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Objective Diffuse large B-cell lymphoma (DLBCL) is an aggressive type of lymphoma. Circulating cell-free DNA (cfDNA) is the promising liquid biopsy in malignancy.Method Assessment of cfDNA concentration and DNA integrity index (DII) in 30 newly diagnosed DLBCL patients and correlating them with the international prognostic index.Results Circulating cfDNA concentration was significantly higher in newly diagnosed DLBCL patients than controls, while DII was significantly lower. There was a significant correlation between cfDNA concentration and adverse prognostic factors.Conclusion cfDNA concentration is potentially strong diagnostic and prognostic marker of DLBCL, however DII is only useful as a diagnostic biomarker.

Aim: There is limited data on prognostic value of baseline plasma cell free DNA (cfDNA) in advanced squamous non-small cell lung cancer (sq-NSCLC). This prospective observational study aimed to assess change in plasma cfDNA levels in locally-advanced/metastatic sq-NSCLC with chemotherapy and its correlation with symptom-scores and radiological-responses. Methods: Chemotherapy-naive patients with stages-IIIB/IIIC/IV sq-NSCLC (n = 59), smokers with chronic obstructive pulmonary disease [COPD, COPD-controls (CC); n = 27] and healthy-controls (n = 25) were enrolled. Respiratory symptom burden (RSB) and total symptom burden (TSB) were calculated from mean visual-analog-scores (VAS) of dyspnoea, cough, chest pain, hemoptysis RSB, anorexia and fatigue (all six for TSB). cfDNA was isolated from peripheral blood. All patients received platinum-doublet chemotherapy. RSB/TSB/cfDNA assessment and contrast-enhanced computed tomography (CECT)-thorax scans were done at baseline and post-chemotherapy. Results: At baseline, 13/59 (22%) sq-NSCLC, 3/27 (11%) CC and none (0%) healthy-controls had detectable cfDNA. All three CC were heavy smokers with no evidence of malignancy and undetectable cfDNA levels on repeat testing. In sq-NSCLC group, majority were males (95%), current-smokers (88%), heavy-smokers (70%), had metastatic disease (59%) with median age of 65 years. Eastern Co-operative Oncology Group (ECOG) performance status (PS) was 0–1 (56%) and 2 (42%). Median RSB- and TSB-scores were 9 [interquartile range (IQR) = 5–14] and 16 (IQR = 9–23), respectively. Of the 59 patients, 54 received ≥ 1 cycle while 27 underwent post-C4 evaluation with detectable cfDNA levels in 18/27 (66.7%). No baseline characteristic correlated with cfDNA detectability. Median overall survival (OS) and progression-free survival (PFS) were 262 days and 167 days, respectively. ECOG PS ≥ 2, RSB-score > 9 and TSB-score > 16 were all associated with worse OS and PFS as was cfDNA detectability [median OS = 97 days vs. 298 days and median PFS = 97 days vs. 197 days; P = 0.025; hazard ratio (HR) = 2.17]. Conclusions: Baseline cfDNA detectability is independently associated with poor OS and PFS in patients with advanced sq-NSCLC on chemotherapy.

Precision medicine has emerged as a cornerstone in cancer treatment revolutionizing our approach across malignancies. Molecular profiling of biliary tract cancers (BTCs) has changed the treatment landscape positively by prolonging survival in an aggressively fatal malignancy in its advanced stages. The acquisition of tissue tumor DNA for genomic analysis in BTC is often anatomically challenging, limited by quantity and quality. In response, ctDNA has emerged as a noninvasive means of molecular profiling. The utility of both plasma and bile ctDNA has been explored in several studies demonstrating the high mutation detection rates and the ability to isolate targetable mutations when present. In addition, the concordance between plasma and tissue DNA provides validity in utilizing ctDNA results to infer treatment decisions. Analysis of ctDNA in BTC has also provided prognostic information and facilitated evaluation of clonal evolution with ease of serial measurements. Insight into novel mechanisms of resistance to targeted therapies are being uncovered in ctDNA. As research endeavors continue to deepen our understanding in the field particularly in the space of ctDNA surveillance after curative intent, the tremendous progress made so far has enabled integration of ctDNA into the clinical practice of BTCs.

Background: Esophageal cancer (EC) is a prevalent malignancy characterized by a low 5-year survival rate, primarily attributed to delayed diagnosis and limited therapeutic options. Currently, early detection of EC heavily relies on endoscopy and pathological examination, which pose challenges due to their invasiveness and high costs, leading to low patient compliance. The detection of DNA methylation offers a non-endoscopic, cost-effective, and secure approach that holds promising prospects for early EC detection. Methods: To identify improved methylation markers for early EC detection, we conducted a comprehensive review of relevant literature, summarized the performance of DNA methylation markers based on different input samples and analytical methods in EC early detection and screening. Findings: This review reveals that blood cell free DNA methylation-based method is an effective non-invasive method for early detection of EC, although there is still a need to improve its sensitivity and specificity. Another highly sensitive and specific non-endoscopic approach for early detection of EC is the esophageal exfoliated cells based-DNA methylation analysis. However, while there are substantial studies in esophageal adenocarcinoma, further more validation is required in esophageal squamous cell carcinoma. Conclusion: In conclusion, DNA methylation detection holds significant potential as an early detection and screening technology for EC. [ABSTRACT FROM AUTHOR]

Novel blood-circulating molecules, as potential biomarkers for glioblastoma multiforme (GBM) diagnosis and monitoring, are attracting particular attention due to limitations of imaging modalities and invasive tissue biopsy procedures. This study aims to assess the diagnostic and prognostic values of circulating cell-free DNA (cfDNA) in relation to inflammatory status in GBM patients and to determine the concentration and average size of DNA fragments typical of tumour-derived DNA fractions. Preoperative plasma samples from 40 patients (GBM 65.0 ± 11.3 years) and 40 healthy controls (HC 70.4 ± 5.4 years) were compared. The cfDNA concentrations and lengths were measured using the electrophoresis platform, and inflammatory indices (NLR, PLR, LMR, and SII) were calculated from complete blood cell analysis. More fragmented cfDNA and 4-fold higher 50–700 bp cfDNA concentrations were detected in GBM patients than in healthy controls. The average cfDNA size in the GBM group was significantly longer (median 336 bp) than in the HC group (median 271 bp). Optimal threshold values were 1265 pg/μL for 50–700 bp cfDNA (AUC = 0.857) and 290 bp for average cfDNA size (AUC = 0.814). A Kaplan–Meier survival curves analysis also demonstrated a higher mortality risk in the GBM group with a cut-off >303 bp cfDNA. This study is the first to have revealed glioblastoma association with high levels of cfDNA > 1000 pg/μL of 50–700 bp in length, which can be aggravated by immunoinflammatory reactivity. [ABSTRACT FROM AUTHOR]

Amoebic liver abscess (ALA) is commonly seen in tropical countries and diagnosis of ALA relies mainly on non-specific serological and imaging techniques as well as PCR from pus. This study evaluated the potential of using cell free DNA (cfDNA) from serum and urine for diagnosing ALA. We prospectively evaluated quantitative PCR (qPCR) for detection of cf DNA in serum and urine sample in all liver abscess patients. The samples were collected from patients reporting to emergency ward of Postgraduate Institute of Medical Education and Research, Chandigarh, India with symptoms suggestive of liver abscess. Real time PCR was done to detect cf DNA in serum and urine by targeting 99-bp unit of small subunit rRNA of Entamoeba histolytica and conventional PCR for pus. A total 113 samples (serum and urine) and 100 pus samples were analysed. A total of 62 ALA patients were confirmed; with maximum 57 patients detected by qPCR for cfDNA in the serum, 55 patients by PCR on pus aspirate and 50 ALA patients by qPCR for cfDNA in urine sample. Therefore, the sensitivity of qPCR for detection of cf DNA in serum was 91.94% and for urine was 80.65%. A total of 11.2% of ALA patients were diagnosed only through detection of E. histolytica cf DNA in their serum and urine. Detection of cfDNA from serum, urine of ALA has a potential role in future especially for developing countries as it is a rapid, sensitive and patient friendly diagnostic approach. [ABSTRACT FROM AUTHOR]

• Our systematic review of publications related to methylation biomarkers in breast cancer identifies potential genes that can be evaluated for non-invasive early detection of breast cancer. • Evaluation of the literature demonstrates that comprehensive prospective studies are yet needed to bring these findings to the patients. Early detection of breast cancer would help alleviate the burden of treatment for early-stage breast cancer and help patient prognosis. There is currently no established gene panel that utilizes the potential of DNA methylation as a molecular signature for the early detection of breast cancer. This systematic review aims to identify the optimal methylation biomarkers for a non-invasive liquid biopsy assay and the gaps in knowledge regarding biomarkers for early detection of breast cancer. Following the PRISMA-ScR method, Pubmed and Google Scholar was searched for publications related to methylation biomarkers in breast cancer over a five-year period. Eligible publications were mined for key data fields such as study aims, cohort demographics, types of breast cancer studied, technologies used, and outcomes. Data was analyzed to address the objectives of the review. Literature search identified 112 studies of which based on eligibility criteria, 13 studies were included. 28 potential methylation gene targets were identified, of which 23 were methylated at the promoter region, 1 was methylated in the body of the gene and 4 were methylated at yet to be identified locations. Our evaluation shows that at minimum APC, RASSFI, and FOXA1 genes would be a promising set of genes to start with for the early detection of breast cancer, based on the sensitivity and specificity outlined in the studies. Prospective studies are needed to optimize biomarkers for broader impact in early detection of breast cancer. [ABSTRACT FROM AUTHOR]

Oropharyngeal cancer (OPC) poses a complex therapeutic dilemma for patients and oncologists alike, made worse by the epidemic increase in new cases associated with the oncogenic human papillomavirus (HPV). In a counterintuitive manner, the very thing which gives patients hope, the high response rate of HPV-associated OPC to conventional chemo-radiation strategies, has become one of the biggest challenges for the field as a whole. It has now become clear that for ~30-40% of patients, treatment intensity could be reduced without losing therapeutic efficacy, yet substantially diminishing the acute and lifelong morbidity resulting from conventional chemotherapy and radiation. At the same time, conventional approaches to de-escalation at a population (selected or unselected) level are hampered by a simple fact: we lack patient-specific information from individual tumors that can predict responsiveness. This results in a problematic tradeoff between the deleterious impact of de-escalation on patients with aggressive, treatment-refractory disease and the beneficial reduction in treatment-related morbidity for patients with treatment-responsive disease. True precision oncology approaches require a constant, iterative interrogation of solid tumors prior to and especially during cancer treatment in order to tailor treatment intensity to tumor biology. Whereas this approach can be deployed in hematologic diseases with some success, our ability to extend it to solid cancers with regional metastasis has been extremely limited in the curative intent setting. New developments in metabolic imaging and quantitative interrogation of circulating DNA, tumor exosomes and whole circulating tumor cells, however, provide renewed opportunities to adapt and individualize even conventional chemo-radiation strategies to diseases with highly variable biology such as OPC. In this review, we discuss opportunities to deploy developing technologies in the context of institutional and cooperative group clinical trials over the coming decade. [ABSTRACT FROM AUTHOR]

Purpose: To report a case of metastatic uveal melanoma treated with immune checkpoint inhibition in which serial circulating tumor DNA (ctDNA) was assessed throughout treatment. Observations: A 33-year-old man was diagnosed with metastatic uveal melanoma and initially had progression of disease following hepatic embolization and nivolumab/ipilimumab. At the time, plasma ctDNA GNA11 and SF3B1 were measurable and repeat ctDNA showed increased variant allele frequency following further progression of disease on vorinostat. Following additional nivolumab/ipilimumab, radiographic response was noted and repeat ctDNA became undetectable and remained so at 27 months follow up. Conclusions and importance: Clearance of cell free DNA in metastatic uveal melanoma may be associated with radiographic response to immune checkpoint inhibitors.

BackgroundEndomyocardial biopsy (EMB), the reference surveillance test for acute rejection (AR) in heart transplant (HTx) recipients, is invasive, costly, and shows significant interobserver variability. Recent studies indicate that donor-derived cell-free DNA (dd-cfDNA), obtained non-invasively from blood, is associated with AR and could reduce the frequency of EMB surveillance. The aim of this study was to examine the performance characteristics of a novel test for detecting AR in adult HTx recipients.MethodsPlasma samples with contemporaneous EMBs were obtained from HTx recipients. A clinically available SNP-based massively multiplexed-PCR dd-cfDNA assay was used to measure dd-cfDNA fraction. dd-cfDNA fractions were compared with EMB-defined rejection status and test performance was assessed by constructing ROC curves and calculating accuracy measures.ResultsA total of 811 samples from 223 patients with dd-cfDNA testing and contemporaneous EMB were eligible for the study. dd-cfDNA fraction was significantly higher in AR (median 0.58%, IQR, 0.13%-1.68%) compared to non-AR (median 0.04%, IQR, 0.01%-0.11%, pc < 0.001). ROC analysis produced an area under the curve (AUC-ROC) of 0.86 (95% CI, 0.77-0.96). Defining samples with dd-cfDNA fraction ≥0.15% as AR yielded 78.5% sensitivity (95% CI, 60.7%-96.3%) and 76.9% specificity (95% CI, 71.1%-82.7%). Positive and negative predictive values were 25.1% (95% CI, 18.8%-31.5%) and 97.3% (95% CI, 95.1%-99.5%) respectively, calculated using the cohort AR prevalence of 9.0% (95% CI, 5.3%-12.8%) with adjustment for repeat samples.ConclusionsThis novel dd-cfDNA test detects AR in HTx recipients with good accuracy and holds promise as a noninvasive test for AR in HTx recipients.

BACKGROUND: Twin pregnancies consisting of a complete hydatidiform mole and a coexistent fetus (CMCF) are rare and associated with a high rate of maternal-fetal morbidity and mortality. Management of these pregnancies remains controversial and increasingly challenging following the Dobbs versus Jackson Women’s Health decision given the viability of the coexisting twin fetus. CASE: This case looks at the diagnosis, management, and maternal-fetal outcomes of a viable fetus coexisting molar pregnancy at a large academic center in an abortion-restricted state. CONCLUSION: CMCF pregnancies are associated with a high risk of morbidity and mortality and are increasingly difficult to manage following the Dobbs decision. Testing platforms, which identify genetic abnormalities in the first trimester, are increasingly important as access to abortion care in the United States is restricted.

Oropharyngeal cancer (OPC) poses a complex therapeutic dilemma for patients and oncologists alike, made worse by the epidemic increase in new cases associated with the oncogenic human papillomavirus (HPV). In a counterintuitive manner, the very thing which gives patients hope, the high response rate of HPV-associated OPC to conventional chemo-radiation strategies, has become one of the biggest challenges for the field as a whole. It has now become clear that for ~30-40% of patients, treatment intensity could be reduced without losing therapeutic efficacy, yet substantially diminishing the acute and lifelong morbidity resulting from conventional chemotherapy and radiation. At the same time, conventional approaches to de-escalation at a population (selected or unselected) level are hampered by a simple fact: we lack patient-specific information from individual tumors that can predict responsiveness. This results in a problematic tradeoff between the deleterious impact of de-escalation on patients with aggressive, treatment-refractory disease and the beneficial reduction in treatment-related morbidity for patients with treatment-responsive disease. True precision oncology approaches require a constant, iterative interrogation of solid tumors prior to and especially during cancer treatment in order to tailor treatment intensity to tumor biology. Whereas this approach can be deployed in hematologic diseases with some success, our ability to extend it to solid cancers with regional metastasis has been extremely limited in the curative intent setting. New developments in metabolic imaging and quantitative interrogation of circulating DNA, tumor exosomes and whole circulating tumor cells, however, provide renewed opportunities to adapt and individualize even conventional chemo-radiation strategies to diseases with highly variable biology such as OPC. In this review, we discuss opportunities to deploy developing technologies in the context of institutional and cooperative group clinical trials over the coming decade.

BACKGROUND: Chronic heart failure (CHF) is one of the most serious problems in cardiovascular diseases, requiring accurate diagnosis and treatment. The search for new laboratory markers of CHF can improve the accuracy of diagnosis and identify the severity of the patients condition. AIM: Our aim was to study the relationship between the levels of free-circulating DNA (cfDNA) and brain natriuretic peptide (NT-proBNP) in the blood plasma of patients suffering from CHF with ejection fraction (EF), to investigate the relationship between these laboratory markers, and to evaluate the dynamics of changes in the studied parameters against the background of drug therapy. MATERIALS AND METHODS: The study involved 67 patients of both sexes with a diagnosis of CHF, verified by clinical and functional methods. 23 people without established chronic diseases formed the control group. At the stage of inclusion in the study, all patients underwent: physical examination, general blood test, biochemical blood test with determination of lipid profile, glucose, creatinine, NT-proBNP and cfDNA levels, as well as electrocardiography (ECG), electrocardiography (ECHO-CG), radiography of organs chest, ultrasound of the abdominal organs, 6-minute walk test. The level of cfDNA was determined using the method of P.P. Laktionov, S.N. Tamkovich, E.Yu. Rykova (2005). Repeated blood sampling with assessment of cfDNA and NT-proBNP levels was carried out in the group of patients with reduced ejection fraction 57 months from the start of treatment/correction of previous therapy. RESULTS: The study revealed significant differences in the levels of cfDNA in the blood plasma in patients with different EF (less than 40%, 4049%, 50% or more). At the same time, an inverse relationship was established between cfDNA indicators and EF, as well as between the level of NT-proBNP and EF, that is, a progressive decrease in myocardial contractility is accompanied by a combined increase in the levels of the studied markers in the blood, reflecting the severity of the patients condition. In addition, the positive effect of drug therapy on cfDNA and NT-proBNP levels in the group of patients with EF 40% has been proven. CONCLUSION: The identified patterns make it possible to consider the level of cfDNA in blood plasma as a potential biomarker of CHF, and also to use it for dynamic monitoring of patients.

In this prospective study we investigated a cohort after heart transplantation with a novel PCR-based approach with focus on treated rejection. Blood samples were collected coincidentally to biopsies, and both absolute levels of dd-cfDNA and donor fraction were reported using digital PCR. 52 patients (11 children and 41 adults) were enrolled (NCT03477383, clinicaltrials. gov), and 557 plasma samples were analyzed. 13 treated rejection episodes >14 days after transplantation were observed in 7 patients. Donor fraction showed a median of 0.08% in the cohort and was significantly elevated during rejection (median 0.19%, p < 0.0001), using a cut-off of 0.1%, the sensitivity/specificity were 92%/56% (AUC ROC-curve: 0.78). Absolute levels of dd-cfDNA showed a median of 8.8 copies/mL and were significantly elevated during rejection (median 23, p = 0.0001). Using a cut-off of 7.5 copies/mL, the sensitivity/specificity were 92%/43% for donor fraction (AUC ROC-curve: 0.75). The results support the feasibility of this approach in analyzing dd-cfDNA after heart transplantation. The obtained values are well aligned with results from other trials. The possibility to quantify absolute levels adds important value to the differentiation between ongoing graft damage and quiescent situations. [ABSTRACT FROM AUTHOR]

The aim of the study was to study the clinical profile of acute on chronic liver failure (ACLF) and establish Cell-free DNA (Cf DNA) as a predictor of the outcome of ACLF. In this prospective study, those patients who fulfilled EASL criteria were included. Cf DNA was estimated in 30 patients and compared with the CLIF-C ACLF score. The median age of 132 consecutive ACLF patients was 40 years. The most common acute insult were sepsis (30.3%) and alcohol (22%). While alcohol (35.6%) and chronic HBV (14.3%) were the most common etiologies of cirrhosis. The overall mortality was 45.5% and 71.2% at 28 days and 90 days, respectively. Multiple regression analysis using the Cox proportional hazard model showed that heart rate (HR 1.06, 95% CI 1.04–1.08 P = 0.001), lung failure (HR 2.82, 95% CI 1.24–6.44, P = 0.02), and cell-free DNA (HR 2.70, 95% CI 1.17–6.24, P = 0.02) were independent predictors of mortality When Cf DNA was used to predict 28-day mortality, Cf DNA was found to have a higher AUC (AUROC 0.84, 95% CI 0.70-0.98, P = 0.001) than the CLIF-C-ACLF score (AUROC 0.81, 95% 0.66–0.97, P = 0.003). However, when 90-day mortality was compared, CLIF-C-ACLF score had a higher area under the curve (AUROC 0.93, 95% CI 0.83–1.00, P = 0.0001) than Cf DNA (AUROC 0.89, 95% CI 0.77–1.00, P = 0.0001). Alcohol and sepsis remain the most common causes of acute insult. Cf DNA is a better predictor of 28-day mortality, whereas CLIF-C ACLF is more accurate to predict 90-day mortality. [ABSTRACT FROM AUTHOR]

Abnormally high level of cell-free DNA (cfDNA) is one of the important causes of autoimmune diseases, which aggravate the symptoms of rheumatoid arthritis (RA). Recently, the utilization of cationic polymeric nanoparticles for scavenging cfDNA has emerged as a promising therapeutic strategy for the treatment of RA. However, the intravenous introduction of cationic polymeric nanoparticles into the circulation carries a risk of dissociation, causing toxicity. To realize the potential clinical translation, we employed a series of silica particles grafted with poly(2-(dimethylamino) ethyl methacrylate) (PDMA) (SiNP@PDMA) brush, which possess adjustable PDMA content (100, 200, and 300 degree of polymerization (DP)) and particle size (50, 100, and 200 nm diameter), to selectively scavenge cfDNA in inflamed joint cavity. We demonstrate that the binding affinity for cfDNA, cytotoxicity, circulation time in vivo and retention in the inflamed joint cavity are influenced by the core-shell structure of SiNP@PDMA, ultimately impacting therapeutic efficacy. Among them, SiNP@PDMA with 100 nm size and 200 DP of PDMA exhibit enhanced accumulation and prolonged retention time in inflammatory joint cavity, resulting in superior therapeutic effect. Therefore, in this study, applying the precisely tuning size and cation content of SiNP@PDMA, we demonstrated the factors to matter the therapeutic effect of cationic nanoparticles, which deepened the understanding of the anti-inflammatory therapies based on cfDNA scavenger for RA. Inspired by the discovery that cfDNA would induce inappropriate immune responses to exacerbate the progress of RA, we innovatively employed SiNP@PDMA as a cfDNA scavenger to inhibit cfDNA-induced inflammation in RA. Increase in the cation content efficiently strengthened the binding between SiNP@PDMA and cfDNA, leading to an improvement in inhibitory effect of inflammation. In addition, we compared the behaviors of 50, 100 and 200 nm SiNP@PDMA in RA symptom suppression, local cfDNA scavenging and inflammation inhibition. The results demonstrated that SiNP 100 -PDMA 200 outperformed other analogues, corresponding to their more favorable distribution in inflammatory articular cavity. Together, this study revealed the structure-property relationship of cfDNA scavengers for further development of safe and effective cfDNA scavenging system. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction: Assessment of renal tumour masses is based on conventional imaging studies (computer tomography or magnetic resonance), which does not allow characterisation of the histopathological type. Moreover, the prediction of prognosis in localised and metastatic renal cell carcinoma requires improvement as well. Analysis of circulating free DNA (cfDNA) in blood is one of the variants of liquid biopsy that may improve diagnostics and prognosis issues of patients with renal tumour masses suspected to be renal cell carcinoma. The aim of the study was to assess the diagnostic and prognostic role of preoperative cfDNA concentration in the plasma samples of clear cell renal cell carcinoma (ccRCC) patients. Material and methods: The preoperative plasma cfDNA concentration was assessed in ccRCC patients (n = 46) and healthy individuals (control group) (n = 17). The circulating free DNA concentration was reflected by the 90 bp DNA fragments determined by real-time polymerase chain reaction. Results: The median cfDNA concentration was significantly higher in ccRCC patients (n = 46) compared to the control g roup (n = 17) (2588 ±2554 copies/ml vs. 960 ±490 copies/ml, p < 0.01). In multivariate analysis, the preoperative plasma cfDNA concentration was the significant factor increasing the probability of ccRCC detection (OR: 1.003; 95% CI: 1.001-1.005). The median cfDNA concentration depended on the stage of ccRCC; it was higher in metastatic ccRCC patients (n = 11) compared to non-metastatic ccRCC patients (n = 35) (3619 ±4059 copies/ml vs. 2473 ±1378 copies/ml, p < 0.03). Kaplan-Meier survival analysis demonstrated that patients with high cfDNA values (above 2913 copies/ml) had significantly worse cancer-specific survi-val (HR: 4.5; 95% CI: 1.3-16.9, logrank Mantel-Cox test p = 0.015). Conclusions: Preoperative plasma cfDNA concentration has diagnostic and prognostic potential in ccRCC patients. [ABSTRACT FROM AUTHOR]

Recent insights in allorecognition and graft rejection mechanisms revealed a more complex picture than originally considered, involving multiple pathways of both adaptive and innate immune response, supplied by efficient inflammatory synergies. Current pillars of transplant monitoring are serum creatinine, proteinuria, and drug blood levels, which are considered as traditional markers, due to consolidated experience, low cost, and widespread availability. The most diffuse immunological biomarkers are donor-specific antibodies, which are included in routine post-transplant monitoring in many centers, although with some reproducibility issues and interpretation difficulties. Confirmed abnormalities in these traditional biomarkers raise the suspicion for rejection and guide the indication for graft biopsy, which is still considered the gold standard for rejection monitoring. Rapidly evolving new "omic" technologies have led to the identification of several novel biomarkers, which may change the landscape of transplant monitoring should their potential be confirmed. Among them, urinary chemokines and measurement of cell-free DNA of donor origin are perhaps the most promising. However, at the moment, these approaches remain highly expensive and cost-prohibitive in most settings, with limited clinical applicability; approachable costs upon technology investments would speed their integration. In addition, transcriptomics, metabolomics, proteomics, and the study of blood and urinary extracellular vesicles have the potential for early identification of subclinical rejection with high sensitivity and specificity, good reproducibility, and for gaining predictive value in an affordable cost setting. In the near future, information derived from these new biomarkers is expected to integrate traditional tools in routine use, allowing identification of rejection prior to clinical manifestations and timely therapeutic intervention. This review will discuss traditional, novel, and invasive and non-invasive biomarkers, underlining their strengths, limitations, and present or future applications in children. [ABSTRACT FROM AUTHOR]

Background: Cell-free plasma DNA (cfDNA) is circulating extracellular DNA arising from cell death mechanisms (apoptosis, necrosis, etc.). It is commonly existent in healthy individuals, but its ranks increase in diverse clinical circumstances, such as kidney disease, sepsis, myocardial infarction, trauma and cancer. In patients with advanced chronic kidney disease, cfDNA is connected to inflammation, and it has been associated with higher mortality. Caspase-3 plays a dominant role in apoptosis, a mechanism of programmed cell death involved in the pathogenesis and progression of chronic kidney disease (CKD). The aim of this pilot study was the evaluation of cfDNA levels and caspase-3 concentrations in patients with chronic kidney disease, in order to investigate the potential role of these molecules, deriving from inflammatory and apoptotic mechanisms, in the progression of renal damage. Methods: We compared cfDNA and caspase-3 levels in 25 CKD patients and in 10 healthy subjects, evaluating their levels based on CKD stage. We also explored correlations between cfDNA and caspase-3 levels in CKD patients and between cfDNA and caspase-3 levels and serum creatinine and urea in this population. Results: We observed that cfDNA and caspase-3 levels were higher in patients with CKD compared to healthy subjects, in particular in patients with advanced renal disease (CKD stage 5). A positive correlation between cfDNA and caspase-3 levels and between cfDNA and caspase-3 and creatinine and urea were also noticed. Conclusions: Patients with chronic kidney disease show higher levels of cfDNA and caspase-3 levels compared to the control group. Based on these preliminary results, we speculated that the worsening of renal damage and the increase in uremic toxin concentration could be associated with higher levels of cfDNA and caspase-3 levels, thus reflecting the potential role of inflammation and apoptosis in the progression of CKD. Future studies should focus on the validation of these promising preliminary results. [ABSTRACT FROM AUTHOR]

Background: COVID-19 is a worldwide pandemic caused by the highly infective SARS-CoV-2. There is a need for biomarkers not only for overall prognosis but also for predicting the response to treatments and thus for improvements in the clinical management of patients with COVID-19. Circulating cell-free DNA (cfDNA) has emerged as a promising biomarker in the assessment of various pathological conditions. The aim of this retrospective and observational pilot study was to investigate the range of cfDNA plasma concentrations in hospitalized COVID-19 patients during the first wave of SARS-CoV-2 infection, to relate them to established inflammatory parameters as a correlative biomarker for disease severity, and to compare them with plasma levels in a healthy control group. Methods: Lithium-Heparin plasma samples were obtained from COVID-19 patients (n = 21) during hospitalization in the University Medical Centre of Mainz, Germany between March and June 2020, and the cfDNA concentrations were determined by quantitative PCR yielding amplicons of long interspersed nuclear elements (LINE-1). The cfDNA levels were compared with those of an uninfected control group (n = 19). Results: Plasma cfDNA levels in COVID-19 patients ranged from 247.5 to 6,346.25 ng/ml and the mean concentration was 1,831 ± 1,388 ng/ml (± standard deviation), which was significantly different from the levels of the uninfected control group (p < 0.001). Regarding clinical complications, the highest correlation was found between cfDNA levels and the myositis (p = 0.049). In addition, cfDNA levels correlated with the "WHO clinical progression scale". D-Dimer and C-reactive protein (CRP) were the clinical laboratory parameters with the highest correlations with cfDNA levels. Conclusion: The results of this observational pilot study show a wide range in cfDNA plasma concentrations in patients with COVID-19 during the first wave of infection and confirm that cfDNA plasma concentrations serve as a predictive biomarker of disease severity in COVID-19. [ABSTRACT FROM AUTHOR]

Abstract Background Severe COVID-19 entails a dysregulated immune response, most likely inflammation related to a lack of virus control. A better understanding of immune toxicity, immunosuppression balance, and COVID-19 assessments could help determine whether different clinical presentations are driven by specific types of immune responses. The progression of the immune response and tissular damage could predict outcomes and may help in the management of patients. Methods We collected 201 serum samples from 93 hospitalised patients classified as moderately, severely, and critically ill. We differentiated the viral, early inflammatory, and late inflammatory phases and included 72 patients with 180 samples in separate stages for longitudinal study and 55 controls. We studied selected cytokines, P-selectin, and the tissue damage markers lactate dehydrogenase (LDH) and cell-free DNA (cfDNA). Results TNF-α, IL-6, IL-8, and G-CSF were associated with severity and mortality, but only IL-6 increased since admission in the critical patients and non-survivors, correlating with damage markers. The lack of a significant decrease in IL-6 levels in the critical patients and non-survivors in the early inflammatory phase (a decreased presence in the other patients) suggests that these patients did not achieve viral control on days 10–16. For all patients, lactate dehydrogenase and cfDNA levels increased with severity, and cfDNA levels increased in the non-survivors from the first sample (p = 0.002) to the late inflammatory phase (p = 0.031). In the multivariate study, cfDNA was an independent risk factor for mortality and ICU admission. Conclusions The distinct progression of IL-6 levels in the course of the disease, especially on days 10–16, was a good marker of progression to critical status and mortality and could guide the start of IL-6 blockade. cfDNA was an accurate marker of severity and mortality from admission and throughout COVID-19 progression.

Abstract Background Late detection of hepatocellular carcinoma (HCC) results in an overall 5-year survival rate of less than 16%. Liquid biopsy (LB) assays based on detecting circulating tumor DNA (ctDNA) might provide an opportunity to detect HCC early noninvasively. Increasing evidence indicates that ctDNA detection using mutation-based assays is significantly challenged by the abundance of white blood cell-derived mutations, non-tumor tissue-derived somatic mutations in plasma, and the mutational tumor heterogeneity. Methods Here, we employed concurrent analysis of cancer-related mutations, and their fragment length profiles to differentiate mutations from different sources. To distinguish persons with HCC (PwHCC) from healthy participants, we built a classification model using three fragmentomic features of ctDNA through deep sequencing of thirteen genes associated with HCC. Results Our model achieved an area under the curve (AUC) of 0.88, a sensitivity of 89%, and a specificity of 82% in the discovery cohort consisting of 55 PwHCC and 55 healthy participants. In an independent validation cohort of 54 PwHCC and 53 healthy participants, the established model achieved comparable classification performance with an AUC of 0.86 and yielded a sensitivity and specificity of 81%. Conclusions Our study provides a rationale for subsequent clinical evaluation of our assay performance in a large-scale prospective study.

Novel blood-circulating molecules, as potential biomarkers for glioblastoma multiforme (GBM) diagnosis and monitoring, are attracting particular attention due to limitations of imaging modalities and invasive tissue biopsy procedures. This study aims to assess the diagnostic and prognostic values of circulating cell-free DNA (cfDNA) in relation to inflammatory status in GBM patients and to determine the concentration and average size of DNA fragments typical of tumour-derived DNA fractions. Preoperative plasma samples from 40 patients (GBM 65.0 ± 11.3 years) and 40 healthy controls (HC 70.4 ± 5.4 years) were compared. The cfDNA concentrations and lengths were measured using the electrophoresis platform, and inflammatory indices (NLR, PLR, LMR, and SII) were calculated from complete blood cell analysis. More fragmented cfDNA and 4-fold higher 50–700 bp cfDNA concentrations were detected in GBM patients than in healthy controls. The average cfDNA size in the GBM group was significantly longer (median 336 bp) than in the HC group (median 271 bp). Optimal threshold values were 1265 pg/μL for 50–700 bp cfDNA (AUC = 0.857) and 290 bp for average cfDNA size (AUC = 0.814). A Kaplan–Meier survival curves analysis also demonstrated a higher mortality risk in the GBM group with a cut-off >303 bp cfDNA. This study is the first to have revealed glioblastoma association with high levels of cfDNA > 1000 pg/μL of 50–700 bp in length, which can be aggravated by immunoinflammatory reactivity.

In this prospective study we investigated a cohort after heart transplantation with a novel PCR-based approach with focus on treated rejection. Blood samples were collected coincidentally to biopsies, and both absolute levels of dd-cfDNA and donor fraction were reported using digital PCR. 52 patients (11 children and 41 adults) were enrolled (NCT03477383, clinicaltrials.gov), and 557 plasma samples were analyzed. 13 treated rejection episodes >14 days after transplantation were observed in 7 patients. Donor fraction showed a median of 0.08% in the cohort and was significantly elevated during rejection (median 0.19%, p < 0.0001), using a cut-off of 0.1%, the sensitivity/specificity were 92%/56% (AUC ROC-curve: 0.78). Absolute levels of dd-cfDNA showed a median of 8.8 copies/mL and were significantly elevated during rejection (median 23, p = 0.0001). Using a cut-off of 7.5 copies/mL, the sensitivity/specificity were 92%/43% for donor fraction (AUC ROC-curve: 0.75). The results support the feasibility of this approach in analyzing dd-cfDNA after heart transplantation. The obtained values are well aligned with results from other trials. The possibility to quantify absolute levels adds important value to the differentiation between ongoing graft damage and quiescent situations.

ObjectivesNon-invasive prenatal testing (NIPT) is utilized for screening the likelihood of fetal aneuploidy, presenting the benefits of non-invasiveness, high sensitivity, and specificity. Its application in prenatal screening has become ubiquitous. The inquiry into how pregnant women comprehend and determine NIPT screening strategies is paramount. Regrettably, there has been a dearth of research on this subject in China. Consequently, this study scrutinizes pregnant women’s cognizance and perspectives concerning NIPT, furnishing a foundation for advancing its judicious implementation.MethodsFrom February 2021 to December 2022, a questionnaire survey was conducted among pregnant women receiving prenatal care and screening at the Women’s Hospital, School of Medicine, Zhejiang University, who were randomly selected from a pool of individuals exhibiting a high risk of fetal aneuploidy on serological screening. The survey aimed to gather data on participant characteristics, knowledge, perception, and willingness concerning NIPT. The study employed chi-square and Kruskal Wallis tests to analyze subgroup differences.ResultsA total of 226 valid questionnaires were obtained. 83.2% of women pregnant women identified as high risk by serological screening would opt for NIPT, with 66.4% indicating that they would prefer NIPT for fetal aneuploidy screening in future pregnancies. These findings suggest a notable willingness among pregnant women to undergo NIPT. Additionally, the results suggest that various factors, including place of residence, educational level, family income, causes of abortion, and conception method, influence pregnant women’s knowledge about NIPT Accordingly, the level of NIPT knowledge varies among pregnant women.ConclusionThe survey generally revealed that pregnant women were strongly inclined to select NIPT; however, expectant Chinese mothers possess limited knowledge and perception regarding this screening method for fetal aneuploidy. Therefore, the government must implement effective measures to augment public awareness of fetal aneuploidy screening and encourage the judicious utilization of NIPT.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal cancer globally. Over 90% of HCC cases arise in the context of liver cirrhosis, and the severity of the underlying liver disease or advanced tumor stage at diagnosis significantly limits treatment options. Early diagnosis is crucial, and all guidelines stress the importance of screening protocols for HCC early detection as a public health objective. As serum biomarkers are not optimal for early diagnosis, liquid biopsy has emerged as a promising tool for diagnosis, prognostication, and patients’ stratification for personalized therapy in various solid tumors, including HCC. While circulating tumor cells (CTCs) are better suited for personalized therapy and prognosis, cell-free DNA (cfDNA) and extracellular vesicle-based technologies show potential for early diagnosis, HCC screening, and surveillance protocols. Evaluating the added value of liquid biopsy genetic and epigenetic biomarkers for HCC screening is a key goal in translational research. Somatic mutations commonly found in HCC can be investigated in cfDNA and plasma exosomes as genetic biomarkers. Unique methylation patterns in cfDNA or cfDNA fragmentome features have been suggested as innovative tools for early HCC detection. Likewise, extracellular vesicle cargo biomarkers such as miRNAs and long non-coding RNAs may serve as potential biomarkers for early HCC detection. This review will explore recent findings on the utility of liquid biopsy for early HCC diagnosis. Combining liquid biopsy methods with traditional serological biomarkers could improve the overall diagnostic accuracy for early HCC detection.

Background COVID-19 is a worldwide pandemic caused by the highly infective SARS-CoV-2. There is a need for biomarkers not only for overall prognosis but also for predicting the response to treatments and thus for improvements in the clinical management of patients with COVID-19. Circulating cell-free DNA (cfDNA) has emerged as a promising biomarker in the assessment of various pathological conditions. The aim of this retrospective and observational pilot study was to investigate the range of cfDNA plasma concentrations in hospitalized COVID-19 patients during the first wave of SARS-CoV-2 infection, to relate them to established inflammatory parameters as a correlative biomarker for disease severity, and to compare them with plasma levels in a healthy control group. Methods Lithium-Heparin plasma samples were obtained from COVID-19 patients (n = 21) during hospitalization in the University Medical Centre of Mainz, Germany between March and June 2020, and the cfDNA concentrations were determined by quantitative PCR yielding amplicons of long interspersed nuclear elements (LINE-1). The cfDNA levels were compared with those of an uninfected control group (n = 19). Results Plasma cfDNA levels in COVID-19 patients ranged from 247.5 to 6,346.25 ng/ml and the mean concentration was 1,831 ± 1,388 ng/ml (± standard deviation), which was significantly different from the levels of the uninfected control group (p < 0.001). Regarding clinical complications, the highest correlation was found between cfDNA levels and the myositis (p = 0.049). In addition, cfDNA levels correlated with the “WHO clinical progression scale”. D-Dimer and C-reactive protein (CRP) were the clinical laboratory parameters with the highest correlations with cfDNA levels. Conclusion The results of this observational pilot study show a wide range in cfDNA plasma concentrations in patients with COVID-19 during the first wave of infection and confirm that cfDNA plasma concentrations serve as a predictive biomarker of disease severity in COVID-19.

Abstract Background CMV gastroenteritis is common in patients receiving allogeneic hematopoietic stem cell transplantation and it is difficult to distinguish from acute graft-versus-host disease (aGvHD), which has very similar symptoms but needs quite different treatment. CMV gastroenteritis is caused by local infection or reactivation of CMV in the gastrointestinal tract while aGvHD is due to immune rejection. The gold standard of diagnosis of CMV gastroenteritis and aGvHD is gastrointestinal biopsy under endoscopy, which is invasive and can potentially lead to severe side effects. Stool samples testing with quantitative polymerase chain reaction (qPCR) may be an alternative, while the application in trace level measurements and precision are not all satisfactory enough in reported research. Methods In this study, we designed a novel method that extracted the cell free DNA (cfDNA) from the fecal supernatant to perform digital PCR (dPCR) for the detection of CMV, analyzed the performance and compared it with the total DNA extracted by the current procedure. Results Twenty-two paired stool samples using two DNA extraction methods proved that the cfDNA extraction method had markedly higher DNA concentrations and control gene copy number, suggesting that cfDNA may be more informative and more useful for the detection of CMV DNA segment. The dPCR approach in detecting CMV DNA segment also exhibit good linearity (R2 = 0.997) and higher sensitivity (limit of detection at 50% was 3.534 copies/μL). Eighty-two stool samples from 44 immunocompromised patients were analyzed, CMV-positive rate was 28%, indicating that more than one-quarter of the gastrointestinal symptoms within these patients may be caused by CMV infection or reactivation. Conclusion The combined results suggest that detection of CMV by dPCR in cfDNA of stool supernatant is a powerful method to identify CMV gastroenteritis and helps in clinical treatment decision making.

Background: Severe COVID-19 entails a dysregulated immune response, most likely inflammation related to a lack of virus control. A better understanding of immune toxicity, immunosuppression balance, and COVID-19 assessments could help determine whether different clinical presentations are driven by specific types of immune responses. The progression of the immune response and tissular damage could predict outcomes and may help in the management of patients. Methods: We collected 201 serum samples from 93 hospitalised patients classified as moderately, severely, and critically ill. We differentiated the viral, early inflammatory, and late inflammatory phases and included 72 patients with 180 samples in separate stages for longitudinal study and 55 controls. We studied selected cytokines, P-selectin, and the tissue damage markers lactate dehydrogenase (LDH) and cell-free DNA (cfDNA). Results: TNF-α, IL-6, IL-8, and G-CSF were associated with severity and mortality, but only IL-6 increased since admission in the critical patients and non-survivors, correlating with damage markers. The lack of a significant decrease in IL-6 levels in the critical patients and non-survivors in the early inflammatory phase (a decreased presence in the other patients) suggests that these patients did not achieve viral control on days 10–16. For all patients, lactate dehydrogenase and cfDNA levels increased with severity, and cfDNA levels increased in the non-survivors from the first sample (p = 0.002) to the late inflammatory phase (p = 0.031). In the multivariate study, cfDNA was an independent risk factor for mortality and ICU admission. Conclusions: The distinct progression of IL-6 levels in the course of the disease, especially on days 10–16, was a good marker of progression to critical status and mortality and could guide the start of IL-6 blockade. cfDNA was an accurate marker of severity and mortality from admission and throughout COVID-19 progression. [ABSTRACT FROM AUTHOR]

Simple Summary: Medulloblastoma is one of the most common types of brain tumors in children. During and after treatment with surgery, chemotherapy, and/or radiotherapy, children with this disease are monitored with imaging and cerebrospinal fluid analysis for the detection of tumor cells. These methods are not always sensitive or specific enough to confirm or rule out residual disease. Here, we develop a laboratory test based on the genetic makeup of medulloblastomas in 12 children. We analyze liquid biopsies (cerebrospinal fluid and blood plasma) for specific genetic fragments leaking from the individual tumors and find molecular traces of disease in 75% (9/12) of children overall. None of the children had malignant cells in the cerebrospinal fluid. We propose that this test could open up new technical possibilities to track measurable residual disease in children with medulloblastoma in order to further risk-adapt treatment, but first, larger studies of the approach at standardized time points are warranted. Medulloblastoma is a malignant embryonal tumor of the central nervous system (CNS) that mainly affects infants and children. Prognosis is highly variable, and molecular biomarkers for measurable residual disease (MRD) detection are lacking. Analysis of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) using broad genomic approaches, such as low-coverage whole-genome sequencing, has shown promising prognostic value. However, more sensitive methods are needed for MRD analysis. Here, we show the technical feasibility of capturing medulloblastoma-associated structural variants and point mutations simultaneously in cfDNA using multiplexed droplet digital PCR (ddPCR). Assay sensitivity was assessed with a dilution series of tumor in normal genomic DNA, and the limit of detection was below 100 pg of input DNA for all assays. False positive rates were zero for structural variant assays. Liquid biopsies (CSF and plasma, n = 47) were analyzed from 12 children with medulloblastoma, all with negative CSF cytology. MRD was detected in 75% (9/12) of patients overall. In CSF samples taken before or within 21 days of surgery, MRD was detected in 88% (7/8) of patients with localized disease and in one patient with the metastasized disease. Our results suggest that this approach could expand the utility of ddPCR and complement broader analyses of cfDNA for MRD detection. [ABSTRACT FROM AUTHOR]

The purpose of treating alveolar bone cleft is to restore a normal maxilla structure. Multiple factors have been identified that can affect the success of alveolar bone grafting. However, with consistent treatment modifications, the surgical outcomes have been improved, but alveolar bone loss still exists. Thus, a new aspect should be found to solve this problem. As alveolar bone belongs to the periodontal tissues, the mechanism of the alveolar bone loss after bone grafting in patients with alveolar bone cleft may be similar to the development of alveolar bone loss in periodontitis. Cell-free DNA (cfDNA) has been demonstrated as a key promoter of alveolar bone loss during periodontal inflammation. We hypothesized that cfDNA-related innate immune responses could be a major inducement for postoperative bone loss after alveolar bone grafting. In this perspective, we preliminarily proved the potential association between cfDNA, TLR9 pathway, and alveolar bone grafting operation, and it might verify that surgical trauma could accumulate cfDNA, which can further activate cellular TLR9 signaling. [ABSTRACT FROM AUTHOR]

Background: Late detection of hepatocellular carcinoma (HCC) results in an overall 5-year survival rate of less than 16%. Liquid biopsy (LB) assays based on detecting circulating tumor DNA (ctDNA) might provide an opportunity to detect HCC early noninvasively. Increasing evidence indicates that ctDNA detection using mutation-based assays is significantly challenged by the abundance of white blood cell-derived mutations, non-tumor tissue-derived somatic mutations in plasma, and the mutational tumor heterogeneity. Methods: Here, we employed concurrent analysis of cancer-related mutations, and their fragment length profiles to differentiate mutations from different sources. To distinguish persons with HCC (PwHCC) from healthy participants, we built a classification model using three fragmentomic features of ctDNA through deep sequencing of thirteen genes associated with HCC. Results: Our model achieved an area under the curve (AUC) of 0.88, a sensitivity of 89%, and a specificity of 82% in the discovery cohort consisting of 55 PwHCC and 55 healthy participants. In an independent validation cohort of 54 PwHCC and 53 healthy participants, the established model achieved comparable classification performance with an AUC of 0.86 and yielded a sensitivity and specificity of 81%. Conclusions: Our study provides a rationale for subsequent clinical evaluation of our assay performance in a large-scale prospective study. [ABSTRACT FROM AUTHOR]

Liquid biopsies, based on cell free DNA (cfDNA) and proteins, have shown the potential to detect early stage cancers of diverse tissue types. However, most of these studies were retrospective, using individuals previously diagnosed with cancer as cases and healthy individuals as controls. Here, we developed a liquid biopsy assay, named the hepatocellular carcinoma screen (HCCscreen), to identify HCC from the surface antigen of hepatitis B virus (HBsAg) positive asymptomatic individuals in the community population. The training cohort consisted of individuals who had liver nodules and/or elevated serum α-fetoprotein (AFP) levels, and the assay robustly separated those with HCC from those who were non-HCC with a sensitivity of 85% and a specificity of 93%. We further applied this assay to 331 individuals with normal liver ultrasonography and serum AFP levels. A total of 24 positive cases were identified, and a clinical follow-up for 6-8 mo confirmed four had developed HCC. No HCC cases were diagnosed from the 307 test-negative individuals in the follow-up during the same timescale. Thus, the assay showed 100% sensitivity, 94% specificity, and 17% positive predictive value in the validation cohort. Notably, each of the four HCC cases was at the early stage (

Non-invasive prenatal screening (NIPS) is a DNA sequencing-based screening test for fetal aneuploidies and possibly other pathogenic genomic abnormalities, such as large deletions and duplications. Validation and quality assurance (QA) of this clinical test using plasmas with and without targeted chromosomal abnormalities from pregnant women as negative and positive controls are required. However, the positive plasma controls may not be available for many laboratories that are planning to establish NIPS. Limited synthetic positive plasmas are commercially available, but the types of abnormalities and the number/quantity of synthetic plasmas for each abnormality are insufficient to meet the minimal requirements for the initial validation. We report here a method of making synthetic positive plasmas by adding cell-free DNA (cfDNA) isolated from culture media of prenatal cells with chromosomal abnormalities to the plasmas from non-pregnant women. Thirty-eight positive plasmas with various chromosomal abnormalities, including autosomal and sex chromosomal aneuploidies, large deletions and duplications, were synthesized. The synthetic plasmas were characterized side-by-side with real positive plasmas from pregnant women and commercially available synthetic positive plasmas using the Illumina VeriSeq NIPT v2 system. All chromosomal abnormalities in the synthetic plasmas were correctly identified with the same testing sensitivity and specificity as in the real and commercial synthetic plasmas. The findings demonstrate that the synthetic positive plasmas are excellent alternatives of real positive plasmas for validation and QA of NIPS. The method described here is simple and straightforward, and can be readily used in clinical genetics laboratories with accessibility to prenatal cultures. [ABSTRACT FROM AUTHOR]

Objective: There is no consensus for the method of aneuploidy screening in pregnancy. Cell free DNA (cfDNA) is the most sensitive screen for trisomies 21, 13, and 18, however the first trimester screen (FTS) is a marker for other adverse outcomes, such as structural anomalies, growth restriction, and preeclampsia. In 2019, we offered FTS (nuchal translucency (NT) and analytes) with or without cfDNA. The purpose of this study was to assess clinical relevance of abnormal FTS in women with normal cfDNA. Methods: We retrospectively reviewed women undergoing screening in our Fetal Evaluation Unit in 2019. Women included had normal cfDNA and abnormal FTS; consisting of NT >95%, PAPP-A < 0.4 MoM, beta-HCG >2.5 MoM, or overall increased risk of trisomies. Results: 195 patients had abnormal FTS and normal cfDNA. 41 (21%) had adverse maternal outcomes including hypertension, abnormal placentation, and placental abruption. 34 (17%) had adverse fetal outcomes including growth restriction, structural anomalies, fetal demise, polyhydramnios, previable PPROM, necrotizing enterocolitis after a preterm birth, and a balanced translocation. Conclusion: Abnormal FTS predicts adverse outcomes in 33% of women with normal cfDNA. Our data suggests that offering universal FTS with cfDNA may have clinical benefit. [ABSTRACT FROM AUTHOR]

Objective To investigate whether first-trimester maternal plasma fetal fraction is altered in women that subsequently develop preeclampsia (PE) or gestational hypertension (GH) and to examine its potential value in improving the performance of screening for PE and GH by maternal factors and maternal serum pregnancy associated plasma protein-A (PAPP-A), mean arterial pressure (MAP) and uterine artery pulsatility index (UtA-PI). Methods The study population of 10,131 pregnancies undergoing cell free fetal DNA testing at 11–13 weeks’ gestation included 91 (0.9%) cases with preterm-PE, 222 (2.2%) cases with term-PE, 360 (3.6%) with GH and 9,458 (93.4%) cases unaffected by hypertensive disorders. Maternal plasma fetal fraction levels were expressed as multiples of the median (MoM) after adjustment for maternal factors and crown-rump length. The performance of screening for preterm-PE, term PE and GH by maternal factors and MoM values of fetal fraction, PAPP-A, UtA-PI and MAP was evaluated by receiver operating characteristic (ROC) curves. Results The median fetal fraction MoM was significantly lower in the preterm-PE (0.825; IQR 0.689–1.115 MoM, p < .001), term-PE (0.946; IQR 0.728–1.211 MoM, p = .028) and GH (0.928; IQR 0.711–1.182 MoM, p < .001) groups than in the unaffected group (1.002; IQR 0.785–1.251 MoM). However, the performance of screening for PE or GH by maternal factors alone or by maternal factors and PAPP-A, UtA-PI and MAP was not significantly improved by the addition of fetal fraction. Conclusions First trimester maternal plasma fetal fraction is not useful in screening for hypertensive disorders of pregnancy. [ABSTRACT FROM AUTHOR]

Cell-free DNA(cfDNA) methylation profiling is considered promising and potentially reliable for liquid biopsy to study progress of diseases and develop reliable and consistent diagnostic and prognostic biomarkers. There are several different mechanisms responsible for the release of cfDNA in blood plasma, and henceforth it can provide information regarding dynamic changes in the human body. Due to the fragmented nature, low concentration of cfDNA, and high background noise, there are several challenges in its analysis for regular use in diagnosis of cancer. Such challenges in the analysis of the methylation profile of cfDNA are further aggravated due to heterogeneity, biomarker sensitivity, platform biases, and batch effects. This review delineates the origin of cfDNA methylation, its profiling, and associated computational problems in analysis for diagnosis. Here we also contemplate upon the multi-marker approach to handle the scenario of cancer heterogeneity and explore the utility of markers for 5hmC based cfDNA methylation pattern. Further, we provide a critical overview of deconvolution and machine learning methods for cfDNA methylation analysis. Our review of current methods reveals the potential for further improvement in analysis strategies for detecting early cancer using cfDNA methylation.

A review of the literature on DNA transfer and persistence highlights many difficulties that are encountered when conducting research of this nature. One of the main problems highlighted repeatedly in the literature is the prevalence of inherent uncontrolled variation that accompany these studies, and in turn, the results obtained. This work aims to decrease the amount of intrinsic variability associated with DNA transfer and persistence experiments using a realistic proxy solution which is adaptable, of known composition, reproducible, and capable of being standardised. This proxy is composed of three parts: a synthetic fingerprint solution, cellular DNA, and cell free DNA. In this proof-of-concept study the proxy was tested with a small-scale DNA transfer and recovery experiment and the data obtained suggests that the use of a solution that mimics real fingerprint secretions, over an alternative (such as buffer or a body fluid), is important when working with non-donor provided trace DNA samples. This is because the DNA deposit solution likely impacts the transfer of DNA from fingers/hands to a surface as well as the ability to recover the biological material once deposited.

Background: CMV gastroenteritis is common in patients receiving allogeneic hematopoietic stem cell transplantation and it is difficult to distinguish from acute graft-versus-host disease (aGvHD), which has very similar symptoms but needs quite different treatment. CMV gastroenteritis is caused by local infection or reactivation of CMV in the gastrointestinal tract while aGvHD is due to immune rejection. The gold standard of diagnosis of CMV gastroenteritis and aGvHD is gastrointestinal biopsy under endoscopy, which is invasive and can potentially lead to severe side effects. Stool samples testing with quantitative polymerase chain reaction (qPCR) may be an alternative, while the application in trace level measurements and precision are not all satisfactory enough in reported research. Methods: In this study, we designed a novel method that extracted the cell free DNA (cfDNA) from the fecal supernatant to perform digital PCR (dPCR) for the detection of CMV, analyzed the performance and compared it with the total DNA extracted by the current procedure. Results: Twenty-two paired stool samples using two DNA extraction methods proved that the cfDNA extraction method had markedly higher DNA concentrations and control gene copy number, suggesting that cfDNA may be more informative and more useful for the detection of CMV DNA segment. The dPCR approach in detecting CMV DNA segment also exhibit good linearity (R2 = 0.997) and higher sensitivity (limit of detection at 50% was 3.534 copies/μL). Eighty-two stool samples from 44 immunocompromised patients were analyzed, CMV-positive rate was 28%, indicating that more than one-quarter of the gastrointestinal symptoms within these patients may be caused by CMV infection or reactivation. Conclusion: The combined results suggest that detection of CMV by dPCR in cfDNA of stool supernatant is a powerful method to identify CMV gastroenteritis and helps in clinical treatment decision making. [ABSTRACT FROM AUTHOR]

배경: Dagnosis of invasive aspergillosis (IA) is often difficult due to need for invasive procedures. Next-generation sequencing of plasma microbial cell free DNA (cfDNA) can be a novel non-invasive diagnostic method. We evaluated the clinical accuracy and utility of microbial cfDNA whole genome sequencing (WGS) for diagnosis of IA in patients with hematologic malignancy (HM) or coronavirus disease (COVID-19). 방법: A single-center prospective cohort study was conducted in a tertiary hospital in Korea. We enrolled adult patients with HM or severe COVID-19, who suspected of IA and collected plasma samples. cfDNA was extracted from plasma and WGS was performed. IA diagnosis was retrospectively confirmed according to EORTC/MSG definitions in patients with HM, and modified AspICU criteria in patients with severe COVID-19. The final diagnosis of IA was compared to the results of microbial cfDNA WGS. 결과: Between Mar 2021 and Feb 2022, a total of 64 microbial cfDNA WGS was performed on the plasma samples from 37 participants (19 with HM and 19 with COVID-19) and 7 controls who didn’t have any evidence of fungal infection. In participants with HM, aspergillus cfDNA was detected in 100% (1/1) of proven and 84.6% (11/13) of probable IA cases. In participants with COVID-19, 46.7% (7/15) of probable IA showed positive aspergillus cfDNA. Positive agreement of detection of aspergillus cfDNA versus conventional diagnosis of proven/probable IA was significantly higher in participants with HM compared to COVID-19 (85.7% vs. 46.7%, p=0.011). Fifteen of 19 (78.9%) participants with positive aspergillus cfDNA had exposure to antifungal agents. In 7 participants, non-aspergillus pathogens detected by microbial cfDNA WGS were in accordance with those identified through conventional culture. 결론: Detection of aspergillus cfDNA showed high concordance with conventional diagnostic methods of proven/probable IA in patients with HM and could be a helpful non-invasive approach to IA diagnosis in those populations. [ABSTRACT FROM AUTHOR]

Simple Summary: At a genomic level metastatic castrate resistant prostate cancer state is highly heterogeneous and no clear genome-based prognostic or predictive biomarkers exist in practice. We evaluated multiple copy number somatic alterations in two castrate resistant patient cohorts to determine if a genome-based risk score at the copy number level can predict clinical outcomes. The first cohort included patients in a prospective clinical-trial in which abiraterone acetate was given and the other comprised of a real-world hospital-registry. We extracted plasma cell free DNA in both cohorts and performed low pass whole genome sequencing. Copy number alterations were identified for 24 candidate genes and a final composite score developed from 11 genes. This risk score was able to predict survival in castrate resistant patients after adjusting for known clinical biomarkers. Additionally, the multi-gene copy number alteration based risk score algorithm also predicted if abiraterone acetate would be effective in castrate resistant patients. A plasma cell-free DNA (cfDNA) multi-gene copy number alteration (CNA)-based risk score was evaluated to predict clinical outcomes in metastatic castrate resistant prostate cancer (mCRPC) patients. Methods: Plasma specimens from two independent mCRPC patient cohorts (N = 88 and N = 92 patients) were used. A treatment-naïve mCRPC cohort (prospective clinical-trial cohort) included plasma samples before treatment with abiraterone acetate/prednisone and serially at 3-months. A separate real-world hospital-registry (RWHR) mCRPC cohort included a single blood sample collected prior to mCRPC treatments in 92 mCRPC patients following ADT failure. Low pass whole genome sequencing was performed on plasma cell-free DNA (cfDNA) and copy number alterations (CNAs) were identified for 24 candidate genes of interest. Associations of individual gene CNAs with 3 month primary resistance to therapy, progression-free survival (PFS) in the prospective trial cohort and overall survival (OS) in both cohorts was evaluated by Cox regression. A multi-gene risk score was determined for significantly associated candidate CNAs for predicting clinical outcomes. Clinical factors were included in the risk model for survival. Statistical significance for all tests was set at 0.05. Results: In the prospective trial cohort, patients responding to treatment were observed to have a significant copy number decrease in AR (p = 0.001) and COL22A1 (p = 0.037) at 3 months, while the non-responder group showed a significant CNA decrease in NKX3.1 (p = 0.027), ZBTB16 (p = 0.025) and CNA increases in PIK3CB (p = 0.006). Based on the significance level of each gene, CNAs in 11 of the 24 genes (AR, COL22A1, MYC, NCOR1, NKX3.1, NOTCH1, PIK3CA, PIK3CB, TMPRSS2, TP53, ZBTB16) were selected to develop a Cox-regression coefficient-based weighted multi-gene risk score for predicting mCRPC outcomes in both cohorts. A higher multi-gene risk score was observed to have poor OS in mCRPC patients in the prospective trial cohort (p = 0.00019) and for the RWHR cohort, (p < 0.0001). A higher risk score was also associated with poor PFS in the prospective cohort (p = 0.0043). Conclusions: A multi-gene CNAs-based risk score derived from plasma cfDNA may predict treatment response and prognosticate survival in mCRPC and warrants prospective validation of risk-based algorithms. [ABSTRACT FROM AUTHOR]

First-trimester septated cystic hygroma occurs in approximately 1 in 268 pregnancies and has long been associated with a markedly increased risk of fetal aneuploidy and, among euploid fetuses, an increased risk of structural anomalies primarily affecting the cardiac and skeletal systems. Invasive prenatal diagnosis – chorionic villus sampling and/or amniocentesis – encompasses the time-honored clinical tools for the next step in management following prenatal sonographic diagnosis of first-trimester septated cystic hygroma. Currently, prenatal cell-free DNA (cfDNA) screening for fetal aneuploidy with select microdeletions is gradually replacing the considerably less sensitive, and labor-intensive combined first-trimester screening. These new technologies have opened potential new venues in the clinical management of this ominous late first-trimester sonographic diagnosis. Advances in cfDNA technologies are now permitting detection of chromosomal copy number variants (CNV) larger than 7Mb across genome and select serious single-gene disorders (mainly impacting skeletal and neurological development), affecting quality of life and may benefit from medical and/or surgical management. This commentary will address the available non-invasive prenatal screening technologies, which clearly enhance immediate genetic analysis modalities applicable in the presence of the complex sonographic finding of first-trimester septated cystic hygroma. [ABSTRACT FROM AUTHOR]

Simple Summary: Even though the presently employed biomarkers in the detection and management of multiple myeloma are demonstrating encouraging results, the mortality percentage of the malignancy is still elevated. Thus, searching for new diagnostic or prognostic markers is pivotal. Liquid biopsy allows the examination of circulating tumour DNA, cell-free DNA, extracellular RNA, and cell free proteins, which are released into the bloodstream due to the breakdown of tumour cells or exosome delivery. Liquid biopsy can now be applied in clinical practice to diagnose, and monitor multiple myeloma, probably allowing a personalized treatment of the disease. Liquid biopsy is one of the fastest emerging fields in cancer evaluation. Circulating tumour cells and tumour-originated DNA in plasma have become the new targets for their possible employ in tumour diagnosis, and liquid biopsy can define tumour burden without invasive procedures. Multiple Myeloma, one of the most frequent hematologic tumors, has been the target of therapeutic progresses in the last few years. Bone marrow aspirate is the traditional tool for diagnosis, prognosis, and genetic evaluation in multiple myeloma patients. However, this painful procedure presents a relevant drawback for regular disease examination as it requires an invasive practice. Moreover, new data demonstrated that a sole bone marrow aspirate is incapable of expressing the multifaceted multiple myeloma genetic heterogeneity. In this review, we report the emerging usefulness of the assessment of circulating tumour cells, cell-free DNA, extracellular RNA, cell-free proteins, extracellular vesicles, and tumour-educated platelets to evaluate the changing mutational profile of multiple myeloma, as early markers of disease, reliable predictors of prognosis, and as useful tools to perform less invasive monitoring in multiple myeloma. [ABSTRACT FROM AUTHOR]

The work presented herein is the second part of a large-scale persistence project aimed at identifying trends in trace DNA persistence. This study aims to show how different environmental storage conditions and target surface characteristics influence the persistence of cellular and cell free DNA (cfDNA) over time. To eliminate variation within the experiment, we used a proxy DNA deposit consisting of a synthetic fingerprint solution, cellular DNA, and/or cfDNA. Samples were collected and analysed from eight non-metal surfaces over the course of 1 year (27 time points) under three different environmental storage conditions. The results of this experiment show that surface characteristics in conjunction with DNA type greatly influence DNA persistence. Variation in the amount of DNA recovered over time was greatly influenced by surface porosity. CfDNA persisted at significantly higher levels on non-porous surfaces, and cellular DNA persisted at higher levels on porous items. Furthermore, statistically significant differences in DNA persistence were found among the items classified as non-porous surfaces and among the items classified as porous surfaces. Additionally, this study showed that the sample storage environment had a larger impact on DNA persistence than previously observed for metal surfaces [1]. When considering DNA type, cellular DNA was shown to persist for longer than cfDNA and persistence as a whole appears to be better when DNA is deposited alone rather than in mixtures. Unsurprisingly, it was found that the amount of DNA recovered from trace deposits decreased over time. However, DNA decay is highly dependent on the surface type and exhibits higher variability at short time points and on porous surfaces. For each of the surfaces tested, DNA persisted 1 year past deposition (in some combination of DNA type and environmental condition), except for wood, on which DNA did not persist in any capacity past four months. This data is intended to add to our understanding of DNA persistence and the factors which affect it., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Liquid biopsy for CNS tumors is in its nascent phase, hindered by the low levels of circulating tumor DNA (ctDNA). Overcoming this challenge requires highly sensitive molecular techniques. DD-PCR emerges as a standout technique due to its ability to identify rare mutations, copy number variations, and circulating nucleic acids, making it one of the best methods for identifying somatic mutations in cell-free DNA (cfDNA). Despite promising results from various studies demonstrating the feasibility of obtaining informative ctDNA profiles from liquid biopsy samples, challenges persist, including the need to standardize sample collection, storage, and processing methods, define clear assay positivity thresholds, and address the overall low assay sensitivity. Our two-phase study began by assessing DD-PCR efficacy in FFPE tissues, revealing robust concordance with immunohistochemistry. In Phase 1 (85 cases), DD-PCR on FFPE tissues demonstrated 100 % sensitivity and specificity for IDH1 R132H mutations. In Phase 2 (100 cases), analysis extended to cfDNA, maintaining high specificity (100 %) with moderate sensitivity (44.2 %). Overall concordance with immunohistochemistry was 61 %, highlighting liquid biopsy's potential in glioma management. The findings emphasized DD-PCR's clinical utility in both tissue and liquid biopsy, underscoring its role in early detection, diagnosis, and therapeutic monitoring of diffuse gliomas., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier GmbH.)

Genomic profiling of hundreds of cancer-associated genes is now a component of routine cancer care. DNA sequencing can identify mutations, mutational signatures, and structural alterations predictive of therapy response and assess for heritable cancer risk, but it has been less useful for identifying predictive biomarkers of sensitivity to cytotoxic chemotherapies, antibody drug conjugates, and immunotherapies. The clinical adoption of molecular profiling platforms such as RNA sequencing better suited to identifying those patients most likely to respond to immunotherapies and drug combinations will be critical to expanding the benefits of precision oncology. This review discusses the potential advantages of innovative molecular and functional profiling platforms designed to replace or complement targeted DNA sequencing and the major hurdles to their clinical adoption., Competing Interests: Declaration of interests M.F.B.: Consulting: Eli Lilly, AstraZeneca, Paige.AI. Research support: Boundless Bio. Intellectual property rights: SOPHiA Genetics. D.B.S.: Consulting: Pfizer, Fog Pharma, Paige.AI, BridgeBio, Scorpion Therapeutics, FORE Therapeutics, Function Oncology, Pyramid, Elsie Biotechnologies, Meliora Therapeutics, and Rain Pharmaceuticals., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Abstract Background Relative haplotype dosage (RHDO) approach has been applied in noninvasive prenatal diagnosis (NIPD) of Duchenne muscular dystrophy (DMD). However, the RHDO procedure is relatively complicated and the parental haplotypes need to be constructed. Furthermore, it is not suitable for the diagnosis of de novo mutations or mosaicism in germ cells. Here, we investigated NIPD of DMD using a relative mutation dosage (RMD)-based approach—cell-free DNA Barcode-Enabled Single-Molecule Test (cfBEST), which has not previously been applied in the diagnosis of exon deletion. Methods Five DMD families caused by DMD gene point mutations or exon deletion were recruited for this study. After the breakpoints of exon deletion were precisely mapped with multiple PCR, the genotypes of the fetuses from the five DMD families were inferred using cfBEST, and were further validated by invasive prenatal diagnosis. Results The cfBEST results of the five families indicated that one fetus was female and did not carry the familial molecular alteration, three fetuses were carriers and one was male without the familial mutation. The invasive prenatal diagnosis results were consistent with those of the cfBEST procedure. Conclusion This is the first report of NIPD of DMD using the RMD-based approach. We extended the application of cfBEST from point mutation to exon deletion mutation. The results showed that cfBEST would be suitable for NIPD of DMD caused by different kinds of mutation types.