cfDNA Levels Research Articles

Role of cfDNA and DNA integrity index (DII) in colon cancer in response to surgery.

236 Background: Colorectal cancer diagnosis requires new biomarkers for improved detection and monitoring. cfDNA has shown potential in detecting minimal residual disease, evaluating treatment response, and providing prognostic information. Recent studies highlight cfDNA as a promising molecular marker for early disease detection and progression monitoring. This study aims to evaluate the role of cfDNA and DII in assessing surgical response in CRC patients. The primary objective is to assess cfDNA levels pre- and post-curative resection. Secondary objectives include identifying factors influencing baseline cfDNA levels, evaluating factors affecting DII, and comparing cfDNA and CEA levels in response to surgery. Statistical analyses included descriptive statistics, ROC curve, Student T tests and Fisher's exact test. Methods: The study was designed as a prospective investigation conducted over a period from September 2022 to April 2024 at the Regional Cancer Centre. Forty pathologically proven non metastatic colonic carcinoma planned for primary surgery were included and patients with sepsis and chronic diseases were excluded from the study. Blood samples (10 ml) were collected pre-surgery and at four weeks post-surgery. Plasma cfDNA was extracted, quantified, and analyzed via quantitative real-time PCR using human β-actin as a reference. The DII was calculated as the ratio of 394 bp to 99 bp amplicons of β-actin. Results: The mean age was 62.15 years (range 39-85), with 55% over 60 years. High-risk features included poor differentiation (4.2%), preoperative CEA >5 ng/ml (17.5%), LVI (20.8%), and PNI (6.3%). Surgical approaches included 10 laparoscopic, 29 open, and 1 converted from laparoscopic to open due to excessive bleeding. Overall stage grouping showed 18 patients in Stage III, and 11 each in Stage I and II. Median pre- and postoperative cfDNA levels were 24.2 ng/μl and 29.6 ng/μl (p=0.83), and median DNA integrity index levels were 1.1 and 1.0 (p=0.95). Preoperative cfDNA levels averaged 24.2 ng/μl and postoperative levels 29.6 ng/μl (p = 0.83); DII levels were 1.1 preoperatively and 1.0 postoperatively (p = 0.95). Postoperative cfDNA levels decreased in 21 patients and increased in 19, while DII decreased in 20 and increased in 20. Significant associations were noted for cfDNA changes with surgery type and high-risk features. ROC analysis revealed cfDNA cut-off at 275 ng/μl, DII at 1.5, and CEA at 4 µg/L. Sensitivities for cfDNA and DII were 66.67% and 100%, respectively, with combined sensitivity reaching 100%. Conclusions: Persistent high cfDNA levels post-surgery suggest potential minimal residual disease despite radical resection. The residual disease is a harbinger of systemic failure. Monitoring cfDNA and DII can identify patients at risk for recurrence, guiding intensified adjuvant therapy. Further studies with extended follow-up are recommended to validate these findings. Clinical trial information: CTRI/2023/10/058468 .

Cell-free DNA release following psychosocial and physical stress in women and men

Cell-free DNA (cfDNA) is continuously shed by all cells in the body, but the regulation of this process and its physiological functions are still largely unknown. Previous research has demonstrated that both nuclear (cf-nDNA) and mitochondrial (cf-mtDNA) cfDNA levels increase in plasma in response to acute psychosocial and physical stress in males. This study further investigated these findings by testing 31 female participants (16 using oral hormonal contraception and 15 not using oral hormonal contraception), and the results were subsequently compared with those of 16 male participants. In addition, cf-nDNA and cf-mtDNA were comparatively quantified in both plasma and saliva at four time points, 2 min before and 2, 15, and 45 min after stress induction. A novel method was implemented to facilitate the straightforward collection of capillary blood by non-medical personnel for plasma analysis. While cf-mtDNA is readily detectable in body fluids due to its high copy number, the quantification of cf-nDNA is challenging due to its low abundance. To overcome this, a multiplex quantitative polymerase chain reaction (qPCR) protocol targeting L1PA2 elements, which are prevalent in the human genome, was utilized. The analysis indicated significantly elevated levels of cf-nDNA in both plasma and saliva in all participants, irrespective of gender, following psychosocial and physical stress. Conversely, neither plasma nor saliva exhibited a consistent or stress-induced release pattern for cf-mtDNA. CfDNA is a promising biomarker that is consistently released after stress in both men and women and can be detected in both plasma and saliva. However, further research is necessary to elucidate the mechanisms of cfDNA release from specific cells and to understand its biological function in the body.

P0065 Circulating cell-free DNA and mitochondrial DNA differentiate disease activity in paediatric-onset inflammatory bowel disease: Interim analysis from Scotland-wide Mini-MUSIC study (2020-26)

Abstract Background Mitochondrial dysfunction has been implicated in the pathogenesis of inflammatory bowel disease (IBD) particularly paediatric-onset IBD (PIBD).1,2 Circulating cell-free DNA (cfDNA) and mitochondrial DNA (mtDNA) are damage-associated molecular patterns (DAMPS) released into the bloodstream during tissue damage and cell death and can be detected in peripheral blood samples.3 Recent studies have suggested that levels of cfDNA and mtDNA may correlate with disease activity in adult IBD4 but their utility in paediatric populations remains unexplored. This study aims to present initial data on the potential use of cfDNA and mtDNA as biomarkers in PIBD. Methods Mini-MUSIC and GI-DAMPS are ongoing longitudinal and cross-sectional multi-centre translational research studies in Scotland (2020 – 2026) with a primary aim of investigating the inflammatory mechanisms of IBD with a focus on multi-omics, immune- and microbiome-profiling aligned to prospective clinical follow-up. We measured circulating cfDNA (GAPDH) and mtDNA (ND2), using digital droplet PCR, in the peripheral blood of patients diagnosed under 17 years of age (A1 phenotype) and correlated this with phenotypic data and disease activity scores. Results Sixty-five A1 patients were included (39/65 (60%) male; 51 Crohn’s disease (CD), 6 ulcerative colitis (UC), 1 inflammatory bowel disease unclassified (IBDU) and 7 symptomatic non-IBD controls) with a median (IQR) age at diagnosis of 13 (10.0-15.0) years. mtDNA levels were significantly higher in those with highly active disease compared to those in remission (median [IQR] 145.1 (89.5-673.7 ng/μl) vs 64.4 (24.3-167.5 ng/μl), p=0.008) and compared to non-IBD symptomatic controls (median [IQR] 145.1 (89.5-673.7 ng/μl) vs 28.8 (12.6 – 138.7 ng/μl), p=0.036). Circulating cell-free DNA levels were significantly higher in those with highly active disease than non-IBD controls (4.94 (1.1-9.9 ng/ul) vs median [IQR] 0.53 (0.0-1.4 ng/ul), p=0.017). mtDNA levels were positively correlated with endoscopic disease activity (SES-CD) in Crohn’s disease (Pearson r=0.7619, p=0.004). Conclusion Our initial data from ongoing work, demonstrate that the levels of cfDNA and mtDNA in the blood are potentially valuable biomarkers for differentiating disease activity in PIBD and to identify PIBD-patients where mitochondrial dysfunction is dominant and thereby with the potential to stratify future human mechanistic studies and mitochondrial-targeted therapies.

Assessment of cfDNA release dynamics during colorectal cancer surgery.

Approximately two-thirds of patients with colorectal cancer (CRC) undergo resection with curative intent; however, 30% to 50% of these patients experience recurrence. The concentration of cell-free DNA (cfDNA) before and after surgery may be related to the prognosis of patients with CRC, but there is limited information regarding cfDNA levels at the time of surgery. Here, we analyzed surgical cfDNA release using plasma samples from 30 colorectal cancer patients at three key points during surgery: preoperative (immediately before surgery), intraoperative (during surgery), and postoperative (at the end of surgery). Automated electrophoresis was used to analyze cfDNA concentrations and fragment sizes, which were then correlated with clinical variables. Our findings indicate a significant increase in cfDNA release during and after surgery (2.8- and 2.2-fold higher respectively, p < 0.01). Characteristic fragments of cfDNA (<400 bp) predominated at all surgical stages; however, the release of genomic material (>400 bp) was also observed. We found that cfDNA concentration increases during and after surgery in patients over 60 years old (2.9-fold higher intraoperatively than preoperatively and 2.3 folds higher postoperatively than preoperatively, p < 0.01); in patients with comorbidities (3.0-fold higher intraoperatively and 2.3-fold higher postoperatively, p < 0.01); and in patients with CEA levels >5 ng/mL (3.1-fold higher intraoperatively and 1.3-fold higher postoperatively, p < 0.01). Interestingly, cfDNA release during surgery is significantly higher in patients with adverse clinical characteristics. Patients bearing locally advanced tumors or metastasis had a 3.1-fold increase in cfDNA release intraoperatively and 2.4-fold increase postoperatively, p < 0.01. cfDNA concentration also increases intraoperatively in patients with a high score of tumor buds (2.6 folds higher, p < 0.02), patients with perineural invasion (3.4-fold higher, p < 0.02) and in patients with lymphovascular invasion (3.1-fold higher, p < 0.05). Furthermore, we observed that cfDNA concentration may rise in correlation with the duration of the surgery, highlighting its potential as a marker of surgical quality. Taken together, our results suggest that in addition to physiological age, comorbidities and unfavorable clinical traits, intense surgical manipulation from the tumor's extent, may result in greater tissue damage and elevated cfDNA release.

Klinička vrednost nivoa serumske hijaluronske kiseline, prokolagena III, N-terminalnog propeptida, sST2 i CFDNK u predikciji oštećenja miokarda kod dece sa teškom pneumonijom

Background: Severe pneumonia complicated by myocardial damage is a serious condition in children, and early prediction and intervention are crucial for improving outcomes. To explore the clinical value of serum hyaluronic acid, procollagen III N-terminal propeptide, sST2, and cfDNA levels in predicting myocardial damage in children with severe pneumonia. Methods: A case-control study was conducted on 140 children with severe pneumonia, divided into a control group (n=70) and a group with disease complicated by myocardial injury (n=70). Biomarker levels (hyaluronic acid, procollagen III, N-terminal propeptide levels, sST2, and cfDNA) were detected through biochemical analysis, and clinical outcomes were compared between the two groups. Results: Logistic regression analysis identified hypoalbuminaemia, age &lt;3 years, and elevated levels of serum hyaluronic acid, procollagen III N-terminal propeptide, sST2, and cfDNA as independent risk factors for myocardial damage. The AUC of these biomarkers in predicting myocardial injury ranged from 0.68 to 0.78, with sensitivities and specificities of 65-80%. The combined prediction AUC was 0.92, with a sensitivity of 87.6% and a specificity of 77%. The control group had lower serum NT-proBNP, CK, CK-MB, and C-reactive protein levels and better clinical outcomes than the myocardial injury group. Conclusions: Serum hyaluronic acid, procollagen III, NTproBNP, sST2, and cfDNA significantly predict severe pneumonia complicated by myocardial damage. Nursing strategies based on these biomarkers can effectively improve treatment outcomes, demonstrating significant clinical application value.

Analysis of fetal fraction in non-invasive prenatal testing with low-depth whole genome sequencing.

The fetal fraction (FF) is a critical factor influencing the performance of non-invasive prenatal testing (NIPT). Different NIPT methods and sequencing depths can lead to distinct minimum FF thresholds for Trisomy 21 (T21). This study aims to analyze the minimum FF thresholds for detecting T21 in PCR-free NIPT using a low-depth whole genome sequencing method. A total of 71 non-pregnant women and 4827 pregnant women were enrolled for a study investigating the impact of FF on NIPT testing. The results showed a weak negative correlation between cfDNA levels and age in non-pregnant women. There was no statistically significant difference in the ratio of chromosome 13, 18, and 21 between pregnant and non-pregnant women. The minimum FF required for 100% detection of trisomy 21 was determined to be 5% in PCR-free NIPT using a low-depth whole genome sequencing method. Analysis of a large sample revealed that 4.43% of pregnant women exhibited FF below 5%, with those having lower FF showing significantly higher maternal weight compared to those near the median. Furthermore, FF demonstrated a significant negative correlation with the weight of pregnant women; specifically, an FF of 5% at 12-19 weeks corresponded to a weight of 96kg, while at 20-22 weeks it corresponded to a weight of 93kg. These results suggest that the FF below 5%, or pregnant women weighing over 93kg, should be given more attention in clinical genetic counseling.

Methylome profiling of cell-free DNA during the early life course in (un)complicated pregnancies using MeD-seq: Protocol for a cohort study embedded in the prospective Rotterdam periconception cohort.

Placental DNA methylation differences have been associated with timing in gestation and pregnancy complications. Maternal cell-free DNA (cfDNA) partly originates from the placenta and could enable the minimally invasive study of placental DNA methylation dynamics. We will for the first time longitudinally investigate cfDNA methylation during pregnancy by using Methylated DNA Sequencing (MeD-seq), which is compatible with low cfDNA levels and has an extensive genome-wide coverage. We aim to investigate DNA methylation in placental tissues and cfDNA during different trimesters in uncomplicated pregnancies, and in pregnancies with placental-related complications, including preeclampsia and fetal growth restriction. Identified gestational-age and disease-specific differentially methylated regions (DMRs) could lead to numerous applications including biomarker development. Our study design involves three sub-studies. Sub-study 1 is a single-centre prospective, observational subcohort embedded within the Rotterdam Periconception cohort (Predict study). We will longitudinally collect maternal plasma in each trimester and during delivery, and sample postpartum placentas (n = 300). In sub-study 2, we will prospectively collect first and second trimester placental tissues (n = 10 per trimester). In sub-study 3 we will retrospectively collect plasma after non-invasive prenatal testing (NIPT) in an independent validation case-control cohort (n = 30-60). A methylation-dependent restriction enzyme (LpnPI) will be used to generate DNA fragments followed by sequencing on the Illumina NextSeq2000 platform. DMRs will be identified in placental tissues and cell types, and in cfDNA related to gestational-age or placental-related complications. (Paired) placental methylation profiles will be correlated to DMRs in cfDNA to aid tissue-of-origin analysis. We will establish a methylation score to predict associated diseases. This study will provide insights in placental DNA methylation dynamics in health and disease, and could lead to clinical relevant biomarkers.

Quantifying DNA Lesions and Circulating Free DNA: Diagnostic Marker for Electropathology and Clinical Stage of AF.

Atrial fibrillation (AF) persistence is associated with molecular remodeling that fuels electrical conduction abnormalities in atrial tissue. Previous research revealed DNA damage as a molecular driver of AF. This study sought to explore the diagnostic value of DNA damage in atrial tissue and blood samples as an indicator of the prevalence of electrical conduction abnormalities and stage of AF. High-sensitivity long-run real-time PCR was performed on mitochondrial (ND1) and nuclear (P53) DNA from atrial tissue samples from paroxysmal (PAF), persistent (PeAF), and longstanding persistent (LS-PeAF) AF, and sinus rhythm (SR) patients (n=83). PicoGreen assay and quantitative polymerase chain reaction were used on circulating free DNA (cfDNA) markers (total cfDNA, β-globin, ND1, and P53) in blood samples of 70 patients with AF or SR. High-resolution epicardial mapping of the atria (n=48) was conducted to quantify electrical conduction abnormalities. The number of DNA lesions gradually and significantly increased in PAF and PeAF and in patients with<3years of AF compared with SR. In SR, the quantity of nuclear DNA damage significantly correlated with the proportion of fractionated potentials. Mitochondrial DNA lesions correlated with slower conduction velocity and lower potential amplitudes in AF samples. Also, mitochondrial cfDNA levels decreased in patients with >3 years of AF compared with<3 years of AF (P=0.004). The quantity of DNA lesions in atrial tissue samples is associated with atrial conduction abnormalities and stage of AF. Serum DNA damage markers discriminate short- from long-term AF. Therefore, the quantity of DNA damage may have diagnostic value in clinical AF management.

Human Papilloma Virus Circulating Cell-Free DNA Kinetics in Cervical Cancer Patients Undergoing Definitive Chemoradiation.

The human papillomavirus (HPV) is a significant cause of cervical cancer. We hypothesized that detecting viral cell-free HPV DNA (cfDNA) before, during, and after chemoradiation (chemoRT) could provide insights into disease extent, clinical staging, and treatment response. Sixty-six patients with locally advanced cervical cancer were enrolled between 2017 and 2023. 49 received standard-of-care (SOC) treatment and 17 participated in a clinical trial combining a therapeutic HPV vaccine (PDS0101; IMMUNOCERV). Plasma was collected at baseline, weeks 1, 3, and 5 of chemoRT, and 3-4 months after chemoRT. HPV cfDNA was quantified using droplet digital PCR targeting the HPV E6/E7 oncogenes of 13 high-risk types. MRI was performed at baseline and before brachytherapy. The median follow-up was 23 months, with recurrence-free survival (RFS) of 78.4% at 2 years. Baseline nodal disease extent correlated with HPV cfDNA levels. HPV cfDNA levels peaked in week 1 of radiation and decreased through treatment. Patients receiving the PDS0101 vaccine had a higher rate of undetectable HPV type 16 cfDNA compared to SOC. HPV cfDNA clearance correlated with better 2-yr RFS (92.9% vs. 30%, log-rank p=0.0067). The strongest predictor of RFS was HPV cfDNA clearance in follow-up achieving a concordance index (CI) 0.83, which improved when combined with MRI response (CI 0.88). HPV cfDNA levels change dynamically during chemoRT. HPV cfDNA at follow-up predicts RFS. Delivery of therapeutic HPV vaccine with chemoRT was linked to rapid HPV cfDNA decline. Monitoring HPV cfDNA during and after chemoRT may guide tailoring of personalized treatment.

Biomarker potential of plasma cell-free DNA for cholangiocarcinoma.

To prevent the development of cholangiocarcinoma, an effective screening opisthorchiasis viverrini and/or differential diagnosis of and the cholangiocarcinoma is crucial needed. The level and quality of cfDNA in plasma are being investigated for their potential role as biomarkers in cholangiocarcinoma. The study enrolled 43 healthy controls (N), 36 O. viverrini-infected subjects (OV), and 36 cholangiocarcinoma patients (CCA). Plasma cfDNA was quantified by fluorometry (Qubit 4), and qualified analysis, including % tumor fraction, ctDNA, ploidy number, and somatic copy number alteration (SCNA), was conducted using ULP-WGS and analyzed by iChorCNA. The statistical analysis and comparison among the groups were performed. The results showed that cfDNA could effectively differentiate between N and OV from CCA statistically both by DNA amount and quality. Using a cut-off of >20.94ng/ml, the sensitivity and specificity of the cfDNA concentration were determined to be 86.11% and 98.73% for the differential diagnosis of cholangiocarcinoma, respectively. The ULP-WGS with iChorCNA indicated the % tumor fraction of cfDNA (P<0.001) and the values of the ploidy number (P<0.001) of the cholangiocarcinoma group and the other groups were statistically significant. Moreover, the SCNA of the cholangiocarcinoma group was shown to be significantly high in comparison to that of the healthy control group with an odds ratio of 11.688 (P<0.001). The use of cfDNA concentration and ULP-WGS for analyzing DNA quality including % tumor fraction, ctDNA concentration, tumor ploidy, and SCNA are useful for the differential diagnosis of cholangiocarcinoma from opisthorchiasis viverrini and healthy individuals.

Circulating Cell-free Human Papillomavirus DNA as a Tumor Marker in Recurrent or Metastatic Cervical Cancer: A Pilot Study

PURPOSE: Monitoring of circulating human papillomavirus (HPV) cell-free DNA (cfDNA) is a minimally invasive approach for surveillance in HPV-associated cancers, particularly cervical cancer. The aim of this study was to monitor circulating HPV cfDNA levels in patients with recurrent or metastatic cervical cancer during treatment and follow-up to assess the utility of HPV cfDNA as a tumor marker for disease surveillance and in guiding clinical treatment decisions. EXPERIMENTAL DESIGN: In this prospective pilot observational study, levels of HPV cfDNA in serum samples from 28 patients with recurrent or metastatic HPV+ cervical cancer were measured via digital droplet polymerase chain reaction. Results for HPV cfDNA levels were matched to clinical outcomes and to serum levels of squamous cell carcinoma antigen (SCC-Ag) to assess the clinical potential of HPV cfDNA as a tumor marker. RESULTS: HPV cfDNA was detected in all 28 patients (100%). Notably, median baseline HPV cfDNA levels varied according to the metastatic pattern in individual patients (P=0.019). Specifically, patients with a combined multiple-metastasis pattern had higher median baseline HPV cfDNA levels than patients with a single metastasis (P=0.003). All participants exhibited changes in HPV cfDNA levels over a median monitoring period of 2 months (range 0.3-16.9) before evaluations for treatment response or disease progression. Among 26 patients initially diagnosed with squamous cell cervical cancer, the positivity rate was 100% for HPV cfDNA and 69.2% for SCC-Ag (P=0.004, 95% confidence interval, 0-0.391). Among 20 patients longitudinally monitored for squamous cell cervical cancer, the concordance with changes in disease status was 90% for HPV cfDNA and 50% for SCC-Ag (P=0.014, 95% confidence interval, 0.022-0.621). CONCLUSIONS: HPV cfDNA is a promising tumor marker for HPV+ cervical cancer. In the context of precision medicine, HPV cfDNA is poised to play an increasingly pivotal role in monitoring treatment efficacy, providing valuable insights into disease progression, and guiding clinical decisions.

Circulating cell‐free DNA in liver transplantation: A pre‐ and post‐transplant biomarker of graft dysfunction

AbstractBackgroundLiver transplantation (LT) is still limited by organ shortage and post‐transplant monitoring issues. While machine perfusion techniques allow for improving organ preservation, biomarkers like donor‐derived cell‐free DNA (dd‐cfDNA) and mitochondrial cfDNA (mt‐cfDNA) may provide insights into graft injury and viability pre‐ and post‐LT.MethodsA prospective observational cohort study was conducted on LT recipients (n = 45) to evaluate dd‐cfDNA as a biomarker of graft dysfunction during the first 6 months after LT. Dd‐cfDNA was quantified on blood samples collected pre‐LT and post‐LT using droplet digital PCR. In livers undergoing dual hypothermic oxygenated machine perfusion (D‐HOPE), total cfDNA and mt‐cfDNA levels were measured on perfusate samples collected at 30‐min intervals. Correlations with graft function and clinical outcomes were assessed.ResultsDd‐cfDNA levels peaked post‐LT and correlated with transaminase levels and histological injury severity. The longitudinal assessment showed that postoperative complications and rejection were associated with an increase in dd‐cfDNA levels. Mt‐cfDNA levels in D‐HOPE perfusate correlated with graft function parameters post‐LT and were higher in patients with early allograft dysfunction and severe complications.ConclusionsThis study confirms dd‐cfDNA as a marker of graft injury after LT and suggests that perfusate mt‐cfDNA levels during D‐HOPE correlate with graft function and post‐transplant clinical outcome. Integration of these tests into clinical practice may improve transplant management and viability assessment during hypothermic perfusion.

Early Monitoring of Donor-Derived Cell-Free DNA in Kidney Allograft Recipients Followed-Up for Two Years: Experience of One Center.

(1) Background: donor-derived circulating free DNA (dd-cfDNA), an innovative biomarker with great potential for the early identification and prevention of graft damage. (2) Methods: Samples were collected prospectively and the study was performed retrospectively to analyze dd-cfDNA plasma levels in 30 kidney transplant patients during their post-transplant follow-up (15 days, 3, 6, and 9 months), to determine if the result could be of interest in the identification of possible adverse events, especially rejection. The aim was to verify whether the data on sensitivity, specificity, NPV, and PPV compare with reference values and creatinine values. (3) Results: We observed levels of dd cfDNA > 1% in six of nine patients with active rejection (ABMR or TCMR) and elevated values (>0.5%) in two other patients in this rejection group. Our results show low values of sensitivity = 50%, specificity = 61.11%, rejection NPV = 64.71%, and rejection PPV = 46.13% of the technique compared to reference values previously published. With respect to creatinine, only for TCRM, we observed better results for dd-cfDNA in these parameters than in creatinine. Also, our data suggest that dd-cfDNA could help to differentiate those patients with dnDSAs that are going to through rejection better than creatinine, specially at 15 d post transplant. In this study, this appears to have no positive predictive value for borderline rejection (BR) or TCMR IA. (4) Conclusions: plasma levels of dd-cfDNA could be considered an additional or alternative biomarker for graft rejection monitoring in early post-kidney transplant up to several months before its clinical presentation, especially for patients with suspected TCMR or ABMR.

Evaluation of the cell death markers for aberrated cell free DNA release in high altitude pulmonary edema.

The effect of high altitude (HA, altitude >2500 m) can trigger a maladaptive response in unacclimatized individuals, leading to various HA illnesses such as high altitude pulmonary edema (HAPE). The present study investigates circulating cell free (cf) DNA, a minimally invasive biomarker that can elicit a pro-inflammatory response. Our earlier study observed altered cfDNA fragment patterns in HAPE patients and the significant correlation of these patterns with peripheral oxygen saturation levels. However, the unclear release mechanisms of cfDNA in circulation limit its characterization and clinical utility. The present study not only observed a significant increase in cfDNA levels in HAPE patients (27.03 ± 1.37 ng/ml; n = 145) compared to healthy HA sojourners (controls, 14.57 ± 0.74 ng/ml; n = 65) and highlanders (HLs, 15.50 ± 0.8 ng/ml; n = 34) but also assayed the known cell death markers involved in cfDNA release at HA. The study found significantly elevated levels of the apoptotic marker, annexin A5, and secondary necrosis or late apoptotic marker, high mobility group box 1, in HAPE patients. In addition, we observed a higher oxidative DNA damage marker, 8-hydroxy-2'-deoxyguanosine, in HAPE compared with controls, suggestive of the role of oxidative DNA status in promoting the inflammatory potential of cfDNA fragments and their plausible role in manifesting HAPE pathophysiology. Extensive in vitro future assays can confirm the immunogenic role of cfDNA fragments that may act as a danger-associated molecular pattern and associate with markers of cellular stresses in HAPE.

Abstract A035: A real-time PCR method for the detection of cancer-specific methylation patterns in cfDNA

Abstract Non-invasive liquid biopsy tests are proving to be the next frontier in cancer diagnostics with methylation emerging as a reliable biomarker for low levels of circulating tumor DNA (ctDNA). There is a rising interest in sensitive, low-cost liquid biopsy assays to detect low copies of hypermethylated ctDNA in an excess background of non-cancer-associated hypomethylated cell- free DNA (cfDNA). Real-time quantitative methylation-specific PCR (qMSP) allows for the detection of low abundance methylated fragments. qMSP approaches have previously been developed but are predominantly designed to target a specific CpG site. To date we are not aware of any qMSP approaches that target pan-cancer-associated methylation patterns across multiple CpG sites. Harbinger Health has developed a qMSP method with locked nucleic acid (LNA) blockers that target methylation haplotypes, allowing for multiplex enrichment and quantification of cancer-specific methylation patterns. Using targeted bisulfite sequencing, we identified ten pan-cancer-informative CpG-dense regions which showed consistent contiguous elevated methylation states in cancer cfDNA and low levels of methylation in non-cancer cfDNA. We designed qMSP assays for these regions and LNA blockers which bind to unmethylated target CpG sites, outcompeting the hydrolysis probe through a high Tm differential. This prevented non-specific probe hybridization. The qMSP-LNA assays were evaluated in simplex and multiplex using cell line DNA in a titration of hypermethylated “ctDNA” spiked into hypomethylated “cfDNA” to simulate varying ctDNA levels, and then validated with cancer and non-cancer cfDNA samples at 5 ng input, equivalent to 1450 haploid copies. The percentage of methylated DNA was estimated by the ratio of methylated copies (qMSP-LNA) to total copies (internal reference), which were quantified by standard curves. The qMSP-LNA assays detected low abundance methylated DNA fragments down to 23 target copies. LNA blockers completely depleted background signal originating from unmethylated DNA with high specificity, showing no inhibition of methylated DNA detection. When tested in cfDNA samples which varied in conversion efficiency, discordant methylation states, and methylation abundance, the assays reproducibly amplified and detected highly methylated fragments which correspond to cancer signal. Harbinger Health has developed qMSP-LNA assays for contiguous CpG biomarkers which differentiate cancer and non-cancer samples from only ten pan-cancer-informative regions. The qMSP-LNA method detected low abundance fragments with specific methylation patterns with high specificity and sensitivity. The assay reproducibly detected methylated DNA in cfDNA samples that represent a clinically relevant range of methylation signal and will be further validated in a larger cohort of cancer and non-cancer cfDNA samples. Due to the high specificity and sensitivity, this cost-effective PCR method may provide utility and improve access for early cancer detection and monitoring. Citation Format: Emily Neaga, Caitlin Gilley, Sarah Falotico, Mayur Gurnani, Zhenyu Zhang, Jocelyn Charlton, Miguel Williams, Anthony Shuber. A real-time PCR method for the detection of cancer-specific methylation patterns in cfDNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A035.

INNV-15. A PIVOTAL, MULTICENTER TRIAL OF FOCUSED ULTRASOUND FOR BLOOD-BRAIN BARRIER DISRUPTION FOR PLASMA-BASED LIQUID BIOPSY IN PATIENTS WITH GLIOBLASTOMA (LIBERATE)

Abstract BACKGROUND Liquid biopsy in glioblastoma (GBM) is hindered by insufficient requisite circulating-tumor (ct) and cell-free (cf) DNA in blood due to blood-brain barrier (BBB). This limits the identification of blood-based tumor biomarkers along with biomarker-driven systemic therapies. Real-time image-guided low-intensity focused ultrasound (LIFU) plus IV microbubble oscillators, leads to non-invasive BBB disruption. METHODS LIBERATE is an ongoing, prospective, multi-center, self-controlled, pivotal trial investigating safety and technical efficacy of LIFU-based BBB disruption for increasing blood ctDNA and cfDNA levels in adult GBM patients (aged 18-80 years). Patients with suspected GBM planned for biopsy/resection at ten US centers are being enrolled. Patients with multifocal tumors or tumors arising from deep midline, thalamus, cerebellum, or brainstem are excluded. Patients are administered IV microbubbles for enhanced sonication effects, after which MR-guided BBB disruption using 220kHz LIFU device is performed with real-time acoustic-feedback-control for effective microbubble activation. Pre- and post-LIFU, phlebotomy and MRI brain are performed. Primary study endpoint is defined, per subject, as ratio between their blood cfDNA level 1-hour post-LIFU procedure compared to blood cfDNA pre-procedure. Primary study hypothesis is that BBB disruption leads to ≥2-fold rise in blood cfDNA. Secondary hypothesis is that there exists ≥75% agreement between biomarker pattern in blood cfDNA sample obtained 1-hour post-LIFU and biomarker pattern in tumor tissue obtained later. Trial is powered to evaluate both primary and secondary hypotheses. Based on assumed true agreement rate of 91% and one-sided alpha=0.025, an exact test for binomial proportions provides N=50 with 84% power for secondary hypothesis. Exploratory endpoints include (1) sensitivity of detection of specific somatic mutations in ctDNA from blood collected pre- and post-LIFU, (2) ctDNA estimation in samples collected at 30-minutes, 1-hour, 2-hour, and 3-hour post-LIFU to determine time of greatest yield, (3) correlation of MRI parameters related to grading of BBB disruption and ctDNA-based biomarkers from post-LIFU blood samples. Patient enrollment commenced in 2022 and remains ongoing (NCT05383872).

IMMU-20. CIRCULATING CELL-FREE DNA DIRECTLY INHIBITS T CELL RESPONSES AND CONTRIBUTES TO SYSTEMIC IMMUNOSUPPRESSION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with poor survival despite standard of care. Checkpoint inhibitors aimed to revive dysfunctional T cells have significantly improved outcomes in other cancers, but have failed GBM. GBM patients have severe peripheral immunosuppression, including severe lymphopenia, immune organ atrophy, and defective T cell responses. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. Using parabiosis, we demonstrated that serum-derived factors were sufficient to drive hallmark features of immunosuppression, including inhibition of ex vivo T cell proliferation. This factor was nonsteroidal in nature and had molecular weights greater than 100kDa. Mass-spectrometry and pathway analysis of the suppressive serum implicated proteins involved in DNA response elements, cell death, and DNA-histone complexes. We hypothesized that the factor is cell-free DNA (cfDNA). Confirming this, serum levels of cfDNA are increased in glioma-bearing mice. Moreover, exposure to cfDNA from serum of glioma-bearing mice was sufficient to directly and potently inhibit T cell proliferation ex vivo. Interestingly, exposure to genomic DNA was not sufficient to induce proliferation defects in T cells, highlighting the unique features of cfDNA. Importantly, reducing cfDNA content using DNaseI partially restored proliferation capacity. We determined that the T cell proliferation defects were independent of DNA sensing mechanisms through AIM2, implicating a novel pathway of DNA sensing in T cells. Finally, to determine the origin of the cfDNA, we employed methyl-seq. The chromatin signature most closely represented neutrophils. This highlights a potential role for neutrophils-derived cfDNA as a targetable strategy in GBM. Together, we contend that cfDNA induces immunosuppressive effects in GBM and devising strategies to reduce circulating cfDNA could be a therapeutic approach to improve T cell functions and outcomes in GBM patients.

The Imbalance of Homeostasis in Neutrophil Extracellular Traps is Associated with Portal Vein Thrombosis in Patients with Decompensated Cirrhosis.

Portal vein thrombosis (PVT) is a challenging complication in liver cirrhosis, with no currently available sensitive diagnostic markers. This study aimed to investigate the potential of neutrophil extracellular traps (NETs) and Deoxyribonuclease (DNase) as diagnostic indicators for PVT in chronic hepatitis B (CHB)-related decompensated cirrhosis. We analyzed 145 CHB-related decompensated cirrhosis patients from the Ditan study and 33 from the Changgung validation study, categorizing them based on PVT occurrence. Plasma samples were assessed for NET markers, including cell-free DNA (cfDNA) and histone-DNA complexes, along with DNase activity. PVT patients exhibited elevated levels of cfDNA and histone-DNA complexes, and reduced DNase activity. This pattern persisted regardless of hepatocellular carcinoma (HCC) status. Histone-DNA levels, DNase activity, and hemoglobin were identified as independent risk factors for PVT. Receiver operating characteristic curve analysis revealed that high histone-DNA levels may serve as a potential diagnostic marker for PVT, with an area under the curve of 0.8628 in the Ditan study and 0.7521 in the Changgung study. When combined with cfDNA and DNase activity, the area under the curve improved to 0.8774 in the Ditan study and 0.7975 in the Changgung study. Imbalances in NET homeostasis are associated with PVT in CHB-related decompensated cirrhosis, including cases involving HCC. Histone-DNA complexes, a significant risk factor for PVT, show potential as a diagnostic marker for PVT in decompensated cirrhosis, particularly in HBV-related HCC.

Astaxanthin improves assisted reproductive technology outcomes in poor ovarian responders through alleviating oxidative stress, inflammation, and apoptosis: a randomized clinical trial

BackgroundPoor ovarian response (POR) to controlled ovarian stimulation (COS) remains challenging, especially in advanced-age women with diminished ovarian reserve, resulting in low live birth rates. Many patients prefer to conceive with their eggs, underscoring the need for improved treatments. This study explores astaxanthin potential as a COS adjuvant to improve ovarian response and assisted reproductive technology (ART) outcomes, considering its impact on oxidative stress (OS), inflammation, and apoptosis, which are key factors in POR.MethodsIn this randomized, triple-blind, placebo-controlled trial, 60 infertile POR patients from POSEIDON Group 4 (the poorest prognosis category, age > 35 and poor ovarian reserve (anti-müllerian hormone < 1.2 ng/ml or antral follicle count < 5) undergoing intracytoplasmic sperm injection were enrolled. Patients were assigned to receive either 12 mg/day AST or placebo for eight weeks. All patients underwent a gonadotropin-releasing hormone antagonist regimen for COS. ART outcomes were compared between groups. Blood serum and follicular fluid (FF) were analyzed for OS markers (superoxide dismutase [SOD], total antioxidant capacity [TAC], and malondialdehyde [MDA]), and pro-inflammatory cytokines (interleukin-6 [IL-6], interleukin-8 [IL-8], and vascular endothelial growth factor [VEGF]) via enzyme-linked immunosorbent assay kits, and cell-free DNA [cfDNA] (apoptotic marker) via ALU quantitative polymerase chain reaction.ResultsAfter the intervention, the AST group exhibited a significant elevation in serum (P = 0.013) and TAC (P = 0.030), accompanied by a significant reduction in serum MDA (P = 0.005). No significant differences between AST and placebo groups were observed in OS markers in FF. AST group showed significant reductions in the serum IL-6 (P < 0.001), IL-8 (P = 0.001), and VEGF (P = 0.002) levels following AST therapy. In the AST group, FF levels of IL-6 (P = 0 < 001), IL-8 (P = 0.036), VEGF (P = 0.006), and cfDNA (P < 0.001) were significantly lower than in the placebo group. Between-group comparisons showed significant differences in the alterations of serum SOD (P = 0.027), IL-6 (P < 0.001), and IL-8 (P = 0.035) levels between AST and placebo groups. The AST group showed significant increases in the number of retrieved oocytes (P = 0.003), MII oocytes (P = 0.004), frozen embryos (P = 0.037), and high-quality embryos (P = 0.014) compared to the placebo group.ConclusionAST shows promise as a COS adjuvant therapy, potentially enhancing some ART outcomes in POR through alleviating OS, inflammation, and apoptosis.Trial registrationClinical trial registration number: IRCT20230223057510N1, URL: https://irct.behdasht.gov.ir/trial/68870, registration date: 2023 March 16.Graphical

Circulating nucleases degrade high-molecular-weight cell-free DNA in coronary vessels during acute myocardial infarction

Abstract Introduction Extracellular cell-free DNA (cfDNA) originates from various biological processes, including cell turnover, apoptosis, necrosis, NETosis, and other forms of cell death. In healthy individuals, highly fragmented cfDNA is elevated in the bloodstream due to infection, cancer, inflammation and thrombosis. In thrombotic vascular diseases, neutrophils release elongated, high-molecular-weight DNA with bioactive and prothrombotic properties in a process termed NETosis. Circulating nucleases are thought to counteract pathogenic properties of NETs. However, attempts to connect total cfDNA concentration to nuclease activity have failed. Purpose We aimed to elucidate the in vivo connection between extracellular DNA and circulating nucleases in thrombotic vascular diseases by investigating DNA length as a potential novel biomarker in cardiovascular disease. Methods Citrated plasma from healthy volunteers (n=363) and from ST-elevation myocardial infarction (MI) coronary aspirates (n=59) and peripheral arterial sites (n=96) were analyzed for DNA concentration, length, and nuclease activity. Total DNA concentration was measured with a fluorescent DNA binding dye, and elongated DNA assessed using qPCR and Agilent bioanalyzer high-sensitivity DNA chip. Nuclease activity was measured by single radial enzyme-diffusion (SRED) assay. Cardiac MRI assessed infarct size acutely and six months after the event. Results Coronary aspirates exhibited an elevation in total DNA, with a significant rise in high-molecular weight cell-free DNA in addition to an increase in sub-nucleosome fragmentation (Figure 1). Peripheral and coronary plasma samples from individuals with myocardial infarction (MI) showed elevated levels of total and elongated cfDNA. When compared to healthy individuals, the integrity of cfDNA was lower in peripheral samples and higher in coronary aspirates. During myocardial infarction, circulating nuclease activity was elevated systemically but low at the coronary culprit lesion site. Elongated high-molecular-weight cfDNA accumulates primarily in coronary vessels but is also elevated systemically. In coronary aspirates, nuclease activity negatively correlated with elongated DNA and DNA integrity (rs=0.6255, p&amp;lt;0.0001). Conclusions Elongated DNA accumulates during MI at the culprit lesion site. Its inverse correlation with nuclease activity underscores the role of nucleases in cleaving elongated DNA to shorter, soluble fragments. This study establishes DNA length and integrity as novel biomarkers in thrombotic cardiovascular disease.Figure 1:Coronary cfDNA lengthFigure 2:Nuclease activity and cfDNA