to become involved in the disease process at an early stage.

Plasma DNA Mediate Autonomic Dysfunctions and White Matter Injuries in Patients with Parkinson's Disease.

Background. Cardiovascular autonomic dysfunction is well known in Parkinson's disease (PD) presentation and it produces hypoperfusion of vital organs. The association between cardiovascular autonomic dysfunction and oxidative stress was examined in previous animal models. Oxidative stress and neuroinflammation were thought to have roles in PD pathogenesis. Owing to the relative low intrinsic antioxidative properties, brain white matter (WM) is vulnerable to the oxidative stress. This study is conducted to examine possible relationships by using a hypothesis-driven mediation model. Methods. Twenty-nine patients with PD and 26 healthy controls participated in this study, with complete examinations of cardiac autonomic parameters, plasma DNA level, and WM integrity. A single-level three-variable mediation model was used to investigate the possible relationships. Results. The elevated serum oxidative stress biomarkers include plasma nuclear DNA and mitochondrial DNA, and poorer cardiac autonomic parameters and multiple regional microstructural WM changes are demonstrated. Further mediation analysis shows that plasma nuclear DNA served as the mediators between poorer baroreflex sensitivity and mean diffusivity changes in cingulum. Conclusions. These results provide a possible pathophysiology for how the poor baroreflex sensitivity and higher oxidative stress adversely impacted the WM integrity. This model could provide us with a piece of the puzzle of the entire PD pathogenesis.

Torque Teno Virus (TTV) and Epstein–Barr Virus (EBV) Viral Load in Peripheral Blood as a Biomarker of “Net Immunosuppression”: Factors Influencing Viral Load in Adult Patients Awaiting Solid Organ Transplantation

BackgroundQuantitative measurement of DNA levels of persistent highly prevalent viral infections such as Torque Teno Virus (TTV) and Epstein–Barr Virus (EBV) that are highly dependent on T-cell responses for control have been proposed as possible biomarkers of “net immunosuppression.”MethodsWe examined factors influencing TTV in 51 adult patients awaiting solid organ transplantation (15 kidney, 15 liver/small bowel, 10 heart, 11 lung) and 51 healthy age- and sex-matched controls. Samples from all patients were tested for TTV DNA (plasma) using quantitative nucleic acid testing (QNAT), EBV and cytomegalovirus (CMV) serology, CMV DNA (QNAT of plasma, urine, saliva), and EBV DNA (QNAT of whole blood [WB], saliva). Effects of age, sex, organ, CMV serology, and EBV DNA on TTV DNA were explored in univariate and multivariate models.ResultsPatients and controls were well-matched for age (median 48.4 vs. 49.2 years), sex 64.7% M, both CMV seropositivity (45.1% vs. 51%) and EBV seropositivity (94.1% vs. 98.0%), and detection of EBV DNA in saliva (52.9% vs. 50.0%) and WB (28.6% vs. 16.3%). TTV DNA was detected more frequently and at higher levels (Figure 1) in patients (86.3%) vs. controls (66.7%). TTV DNA plasma levels were age-dependent for patients but not controls (<50 years vs. >50 years, P = 0.021), and were only higher in patients >50 years vs. controls (P = 0.003). Only two patients and no controls had CMV DNA detected. Neither CMV serostatus nor sex impacted TTV DNA levels in patients or controls. Patients awaiting lung transplant had lower TTV levels compared with those awaiting liver (P = 0.04) or heart transplant (P = 0.03). A significant correlation between plasma TTV DNA levels and WB EBV DNA (r = 0.28, P = 0.05) but not saliva EBV DNA levels was observed in patients (Figure 2); only WB EBV DNA level was a significant predictor of high (≥3rd quartile of control) TTV DNA levels in patients in multivariate analysis (P = 0.026). Four of the five patients tested before and 27–285 days after transplant had a significant post-transplant rise in TTV DNA levels (Figure 3).ConclusionMeasurement of TTV DNA in plasma alone or combined with EBV DNA in WB may be a useful marker for measuring global immunosuppression including age-related immunosenescence in subjects not receiving exogenous immunosuppression.Disclosures All authors: No reported disclosures.

P2.03b-039 Cell-Free (cf) DNA and cfRNA levels in Plasma of Lung Cancer Patients Indicate Disease Status and Predict Progression: Topic: Biomarkers

Cell-free circulating tumor DNA (ctDNA) and RNA (ctRNA) can be extracted from plasma of cancer patients (pts). Measuring dynamic changes in gene expression, allele-fractions of mutations and levels of nucleic acids per ml of plasma in metastatic patients has shown great potential for monitoring disease state and predicting outcome to antitumoral therapy in advance of imaging. We designed a clinical trial to measure gene levels of plasma ctDNA and ctRNA in metastatic pts with NSCLC, breast, and GI malignancies under treatment and correlate the levels with CT scans done assessing response for one-year clinical trial. CtDNA/ctRNA were extracted from plasma separated from patient whole-blood draws shipped at ambient temperature. CtRNA was reverse transcribed with random primers to cDNA. Levels of ctDNA/ctRNA were determined by real-time qPCR. Results of ctDNA/ctRNA tests were compared with responses (CR, PR, SD or PD) as determined by the CT scans. Levels of PD-L1 expression relative to β–actin were determined by qPCR. We have enrolled 39 pts and we are presenting data from 15 pts with NSCLC. The first-line treatments were Carboplatin (40%, 6/15), Opdivo (26.7%, 4/15), Afatinib (13.3%, 2/15), Ceritinib (6.7%, 1/15), Crizotinib (6.7%, 1/15), and Taxotere/Cyramza (6.7%, 1/15). Histological subtypes were 73.3% (11/15) adeno, 20% (3/15) squamous, and 6.7% (1/15) other. The majority of patients were Caucasian (66.7%, 10/15), Hispanic (26.7%, 4/15), and African American (6.7%, 1/15). Levels of ctDNA and ctRNA were significantly correlated with one another across patient blood draws (r = 0.5781, p < 0.0001). After the first 2 cycles of therapy, 9 of the 15 patients achieved SD, of whom 8 were analyzed for ctDNA/ctRNA levels. In 7/8 of pts who had SD indicated by CT scan, CtDNA/ctRNA levels were stable, while the 2 PD pts showed significant increases in levels of ctDNA/ctRNA 4.8 weeks prior to progression. In the 2 SD pts treated with Opdivo, PD-L1 expression was stable during the cycles when CT scans also indicated an SD status. In almost 90% of the pts, constant ctDNA/ctRNA levels correlated with stable disease status as seen by CT; moreover, in 2 pts changes in the levels of both ct nucleic acids predicted progression about 5 weeks before CT scans. Importantly, the concordance between cDNA and cfRNA suggest that cfRNA can just as effective as cfDNA as a prognostic tool, while adding the extra dimension of gene expression.

Potential biomarkers to follow the progression and treatment response of Huntington's disease.

Huntington's disease (HD) is a rare genetic disease caused by expanded polyglutamine repeats in the huntingtin protein resulting in selective neuronal loss. Although genetic testing readily identifies those who will be affected, current pharmacological treatments do not prevent or slow down disease progression. A major challenge is the slow clinical progression and the inability to biopsy the affected tissue, the brain, making it difficult to design short and effective proof of concept clinical trials to assess treatment benefit. In this study, we focus on identifying peripheral biomarkers that correlate with the progression of the disease and treatment benefit. We recently developed an inhibitor of pathological mitochondrial fragmentation, P110, to inhibit neurotoxicity in HD. Changes in levels of mitochondrial DNA (mtDNA) and inflammation markers in plasma, a product of DNA oxidation in urine, mutant huntingtin aggregates, and 4-hydroxynonenal adducts in muscle and skin tissues were all noted in HD R6/2 mice relative to wild-type mice. Importantly, P110 treatment effectively reduced the levels of these biomarkers. Finally, abnormal levels of mtDNA were also found in plasma of HD patients relative to control subjects. Therefore, we identified several potential peripheral biomarkers as candidates to assess HD progression and the benefit of intervention for future clinical trials.

Neutrophil Extracellular Traps Promote Angiogenesis: Evidence From Vascular Pathology in Pulmonary Hypertension.

Inflammation and dysregulated angiogenesis are features of endothelial dysfunction in pulmonary hypertension. Neutrophil extracellular traps (NETs), produced by dying neutrophils, contribute to pathogenesis of numerous vascular disorders but their role in pulmonary hypertension has not been studied. We sought evidence of (NETs) formation in pulmonary hypertension and investigated the effect of NETs on endothelial function. Plasma and lung tissues of patients with pulmonary hypertension were analyzed for NET markers. The effects of NETs on endothelial function were studied in vitro and in vivo. Patients with chronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension showed elevated plasma levels of DNA, neutrophil elastase, and myeloperoxidase. NET-forming neutrophils and extensive areas of NETosis were found in the occlusive plexiform lesions and vascularized intrapulmonary thrombi. NETs induced nuclear factor κB-dependent endothelial angiogenesis in vitro and increased vascularization of matrigel plugs in vivo. Angiogenic responses were associated with increased release of matrix metalloproteinase-9, heparin-binding epidermal growth factor-like growth factor, latency-associated peptide of the transforming growth factor β1, and urokinase-type plasminogen activator, accompanied by increased endothelial permeability and cell motility. NETs-induced responses depended on myeloperoxidase/H2O2-dependent activation of Toll-like receptor 4/nuclear factor κB signaling. NETs stimulated the release of endothelin-1 in HPAECs (human pulmonary artery endothelial cells) and stimulated pulmonary smooth muscle cell proliferation in vitro. We are the first to implicate NETs in angiogenesis and provide a functional link between NETs and inflammatory angiogenesis in vitro and in vivo. We demonstrate the potential pathological relevance of this in 2 diseases of disordered vascular homeostasis, pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension.

Plasma and urine DNA levels are related to microscopic hematuria in patients with bladder urothelial carcinoma

a) Objective: An increase in cell-free DNA was observed in the plasma of many cancer patients. This major biomarker can be used to differentiate patients with malignant neoplasms from those with benign neoplasms or healthy patients. Depending on the characteristic of the tumor, there are qualitative variations in the circulating cell-free DNA. Today, studies on the concentration of fragments of circulating cell-free DNA and their respective sizes in patients with bladder cancer are not plentiful in the literature. A 100% effective plasma tumor marker, which would help in the diagnosis and follow-up of bladder cancer, is yet to be developed; therefore, cell-free DNA levels in the plasma may represent a valuable biomarker for the diagnosis, prognosis and follow-up of patients with this type of tumor. b) Design and methods: In this study we analyze the kinetics of plasma and urine DNA concentrations in patients with bladder cancer, relating them to the other clinical laboratory variables. c) Results: Patients with hematuria showed a positive correlation with urine DNA. d) Conclusion: An increase in plasma and urine DNA was unprecedentedly reported over time, a fact that may come in handy in the prognosis of patients. Furthermore, microscopic haematuria is correlated with plasma and urinary DNA levels.

Digestion of Chromatin in Apoptotic Cell Microparticles Prevents Autoimmunity

Antibodies to DNA and chromatin drive autoimmunity in systemic lupus erythematosus (SLE). Null mutations and hypomorphic variants of the secreted deoxyribonuclease DNASE1L3 are linked to familial and sporadic SLE, respectively. We report that DNASE1L3-deficient mice rapidly develop autoantibodies to DNA and chromatin, followed by an SLE-like disease. Circulating DNASE1L3 is produced by dendritic cells and macrophages, and its levels inversely correlate with anti-DNA antibody response. DNASE1L3 is uniquely capable of digesting chromatin in microparticles released from apoptotic cells. Accordingly, DNASE1L3-deficient mice and human patients have elevated DNA levels in plasma, particularly in circulating microparticles. Murine and human autoantibody clones and serum antibodies from human SLE patients bind to DNASE1L3-sensitive chromatin on the surface of microparticles. Thus, extracellular microparticle-associated chromatin is a potential self-antigen normally digested by circulating DNASE1L3. The loss of this tolerance mechanism can contribute to SLE, and its restoration may represent a therapeutic opportunity in the disease.

Granulocyte colony-stimulating factor (G-CSF) increases histone-complexed DNA plasma levels in healthy volunteers

Granulocyte colony-stimulating factor (G-CSF) is an activator of neutrophil granulocytes. Neutrophil extracellular traps are a defensive mechanism consisting of neutrophils, platelets, DNA, histones and antimicrobial proteins. This study was performed to determine whether G-CSF increases histone-complexed DNA in the plasma of healthy volunteers. In total, 51 healthy volunteers (25 males and 26 females) were treated with G-CSF (18 with 300 µg single dose i.v., 27 with 5 µg/kg s.c. for 4 days) and six participants received a placebo. Histone-complexed DNA was measured by enzyme immunoassay in plasma samples at predefined time points (0, 2, 4, 6, 24 h after single dose, day 1, day 2 and day 5 after repeated doses). Histone levels were quantified by Western blotting. A single dose of G-CSF rapidly increased hc-DNA by about 50 % (p < 0.05 for 2–24 h). After repeated doses the increase was even more pronounced: hc-DNA increased by about 50 % (3.0 ± 0.9, p < 0.001 after 24 h and about fourfold after 96 h (p < 0.001)). A statistical significant increase in histone levels was detected as early as 4 h after G-CSF injection (0.43 ± 0.2 vs. 1.08 ± 0.3 µg/ml; p = 0.034). In the placebo group no significant changes occurred. Moreover, significantly higher levels of hc-DNA were measured in male compared to female subjects (226 ± 43 vs. 84 ± 19, p < 0.001). G-CSF injection substantially increases hc-DNA levels in healthy volunteers. There is a significant gender difference in hc-DNA at the baseline.

Genomic Alterations in Liquid Biopsies from Patients with Bladder Cancer

BackgroundAt least half of the patients diagnosed with non–muscle-invasive bladder cancer (NMIBC) experience recurrence and approximately 15% will develop progression to muscle invasive or metastatic disease. Biomarkers for disease surveillance are urgently needed. ObjectiveDevelopment of assays for surveillance using genomic variants in cell-free tumour DNA from plasma and urine. Design, setting, and participantsRetrospective pilot study of 377 samples from 12 patients with recurrent or progressive/metastatic disease. Three next-generation sequencing methods were applied and somatic variants in DNA from tumour, plasma, and urine were subsequently monitored by personalised assays using droplet digital polymerase chain reaction (ddPCR). Samples were collected from 1994 to 2015, with up to 20 yr of follow-up. Outcome measurements and statistical analysisProgression to muscle-invasive or metastatic bladder cancer; t test for ddPCR data. Results and limitationsWe developed from one to six personalised assays per patient. Patients with progressive disease showed significantly higher levels of tumour DNA in plasma and urine before disease progression, compared with patients with recurrent disease (p=0.032 and 1.3×10−6, respectively). Interestingly, tumour DNA was detected in plasma and urine in patients with noninvasive disease, being no longer detectable in disease-free patients. A significant level of heterogeneity was observed for each patient; this could be due to tumour heterogeneity or assay performance. ConclusionsCell-free tumour DNA can be detected in plasma and urine, even in patients with noninvasive disease, with high levels of tumour DNA detectable before progression, especially in urine samples. Personalised assays of genomic variants may be useful for disease monitoring. Patient summaryTumour DNA can be detected in blood and urine in early and advanced stages of bladder cancer. Measurement of these highly tumour-specific biomarkers may represent a novel diagnostic tool to indicate the presence of residual disease or to discover aggressive forms of bladder cancer early in the disease course.

Epstein-Barr Virus and Classical Hodgkin’s Lymphoma

Among oncogenic human viruses, the Epstein-Barr virus (EBV) drew special attention due to its unique properties. Being widespread among the population of the planet, the virus is also a leader in the number of associated different benign and malignant neoplasms of lymphoid and epithelial origin. The oncogenic potential of EBV is related to its ability to infect and transform human lymphocytes. In cases, when the interaction between reproduction of EBV, its latent state and immune control of the body is impaired, conditions for long-term proliferation of EBV-infected cells and their malignant transformation are formed. According to some investigators, the molecular mechanisms of EBV-associated carcinogenesis are due to the ability of the viral genome to promote the expression of series of products that simulate a number of growth factors and transcription and produce an anti-apoptotic effect. These products impair EBV-encoded signaling pathways that regulate a variety of cellular functions of homeostasis giving a cell the ability to proliferate indefinitely. However, the exact mechanism by which the EBV initiates tumor formation is not clear. The review provides summarized information on the structure and oncogenic potential of EBV, morphological and clinical cases of Hodgkin’s lymphoma (HL), and the role of EBV in the pathogenesis of types of HL associated with the virus. The review also dwells on the latest data on the use of EBV DNA plasma levels of patients with HL as a biomarker reflecting the effectiveness of the treatment performed and the prognosis of the disease.

Plasma Mitochondrial DNA Levels as a Biomarker of Lipodystrophy Among HIV-infected Patients Treated with Highly Active Antiretroviral Therapy (HAART).

Lipodystrophy is a common complication in HIV-infected patients taking highly active antiretroviral therapy. Its early diagnosis is crucial for timely modification of antiretroviral therapy. We hypothesize that mitochondrial DNA in plasma may be a potential marker of LD in HIV-infected individuals. In this study, we compared plasma mitochondrial DNA levels in HIV-infected individuals and non-HIV-infected individuals to investigate its potential diagnostic value.Total plasma DNA was extracted from 67 HIV-infected patients at baseline and 12, 24 and 30 months after initiating antiretroviral therapy. Real-time quantitative PCR was used to determine the mitochondrial DNA levels in plasma. Lipodystrophy was defined by the physician-assessed presence of lipoatrophy or lipohypertrophy in one or more body regions.The mitochondrial DNA levels in plasma were significantly higher at baseline in HIV-infected individuals than in non-HIV-infected individuals (p<0.05). At month 30, 33 out of 67 patients (49.2%) showed at least one sign of lipodystrophy. The mean plasma mitochondrial DNA levels in lipodystrophy patients were significantly higher compared to those without lipodystrophy at month 24 (p<0.001). The receiver operating curve analysis demonstrated that using plasma mitochondrial DNA level (with cut-off value >5.09 log10 copies/ml) as a molecular marker allowed identification of patients with lipodystrophy with a sensitivity of 64.2% and a specificity of 73.0%.Our data suggest that mitochondrial DNA levels may help to guide therapy selection with regards to HIV lipodystrophy risk.

Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome

Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA–based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P < .05). Branched DNA–based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

Neutrophil Extracellular Traps Induce Trypsin Activation, Inflammation, and Tissue Damage in Mice With Severe Acute Pancreatitis

Neutrophils are involved in the development of acute pancreatitis (AP), but it is not clear how neutrophil-induced tissue damage is regulated. In addition to secreting antimicrobial compounds, activated neutrophils eliminate invading microorganisms by expelling nuclear DNA and histones to form extracellular web-like structures called neutrophil extracellular traps (NETs). However, NETs have been reported to contribute to organ dysfunction in patients with infectious diseases. We investigated whether NETs contribute to the development of AP in mice. AP was induced in C57BL/6 mice by infusion of taurocholate into the pancreatic duct or by intraperitoneal administration of L-arginine. Pancreata were collected and extracellular DNA was detected by Sytox green staining, levels of CXC chemokines, histones, and cytokines also were measured. Cell-free DNA was quantified in plasma samples. Signal transducer and activator of transcription 3 phosphorylation and trypsin activation were analyzed in isolated acinar cells. NETs were depleted by administration of DNase I to mice. Plasma was obtained from healthy individuals (controls) and patients with severe AP. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice and increased levels of cell-free DNA in plasma. Neutrophil depletion prevented taurocholate-induced deposition of NETs in the pancreas. Administration of DNase I to mice reduced neutrophil infiltration and tissue damage in the inflamed pancreas and lung, and decreased levels of blood amylase, macrophage inflammatory protein-2, interleukin 6, and high-mobility groups protein 1. In mice given taurocholate, DNase I administration also reduced expression of integrin α M (macrophage-1 antigen) on circulating neutrophils. Similar results occurred in mice with L-arginine-induced AP. Addition of NETs and histones to acinar cells induced formation of trypsin and activation of signal transducer and activator of transcription 3; these processes were blocked by polysialic acid. Patients with severe AP had increased plasma levels of NET components compared with controls. NETs form in the pancreata of mice during the development of AP, and NET levels are increased in plasma from patients with AP, compared with controls. NETs regulate organ inflammation and injury in mice with AP, and might be targeted to reduce pancreatic tissue damage and inflammation in patients.

Tissue factor-dependent and -independent pathways of systemic coagulation activation in acute myeloid leukemia: a single-center cohort study.

BackgroundIn acute myeloid leukemia (AML), disseminated intravascular coagulation (DIC) contributes to morbidity and mortality, but the underlying pathomechanisms remain incompletely understood.MethodsWe conducted a prospective study on 69 patients with newly diagnosed AML to further define the correlates of systemic coagulation activation in this hematological malignancy. Tissue factor procoagulant activity (TF PCA) of isolated peripheral blood mononuclear cells (PBMCs) and TF expression by circulating microparticles (MPs) were assessed by single-stage clotting and thrombin generation assay, respectively. Soluble plasma TF antigen and secretion of vascular endothelial growth factor (VEGF) by cultured PBMCs were measured by ELISA. Cell-free plasma DNA was quantified by staining with a fluorescent dye.ResultTF PCA of PBMCs was significantly increased in AML patients as compared to healthy controls. Furthermore, TF PCA was significantly associated with decompensated DIC at presentation, as defined by a plasma fibrinogen level of ≤1 g/L (n = 11). In addition to TF PCA and circulating blasts, serum lactate dehydrogenase, a surrogate marker for leukemic cell turnover, correlated with plasma D-Dimer in the total patient cohort and was significantly increased in DIC patients, suggesting a role for myeloblast apoptosis/necrosis in activation of the TF-dependent coagulation pathway. Consistently, TF-bearing plasma MPs were more frequently detected and levels of soluble TF antigen were significantly higher in DIC vs. non-DIC patients. No association was found between TF PCA expression and VEGF secretion by isolated PBMCs, but significantly increased levels of cell-free plasma DNA pointed to a contribution of the intrinsic contact pathway to systemic coagulation activation in the total patient cohort and in patients with lower TF PCA expression. While PBMC-associated TF PCA had no effect on long-term survival, DIC occurrence at presentation increased the risk of early mortality.ConclusionIn newly diagnosed AML, TF expression by PBMCs and shedding of TF-bearing plasma MPs are central to the pathogenesis of DIC, but additional pathways, such as DNA liberation, may contribute to systemic coagulation activation.

Detection of total and covalently closed circular HBV DNA in liver transplant recipients due to HBV-associated liver diseases

To investigate the levels of hepatitis B virus (HBV) total DNA and covalently closed circular (ccc) DNA in liver transplant recipients due to HBV-associated liver diseases and detemaine their clinical significance. Sixty patients undergoing liver transplantation (LT) due to HBV-associated liver diseases were enrolled for the study. Levels of HBV total and ccc DNA in plasma, liver and PBMCs were measured by RT-PCR. The ratio of male:female participants was 48:12. The mean age was 52.98+/-9.40 years old, and the median duration post-LT was 72 (25-128) months. 59 of the patients had no detectable HBV DNA in plasma.Four patients had detectable levels of total HBV DNA in PBMCs, but no detectable ccc DNA. Five patients had detectable levels of total HBV DNA in liver, and two of those also had detectable levels of ccc DNA. One patients who had detectable HBV DNA in PBMCs suffered HBV recurrence. The liver transplant recipients with detectable levels of HBV total and ccc DNA in PBMCs and liver should be considered high risk for HBV recurrence following LT.

Diagnostic significance of DNA and antibodies against capsid antigens of anti-Epstein–Barr virus antibodies levels in blood plasma of nasopharyngeal carcinoma patients from non-endemic region

Epstein–Barr virus (EBV), a representative of the herpesvirus family, is the etiological agent for a number of benign and malignant human neoplasms. Among the latter, the nasopharyngeal carcinoma (NPC) occupies a special place. In NPC development EBV plays a key role stimulating the progression of the pathological process from precancerous lesions to the cancer development. For most NPC patients, elevated levels of humoral IgG and IgA antibodies against capsid and early EBV antigens are characteristic and their antibody titers rise to high levels long before the diagnosis of cancer. Using this phenomenon, virus-specific antibodies are used for many years as markers for NPC screening, especially in cases of undiagnosed primary lesion. In recent years, in endemic for NPC regions (South China, South-East Asia) a great attention has been paid to the use of quantitative determination of EBV DNA copies in the blood plasma of patients with NPC as a method of early cancer detection and monitoring. The aim of this study was to compare clinical significance of EBV DNA and humoral antibodies levels in blood plasma of NPC patients in non-endemic region, Russia. The results obtained indicate that both markers DNA / EBV and IgA antibodies against capsid EBV antigens can be successfully used for diagnosis of NPC in non-endemic region. However, in comparison with the virus-specific antibody titers, the viral DNA levels in the patients plasma are more sensitive and specific as NPC marker reflecting the efficacy of the therapy, and the state of remission or relapse.