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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., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Purpose: To highlight innovations in ophthalmic oncology through histiocytosis advancements., Design: Perspective and retrospective review., Methods: The literature outlining the recent advancements in histiocytosis and ocular oncology were reviewed and combined with trial data and personal recollection. Intersections between these two fields were discussed., Results: The understanding of genetic mutations in disease-both in which cells they occur and the timing of mutation development-has expanded in tandem for the fields of ophthalmic oncology and histiocytosis. Similarly, advancements in diagnostic and treatment technology in one field can help patients in the other. For example, in one study, cell-free DNA testing reliably detected mutations in 14 of 18 (78%) patients with suspected histiocytosis. This technique has also been used in ophthalmic oncology as an alternative to invasive biopsy to avoid the risk of tumor externalization, vision impairment, and other side effects. These and other advancements, have allowed both fields to utilize targeted agents to successfully treat diseases with an actionable mutation; or deliver more targeted chemotherapy via the intraarterial technique., Conclusions: The explosion of molecular genetics technology and targeted therapies has revolutionized cancer treatment, including histiocytosis and ophthalmic oncology. Recent progress in both fields has shown how these seemingly disparate areas have many intersections, and this speaks to the collaborative spirit that is inherent in clinical research., Competing Interests: Declaration of competing interest J.H.F. has no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

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.)

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.)

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.)

Early detection of cancer is vital for increasing patient survivability chances. The three major techniques used to diagnose cancers are instrumental examination, tissue biopsy, and tumor biomarker detection. Circulating tumor DNA (ctDNA) has gained much attention in recent years due to advantages over traditional technology, such as high sensitivity, high specificity, and noninvasive nature. Through the mechanism of apoptosis, necrosis, and circulating exosome release in tumor cells, ctDNA can spread throughout the circulatory system and carry modifications such as methylations, mutations, gene rearrangements, and microsatellite instability. Traditional gene-detection technology struggles to achieve real-time, low-cost, and portable ctDNA measurement, whereas electrochemical biosensors offer low cost, high specificity alongside sensitivity, and portability for the detection of ctDNA. Therefore, this review focuses on describing the recent advancements in ctDNA biomarkers for various cancer types and biosensor developments for real-time, noninvasive, and rapid ctDNA detection. Further in the review, ctDNA sensors are also discussed in regards to their selections of probes for receptors based on the electrode surface recognition elements.

Gene overexpression by transient transfection of in vitro cultured model cell lines with plasmid DNA is a commonly used method for studying molecular aspects of human biology and pathobiology. However, there is accumulating evidence suggesting that human cells may actively secrete fragments of DNA and the implications of this phenomenon for in vitro cultured cells transiently transfected with foreign nucleic acids has been overlooked. Therefore, in the current study we investigated whether a cell-to-cell transmission of acquired plasmid DNA takes place in a commonly used human cell line model. We transiently transfected HEK293 cells with EGFP encoding plasmids to serve as donor cells and either co-cultured these with stably mCherry expressing recipient cells in different set-ups or transferred their culture medium to the recipient cells. We found that recipient cells produced EGFP after being co-cultured with donor cells but not when they were exposed to their culture medium. The employment of different co-culture set-ups excluded that the observed effect stemmed from technical artefacts and provided evidence that an intercellular plasmid transfer takes place requiring physical proximity between living cells. This phenomenon could represent a significant biological artefact for certain studies such as those addressing protein transmissions in prion diseases., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Inc.)

Background and Objectives: The role of cell-free DNA (cfDNA) in operable nonsmall cell lung cancer (NSCLC) is unclear. This study was aimed to evaluate the feasibility for identification of cfDNA in pleural lavage fluid and its correlation with plasma in resectable NSCLCs., Methods: Consecutively resected NSCLCs were evaluated for cfDNA levels in preoperative plasma (PLS1), intraoperative pleural-lavage (PLV) and postoperative (at 1 month) plasma sample (PLS2). CfDNA was isolated and measured quantitatively by qPCR in a TaqMan probe-detection approach using the human β-actin gene as the amplifying target., Results: All (n = 34) except one were negative for malignant cells in PLV cytology. CfDNA could be isolated from all the three samples (PLS1, PLV, and PLS2) successfully in each patient. The median cfDNA levels in PLS1, PLV and PLS2 were 118 ng/mL (IQR 61-158), 167 ng/mL (IQR 59.9-179.9) and 103 ng/mL (IQR 66.5-125.4) respectively. The median follow-up was 34.1 months (IQR 25.2-41.6). A significant overall-survival (OS) and disease-free survival (DFS) were recorded for patients with cfDNA level cut-offs at 125, 170, and 100 ng/mL, respectively for PLS1, PLV, and PLS2. Patients with raised cfDNA in PLS1 (>125 ng/mL) and PLV (>170 ng/mL) had significantly poorer 2-year OS, p = 0.005 and p = 0.012, respectively. The hazards (OS) were also higher for those with raised cfDNA in PLV (HR = 5.779, 95% CI = 1.162-28.745, p = 0.032). PLV (>170 ng/mL) had increased pleural recurrences (p = 0.021) and correlated significantly with poorer DFS at 2-years (p = 0.001) with increased hazards (HR = 9.767, 95% CI = 2.098-45.451, p = 0.004). Multivariable analysis suggested higher cfDNA in PLV as a poor prognostic factor for both OS and DFS., Conclusions: Among patients with operable NSCLC, it is feasible to identify cfDNA in pleural lavage and correlate PLV cfDNA with pleural recurrences and outcomes., (© 2024 Wiley Periodicals LLC.)

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., Competing Interests: All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xu, Wang, Pei, Wang, Xue and Zhao.)

It is imperative for proper evidence triage that forensic biologists understand what kind of results to expect from certain evidence types submitted for DNA analysis. The persistence of trace DNA has been insufficiently investigated and there is little data available pertaining to the persistence of DNA in different environmental conditions and on different materials. The goal of this study is to increase the available data on this topic which would, in turn, help forensic biologists manage expectations when submitting specific evidence types for DNA testing. The work presented herein is a large-scale persistence project aimed to identify trends in the persistence of trace DNA and indicate 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 7 metals over the course of 1 year (27 time points) under 3 different environmental storage conditions. The results of this experiment show that metal type greatly influences DNA persistence. For instance, copper exhibited an expected poor DNA persistence (up to 4 h) which a purification step did not help increase the DNA yield. Alternatively, DNA can persist for up to a year on lead at levels potentially high enough to allow for forensic DNA testing. Additionally, this study showed that the sample storage environment had no impact on DNA persistence in most cases. When considering DNA type, cfDNA was shown to persist for longer than cellular DNA and persistence as a whole appears to be better when DNA is deposited as mixtures over when deposited alone. Unsurprisingly, it can be expected that DNA recovery rates from trace deposits will decrease over time. However, DNA decay is highly dependent on the metal surface and extremely variable at short time points but slightly less variable as time since deposition increases. 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.)

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.

Background: 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., Objective: This study evaluated the potential of using cell free DNA (cfDNA) from serum and urine for diagnosing ALA., Methods: 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., Results: 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%., Conclusion: 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.

Background: 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., Methods: 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., Results: 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., Conclusions: 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., Competing Interests: Declaration of Competing Interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023. Published by Elsevier Inc.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sandulache, Kirby and Lai.)

Head and neck squamous cell carcinoma (HNSCC) is the seventh most commonly diagnosed cancer globally. HNSCC develops from the mucosa of the oral cavity, pharynx and larynx. Methylation levels of septin 9 ( SEPT9 ) and short stature homeobox 2 ( SHOX2 ) genes in circulating cell‑free DNA (ccfDNA) are considered epigenetic biomarkers and have shown predictive value in preliminary reports in HNSCC. Liquid biopsy is a non‑invasive procedure that collects tumor‑derived molecules, including ccfDNA. In the present study, a droplet digital PCR (ddPCR)‑based assay was developed to detect DNA methylation levels of circulating SEPT9 and SHOX2 in the plasma of patients with HNSCC. The assay was first set up using commercial methylated and unmethylated DNA. The dynamic changes in the methylation levels of SEPT9 and SHOX2 were then quantified in 20 patients with HNSCC during follow‑up. The results highlighted: i) The ability of the ddPCR‑based assay to detect very low copies of methylated molecules; ii) the significant decrease in SEPT9 and SHOX2 methylation levels in the plasma of patients with HNSCC at the first time points of follow‑up with respect to T 0 ; iii) a different trend of longitudinally DNA methylation variations in small groups of stratified patients. The absolute and precise quantification of SEPT9 and SHOX2 methylation levels in HNSCC may be useful for studies with translational potential.

Background: Stress is a pervasive issue in modern life, affecting both physical and mental health. Identifying biomarkers like cell-free DNA (cfDNA) could provide insights into stress response and help detect individuals at risk for stress-related disorders., Objective: The aim of this study is to investigate the potential use of cfDNA as a diagnostic biomarker in individuals experiencing stress., Methodology: A case-control analysis was conducted using convenient sampling on university participants (N = 285 cases, N = 500 controls) aged 18-24. The study assessed haematological and lipid profile parameters using the Sysmex XP-300TM automated analyzer and an automated biochemistry analyzer, and cfDNA was extracted using a standardized in house developed Phenol-Chloroform protocol and estimated using Agarose Gel Electrophoresis and Nanodrop. Statistical analysis was performed using SPSS ver. 21.0., Results: The results indicated a significant difference between stressed individuals and healthy controls in demographic, haematological and biochemical parameters. Specifically, stressed cases had significantly higher levels of cholesterol, LDL cholesterol, triglycerides, glucose, VLDL cholesterol, and lower levels of HDL compared to healthy controls. Stressed cases also showed significantly elevated levels of circulating cfDNA relative to healthy controls., Conclusion: These findings suggest that cfDNA may have potential as a diagnostic biomarker for stress., Competing Interests: 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., (© 2024 The Author(s).)

Heart failure, particularly in its advanced stages, significantly impacts quality of life. Despite progress in Guideline-Directed Medical Therapy (GDMT) and invasive treatments, heart transplantation (HT) remains the primary option for severe cases. However, complications such as graft rejection present significant challenges that necessitate effective monitoring. Endomyocardial biopsy (EMB) is the gold standard for detecting rejection, but its invasive nature, associated risks, and healthcare costs have shifted interest in non-invasive techniques. Donor-derived cell-free DNA (dd-cfDNA) has gained attention as a promising non-invasive biomarker for monitoring graft rejection. Compared to EMB, dd-cfDNA detects graft rejection early and enables clinicians to adjust immunosuppression promptly. Despite its advantages, dd-cfDNA testing faces challenges, such as the need for specialized technology and potential inaccuracies due to other clinical conditions. Additionally, dd-cfDNA cannot yet differentiate between types of graft rejection, and its effectiveness in chronic rejection remains unclear. Research is ongoing to set precise standards for dd-cfDNA levels, which would enhance its diagnostic accuracy and help in clinical decisions. The article also points to the future of HT monitoring, which may involve combining dd-cfDNA with other biomarkers and integrating artificial intelligence to improve diagnostic capabilities and personalize patient care. Furthermore, it emphasizes both global and racial inequalities in dd-cfDNA testing and the ethical issues related to its use in transplant medicine., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Borkowski et al.)

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., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2024.)

Background: Cell-free DNA (cfDNA) profiles of 5-hydroxymethylcytosine (5-hmC), an epigenetic marker of open chromatin and active gene expression, are correlated with metastatic disease burden in patients with neuroblastoma. Neuroblastoma tumors are comprised of adrenergic (ADRN) and mesenchymal (MES) cells, and the relative abundance of each in tumor biopsies has prognostic implications. We hypothesized that ADRN and MES-specific signatures could be quantified in cfDNA 5-hmC profiles and would augment the detection of metastatic burden in patients with neuroblastoma., Methods: We previously performed an integrative analysis to identify ADRN and MES-specific genes (n = 373 and n = 159, respectively). Purified DNA from cell lines was serial diluted with healthy donor cfDNA. Using Gene Set Variation Analysis (GSVA), ADRN and MES signatures were optimized. We then quantified signature scores, and our prior neuroblastoma signature, in cfDNA from 84 samples from 46 high-risk patients including 21 patients with serial samples., Results: Samples from patients with higher metastatic burden had increased GSVA scores for both ADRN and MES gene signatures (p < .001). While ADRN and MES signature scores tracked together in serially collected samples, we identified instances of patients with increases in either MES or ADRN score at relapse., Conclusions: While it is feasible to identify ADRN and MES signatures using 5-hmC profiles of cfDNA from neuroblastoma patients and correlate these signatures to metastatic burden, additional data are needed to determine the optimal strategies for clinical implementation. Prospective evaluation in larger cohorts is ongoing., (© 2023 The Authors. Pediatric Blood & Cancer published by Wiley Periodicals LLC.)

Aim: 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., Methods: 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., Results: 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)., Conclusions: 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., (© 2023 Indian National Association for Study of the Liver. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Böhmer, Wasslavik, Andersson, Ståhlberg, Jonsson, Wåhlander, Karason, Sunnegårdh, Nilsson, Asp, Dellgren and Ricksten.)

Objectives: Non-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., Methods: From 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., Results: A 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., Conclusion: The 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhao, Xue, Geng, Zhu, Hu and Jiang.)

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. STATEMENT OF SIGNIFICANCE: 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., 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 © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Manea, Iacob, Iacob, Cerban, Dima, Oniscu, Popescu and Gheorghe.)

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., Competing Interests: The authors declare that they have no competing interests., (© 2023 Hoeter et al.)

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., (© 2023. The Author(s).)

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.

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., Competing Interests: 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., (© 2023 The Authors.)

Sepsis is a disease caused by infection, which is characterized by a dysregulated immune response in the host and affects more than 30 million people worldwide each year. However, the current single therapeutic approaches are not effective in controlling the progression of sepsis. Here, we synthesize a nanoparticle (TMP) containing tannic acid (TA), Polymyxin B (PMB), and Mn 2+ (Mn) by a simple one-pot method. TMP has the following characteristics: (1) All components have good biocompatibility; (2) simple preparation process without subsequent processing; (3) antibacterial and remove multiple inflammatory mediators; and (4) effectively mitigating cytokine storm both in the acute lung injury (ALI) and the cecal ligation and puncture (CLP) model. Our results demonstrate the critical role of targeting multiple mediators to mitigate cytokine storms for the treatment of sepsis.

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., (© 2023. The Author(s).)

Liquid biopsy (LB) is clinically utilized to detect minute amounts of genetic material or protein shed by cancer cells, most commonly cell free DNA (cfDNA), as a noninvasive precision oncology tool to assess genomic alterations to guide cancer therapy or to detect the persistence of tumor cells after therapy. LB is also being developed as a multi-cancer screening assay. The use of LB holds great promise as a tool to detect lung cancer early. Although lung cancer screening (LCS) with low-dose computed tomography (LDCT) substantially reduces lung cancer mortality in high-risk individuals, the ability of current LCS guidelines to reduce the public health burden of advanced lung cancer through early detection has been limited. LB may be an important tool to improve early lung cancer detection among all populations at risk for lung cancer. In this systematic review, we summarize the test characteristics, including sensitivity and specificity of individual tests, as they pertain to the detection of lung cancer.  We also address critical questions in the use of liquid biopsy for early detection of lung cancer including: 1. How might liquid biopsy be used to detect lung cancer early; 2. How accurate is liquid biopsy in detecting lung cancer early; and 3. Does liquid biopsy perform as well in never and light-smokers compared with current and former smokers., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Qi and Yu.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Huang, Yang and Shi.)

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 demon-strated that patients with high cfDNA values (above 2913 copies/ml) had significantly worse cancer-specific survival (HR: 4.5; 95% CI: 1.3-16.9, log-rank Mantel-Cox test p = 0.015)., Conclusions: Preoperative plasma cfDNA concentration has diagnostic and prognostic potential in ccRCC pa-tients., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 Termedia.)

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.

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.

Background: The inadequate early detection strategies makes hepatocellular carcinoma (HCC) patients with poor prognisis. Therefore, more effective detection methods are urgently needed for early detection and early intervention of HCC. Methods: 17 cases of suspected HCC patients and 11 cases of HBV-related decompensated cirrhosis (HBV-DeCi) patients were enrolled. For each patient, 5 ml blood sample was separated into circulating tumor cells (CTCs) and plasma, CTCs were stained with Diff staining for counting. Plasma was used for extracting cell free DNA (cfDNA) and then analyzed by qMSP assay. Ct values were recorded for GNB4 and Riplet as target genes and β-actin as an endogenous reference gene. Finally, clinical efficacy of CTC count combined with GNB4/Riplet methylation detection for early diagnosis of HCC was analyzed. Results: The CTC of HCC patients has pleomorphic characteristics, but it is difficult to distinguish from other blood cells with non-obviously pleomorphic of CTC. Although a small number of CTCs can also be detected in HBV-DeCi patients (control group), the number is significantly lower than that in HCC patients, the sensitivity and specificity of CTC for HCC detection were 70.6% and 90.9% (AUC = 0.81). The Ct values of GNB4 and Riplet methylation were significantly different between HCC patients and control group patients. When CTC combined with two genes, the AUC value was significantly increased to 0.98, the sensitivity was 88.2%, and the specificity was 100%. Conclusion: Our study has developed a novel test that CTC count combined with GNB4/Riplet methylation detection and showed its high performance for early diagnosis of HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liang, Xu, Kong, Huang, Xiao, Zhou, Ye and Ye.)

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 (R 2  = 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., (© 2022. The Author(s).)

Background: Exosomes are ubiquitous extracellular nanovesicles secreted from almost all living cells that are thought to be involved in several important cellular processes, including cell-cell communication and signaling. Exosomes serve as a liquid biopsy tool for clinical and translational research. Although many techniques have been used to isolate exosomes, including ultracentrigation, size-exclusion chromatography, and immunocapturing-based techniques, these techniques are not convenient, they require expensive instrumentation, and they are unhandy for clinical samples. Precipitation techniques from available commercial kits that contain polyethelene glycol (PEG) are now widely used, but these kits are expensive, especially if a large number of biological samples are to be processed., Objective: the purpose of this study is to compare and optimize the efficacy of different concentrations of PEG with two commercial kits ExoQuick (SBI) and Total Exosome Isolation (TEI) from Invitrogen in human plasma., Methods and Materials: we determined exosome quantity, size distribution, marker expression, and downstream application., Results: among the precipitation methods, we found the size of particles and concentrations with 10-20% PEG are similar to ExoQuick and better than TEI. Interestingly, we detected cfDNA with ExoQuick and 10-20% PEG but not TEI and 5% PEG. Moreover, 10% PEG detection of miR-122 and miR-16 expression was superior to ExoQuick and TEI. Furthermore, in proteomics results it also found the identified proteins better than commercial kits but there was a high level of contamination of other proteins in serum., Conclusions: together, these findings show that an optimal concentration of 10% PEG serves as a guide for use with clinical samples in exosome isolation for downstream applications., Competing Interests: The authors declare no conflict of interest.

Background: Lack of biomarkers for treatment selection and monitoring in small cell lung cancer (SCLC) patients with the limited therapeutic options, result in poor outcomes. Therefore, new prognostic biomarkers are needed to improve their management. The prognostic value of cell-free DNA (cfDNA) and circulating tumor cells (CTCs) have been less explored in SCLC., Methods: We quantified cfDNA in 46 SCLC patients at different times during first-line of chemotherapy or chemo-immunotherapy. Moreover, CTCs were analyzed in 21 patients before therapy onset using CellSearch ® system. The possible association between both biomarkers and patients' outcomes was investigated in order to develop a prognostic model., Results: High cfDNA levels before therapy were associated with shorter progression-free survival (PFS) and overall survival (OS). Furthermore, cfDNA levels at 3 weeks and at progression disease were also associated with patients' outcomes. Multivariate analyses confirmed the independence of cfDNA levels as a prognostic biomarker. Finally, the three-risk category prognostic model developed included Eastern Cooperative Oncology Group Performance Status (ECOG PS), gender and baseline cfDNA levels was associated with a higher risk of progression and death., Conclusions: We confirmed the prognostic utility of cfDNA quantitative analysis in SCLC patients before and during therapy. Our novel risk prognostic model in clinical practice will allow to identify patients who could benefit with actual therapies., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-273/coif). JGG reports consultant or advisory role fees from AstraZeneca, Boehringer-Ingelheim, Bristol-Myers Squibb, MSD, Novartis, Roche, Sanofi, and Takeda; honoraria for presentations and speaker bureaus from AstraZeneca, Bristol-Myers Squibb, Pierre-Fabre, Roche, Rovi, and Takeda; non-financial support from AstraZeneca, Bristol-Myers Squibb, Ippsen Pharma, Lilly, MSD, Roche, and Sanofi outside the submitted work. SA reports grants or contracts from Merk, BMS and MSD; consulting fees from Merk, BMS and MSD; payment or honoraria for lectures from BMS and MSD; support for attending meeting from MSD and participation on a data safety monitoring board from Merk and BMS outside the submitted work. LLM receives honoraria for lectures from Pfizer, Boehringer, Novartis, AstraZeneca, Sanofi, Bristol, MSD, Takeda; for advisory board from Sanofi, Lilly, Novartis, Boehringer, Amgen and receives support for attending meetings from MSD and AstraZeneca, outside the submitted work. RLL reports grants from Astellas, Janssen, Sanofi, Bayer, Ipsen, Roche, Novartis, Pfizer; consulting fees from Sanofi, Janssen, Astellas, Pfizer, Bayer, Roche, Ipsen, Novartis, Eisai, EUSA Pharma, BMS; payment or honoraria for lectures from Astellas, Janssen, Sanofi, Bayer, Ipsen, Pfizer, Roche, Novartis; travel, accommodations and expenses from Pharmamar, Roche, BMS and Pfizer; honoraria for participation in advisory boards from Roche, AstraZeneca, Merck, MSD, Bayer, BMS, Novartis, Janssen, Lilly, Pfizer and Leo, outside the submitted work. The other authors have no conflicts of interest to declare., (2022 Translational Lung Cancer Research. All rights reserved.)

Prostate cancer (PCa) accounted for more than 34,000 deaths in US males [...].

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., Competing Interests: The authors declare no conflict of interest.

Background: Endomyocardial 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., Methods: Plasma 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., Results: A 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%, p c < 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., Conclusions: This novel dd-cfDNA test detects AR in HTx recipients with good accuracy and holds promise as a noninvasive test for AR in HTx recipients., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)