Total Plasma DNA Research Articles

Detection of recurrent colorectal cancer with high specificity using a reporting threshold for circulating tumor DNA methylated in BCAT1 and IKZF1.

A blood assay measuring methylated BCAT1 and IKZF1 can detect recurrent colorectal cancer (CRC) with high sensitivity but suboptimal specificity. This study aimed to establish an upper reference limit (URL) of these biomarkers in a reference population without CRC, apply that threshold to detecting clinical recurrence in patients who had undergone definitive therapy for CRC, and compare the performance of the biomarkers with carcinoembryonic antigen (CEA). The level of methylation was reported as the aggregate methylated BCAT1 and IKZF1 expressed as a percentage of total plasma DNA. A reference population of patients confirmed to have no colorectal neoplasia (n = 857) was used to determine the URL. Test accuracy for clinical recurrence was determined in a post-treatment surveillance population (n = 549; 77 recurrence cases). A methylation level of 0.07%, corresponding to the 98th percentile in the reference population, was set as the URL. In the surveillance population, 60 patients had methylation levels above 0.07%, and 81.7% of these had recurrence. In comparison with no minimum threshold being applied, assay sensitivity with a URL of 0.07% yielded similar sensitivity (63.6% [CI, 51.9%-74.3%] vs 64.9% [CI, 53.8%-74.7%]; P = .87) and higher specificity (97.7% [CI, 95.9%-98.8%] vs 91.3% [CI, 88.4%-93.5%]; P < .001). The BCAT1/IKZF1 test was 2.5-fold more sensitive than CEA for detecting recurrences considered amenable to surgery with curative intent (50.0% vs 20.8%; P = .016). Applying a threshold for positivity to the methylated BCAT1/IKZF1 blood assay improved the specificity for CRC recurrence without compromising sensitivity. Both the sensitivity and the specificity were superior to those of CEA.

Repurposing non-invasive prenatal testing data: Population study of single nucleotide variants associated with colorectal cancer and Lynch syndrome.

In our previous work, genomic data generated through non-invasive prenatal testing (NIPT) based on low-coverage massively parallel whole-genome sequencing of total plasma DNA of pregnant women in Slovakia was described as a valuable source of population specific data. In the present study, these data were used to determine the population allele frequency of common risk variants located in genes associated with colorectal cancer (CRC) and Lynch syndrome (LS). Allele frequencies of identified variants were compared with six world populations to detect significant differences between populations. Finally, variants were interpreted, functional consequences were searched for and clinical significance of variants was investigated using publicly available databases. Although the present study did not identify any pathogenic variants associated with CRC or LS in the Slovak population using NIPT data, significant differences were observed in the allelic frequency of risk CRC variants previously reported in genome-wide association studies and common variants located in genes associated with LS. As Slovakia is one of the leading countries with the highest incidence of CRC among male patients in the world, there is a need for studies dedicated to investigating the cause of such a high incidence of CRC in Slovakia. The present study also assumed that extensive cross-country data aggregation of NIPT results would represent an unprecedented source of information concerning human genome variation in cancer research.

The cGAS-STING pathway connects mitochondrial damage to inflammation in burn-induced acute lung injury in rat

ObjectiveDamage associated molecular patterns (DAMPs) are pathological mediators linking local tissue damage to systemic inflammation in various diseases. Some DAMPs, such as mitochondrial DNA (mtDNA), can be recognized by the cytoplasmic cGAS protein to trigger the activation of the stimulator of interferon genes (STING)-dependent innate immune pathway responsible for infection or sterile inflammation. The objective of our study was to evaluate the association between circulating mtDNA and cGAS-STING pathway activation in mediating inflammation following burn injury. Methods48 adult Sprague-Dawley male rats were divided into eight groups (Sham, 2, 4, 8, 12, 24, 48, 72 h after burn injury). The animals underwent 40% total body surface area scald injury to produce a full-thickness burn. Plasma samples were collected via cardiac puncture under deep anesthesia. Tissues were harvested and placed in formalin, followed by paraffin embedment. Total plasma DNA was isolated followed by measurement of mtDNA using quantitative polymerase chain reaction. Haemotoxylin-Eosin stain and Western blot was used for lung histology and protein assays, respectively. Statistical analyses were performed using ANOVA and student’s t-test and represented as mean ± s.d. ResultsPlasma mtDNA trended upward at early time-points following burn injury with peak levels at 8 h after burn when compared to the control group (345 ± 83.4 copies/μl vs. 239 ± 43.1 copies/μl, p = 0.07) and followed a bell-shaped distribution. Lung slices from burned rats showed acute injury marked by increased inflammatory infiltrate, with the maximum changes seen at 24 h, accompanied with significant upregulation of neutrophil elastase (p = 0.04). Compared with sham animals, cGAS and STING protein levels in lung tissue were up-regulated at 4 and 8 h after burn (p = 0.03 and p < 0.001, respectively). ConclusionActivation of the cGAS-STING pathway by increased plasma mtDNA is an important pathway driving neutrophil infiltration in burn-induced acute lung injury in rats. A further understanding of the STING-mediated immunopathology in lung and other susceptible organs may be important for the development of novel therapies for burn injury.

Identification of Structural Variation from NGS-Based Non-Invasive Prenatal Testing.

Copy number variants (CNVs) are an important type of human genome variation, which play a significant role in evolution contribute to population diversity and human genetic diseases. In recent years, next generation sequencing has become a valuable tool for clinical diagnostics and to provide sensitive and accurate approaches for detecting CNVs. In our previous work, we described a non-invasive prenatal test (NIPT) based on low-coverage massively parallel whole-genome sequencing of total plasma DNA for detection of CNV aberrations ≥600 kbp. We reanalyzed NIPT genomic data from 5018 patients to evaluate CNV aberrations in the Slovak population. Our analysis of autosomal chromosomes identified 225 maternal CNVs (47 deletions; 178 duplications) ranging from 600 to 7820 kbp. According to the ClinVar database, 137 CNVs (60.89%) were fully overlapping with previously annotated variants, 66 CNVs (29.33%) were in partial overlap, and 22 CNVs (9.78%) did not overlap with any previously described variant. Identified variants were further classified with the AnnotSV method. In summary, we identified 129 likely benign variants, 13 variants of uncertain significance, and 83 likely pathogenic variants. In this study, we use NIPT as a valuable source of population specific data. Our results suggest the utility of genomic data from commercial CNV analysis test as background for a population study.

Ammonia-Induced Brain Edema Requires Macrophage and T Cell Expression of Toll-Like Receptor 9

Background & AimAmmonia is central in the pathogenesis of brain edema in acute liver failure (ALF) with infection and systemic inflammation expediting development of intracranial hypertension (ICH). Patients with acetaminophen-induced ALF have increased neutrophil TLR9 expression which can be induced by ammonia. We determined whether ammonia-induced brain edema and immune dysfunction are mediated by TLR9 and if this could be prevented in a TLR9-deficient mouse model.MethodsAmmonium acetate (NH4-Ac; 4mmol/kg) was injected intraperitoneally in wild type (WT), Tlr9-/- and Lysm-Cre Tlr9fl/fl mice (TLR9 absent in neutrophils and macrophages including Kupffer cells) and compared to controls. Six hours after NH4-Ac injection, intracellular cytokine production was determined in splenic macrophages, CD4+ and CD8+ T cells. Brain water (BW) and total plasma DNA (tDNA) were also measured. The impact of the TLR9 antagonist ODN2088 (50μg/mouse) was evaluated.ResultsFollowing NH4-Ac injection, BW, macrophage and T cell cytokine production increased (P < .0001) in WT but not Tlr9-/- mice (P < .001). ODN2088 inhibited macrophage and T cell cytokine production (P < .05) and prevented an increase in BW (P < .0001). Following NH4-Ac injection, macrophage cytokine production and BW were ameliorated in Lysm-Cre Tlr9fl/fl mice compared to WT mice (P < .05) but there was no difference compared to Tlr9-/- mice. Following NH4-Ac injection, plasma tDNA levels increased in WT and Tlr9-/- mice (P < .05) suggesting that TLR9 may be activated by DNA released from ammonia-stimulated cells.ConclusionAmmonia-induced brain edema requires macrophage and T cell expression of TLR9. Amelioration of brain edema and lymphocyte cytokine production by ODN2088 supports exploration of TLR9 antagonism in early ALF to prevent progression to ICH.

PS02.195: CIRCULATING TUMOUR DNA HAS UTILITY IN THE CLINICAL MANAGEMENT OF ESOPHAGEAL CANCER

Abstract Background Cell-free DNA, a consequence of normal cell death and apoptosis, is found in the blood of all individuals. In cancer patients, a proportion of circulating cell free DNA will be derived from tumour cells, and is termed circulating tumour DNA (ctDNA).We have developed a blood-based ‘liquid biopsy’ to detect ctDNA in plasma of esophageal cancer patients. Methods Tumour, buffy-coat (germline) and plasma samples were obtained from 28 esophageal cancer patients at the time of diagnosis. Serial blood samples were collected from 8 patients. Somatic DNA mutations in 9 genes (TP53, ARID1A, SMAD4, CDKN2A, SMARCA4, NRG1, APC, PIK3CA and KRAS) were evaluated in tumour biopsies and plasma using targeted sequencing. Tumour specific mutations were confirmed by droplet digital PCR, which was used to track patient-specific ctDNA in plasma from serially collected blood samples. Results Somatic mutations in at least one of the targeted genes were detected in 19/28 (68%) tumour biopsies. The same mutations were detected in ctDNA from plasma in 9/19 (48%) patients. Additional mutations that were not detected in the tumour biopsies were detected in plasma DNA from 2 patients, highlighting the issues of intra-tumoural heterogeneity and sampling bias of tumour biopsies. ctDNA was more frequently detected in patients with advanced disease where it represented a greater proportion of the total cell-free plasma DNA than in patients with localised disease. In patients with serial samples, levels of ctDNA correlated with response to treatment, and rises in ctDNA could be detected prior to clinical evidence of relapse. Conclusion Detection of ctDNA using targeted sequencing and digital PCR is feasible in the majority of esophageal cancer patients, particularly those with advanced disease. Dynamic changes in ctDNA levels reflect response to treatment and disease relapse as determined by conventional clinical imaging. We conclude that ctDNA levels can provide supplemental evidence regarding disease staging, as well as prognostic and predictive information that can inform therapeutic pathway decisions in esophageal cancer patients. Disclosure All authors have declared no conflicts of interest.

Cell-Free DNA Kinetics in a Pre-Clinical Model of Head and Neck Cancer

In cancer patients, circulating tumour-derived DNA (ctDNA) levels imperfectly reflect disease burden apparent on medical imaging. Further evaluation of ctDNA levels over time is needed to better understand the correlation with tumour growth and therapeutic response. We describe ctDNA kinetics within an orthotopic, immunocompetent preclinical rabbit model of local-regionally advanced head and neck squamous cell carcinoma (HNSCC). Monitoring primary tumour and metastatic lymph node volume by computed tomography (CT), we observed a correlation between ctDNA levels and tumour burden. We found that ctDNA detection could precede evidence of tumour on CT. Sensitivity and specificity of ctDNA detection in this model was 90.2% (95% C.I.: 76.9–97.3%) and 85.7% (95% C.I.: 67.3–96.0%), respectively. Rapid tumour growth followed by auto-necrosis and tumour volume contraction produced a spike in ctDNA levels, suggesting that viable tumour cells may be required for sustained ctDNA release. Following surgical resection, both ctDNA and total plasma DNA were correlated with recurrent tumour volume. Our results reveal the complex kinetic behaviour of ctDNA and total plasma DNA upon tumour growth or surgery. This pre-clinical model could be useful for future studies focused on elucidating mechanisms of ctDNA release into the circulation from primary and metastatic sites.

Abstract 2758: Comparison of cell-free DNA blood collection tubes

Abstract Critical biomarkers such as cell-free DNA (cfDNA) derived from tumors and circulating tumor cells (CTCs) can be detected and analyzed from a simple blood draw. These analytes are fragile, prone to degradation, and present in extremely low quantities. Therefore, proper preservation of these analytes is necessary to ensure accuracy of test results. Several blood collection tubes are commercially available for cfDNA applications, and selecting the ideal blood collection tube for cfDNA impacts test results, sample collection logistics and costs. However, no comprehensive and systematic evaluation of performance among these tubes is available. This study evaluates five commercial blood collection tubes: LBgard™ Blood Tube (Biomatrica), Streck cfDNA BCT® (Streck), PAXgene Blood ccfDNA Tube (PreAnalytiX), Cell-Free DNA Collection Tube (Roche), and EDTA (BD). Healthy donor blood samples were collected in each tube type and incubated over several days at different temperatures. Total plasma DNA was subsequently isolated, and the yield, fold increase versus time 0 and quality of purified DNA were compared. cfDNA controls were also spiked into blood samples collected in each tube type and measured by droplet digital PCR to determine the mutant allele frequencies over time. Finally, the yield and quality of cfDNA isolated from stage IV colorectal cancer blood collected in LBgard Blood Tube, Streck cfDNA BCT and EDTA were compared. Additionally, the inhibition of hemolysis and CTC stabilization were also assessed. Our results show that LBgard Blood Tube, Streck cfDNA BCT, PAXgene ccfDNA Tube and Cell-Free DNA Collection Tube are superior to EDTA tubes in maintaining cfDNA yield and quality over 7 days at ambient temperature. LBgard Blood Tube out-performs all tested blood tubes in inhibiting genomic DNA release for the longest duration (14 days) and across the widest temperature range (4°C, 25°C and 37°C). LBgard Blood Tube shows equivalent inhibition of genomic DNA release to Streck cfDNA BCT for clinical samples, and both tubes out-perform EDTA blood tubes. LBgard Blood Tube also consistently shows better CTC stabilization and inhibition of hemolysis over Streck cfDNA BCT. This comprehensive and systematic study of blood collection tube performance provides a quantitative and exhaustive assessment of blood sample stability, allowing researchers to make informed decisions based on their sample stabilization needs. Citation Format: Cecille D. Browne, Margrith E. Mattmann, Marc J. Wycoco, Sheila N. Chen, Rajeswari Ravichandran, Joel Desharnais, Laura J. Varela, Jonathan D. Browne, Vasco Liberal, Florence Lee. Comparison of cell-free DNA blood collection tubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2758. doi:10.1158/1538-7445.AM2017-2758

Abstract 2745: Evaluation of a new system for collection, stabilization, and purification of circulating tumor DNA

Abstract Introduction: The current preanalytical workflows for circulating tumor DNA (ctDNA) analysis have limitations that affect the accurate detection and quantification of these plasma cancer biomarkers. Release of genomic DNA (gDNA) from white blood cells (WBCs) due to cell lysis or apoptosis during whole blood storage in EDTA tubes creates higher gDNA background levels, affecting the sensitivity of ctDNA assays. Current tubes that stabilize WBCs often contain crosslinking reagents, which have negative effects on sensitive downstream assays, including methylation-based assays. Using ctDNA assays, the new PAXgene® Blood ccfDNA System*, consisting of a blood collection tube with unique, non-crosslinking chemistry and an automated circulating cell-free DNA (ccfDNA) extraction kit, was evaluated in three research studies. Methods: Blood samples were collected into paired PAXgene and EDTA tubes and stored for 7 days at room temperature (RT). ccfDNA was isolated from plasma using the PAXgene kit or the QIAGEN QIAamp® Circulating Nucleic Acid Kit. Study 1: The ccfDNA from lung cancer patients was quantified by real-time PCR for the amount of the ERV sequence (as a measure of the total plasma DNA quantity) and after bisulfte treatment for mSHOX2 as a marker for ctDNA. Study 2: Blood from healthy donors was spiked with fragmented, fully-methylated CpGenome DNA. During storage, tubes were intermittently inverted to simulate tubes in transit. Subsequent to bisulfite conversion, PCR assays targeting ACTB and methylated BCAT1 and IKZF1 DNA were used to determine the yields of ccfDNA and ctDNA, respectively. Study 3: Restriction enzyme treated EGFR DNA containing exon 19 deletions and exon 20 and 21 substitutions (T790M, L858R) were spiked into healthy donors’ blood. ccfDNA was tested with the QIAGEN therascreen® EGFR Plasma RGQ PCR Kit*. Results: Both study 1 and 2 demonstrated constant levels of the methylation ctDNA markers, SHOX2, BCAT1 and IKZF1, over the investigated time course. There was no significant release of gDNA in the PAXgene tube whereas a significant release of gDNA was detected in EDTA samples. Likewise, study 3 showed constant EGFR Ct values in the PAXgene system with reliable mutation detection, whereas the high DNA concentration from the EDTA system resulted in false-positive callings. Conclusions: The new system allows researchers to accurately detect and quantify plasma cancer biomarkers from blood samples that have been stored in the tube for up to 7 days at RT. This includes challenging assays based on methylated ctDNA. The system provides the required assay sensitivity to allow the correct assay interpretation beyond the typical 3–6 hour storage limit for EDTA tubes. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Michael Fleischhacker, Bernd Schmidt, Rohan Baker, Susanne Pedersen, Natasha Cant, Maryam Zahedi-Nejad, Thorsten Voss, Andrea Ullius, Daniel Groelz. Evaluation of a new system for collection, stabilization, and purification of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2745. doi:10.1158/1538-7445.AM2017-2745

Deoxyribonuclease partially ameliorates thioacetamide-induced hepatorenal injury.

Several recent studies have shown that liver injury is associated with the release of DNA from hepatocytes. This DNA stimulates innate immunity and induces sterile inflammation, exacerbating liver damage. Similar mechanisms have been described for acute renal injury. Deoxyribonuclease degrades cell-free DNA and can potentially prevent some of the induced tissue damage. This study analyzed the effects of thioacetamide-induced hepatorenal injury on plasma DNA in rats. Plasma DNA of both nuclear and mitochondrial origin was higher in thioacetamide-treated animals. Administration of deoxyribonuclease resulted in a mild, nonsignificant decrease in total plasma DNA and plasma DNA of mitochondrial origin but not of nuclear origin. This was accompanied by a decrease in bilirubin, creatinine, and blood urea nitrogen as markers of renal function. In conclusion, the study confirmed the hepatotoxic and nephrotoxic effect of thioacetamide. The associated increase in cell-free DNA seems to be involved in hepatorenal pathogenesis because treatment with deoxyribonuclease resulted in a partial prevention of hepatorenal injury. Further experiments will focus on the effects of long-term treatment with deoxyribonuclease in other clinically more relevant models. Clinical studies should test endogenous deoxyribonuclease activity as a potential risk determinant for kidney or liver failure.NEW & NOTEWORTHY Thioacetamide-induced hepatorenal injury resulted in higher plasma cell-free DNA. Deoxyribonuclease decreased average cell-free DNA of mitochondrial origin but not nuclear origin. Deoxyribonuclease partially prevented hepatorenal injury in rats.

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA.

BackgroundCirculating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application.MethodsWe designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients.ResultsCompared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R2=0.916, P<0.0001).ConclusionsThis duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.

Debates on fetal fraction measurement and DNA-based noninvasive prenatal screening: time for standardisation?

Debates on fetal fraction measurement and DNA-based noninvasive prenatal screening: time for standardisation?

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.

Real-time monitoring efficiency and toxicity of chemotherapy in patients with advanced lung cancer.

BackgroundThe Response Evaluation Criteria in Solid Tumors (RECIST) guideline and Common Terminology Criteria for Adverse Events (CTCAE) criteria are used to assess chemotherapy efficiency and toxicity in patients with advanced lung cancer. However, no real-time, synchronous indicators that can evaluate chemotherapy outcomes are available. We wanted to evaluate tumor response and toxicity in advanced lung cancer chemotherapy by using a novel synchronous strategy.ResultsWe enrolled 316 patients with advanced lung cancer who were treated with cisplatin-based therapy and followed up them for 3 years. Plasma was obtained before and after every chemotherapy cycle. We quantitative assayed total plasma DNA and methylation of the APC/RASSF1A genes. Four parameters were assessed: methylation level before chemotherapy (meth0 h), methylation level 24 h after chemotherapy (meth24 h), total plasma DNA concentration before chemotherapy (DNA0 h), and total plasma DNA concentration 24 h after chemotherapy (DNA24 h). When meth24 h > meth0 h of at least one gene was used to predict tumor response, the correct prediction rate was 82.4 %. Additionally, patients for whom DNA24 h/DNA0 h ≤ 2 had mild toxicities. Therefore, meth24 h > meth0 h and DNA24 h/DNA0 h ≤ 2 were defined as criteria for better tumor response and fewer adverse events with a high correct prediction rate (84.7 %).ConclusionsQuantitative analysis of total plasma DNA and plasma APC/RASSF1A methylation provide a real-time synchronous rapid monitoring indicator for therapeutic outcomes of advanced lung cancer, which could be a reference or supplementary guidelines in evaluating chemotherapy effects.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0150-9) contains supplementary material, which is available to authorized users.

Abstract 5241: Clinical relevance of circulating tumor DNA in plasma from pancreatic cancer patients

Abstract Background: Circulating tumor DNA (ctDNA) may originate from necrotic or apoptotic tumor cells in the primary tumor, metastatic lesions or in the circulation. There is evidence that the ctDNA level may reflect the total tumor burden in a patient. We wanted to investigate whether changes in the ctDNA level might be used to monitor disease progression in pancreatic cancer patients with locally or advanced disease. Methods: Blood samples (9 mL in EDTA tubes) were collected from 15 prospectively recruited patients with locally advanced and/or metastatic pancreatic cancer before initiation of treatment, and subsequently every month during chemotherapy. The samples were processed by LymphoprepTM (Axis Shield) density centrifugation before plasma DNA isolation using the QIAamp Circulating Nucleic Acid kit (Qiagen). A high-fidelity polymerase-based PNA clamp PCR method (Gilje et al., 2008, Oltedal et al., 2010) was then used for the detection of KRAS mutations in exon 12 and 13, as a surrogate marker for ctDNA. KRAS mutations have previously been reported to be present in &amp;gt;90% of the pancreatic cancers. Plasma samples from 29 healthy individuals were also analysed as a reference group for the PNA clamp PCR method. The results were compared with conventional biochemical and radiological monitoring measures. Results: The majority of the patients (80%) had metastatic disease and they were treated either by gemcitabine or FOLFORINOX. Nine (60%) patients had a positive KRAS status in the plasma samples obtained before initiation of chemotherapy, indicating presence of ctDNA. Moreover, 11 of the 15 included patients had ≥2 follow-up samples, and in several of these patients the ctDNA level changed substantially during the course of chemotherapy. In total, 17/38 (44.7%) patient samples were positive for plasma DNA KRAS mutations during chemotherapy. Changes in the ctDNA level seemed to correspond both to radiological follow-up data and changes in CA19-9 for several patients. Analyses of the total plasma DNA fraction with regard to disease monitoring, gave more inconclusive results in this small pilot study. Conclusion: Our pilot study gives support to the hypothesis that ctDNA may be used as a marker for monitoring treatment efficacy and disease progression in pancreatic cancer patients. This will be further investigated. Citation Format: Kjersti Tjensvoll, Morten Lapin, Tove Buhl, Satu Oltedal, Katrine Steen-Ottosen Berry, Bjørnar Gilje, Jon Arne Søreide, Millind Javle, Oddmund Nordgård, Rune Smaaland. Clinical relevance of circulating tumor DNA in plasma from pancreatic cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5241. doi:10.1158/1538-7445.AM2015-5241

A new blood collection device minimizes cellular DNA release during sample storage and shipping when compared to a standard device.

BackgroundCell‐free DNA (cfDNA) circulating in blood is currently used for noninvasive diagnostic and prognostic tests. Minimizing background DNA is vital for detection of low abundance cfDNA. We investigated whether a new blood collection device could reduce background levels of genomic DNA (gDNA) in plasma compared to K 3 EDTA tubes, when subjected to conditions that may occur during sample storage and shipping.MethodsBlood samples were drawn from healthy donors into K 3 EDTA and Cell‐Free DNA™ BCT (BCT). To simulate shipping, samples were shaken or left unshaken. In a shipping study, samples were shipped or not shipped. To assess temperature variations, samples were incubated at 6°C, 22°C, and 37°C. In all cases, plasma was harvested by centrifugation and total plasma DNA (pDNA) assayed by quantitative real‐time polymerase chain reaction (qPCR).ResultsShaking and shipping blood in K 3 EDTA tubes showed significant increases in pDNA, whereas no change was seen in BCTs. Blood in K 3 EDTA tubes incubated at 6°C, 22°C, and 37°C showed increases in pDNA while pDNA from BCTs remained stable.Conclusions BCTs prevent increases in gDNA levels that can occur during sample storage and shipping. This new device permits low abundance DNA target detection and allows accurate cfDNA concentrations.

Circulating cell-free and Epstein–Barr virus DNA in pediatric B-non-Hodgkin lymphomas

Tumor-derived DNA is elevated in the plasma of patients with cancer. The analysis of circulating DNA may be useful for diagnosis, prognosis evaluation, and early detection of disease recurrence. In order to investigate cf-DNA as a marker during treatment, we serially quantified total cell-free (cf) and EBV plasma DNA in 30 cases of pediatric B-non-Hodgkin lymphoma by real-time PCR. The cf-DNA levels were significantly increased in patient samples at diagnosis as compared with the healthy controls (p < 0.001). At the end of treatment, a significant decrease in plasma DNA concentration was observed as compared with values observed at diagnosis (median: 94.0 copies/mL, p = 0.001). EBV was detected by ISH in 7/30 patients. Plasma EBV DNA levels were obtained from seven EBV-positive patients (median: 1278 copies/mL), while EBV DNA was not detected in 23 EBV-negative patients and 10 healthy controls. The association between the two methods of detection was statistically significant, with 100% correlation (Kappa coefficient, p = 1). In addition, the decrease of EBV viral load was associated with therapy response. Quantification of plasma EBV DNA may become a valuable source for disease detection of pediatric EBV-associated lymphomas and for monitoring treatment response.

Plasma DNA level in predicting therapeutic efficacy in advanced nonsmall cell lung cancer

Assessment of total plasma DNA can be a promising noninvasive tool for monitoring the effect of cytotoxic therapies in order to predict therapeutic efficacy at an early stage. Cell-free plasma DNA levels were quantified before the first, second and third cycle of chemotherapy in 42 patients with advanced nonsmall cell lung cancer and correlated with response to therapy, as assessed by computed tomography following the third chemotherapy cycle. A significantly lower plasma DNA level, measured before various treatment cycles, was found in patients with remission or stable disease than in those with progression. Higher levels and insufficient decrease in plasma DNA levels during the course of chemotherapy indicated poor outcome. For predicting insufficient therapy response, a sensitivity of 26.9% was achieved at 100% specificity using plasma DNA levels before the first therapy cycle. Prediction of disease progression was achieved with a sensitivity of 35.7% at 100% specificity using plasma DNA levels before the first therapy cycle. Monitoring of plasma DNA levels during the course of chemotherapy could identify patients who are likely to exhibit an insufficient therapeutic response and disease progression at an early stage. This may help in individualising treatment, and could lead to better management of advanced-stage lung cancer.

Share of extracellular mitochondrial DNA with a mutations increases in the plasma of patients lung cancer after radiotherapy

Quantitative and qualitative changes in circulating extracellular DNA (ec-DNA) of blood plasma are considered as markers for diagnosis and prognostic of tumor pathology. We investigated the content of mutant copies of the circulating extracellular mitochondrial DNA (ec-mtDNA) in blood plasma (using the enzymatic method, based on the cleavage of DNA with unpaired bases by CEL-I endonuclease) in 8 patients with lung cancer before and after radiotherapy, as well as in healthy young and elderly donors. It was found that in the plasma of healthy elderly donors share of ec-mtDNA with mutations (consisting of total circulating DNA) is much greater, than that of young donors. On the other hand, in the plasma of lung cancer patients (aged 70-76 years) before radiotherapy a substantial increase in the share of ec-mtDNA with mutations, compared with that of healthy elderly donors. Following radiotherapy, patients with lung cancer found a twofold increase of the proportion of ec-mtDNA with mutations in the total circulating plasma DNA. This increase is largely, perhaps due to the release of ec-mtDNA with mutations from dying tumor cells and cells damaged by normal tissues.

Quantification of free total plasma DNA and minimal residual disease detection in the plasma of children with acute lymphoblastic leukemia

The analysis of total plasma DNA and the monitoring of leukemic clone-specific immunoglobulin and/or T-cell receptor gene rearrangements for the evaluation of minimal residual disease (MRD) in the plasma may be useful tools for prognostic purposes or for early detection of subclinical disease recurrence in children with acute lymphoblastic leukemia (ALL). The aim of this paper is to establish reference ranges for total plasma DNA concentrations and to test the feasibility of MRD measurements employing plasma DNA from children with ALL by using real-time quantitative (RQ)-PCR. Despite wide inter-individual variation, the median concentrations of total plasma DNA for 12 healthy donors (57 ng/ml), 21 children with ALL after day 4 of treatment initiation (62 ng/ml) and 13 children with other malignancies (76 ng/ml) were similar. However, ALL patients had significantly higher concentrations at diagnosis (277 ng/ml) and on treatment day 3 (248 ng/ml) before returning to normal afterwards. Early plasma DNA MRD kinetics could be established for 15 ALL patients and showed good concordance with bone marrow MRD. Plasma DNA was higher in children with ALL at diagnosis but returned to normal within the first four treatment days. Despite low concentrations of DNA, it is feasible to measure MRD kinetics in plasma DNA during ALL induction therapy by adapted real-time PCR methodologies.