Cell-free DNA Concentration Research Articles

Plasma cell-free DNA integrity: a potential biomarker to monitor the response of breast cancer to neoadjuvant chemotherapy

Background: Although the clinical significance of neoadjuvant chemotherapy (NACT) is widely recognized, there is still no effective means to monitor the therapeutic response in real time. The present study aimed to investigate the significance of the cell-free DNA (cfDNA) concentration and integrity (cfDI) to monitor the response of breast cancer to NACT. Methods: Twenty-nine patients with breast cancer receiving NACT were included in this study. Patients’ peripheral blood was drawn before, in the mid-term, and at the end of chemotherapy. The cfDNA concentration and cfDI were assessed using absolute quantitative PCR. Results: The results showed that the cfDNA concentration and cfDI pre-NACT were not obviously correlated with the patients’ clinical characteristics. The mean cfDI value increased significantly when the patients received NACT (P Conclusions: This study suggested that cfDI could be used as an indicator to monitor the therapeutic response to NACT; however, more research is needed to confirm this conclusion.

Comparison for immunophysiological responses of Jeju and Thoroughbred horses after exercise.

ObjectiveThe study was conducted to investigate variations in the immunophysiological responses to exercise-induced stress in Jeju and Thoroughbred horses.MethodsBlood samples were collected from the jugular veins of adult Jeju (n = 5) and Thoroughbred (n = 5) horses before and after 30 min of exercise. The hematological, biochemical, and immunological profiles of the blood samples were analyzed. Blood smears were stained and observed under a microscope. The concentration of cell-free (cf) DNA in the plasma was determined using real time polymerase chain reaction (PCR). Peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells were separated using Polymorphprep, and the expression of various stress-related and chemokine receptor genes was measured using reverse transcriptase (RT) and real-time PCR.ResultsAfter exercise, Jeju and Thoroughbred horses displayed stress responses with significantly increased rectal temperatures, cortisol levels, and muscle catabolism-associated metabolites. Red blood cell indices were significantly higher in Thoroughbred horses than in Jeju horses after exercise. In addition, exercise-induced stress triggered the formation of neutrophil extracellular traps (NETs) and reduced platelet counts in Jeju horses but not in Thoroughbred horses. Heat shock protein 72 and heat shock protein family A (Hsp70) member 6 expression is rapidly modulated in response to exercise-induced stress in the PBMCs of Jeju horses. The expression of CXC chemokine receptor 4 in PBMCs was higher in Thoroughbred horses than in Jeju horses after exercise.ConclusionIn summary, the different immunophysiological responses of Jeju and Thoroughbred horses explain the differences in the physiological and anatomical properties of the two breeds. The physiology of Thoroughbred horses makes them suitable for racing as they are less sensitive to exercise-induced stress compared to that of Jeju horses. This study provides a basis for investigating the link between exercise-induced stresses and the physiological alteration of horses. Hence, our findings show that some of assessed parameters could be used to determine the endurance performance of horses.

Multiplexed real-time polymerase chain reaction cell-free DNA assay as a potential method to monitor stage IV colorectal cancer

BackgroundLiquid biopsy is a new area in cancer diagnostics that measures cell-free DNA in plasma from tumor that may serve as a monitoring tool in colorectal cancer patients. MethodsMultiplexed real-time polymerase chain reaction based on multicopy retro-transposable elements (targeting 80 base pair and 265 base pair sequences and an internal-positive-control) was used to evaluate the ability of cell-free DNA concentration and DNA Integrity Index to discriminate cancer from healthy patients. A cohort of 40 healthy controls and 39 stage IV colorectal patient's plasma were interrogated. The potency of each biomarker was measured by using receiver operating characteristic curves and derived area under the curve measures. ResultsSignificant differences in cell-free DNA concentration and DNA integrity index were observed between controls and stage IV patients with a limit of detection <0.1 pg/μL. Investigation of the ability of both biomarker candidates to differentiate cancer from healthy patients showed an area under the curve of 0.9891 and 0.9859 for 80 base pair and 265 amplicons respectively and 0.8603 for DNA integrity index-265/80. ConclusionsAfter establishing differences in cell-free DNA levels between healthy and treated and untreated stage IV patients, the multiplexed real-time polymerase chain reaction measurements of retro-transposable elements in cancer patient plasma potentially possess the ability to monitor therapy responsiveness in near real time.

Plasma cell-free DNA integrity: a potential biomarker to monitor the response of breast cancer to neoadjuvant chemotherapy.

BackgroundAlthough the clinical significance of neoadjuvant chemotherapy (NACT) is widely recognized, there is still no effective means to monitor the therapeutic response in real time. The present study aimed to investigate the significance of the cell-free DNA (cfDNA) concentration and integrity (cfDI) to monitor the response of breast cancer to NACT.MethodsTwenty-nine patients with breast cancer receiving NACT were included in this study. Patients’ peripheral blood was drawn before, in the mid-term, and at the end of chemotherapy. The cfDNA concentration and cfDI were assessed using absolute quantitative PCR.ResultsThe results showed that the cfDNA concentration and cfDI pre-NACT were not obviously correlated with the patients’ clinical characteristics. The mean cfDI value increased significantly when the patients received NACT (P<0.05), and an increasing cfDI was associated with tumor shrinkage and reduced Ki67 levels (P<0.05). In addition, the cfDI after NACT was inversely correlated with the number of metastatic lymph nodes, and the cfDI value of patients with a pathologically complete response was significantly higher than that of patients with distant metastasis after surgery.ConclusionsThis study suggested that cfDI could be used as an indicator to monitor the therapeutic response to NACT; however, more research is needed to confirm this conclusion.

Tumor mutation burden and circulating tumor DNA in combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma \u2013 results of a prospective biomarker study

BackgroundMetastasized or unresectable melanoma has been the first malignant tumor to be successfully treated with checkpoint inhibitors. Nevertheless, about 40–50% of the patients do not respond to these treatments and severe side effects are observed in up to 60%. Therefore, there is a high need to identify reliable biomarkers predicting response.Tumor Mutation Burden (TMB) is a debated predictor for response to checkpoint inhibitors and early measurement of ctDNA can help to detect treatment failure to immunotherapy in selected melanoma patients. However, it has not yet been clarified how TMB and ctDNA can be used to estimate response to combined CTLA-4 and PD-1 antibody therapy in metastatic melanoma.Patients and methodsIn this prospective biomarker study, we included 35 melanoma patients with ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) therapy. In all patients, a tumor panel of 710 tumor-associated genes was applied (tumor vs. reference tissue comparison), followed by repetitive liquid biopsies. Cell-free DNA was extracted and at least one driver mutation was monitored. Treatment response was evaluated after about three months of therapy.ResultsTMB was significantly higher in responders than in nonresponders and TMB > 23.1 Mut/Mb (TMB-high) was associated with a survival benefit compared to TMB ≤ 23.1 Mut/Mb (TMB-low or TMB-intermediate). Furthermore, a > 50% decrease of cell-free DNA concentration or undetectable circulating tumor DNA (ctDNA), measured by tumor-specific variant copies/ml of plasma at first follow-up three weeks after treatment initiation were significantly associated with response to combined immunotherapy and improved overall survival, respectively. It is noticeable that no patient with TMB ≤ 23.1 Mut/Mb and detectable or increasing ctDNA at first follow-up responded to immunotherapy.ConclusionHigh TMB, > 50% decrease of cell-free DNA concentration, and undetectable ctDNA at first follow-up seem to be associated with response and overall survival under combined immunotherapy. The evaluation of ctDNA and cell-free DNA three weeks after treatment initiation may be suitable for early assessment of efficacy of immunotherapy.

The influence of fetal gender and maternal characteristics on fetal cell-free DNA in maternal plasma

ObjectiveTo examine the possible effects of fetal gender and maternal characteristics on concentration of fetal cell-free DNA (cfDNA). MethodsMaternal plasma that collected from 2638 singleton pregnancies women were analyzed using non-invasive prenatal testing for aneuploidy by next generation sequence technology. The effects of fetal gender and maternal BMI on fetal cfDNA was measured by Pearson correlation analysis. ResultsThe proportion of fetal cfDNA was positively correlated with gestational age (regression equation: Y=16.2483+6.8856X, r=0.1660, p<0.0001); and negatively correlated with BMI (Body Mass Index) (regression equation: Y=25.6342–19.0065X, r=−0.2146, p<0.0001); Concentration of female fetal cfDNA (mean fetal cfDNA is 13.07%, p<0.0001) is higher than male fetal cfDNA (mean fetal cfDNA is 8.37%, p<0.0001). ConclusionsFetal cfDNA increases stably between 12 and 20 weeks of gestation, and increases in a higher rate after 20 weeks. The maternal BMI is an important factor affecting fetal cfDNA, should be paid enough attention in clinical application.

Development of Reference Materials for Noninvasive Prenatal Aneuploidy Testing by Massively Parallel Sequencing: A Proof-of-Concept Study

Noninvasive prenatal aneuploidy testing (NIPT) represents the first large-scale clinical application of massively parallel sequencing technology. However, no NIPT reference material (RM) has yet been widely adopted, impeding the development of quality management systems and standardization. Developing an NIPT RM from a biological sample is complicated by the low concentration of cell-free DNA (cfDNA), which implies pooling specimens and frequent resampling. We tested the feasibility of using DNA from immortalized cell lines of a woman and her aneuploid offspring to spike an artificial plasma matrix. Enzymatic fragmentation of extracted DNA was optimized to achieve fragment size profiles with a mode of 150 to 200 bp, similar to biological cfDNA. This synthetic material was compared with routine biological samples from pregnant women by a targeted NIPT assay in a multiplex sequencing run on a Proton platform. Sequencing statistics were similar between artificially prepared material and routine biological samples, as well as relative chromosomal representation, and no matrix effects could be detected. Estimate of fetal fraction (FF) was within the range of expected value, and aneuploidy detection statistic (z-score) was also comparable between both types of samples. Artificial plasma spiked with DNA from cell lines of mother and offspring is a promising strategy for developing NIPT RM. This type of material would offer the advantage of a constant and stable composition, allowing for greater standardization of NIPT assays. Moreover, it preserves the parental relatedness used by targeted assay to estimate FF by identification of paternal alleles in single-nucleotide polymorphisms or other variable regions.

Lactic Acid Accumulation During Exhaustive Exercise Impairs Release of Neutrophil Extracellular Traps in Mice.

Lactic acid (LA) is a sensitive indicator of exercise intensity and duration. A single bout of prolonged and intensive exercise can cause transient immunosuppression through the interaction of cellular, humoral, and hormone factors. Exercise-induced influences on neutrophil extracellular traps (NETs) release have been reported, but the underlying mechanism is unknown. This study investigated NETs release, cell-free DNA (cf-DNA), and LA concentration in mice after 60 and 145 min of intensive, graded treadmill running. The concentration of LA and cf-DNA increased, while the level of myeloperoxidase-DNA (MPO-DNA) (an indicator of NETs release) decreased during 145 min of exhaustive running. LA was positively and negatively correlated with cf-DNA and MPO-DNA (R2 = 0.57 and 0.53, respectively, both p < 0.001). Subsequent in vitro experiments were conducted with neutrophils activated by phorbol myristate acetate (PMA) in the presence of LA at different concentrations. Increasing LA concentrations were associated with decreases in NETs release and reactive oxygen species (ROS) formation. Taken together, this work furthers our understanding of how NETs and oxidative reaction respond to one bout of prolonged and intensive running. The data support a negative relationship between LA accumulation and NETs release after heavy exertion.

Sequencing shorter cfDNA fragments improves the fetal DNA fraction in noninvasive prenatal testing

Sequencing cell-free DNA in maternal plasma is an effective noninvasive prenatal testing technique that has been used in fetal aneuploidy screening worldwide. However, its clinical application is limited by the low fetal fraction (<4%) of cell-free DNA in many singleton pregnancies, which usually results in screen failures or no calls. In addition, dizygotic twin contributions of cell-free DNA into the maternal circulation can vary by 2-fold, complicating the quantitative diagnosis of fetal aneuploidy. We performed semiconductor sequencing of shorter fragments (107-145 bp) of circulating cell-free DNA to improve the fetal DNA fraction at lower uniquely mapped reads (1-8.5 MB) to reduce the probability of no calls. We identified 2903 plasma samples from pregnant women, including 86 dizygotic twin pregnancy, that were collected at a single prenatal diagnostic center between October 2015 and July 2018. Size-selection noninvasive prenatal testing for fetal aneuploidy was applied to 2817 plasma samples (1409 male and 1408 female fetuses) and 86 dizygotic twins using noninvasive prenatal testing with and without size selection. Shorter fragment size was the key factor affecting fetal fraction in multivariable linear regression models as well as to validate the accuracy of the size selection for noninvasive prenatal testing. Analysis of 1409 male fetuses by multivariable linear regression showed that maternal age, body mass index, number of pregnancies, average cell-free DNA size, maternal plasma cell-free DNA concentration, library concentration, and multiple gestation were negatively correlated with fetal fraction. Conversely, gestational age and uniquely mapped reads were positively correlated with fetal fraction. Compared with ≤120 bp cell-free DNA fragments, mean fetal fraction differences were -3.57% (95% confidence interval, -5.95% to -1.19%), for 121-130 bp, -9.52% (95% confidence interval, -11.89% to -7.14%) for 131-140 bp, and -14.47% (95% confidence interval, -18.37% to -10.58%) for ≥141 bp (Ptrend < .0001). These results were statistically significant after multivariable adjustments in models for fetal fraction. Meanwhile, results from 86 dizygotic twins showed that the size selection increased the fetal fraction by ∼3.2-fold, with 98.8% of samples reaching a fetal fraction >10%. Improved detection accuracy was also achieved. Sequencing shorter cell-free DNA fragments is a reasonable strategy to reduce the probability of no calls results because of low fetal fraction and should be recommended to pregnant subjects.

Seminal cell free DNA concentration levels discriminate between prostate cancer and benign prostatic hyperplasia

Seminal cell free DNA concentration levels discriminate between prostate cancer and benign prostatic hyperplasia

The value of the plasma circulating cell-free DNA concentration and integrity index as a clinical tool for prostate cancer diagnosis: a prospective case-control cohort study in an Iranian population.

Introduction: Prostate cancer (PCa) is the most common cancer among men and the second cause of cancer death among men. For early detection and differentiating PCa from benign prostate hyperplasia (BPH) tissue biopsy has been used for decades. However, circulating cell-free DNA (ccfDNA) testing is a noninvasive, fast, easily repeatable, and sensitive liquid biopsy for cancer detection. Hence, we aimed to investigate the value of the ccfDNA concentration and integrity index in peripheral blood of a population of Iranian prostatic patients for early diagnosis of the disease.Materials and methods: 100 subjects including 30 PCa, 40 BPH, and 30 healthy individuals were selected. ccfDNA was extracted from fresh blood plasma, and its total concentration and the integrity index were estimated by amplification of ALU115 and ALU247 repeat elements using quantitative real-time PCR.Results: In the PCa group, the ccfDNA concentration and its integrity were significantly higher than that of the BPH and healthy groups (P-value <0.001 and P-value <0.001). The ccfDNA concentration and its integrity were higher in BPH compared to the healthy group, although it was not statistically significant (P-value =0.836 and P-value =0.053, respectively).Conclusion: A significant relation between ccfDNA concentration, its integrity, and PCa suggests that the liquid biopsy can be used as a noninvasive early diagnostic biomarker. Determination of a cutoff or a diagnostic range value of the measured parameters for healthy, BPH, and PCa subjects in more samples of Iranian population results in timely, correct, and early detection, which results in better treatment outcomes. Moreover, this method may reduce overdiagnosis and overtreatment procedures.

Cell-Free DNA Variant Sequencing Using CTC-Depleted Blood for Comprehensive Liquid Biopsy Testing in Metastatic Breast Cancer.

Liquid biopsy analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for personalized treatment. Since cfDNA and CTCs are considered to give additive information and blood specimens are limited, isolation of cfDNA and CTC in an “all from one tube” format is desired. We investigated whether cfDNA variant sequencing from CTC-depleted blood (CTC-depl. B; obtained after positive immunomagnetic isolation of CTCs (AdnaTest EMT-2/Stem Cell Select, QIAGEN)) impacts the results compared to cfDNA variant sequencing from matched whole blood (WB). Cell-free DNA was isolated using matched WB and CTC-depl. B from 17 hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) metastatic breast cancer patients (QIAamp MinElute ccfDNA Kit, QIAGEN). Cell-free DNA libraries were constructed (customized QIAseq Targeted DNA Panel for Illumina, QIAGEN) with integrated unique molecular indices. Sequencing (on the NextSeq 550 platform, Illumina) and data analysis (Ingenuity Variant Analysis) were performed. RNA expression in CTCs was analyzed by multimarker quantitative PCR. Cell-free DNA concentration and size distribution in the matched plasma samples were not significantly different. Seventy percent of all variants were identical in matched WB and CTC-depl. B, but 115/125 variants were exclusively found in WB/CTC-depl. B. The number of detected variants per patient and the number of exclusively detected variants per patient in only one cfDNA source did not differ between the two matched cfDNA sources. Even the characteristics of the exclusively detected cfDNA variants in either WB or CTC-depl. B were comparable. Thus, cfDNA variants from matched WB and CTC-depl. B exhibited no relevant differences, and parallel isolation of cfDNA and CTCs from only 10 mL of blood in an “all from one tube” format was feasible. Matched cfDNA mutational and CTC transcriptional analyses might empower a comprehensive liquid biopsy analysis to enhance the identification of actionable targets for individual therapy strategies.

Progress in non-invasive prenatal testing failure due to less cell-free fetal DNA

Non-invasive prenatal testing (NIPT) has been extensively applied in fetal chromosomal aneuploidy screening with high sensitivity and specificity. The concentration of cell-free fetal DNA (cffDNA) is an important factor related to the accuracy of NIPT. When cffDNA concentration is lower than 4%, accurate results are often unavailable, resulting in failure of NIPT. The factors influencing cffDNA concentration in maternal blood were reviewed and it was found that the concentration of cffDNA was positively correlated with the gestational week at the time of sampling, but negatively correlated with the maternal weight/body mass index. Moreover, gestational complications (such as preeclampsia and polycystic ovary syndrome), maternal age, results of serological screening for Down's syndrome in second trimester, fetal chromosomal karyotype and nuchal translucency thickness were also potential influencing factors. Analysis of the influencing factors of cffDNA concentration helps to improve the NIPT specifications, which is significant for prenatal genetic counselling. Key words: Aneuploidy; Prenatal diagnosis; Cell-free system; DNA; Fetus

Genotyping of Single Nucleotide Polymorphisms Using Allele-Specific qPCR Producing Amplicons of Small Sizes Directly from Crude Serum Isolated from Capillary Blood by a Hand-Powered Paper Centrifuge

The cell-free genomic DNA (gDNA) concentration in serum ranges from 1500 to 7500 copies/mL within 2 h after phlebotomy (6–24 times the concentration observed in plasma). Here, we aimed to evaluate the gDNA size distribution in serum with time after coagulation and to test if crude serum can be directly used as a source of gDNA for qPCR. Next, we investigated if single nucleotide polymorphisms (SNPs) could be genotyped directly from the crude serum isolated from capillary blood using a hand-powered paper centrifuge. All tested PCR targets (65, 100, 202 and 688 base pairs) could be successfully amplified from DNA extracted from serum, irrespective of their amplicon size. The observed qPCR quantitation cycles suggested that the genomic DNA yield increased in serum with incubation at room temperature. Additionally, only 65 and 101 base pair qPCR targets could be amplified from crude serum soon after the coagulation. Incubation for 4 days at room temperature was necessary for the amplification of PCR targets of 202 base pairs. The 688 base pair qPCR target could not be amplified from serum directly. Lastly, serum was successfully separated from capillary blood using the proposed paper centrifuge and the genotypes were assigned by testing the crude serum using allele-specific qPCR, producing small amplicon sizes in complete agreement with the genotypes assigned by testing the DNA extracted from whole blood. The serum can be used directly as the template in qPCR for SNP genotyping, especially if small amplicon sizes are applied. This shortcut in the SNP genotyping process could further molecular point-of-care diagnostics due to elimination of the DNA extraction step.

Bioinformatics Pipeline for Accurate Quantification of Fetal DNA Fraction in Maternal Plasma.

The discovery of circulating cell-free fetal DNA has profoundly transformed the landscape of noninvasive prenatal testing (NIPT) and rapidly found its way into global clinical applications. The fractional concentration of cell-free fetal DNA in plasma DNA of a pregnant woman is an important parameter for understanding and interpreting analytical results of NIPT. Thus, the accurate quantification of fetal DNA fraction is indispensable when NIPT is involved. In this protocol, we describe the bioinformatics workflow to calculate fetal DNA fraction using two programs developed by our group, which provide accurate estimation.

Value of the concentration and integrity of serum cell-free DNA for the clinical diagnosis of esophageal carcinoma

Objective: To explore the diagnostic value of serum cell-free DNA (cfDNA) concentration and integrity for esophageal carcinoma. Methods: Venous blood samples from 68 patients with esophageal cancer, 36 patients with benign esophageal lesions and 45 healthy subjects were collected. Circulating cfDNA was verified through quantitative real-time PCR (Alu-qPCR) using Alu-115 and Alu-247 primers. DNA integrity index was calculated as the ratio of Alu-qPCR results (Alu247/115). Concentrations of carcino-embryonic antigen (CEA) and squamous cell carcinoma associated antigen (SCC) were detected by chemiluminescence analyzer assay. Statistical analysis was performed using Mann-Whitney U test, Kruskal-Wallis H test and Spearman correlation test. The Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficiency of each index to esophageal carcinoma. Results: The median absolute serum Alu115 and the Alu247/115 index (1 162.0 ng/ml, 0.57) in esophageal cancer group were significantly higher than those in benign esophageal disease group (496.7 ng/ml, 0.43) and in healthy control group (432.3 ng/ml, 0.42) (all P<0.01, respectively). The Alu115 and Alu247/115 index of serum DNA in benign esophageal disease group were no statistically different from those in the healthy control group (all P>0.05, respectively). The levels of cfDNA and its integrity were not significantly correlated with age, gender, tumor differentiation, or disease stage according to American Joint Committee on Cancer (AJCC) staging system in the esophageal cancer group (all P>0.05). The serum Alu247/115 index of Stage Ⅲ patients was higher than that of Stage Ⅰ~Ⅱ patients(P<0.05). The serum Alu247/115 index of Stage Ⅳ was higher than that of Stage Ⅲ(P<0.05). In the esophageal cancer group, both of serum Alu115 and Alu247/115 index had no correlation with CEA or SCC (all P>0.05). The area under the ROC curve (AUC) of Alu115 and Alu247/115 index were 0.867 and 0.854, respectively, which were both higher than that of CEA (0.622) and SCC (0.753). The addition of Alu115 or Alu247/115 index to CEA and SCC detection increased the sensitivity of the diagnosis of esophageal cancer by 95.6% and 94.1%, respectively. Conclusions: The detection of serum cfDNA concentration and integrity is helpful to the early diagnosis and monitoring of esophageal cancer. Their diagnostic value of esophageal cancer is better than that of the traditional tumor markers CEA and SCC.

Circulating Cell-Free DNA in Physical Activities.

The interest about circulating cell-free DNA (cfDNA) concentration increased from several years because of its correlation with various conditions like osteoarthritis, cancers, stroke, and sepsis; recently it has become an important marker for overtraining syndrome or performance diagnostics.Several studies have demonstrated that cfDNA increases in vigorous and exhausting exercise but also endurance exercise. Acute effect of exercise on cfDNA concentration seems to be correlated to stress factor, while chronic effect is associated with necrosis and apoptosis.The intensity and duration seem to have effects on the variation of cfDNA concentration that is strongly correlated with other metabolic markers like acid lactate and creatine kinase, recognized as markers of muscle damage. Variation of cfDNA value could be used to predict overtraining syndrome.

A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time.

This study was undertaken to evaluate a novel method for stabilizing and preserving the original proportion of cell-free fetal DNA (cffDNA) in maternal blood for extended periods of time without using crosslinking agents, such as formaldehyde, which compromise DNA integrity and extraction efficiency. Blood was drawn from pregnant donors into K3EDTA and Blood Exo DNA ProTeck® (ProTeck) tubes. Blood drawn into both tubes were aliquoted and stored at three different temperatures. At indicated times sample aliquots were processed for cell-free DNA (cfDNA) extraction. Plasma cfDNA and cffDNA quantified by droplet digital PCR (ddPCR) assay which amplify RASSF1A gene promoter region. ProTeck reagent is formaldehyde free and inhibits blood cell metabolism in blood samples during storage. Cell-free DNA concentration increased over time in blood plasma stored in K3EDTA tubes at 4, 22 and 30°C. Blood stored in ProTeck tubes, cfDNA concentration was stable at 4, 22 and 30°C for 21, 28 and 7 days, respectively. In K3EDTA tubes cffDNA proportion decreases steadily over time whereas in ProTeck tubes cffDNA proportion remained stable. This novel technology stabilizes cffDNA proportion in maternal blood samples at 4, 22 and 30°C for 21, 28 and 7 days, respectively.

Circulating free plasma tumor DNA in patients with advanced gastric cancer receiving systemic chemotherapy

BackgroundAdvanced gastric cancers are usually associated with incurable conditions for which systemic treatments are indicated. Recent studies suggest that circulating cell-free plasma DNA of tumour origin (tDNA) is a promising non-invasive biomarker that can be used to predict the prognosis and monitor the efficacy of systemic treatments in patients with certain types of cancer. We conducted a pilot study to analyse the potential role of tDNA as a biomarker in patients with advanced gastric cancer.MethodsWe included 30 patients with locally advanced unresectable or metastatic gastric cancer. We obtained samples (10 mL of total blood) from each patient every 3 months and performed concomitant CT until disease progression or death. Total cell-free circulating DNA (cfDNA) samples were measured using GeneQuant RNA/DNA Calculator-Amersham Pharmacia Biotech (Biochrom) Ltd. The cfDNA was used to evaluate the ALU DNA sequences 247 and 115. The level of tDNA was calculated from the ratio of the expression of ALU DNA sequences and the concentration of total cell-free DNA. We utilized the RECIST criteria 1.1 to evaluate the tumour response.ResultsPatients with advanced gastric cancer had significantly higher concentrations of cfDNA compared with normal controls (p = 0.00015), which allowed us to conclude that the cfDNA in the patients originated from the tumour. We did not find any significant correlation between the level of tDNA and OS or tumour response. However, after the first cycles of chemotherapy (at 3 months), we observed that patients with lower tDNA levels had significantly longer DFS compared with those with higher levels (Cox Regression p = 0.0228).ConclusionsAt 3 months after the beginning of chemotherapy, the tDNA levels are correlated with DFS in patients with advanced gastric cancer who receive systemic chemotherapy. tDNA may be a specific, non-invasive and cost effective new biomarker for these patients.

Microfluidic Assay Measures Increased Neutrophil Extracellular Traps Circulating in Blood after Burn Injuries

Cell-free DNA (cf-DNA) concentration in human plasma is often increased after burn and trauma injuries. Two major sources of cf-DNA are the parenchymal cells damaged by the injury and various circulating cells indirectly altered by the response to injury. The cf-DNA originating from neutrophils, also known as circulating neutrophil extracellular traps (cNETs), is of notable interest because cNETs have been associated with pathological processes in other conditions, including cancer, autoimmunity, etc. Both intact chromatin and oligonucleotides, which are the by-product of cf-DNA degradation, are assumed to contribute to the cf-DNA in patients. However, traditional assays for cf-DNA quantification do not distinguish between cNETs and cf-DNA of other origins and do not differentiate between intact chromatin and oligonucleotides. Here we measure the amount of intact cNETs in the circulation, using a microfluidic device that mechanically traps chromatin fibers directly from blood and an immunofluorescence protocol that detects neutrophil-specific proteins associated with chromatin. In a rat model of burn injury, we determined that the chromatin fibers in the circulation after injury originate exclusively from neutrophils and are cNETs. We found that the concentration of cNETs surges the first day after injury and then decreases slowly over several days. In a secondary sepsis model, which involved a burn injury followed by cecal-ligation-puncture, we measured additional increases in cNETs in the days after sepsis was induced. These results validate a microfluidic assay for the quantification of cNETs and will facilitate fruther studies probing the contribution of cNETs to complications after burns and sepsis.