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In clinical oncology, cell-free DNA (cfDNA) has shown immense potential in its ability to noninvasively detect cancer at various stages and monitor the progression of therapy. Despite the rapid improvements in cfDNA liquid biopsy approaches, achieving the required sensitivity to detect rare tumor-derived cfDNA still remains a challenge. For next-generation sequencing, the perceived presentation of cfDNA is strongly linked to the extraction and library preparation protocols. Conventional double-stranded DNA library preparation (dsDNA-LP) focuses on assessing ~167bp double-stranded mononucleosomal (mncfDNA) and its other oligonucleosomal cell-free DNA counterparts in plasma. However, dsDNA-LP methods fail to include short, single-stranded, or nicked DNA in the final library preparation, biasing the representation of the actual cfDNA populations in plasma. The emergence of single-stranded library preparation (ssDNA-LP) strategies over the past decade has now allowed these other populations of cfDNA to be studied from plasma. With the use of ssDNA-LP, single-stranded, nicked, and ultrashort cfDNA can be comprehensively assessed for its molecular characteristics and clinical potential. In this review, we overview the current literature on applications of ssDNA-LP on plasma cfDNA from a potential cancer liquid biopsy perspective. To this end, we discuss the molecular principles of single-stranded DNA adapter ligation, how library preparation contributes to the understanding of native cfDNA characteristics, and the potential for ssDNA-LP to improve the sensitivity of circulating tumor DNA detection. Additionally, we review the current literature on the newly reported species of plasma ultrashort single-stranded cell-free DNA plasma, which appear biologically distinct from mncfDNA. We conclude with a discussion of future perspectives of ssDNA-LP for liquid biopsy endeavors.

Background Methods Results Conclusions Placental‐originated cell‐free DNA (cfDNA) provides unique opportunities to study (epi)genetic placental programming remotely, but studies investigating the cfDNA methylome are scarce and usually technologically challenging. Methylated DNA sequencing (MeD‐seq) is well compatible with low cfDNA concentrations and has a high genome‐wide coverage. We therefore aim to investigate the feasibility of genome‐wide methylation profiling of first trimester maternal cfDNA using MeD‐seq, by identifying placental‐specific methylation marks in cfDNA.We collected cfDNA from nonpregnant controls (female n = 6, male n = 12) and pregnant women (n = 10), first trimester placentas (n = 10), and paired preconceptional and first trimester buffy coats (total n = 20). Differentially methylated regions (DMRs) were identified between pregnant and nonpregnant women. We investigated placental‐specific markers in maternal cfDNA, including RASSF1 promoter and Y‐chromosomal methylation, and studied overlap with placental and buffy coat DNA methylation.We identified 436 DMRs between cfDNA from pregnant and nonpregnant women, which were validated using male cfDNA. RASSF1 promoter methylation was higher in maternal cfDNA (fold change 2.87, unpaired t‐test p < .0001). Differential methylation of Y‐chromosomal sequences could determine fetal sex. DMRs in maternal cfDNA showed large overlap with DNA methylation of these regions in placentas and buffy coats. Sixteen DMRs in maternal cfDNA were specifically found only in placentas. These novel potential placental‐specific DMRs were more prominent than RASSF1.MeD‐seq can detect (novel) genome‐wide placental DNA methylation marks and determine fetal sex in maternal cfDNA. Our results indicate a placental and immune‐cell contribution to the pregnancy‐specific cfDNA methylation signature. This study supports future research into maternal cfDNA methylation. [ABSTRACT FROM AUTHOR]

Cervical cancer remains one of the most lethal gynecological malignancies. However, biomarkers for more precise patient care are an unmet need. Herein, the concentration of 285 plasma cell‐free DNA (cfDNA) samples are analyzed from 84 cervical patients and the clinical significance of cfDNA fragmentomic characteristics across the neoadjuvant chemotherapy (NACT) treatment. Patients with poor NACT response exhibit a significantly greater escalation in cfDNA levels following the initial cycle of treatment, in comparison to patients with a favorable response. Distinctive end motif profiles and promoter coverages of cfDNA in initial plasma are observed between patients with differing NACT responses. Notably, the DNASE1L3 analysis further demonstrates the intrinsic association between cfDNA characteristics and chemotherapy resistance. The cfDNA and motif ratios show a good discriminative capacity for predicting non‐responders from responders (area under the curve (AUC) > 0.8). In addition, transcriptional start sites (TSS) coverages around promoters discern the alteration of biological processes associated with chemotherapy resistance and reflect the potential value in predicting chemotherapy response. These findings in predictive biomarkers may optimize treatment selection, minimize unnecessary treatment, and assist in establishing personalized treatment strategies for cervical cancer patients. [ABSTRACT FROM AUTHOR]

The gold standard for precise diagnostic classification of brain tumors requires tissue sampling, which carries relevant procedural risks. Brain biopsies often have limited sensitivity and fail to address tumor heterogeneity, because small tissue parts are being examined. This study aims to explore the detection and quantification of diagnostically relevant somatic copy number aberrations (SCNAs) in cell-free DNA (cfDNA) extracted from cerebrospinal fluid (CSF) in a real-world cohort of patients with defined brain tumor subtypes. A total of 33 CSF samples were collected from 30 patients for cfDNA extraction. Shallow whole-genome sequencing was conducted on CSF samples containing > 3ng of cfDNA and corresponding tissue DNA from nine patients. The sequencing cohort encompassed 26 samples of 23 patients, comprising 12 with confirmed CNS cancer as compared to 11 patients with either ambiguous CNS lesions (n = 5) or non-cancer CNS lesions (n = 6). After mapping and quality filtering SCNAs were called by depth-of-coverage analyses with a binning of 5.5 Mbp. SCNAs were exclusively identified in CSF cfDNA from brain tumor patients (10/12, 83%). In tumor patients, SCNAs were detectable in cfDNA from all patients with, but also in five of seven patients without tumor cells detected by CSF cytopathology. A substantial number of shared SCNAs were traceable between tissue and CSF in matched pair analyses. Additionally, some SCNAs unique to either CSF or tissue indicating spatial heterogeneity or tumor evolution. Also, diagnostically relevant genomic alterations as well as essential and desirable SCNAs as implemented in the current WHO classification of CNS tumors for certain primary brain tumor subtypes were traceable. In summary, this minimally invasive cfDNA-based LB approach employing shallow whole genome sequencing demonstrates potential for providing a molecularly informed diagnosis of CNS cancers, mapping tumor heterogeneity, tracking tumor evolution, and surveilling tumor patients. Further prospective trials are warranted. [ABSTRACT FROM AUTHOR]

Central nervous system germ cell tumors (CNS-GCT) are malignant neoplasms that arise predominantly during adolescence and young adulthood. These tumors are typically sensitive to treatment, but resulting long-term health deficits are common. Additional clinical challenges include surgical risks associated with tumor biopsy, and need to determine treatment response for adapting radiotherapy protocols. The aim of this study was to establish the detectability of circulating-tumor DNA (ctDNA) from cerebrospinal fluid (CSF) of children with CNS-GCT as a potential biomarker. We obtained CSF from patients with CNS-GCT by lumbar puncture or intra-operatively. Cell-free DNA (cfDNA) was extracted and subjected to low-pass whole genome sequencing (LP-WGS). Copy-number alterations (CNAs) were inferred and served as a marker of measurable residual disease (MRD). Comparisons with imaging findings and tumor marker levels were made. A total of 29 CSF samples from 21 patients (16 with germinoma, 5 with non-germinomatous GCT) were sequenced. Twenty samples from 19 patients were collected at diagnosis, and 9 samples from 7 patients were collected during or after therapy. Among the diagnostic samples, CNAs were detected in samples from 17/19 patients (89%), which included 8 with marker-negative tumors. Specific clinical scenarios suggested that serial cfDNA analysis may carry utility in tracking treatment responses as well as clarifying indeterminate imaging findings. Our results provide evidence for the high-sensitivity in detecting ctDNA from CSF of CNS-GCT patients using LP-WGS, with potential utility for non-invasive diagnosis and disease monitoring in upcoming CNS-GCT studies. [ABSTRACT FROM AUTHOR]

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

Introduction: Cancer is a genetic disease that affects people worldwide, and breast cancer is the most common cancer in women. Studies have been conducted on molecular parameters to predict tumor behavior and develop therapeutic strategies. Telomeres, which are at the end of chromosomes, have been studied for their relationship with breast cancer, but more research is needed to understand their role in the disease. Circulating‐free DNA (cfDNA) is DNA that is free in the bloodstream and is considered a promising target for early cancer detection, treatment response monitoring, and prognosis assessment. This study is aimed at comparing cfDNA telomere length of breast cancer patients and healthy individuals and analyzing the impact of neoadjuvant chemotherapy on telomere length in cfDNA. Materials and Methods: Blood samples were collected from 33 breast cancer patients undergoing neoadjuvant chemotherapy before and after treatment. The quantitative PCR method is used to measure the average telomere lengths. Results: This study found that the telomere length of cfDNA in breast cancer patients before and after treatment is significantly shorter than in the control group. Neoadjuvant chemotherapy is found to shorten the cfDNA telomere length, especially in the treatment‐responsive group. Conclusion: Our study suggests that telomere length in cfDNA may be a useful biomarker for predicting therapy response and possible reoccurrence of the disease in breast cancer patients. [ABSTRACT FROM AUTHOR]

Background: In locally advanced rectal cancer, the prediction of tumor response during and after neoadjuvant treatment remains challenging. In terms of organ preservation, adaptive radiotherapy, and intensified (total) neoadjuvant therapies, biomarkers are desirable for patient stratification. Methods: In 16 patients, weekly blood samples (n = 86) to detect cell-free tumor DNA (ctDNA) during long-course neoadjuvant chemoradiotherapy were analyzed. Data were correlated with initial tumor volumes, MRI response in week 2 and 5 of radiotherapy as well as with pathologic tumor response after resection and outcome parameters. Results: Most patients showed decreasing ctDNA during the course of radiochemotherapy. However, we found heterogenous dynamics of ctDNA and could identify three groups: (1) decline (2) no clear decline and/or late shedding (3) persistence of ctDNA. In seven patients we could detect significant amounts of ctDNA in week 5 or week 6 of treatment. In our pilot cohort, we did not find significant correlations of ctDNA dynamics with pathologic response or outcome parameters. However, patients with distinct decline of ctDNA had larger tumor volumes prior to treatment, and MRI imaging in week 2 and 5 revealed bigger absolute decrease of tumor volumes. If significant levels of ctDNA were found in week 5 and / or 6, patients showed less absolute tumor volume decrease in week 2 and 5. Conclusions: Weekly measurement of ctDNA during radiochemotherapy is feasible and might represent a promising biomarker. Bigger initial primary tumors showed different ctDNA shedding profiles compared with smaller primary tumors and correlations of ctDNA dynamics with early imaging response were found. [ABSTRACT FROM AUTHOR]

Introduction: Plasma cell-free DNA (cfDNA) estimation offers a non-invasive method to potentially diagnose, monitor, and prognosticate patients with malignancy. This prospective study aimed to assess plasma cfDNA levels in patients with differentiated thyroid cancer (DTC) to determine its role in predicting disease status in the post-operative setting. Materials and methods: This was a single-center prospective observational study conducted at a public medical research university and hospital in New Delhi, India. 254 patients with DTC in the post-operative setting were included: 95 in Group 1 (active structural disease) and 159 in Group 2 (disease-free). Blood samples were collected for plasma separation and cfDNA extraction. The cfDNA concentrations were quantified and compared across various disease states. Results: Median values of plasma cfDNA (ng/µL) in groups 1 and 2 were found to be 0.272 (IQR: 0.137-0.442) and 0.222 (IQR: 0.123-0.398), respectively with no significant difference (p=0.122). cfDNA levels were significantly higher in patients in the age group ≥55 years (p=0.016). However, the cfDNA levels were not significantly associated with any of the other known prognostic markers of DTC. Discussion: Based on the results of this study, plasma cfDNA levels did not significantly predict disease status in patients with DTC in the post-operative setting. [ABSTRACT FROM AUTHOR]

Enhancing the effectiveness of utilizing circulating cell‐free DNA (cfDNA) for disease screening remains a challenge, necessitating improved sensitivity, specificity, cost‐efficiency, and patient adherence. We present here LAMP‐MS, an innovative technology that integrates linear amplification with single‐base quantitative nucleic acid mass spectrometry on silicon chips. This approach overcomes several limitations in utilizing cfDNA 5‐methylcytosine (5 mC) status for colorectal cancer (CRC) screening. LAMP‐MS enables unbiased amplification of as little as 1 ng of cfDNA, site‐specifically quantify methylation levels of multiple 5 mC sites, thereby facilitating cost‐effective, high‐resolution quantitative detection of cfDNA methylation markers. We have validated the accuracy of DNA methylation determination using DNA probes and cfDNA from patient plasma samples, confirmed by mass spectrometric peak areas. Additionally, we have further shown this Mass Array technology could be expanded to also quantify RNA m6A modification sites. Combining the ability to work with ultra‐low input materials and a visually interpretable output, LAMP‐MS stands out as a promising method for real‐world applications in clinics and laboratories for nucleic acid methylation detection and quantification. [ABSTRACT FROM AUTHOR]

Peptidylarginine deiminase 4 (PAD4)-dependent neutrophil extracellular trap (NET) formation is a new neutrophil death mechanism. Increased NET formation has been demonstrated to be associated with gouty inflammation. Macrophages release proinflammatory mediators and chemokines in acute gouty inflammation and subsequently lead to inflammatory cascades. However, whether NETs regulate macrophage function and polarization and further contribute to gout development remains unclear. Herein, we investigated the relationship between monosodium urate (MSU) crystal-induced NETs and macrophages and the associated mechanisms in gouty inflammation. Elevated NET formation and CD86+ macrophage infiltration were observed in human gouty arthritis (GA). In vitro , MSU crystal-induced NETs or NET-associated histone H3 treatments modulated nod-like receptor protein 3 (NLRP3) inflammasome activation, M1 polarization, and metabolic changes in macrophages. These effects were eliminated by hexokinase-2 (HK-2) silencing. Moreover, NET formation and inflammation were significantly reduced in PAD4−/− GA mice. Pharmacological inhibition of NET formation with Cl-Amidine or NET degradation with DNase Ⅰ significantly reduced M1 polarization of macrophages and ameliorated inflammation in GA mice. In sum, MSU crystal-induced NETs promote M1 polarization and NLRP3 activation in macrophages via targeting HK-2. Cell-free DNA and histone H3 may be the driving elements behind the NET-induced M1 macrophage polarization, NLRP3 activation, and metabolic changes. Targeting NETs could be a potential therapeutic strategy for gout flare. [Display omitted] • NETs promote macrophage activation and M1 polarization via targeting HK-2 in vitro. • Cell-free DNA and histone H3 are the driving elements behind the NET-induced macrophage M1 polarization. • NET inhibition represses M1 macrophage polarization and gouty inflammation in vivo. • Targeting NETs could be a potential therapeutic strategy for gout flare. [ABSTRACT FROM AUTHOR]

Background/Objectives: Sarcomas are a heterogeneous group of cancers, many with high rates of recurrence and metastasis, leading to significant morbidity and mortality. Due to a lack of early diagnostic biomarkers, by the time recurrent disease can be clinically detected, it is often extensive and difficult to treat. Here, we sought to investigate methods of detecting ctDNA in sarcoma patient plasma to potentially monitor disease recurrence, progression, and response to treatment. Methods: Whole-exome sequencing of matched tumor and blood samples revealed patient-specific mutations, which were used to develop personalized assays to detect ctDNA in patient plasma. Since ctDNA is present in extremely low quantities, detection requires highly sensitive methodologies. Droplet digital PCR is highly sensitive; however, it is limited in that it can only be used to target one tumor variant at a time. Therefore, a protocol combining multiplex PCR and targeted amplicon sequencing was developed. Results: ddPCR was successfully able to detect tumor-specific mutations in plasma, confirming the presence of ctDNA in sarcoma patients. Multiplex PCR followed by amplicon sequencing was able to detect multiple tumor variants simultaneously, although it was not as sensitive as ddPCR. Additionally, ctDNA was detected in patient plasma collected at two different time points. Conclusions: This work demonstrates that although there is a lack of recurrent biomarkers, personalized assays detecting ctDNA have the potential to be used to monitor disease progression in sarcoma. [ABSTRACT FROM AUTHOR]

Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics. [ABSTRACT FROM AUTHOR]

Biomarkers in colorectal cancer (CRC) are of great interest in the current literature due to improvements in techniques such as liquid biopsy and next-generation sequencing (NGS). However, screening methods vary globally, with multi-target stool DNA (mt-sDNA) predominantly used in the USA and, more recently, the Cologuard Plus; biomarkers such as the Galectins family and septins show promise in early detection. Gut microbiome assessments, such as Fusobacterium nucleatum, are under intense exploration. Diagnostic tests, such as circulating DNA analysis via NGS, exhibit effectiveness and are being increasingly adopted. Circulating tumor cells emerge as potential alternatives to traditional methods in terms of diagnosis and prognosis. Predictive biomarkers are well established in guidelines; nonetheless, with the aid of machine learning and artificial intelligence, these biomarkers may be improved. This review critically explores the actual dynamic landscape of CRC biomarkers and future, promising biomarkers involved in screening, diagnosis, and prognosis. [ABSTRACT FROM AUTHOR]

Circulating tumor DNA (ctDNA) is a promising cancer biomarker, but accurately detecting tumor mutations in cell-free DNA (cfDNA) is challenging due to their low frequency and sequencing errors. Our study benchmarked Mutect2, VarScan2, shearwater, and DREAMS-vc using deep targeted sequencing of cfDNA with Unique Molecular Identifiers (UMIs) from 111 colorectal cancer patients. Performance was assessed at both the mutation level (distinguish tumor variants from errors) and the sample level (detect if an individual has cancer). Additionally, we investigated the effects of various UMI grouping and consensus strategies. The shearwater-AND variant calling method demonstrated the highest precision in detecting tumor-derived mutations from plasma, and reached the highest ROC-AUC of 0.984 for sample classification in tumor-informed cfDNA analyses. DREAMS-vc exhibited the highest ROC-AUC of 0.808 for sample classification in tumor-agnostic studies. We also found that sequencing depth differences in PBMCs could lead to false positives, particularly with VarScan2 and Mutect2, which was addressed by downsampling to equivalent mean depths. Additionally, network-based UMI grouping methods outperformed those using identical UMIs when all reads were retained. Our findings emphasize that the optimal variant caller depends on the study context—whether focused on mutation or sample classification, and whether conducted under tumor-informed or tumor-agnostic conditions. [ABSTRACT FROM AUTHOR]

Background/Objectives: Biliary tract cancer (BTC) is a rare but aggressive malignancy that requires surgical treatment. However, postoperative recurrence rates are high, and reliable predictors of recurrence are limited. This study aimed to investigate the effectiveness of cell-free DNA (cfDNA) and circulating tumor cells (CTCs) in predicting early recurrence after curative surgery and complete adjuvant therapy in patients with BTC. Methods: Twenty-four patients who underwent R0 and R1 resections and completed adjuvant therapy for BTC between September 2019 and March 2022 were followed up until March 2024. Patients were categorized into early recurrence (ER) and non-ER groups, using one year as the cutoff for recurrence. Results: The combination score derived from ultrashort fragments of cfDNA, vimentin-positive CTCs, and carbohydrate antigen (CA) 19-9 levels showed a statistically significant difference between the ER and non-ER groups (p-value < 0.001). The receiver operating characteristic curve from the combination score and CA 19-9 levels yielded areas under the curve of 0.891 and 0.750, respectively. Conclusions: Although further research is required, these findings suggest that cfDNA and CTCs may increase the accuracy of predicting postoperative recurrence in patients with BTC. [ABSTRACT FROM AUTHOR]

Leptomeningeal melanoma metastases (LMM) are associated with poor survival. Diagnosis is based on clinical presentation, brain MRI and cerebrospinal fluid (CSF) analysis. Inconclusive findings at initial presentation can delay treatment. In this single‐center case series, detection of BRAFV600‐ and NRASQ61‐mutant cell‐free tumor DNA (cfDNA) in CSF was evaluated as a complementary diagnostic biomarker. In 12 patients with clinical suspicion of LMM, a retrospective analysis of MRI, CSF cytology and cfDNA analysis on 1 mL of CSF using the Idylla® platform was carried out. Nine patients displayed MRI abnormalities suggesting LMM. CSF analysis identified malignant cells in three patients (including one without MRI abnormalities). BRAFV600‐ or NRASQ61‐mutant cfDNA was detected in CSF of nine patients (eight with and one without MRI abnormalities; all patients with positive CSF cytology). Subsequent follow‐up confirmed LMM in all patients with positive and in one patient with a negative CSF cfDNA analysis (sensitivity 81.8%; specificity 100%). Our findings suggest that analyzing BRAFV600‐ and NRASQ61‐mutant cfDNA in CSF using the Idylla® platform holds promise as a sensitive and specific complementary diagnostic biomarker for LMM, particularly in case of inconsistency between imaging and CSF cytology. The 110‐min analysis can facilitate urgent treatment decisions. [ABSTRACT FROM AUTHOR]

Background: Human papillomavirus (HPV) has emerged as a significant contributor to cancer incidence globally, particularly in the context of oropharyngeal squamous cell carcinoma (OPSCC) and cancer of unknown primary (HNCUP). This study aimed to develop and validate droplet digital PCR (ddPCR) assays for the detection of circulating tumor HPV DNA (ctHPV-DNA) in plasma, focusing on high-risk HPV genotypes associated with these cancers. Methods: ddPCR assays for HPV16, 18, 33, 35, 56, and 59 were developed and tested using gBlocks, HPV cell-free DNA, fragmented tumor HPV+ DNA, and plasma samples from patients with HPV+ OPSCC (n = 110) and HNCUP (n = 9). Results: Assays demonstrated robust technical sensitivity across all tested HPV genotypes. Clinical application of the assays on a cohort of patients with HPV+ OPSCC and HNCUP revealed high sensitivity (91.6%) and wide variability in ctHPV-DNA levels. Analyses revealed correlations between ctHPV-DNA levels and TNM stage and tumor viral load. The association between ctHPV-DNA and tumor viral load persisted even after adjusting for TNM stage. At posttreatment, 72.5% of samples had reached undetectable ctHPV-DNA levels. Having detectable ctHPV-DNA posttreatment was associated with a higher ctHPV-DNA level at diagnosis and higher viral load at diagnosis. Conclusion: The findings underscore the potential of ctHPV-DNA as a biomarker for monitoring HPV+ cancers and offer insights into tumor dynamics. Implementation of these assays in clinical practice could enhance no-invasive treatment monitoring and recurrence detection in HPV-associated cancers. Clinical Trials: NCT05904327. [ABSTRACT FROM AUTHOR]

Introduction: Comprehensive next-generation sequencing (NGS) of non-small-cell lung cancer specimens can identify oncogenic driver mutations and their corresponding targeted therapies. Plasma cell-free DNA (cfDNA) genotyping is easy to perform; however, false negatives cannot be overlooked. We explored malignant pleural effusion (MPE), a rich source of cfDNA, as a non-inferior alternative to tumor tissues for genotyping. Methods: We conducted a prospective trial including 39 patients with newly diagnosed stage IV lung adenocarcinoma who presented with MPE. Tissue tests matching hotspot variants, including EGFR, ALK, and ROS1, were compared with the AlphaLiquid100 of PE-cfDNA. Results: Among the 39 PE-cfDNA samples successfully sequenced, 32 (82.1%) had a PE cell-block tumor content of < 10%. Standard tissue or cell-block testing for EGFR, ALK, and ROS1 identified 20 mutations (51.3%), whereas PE cfDNA identified 25 mutations (64.1%). Five EGFR mutations were observed in PE cfDNA but not in Cobas EGFR owing to coverage or insufficient tumor content issues. The overall rate of oncogenic mutations identified in the PE cfDNA was 92.3%, and the mutation distribution was as follows: even with a very low cfDNA input, high detection rates could be achieved. Otherwise, most patients harbored co-mutations. Comparison of pleural fluid NGS with traditional testing revealed differences in accuracy. We also followed up with patients with EGFR-sensitizing mutations who had a treatment response rate of 97.2% after 3 months. Conclusions: Genotyping of MPE supernatant cfDNA is feasible in clinical practice, in addition to plasma and tumor testing, to improve diagnostic yield and extend patients' benefit from targeted therapies. [ABSTRACT FROM AUTHOR]

A central issue in immunology is the immunological response against nonself. The prerequisite for a specific immunological response is the exposure to the immune system of a nonself antigen. Mathematical equations are presented, which define the fraction of all outcomes with a nonself allele in biallelic systems at the population level in pregnancy and transfusion/transplantation medicine. When designing assays, the mathematical descriptions can be used for estimating the number of genetic markers necessary to obtain a predetermined probability level in detecting nonself alleles of a given frequency. For instance, the equations can be helpful in the design of assays, where the nonself allele can be detected by analysis of cfDNA in plasma from pregnant women, to estimate fetal fraction or to monitor changes in cfDNA in plasma of transplantation patients. The equations give exact, quantitative descriptions of all nonself situations in pregnancy and transfusion/transplantation. [ABSTRACT FROM AUTHOR]

Background: Adverse pregnancy outcomes, which can be caused by multiple factors, present a significant threat to the health of mothers and their babies. Cell‐free fetal DNA (cffDNA) from placental trophoblast cells might be able to reflect placental and fetal status. Previous studies have yielded controversial results regarding the association of FF or cffDNA with various adverse pregnancy outcomes. A previous study has attempted to systematically assess the association between low fetal fraction (FF) and adverse pregnancy outcomes, but it failed to perform quantitative analyses due to the few studies included. In the present study, we attempted to quantitatively assess the association of FF (or cffDNA) with adverse pregnancy outcomes and further analyze the causes of heterogeneity. Objectives: To investigate the association of high/low FF or cffDNA with adverse pregnancy outcomes. Search Strategy: We searched the databases of PubMed, Embase, Cochrane, and Web of Science from January 1, 1990, to June 15, 2022 in this meta‐analysis. Selection Criteria: Studies on the relationships of adverse pregnancy outcomes in women with FF or cell free DNA were included. Non‐English literature was excluded. Data Collection and Analysis: Data about pregnancy outcomes and cell free DNA were extracted and meta‐analyzed. Subgroup analysis was performed by different outcomes. Main Results: There were 11 studies included involving 8280 participants. No significant heterogeneity was observed among the studies (I2 = 27%, 25%), and a fixed‐effect model was used for weighted quantitative analysis. The results revealed that the FF or cffDNA during pregnancy was significantly associated with adverse pregnancy outcomes in pregnant women (OR = 1.57, 95% CI [1.24, 1.99], P = 0.233). The overall incidence of the maternal adverse outcomes was 8% (95% CI: 5–13). Subgroup analysis of different outcomes showed an evident association between low FF or cffDNA and hypertensive disorders of pregnancy (HDP) (OR = 1.76, 95% CI [1.36, 2.27], P = 0.581). There was no evidence that the occurrence of spontaneous preterm birth (sPTB) and placental abnormality was associated with FF or cffDNA. No association was observed between low FF or cffDNA during pregnancy and adverse outcomes in fetuses (OR = 1.39, 95% CI [0.99, 1.94], P = 0.242). The overall incidence of adverse outcomes in fetuses was 8% (95% CI: 6–11). There were controversies over the association between high FF or cffDNA and HDP, and sPTB and small for gestational age infant, among different studies. Conclusions: Pregnant women with low FF or cffDNA during the first or second trimester of pregnancy have an overall increased risk of adverse pregnancy outcomes, especially HDP. However, the association between FF and various pregnancy outcomes needs to be further explored by more prospective studies. Synopsis: Pregnant women with low FF or cffDNA have an overall increased risk of adverse pregnancy outcomes, especially HDP. [ABSTRACT FROM AUTHOR]

Early cancer diagnosis from bisulfite‐treated cell‐free DNA (cfDNA) fragments requires tedious data analytical procedures. Here, we present a deep‐learning‐based approach for early cancer interception and diagnosis (DECIDIA) that can achieve accurate cancer diagnosis exclusively from bisulfite‐treated cfDNA sequencing fragments. DECIDIA relies on transformer‐based representation learning of DNA fragments and weakly supervised multiple‐instance learning for classification. We systematically evaluate the performance of DECIDIA for cancer diagnosis and cancer type prediction on a curated dataset of 5389 samples that consist of colorectal cancer (CRC; n = 1574), hepatocellular cell carcinoma (HCC; n = 1181), lung cancer (n = 654), and non‐cancer control (n = 1980). DECIDIA achieved an area under the receiver operating curve (AUROC) of 0.980 (95% CI, 0.976–0.984) in 10‐fold cross‐validation settings on the CRC dataset by differentiating cancer patients from cancer‐free controls, outperforming benchmarked methods that are based on methylation intensities. Noticeably, DECIDIA achieved an AUROC of 0.910 (95% CI, 0.896–0.924) on the externally independent HCC testing set in distinguishing HCC patients from cancer‐free controls, although there was no HCC data used in model development. In the settings of cancer‐type classification, we observed that DECIDIA achieved a micro‐average AUROC of 0.963 (95% CI, 0.960–0.966) and an overall accuracy of 82.8% (95% CI, 81.8–83.9). In addition, we distilled four sequence signatures from the raw sequencing reads that exhibited differential patterns in cancer versus control and among different cancer types. Our approach represents a new paradigm towards eliminating the tedious data analytical procedures for liquid biopsy that uses bisulfite‐treated cfDNA methylome. [ABSTRACT FROM AUTHOR]

The emergence of targeted therapies has transformed ovarian cancer treatment. However, biomarker profiling for precision medicine is limited by access to quality, tumour‐enriched tissue samples. The use of cell‐free DNA (cfDNA) in ascites presents a potential solution to this challenge. In this study, next‐generation sequencing was performed on ascites‐derived cfDNA samples (26 samples from 15 human participants with ovarian cancer), with matched DNA from ascites‐derived tumour cells (n = 5) and archived formalin‐fixed paraffin‐embedded (FFPE) tissue (n = 5). Similar tumour purity and variant detection were achieved with cfDNA compared to FFPE and ascites cell DNA. Analysis of large‐scale genomic alterations, loss of heterozygosity and tumour mutation burden identified six cases of high genomic instability (including four with pathogenic BRCA1 and BRCA2 mutations). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Ascites cfDNA identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. This advocates for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a systemic autoimmune disease primarily characterized by erosive and symmetric polyarthritis. As a pivotal axis in the regulation of type I interferon (IFN-I) and innate immunity, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway has been implicated in the pathogenesis of RA. This pathway mainly functions by regulating cell survival, pyroptosis, migration, and invasion. Therefore, understanding the sources of cell-free DNA and the mechanisms underlying the activation and regulation of cGAS-STING signaling in RA offers a promising avenue for targeted therapies. Early detection and interventions targeting the cGAS-STING signaling are important for reducing the medical burden on individuals and healthcare systems. Herein, we review the existing literature pertaining to the role of cGAS-STING signaling in RA, and discuss current applications and future directions for targeting the cGAS-STING signaling in RA treatments. [ABSTRACT FROM AUTHOR]

Inflammation plays a pivotal role in the pathogenesis of primary and post-essential thrombocythemia or post-polycythemia vera myelofibrosis (MF) in close cooperation with the underlying molecular drivers. This inflammatory state is induced by a dynamic spectrum of inflammatory cytokines, although recent evidence points to the participation of additional soluble inflammatory mediators. Damage-associated molecular patterns (DAMPs) represent endogenous signals released upon cell death or damage which trigger a potent innate immune response. We assessed the contribution of two prototypical DAMPs, HMGB1 and S100A8/A9, to MF inflammation. Circulating HMGB1 and S100A8/A9 were elevated in MF patients in parallel to the degree of systemic inflammation and levels increased progressively during advanced disease stages. Patients with elevated DAMPs had higher frequency of adverse clinical features, such as anemia, and inferior survival, suggesting their contribution to disease progression. Monocytes, which are key players inMF inflammation, were identified as a source of S100A8/A9 but not HMGB1 release, while both DAMPs correlated with cell death parameters, such as serumLDH and cell-free DNA, indicating that passive release is an additional mechanism leading to increased DAMPs. HMGB1 and S100A8/A9 promote inflammation through binding to Toll-like receptor (TLR) 4, whereas the former also binds TLR2. Monocytes from MF patients were shown to be hyperactivated at baseline, as reflected by higher CD11b and tissue factor exposure and increased expression levels of proinflammatory cytokines IL-1b and IL-6. Patient monocytes showed preserved TLR4 and TLR2 expression and were able to mount normal or even exacerbated functional responses and cytokine upregulation following stimulation of TLR4 and TLR2. Elevated levels of endogenous TLR ligands HMGB1 and S100A8/A9 coupled to the finding of preserved or hyperreactive TLR-triggered responses indicate that DAMPs may promote monocyte activation and cytokine production in MF, fueling inflammation. Plasma IL-1b and IL-6 were elevated in MF and correlated with DAMPs levels, raising the possibility that DAMPs could contribute to cytokine generation in vivo. In conclusion, this study highlights that, in cooperation with classic proinflammatory cytokines, DAMPs represent additional inflammatory mediators that may participate in the generation of MF inflammatory state, potentially providing novel biomarkers of disease progression and new therapeutic targets. [ABSTRACT FROM AUTHOR]

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

Background: Biosampling studies in critically ill patients traditionally involve bedside collection of samples followed by local processing (ie. centrifugation, aliquotting, and freezing) and storage. However, community hospitals, which care for the majority of Canadian patients, often lack the infrastructure for local processing and storage of specimens. A potential solution is a "simplified" biosampling protocol whereby blood samples are collected at the bedside and then shipped to a central site for processing and storage. One potential limitation of this approach is that delayed processing may alter sample characteristics. Objective: To determine whether delays in blood sample processing affect the stability of cytokines (IL-6, TNF, IL-10, IFN-γ), chemokines (IL-8, IP-10, MCP-1, MCP-4, MIP-1α, MIP-1β), cell-free DNA (cfDNA) (released by dying cells), and blood clotting potential in human blood samples. Methods: Venous blood was collected into EDTA and citrate sample tubes and stored at room temperature (RT) or 4°C for progressive intervals up to 72 hours, prior to processing. Plasma cytokines and chemokines were quantified using single or multiplex immunoassays. cfDNA was measured using Picogreen DNA Quantification. Blood clotting potential was measured using a thrombin generation assay. Results: Blood samples were collected from 9 intensive care unit (ICU) patients and 7 healthy volunteers. Admission diagnoses for the ICU patients included sepsis, trauma, ruptured abdominal aortic aneurysm, intracranial hemorrhage, gastrointestinal bleed, and hyperkalemia. After pre-processing delays of up to 72 hours at RT or 4°C, no significant changes were observed in plasma cytokines, chemokines, cfDNA, or thrombin formation. Conclusions: Delayed sample processing for up to 72 hours at either RT or 4°C did not significantly affect cytokines, chemokines, cfDNA, or blood clotting potential in plasma samples from healthy volunteers and ICU patients. A "simplified" biosampling protocol is a feasible solution for conducting biosampling research at hospitals without local processing capacity. [ABSTRACT FROM AUTHOR]

Background: Accurate differentiation between malignant and benign pulmonary nodules, especially those measuring 5–10 mm in diameter, continues to pose a significant diagnostic challenge. This study introduces a novel, precise approach by integrating circulating cell-free DNA (cfDNA) methylation patterns, protein profiling, and computed tomography (CT) imaging features to enhance the classification of pulmonary nodules. Methods: Blood samples were collected from 419 participants diagnosed with pulmonary nodules ranging from 5 to 30 mm in size, before any disease-altering procedures such as treatment or surgical intervention. High-throughput bisulfite sequencing was used to conduct DNA methylation profiling, while protein profiling was performed utilizing the Olink proximity extension assay. The dataset was divided into a training set and an independent test set. The training set included 162 matched cases of benign and malignant nodules, balanced for sex and age. In contrast, the test set consisted of 46 benign and 49 malignant nodules. By effectively integrating both molecular (DNA methylation and protein profiling) and CT imaging parameters, a sophisticated deep learning-based classifier was developed to accurately distinguish between benign and malignant pulmonary nodules. Results: Our results demonstrate that the integrated model is both accurate and robust in distinguishing between benign and malignant pulmonary nodules. It achieved an AUC score 0.925 (sensitivity = 83.7%, specificity = 82.6%) in classifying test set. The performance of the integrated model was significantly higher than that of individual methylation (AUC = 0.799, P = 0.004), protein (AUC = 0.846, P = 0.009), and imaging models (AUC = 0.866, P = 0.01). Importantly, the integrated model achieved a higher AUC of 0.951 (sensitivity = 83.9%, specificity = 89.7%) in 5–10 mm small nodules. These results collectively confirm the accuracy and robustness of our model in detecting malignant nodules from benign ones. Conclusions: Our study presents a promising noninvasive approach to distinguish the malignancy of pulmonary nodules using multiple molecular and imaging features, which has the potential to assist in clinical decision-making. Trial registration: This study was registered on ClinicalTrials.gov on 01/01/2020 (NCT05432128). https://classic.clinicaltrials.gov/ct2/show/NCT05432128. [ABSTRACT FROM AUTHOR]

Monitoring the status of grafts and the occurrence of postoperative complications, such as rejection, is crucial for ensuring the success and long-term survival of organ transplants. Traditional histopathological examination, though effective, is an invasive procedure and poses risks of complications, making frequent use impractical. In recent years, graft-derived cell-free DNA (gd-cfDNA) has emerged as a promising non-invasive biomarker. It not only provides early warnings of rejection and other types of graft injury but also offers important information about the effectiveness of immunosuppressive therapy and prognosis. gd-cfDNA shows potential in the monitoring of organ transplants. The early, real-time information on graft injury provided by gd-cfDNA facilitates timely individualized treatment and improves patient outcomes. However, the progress of research on gd-cfDNA varies across different organs. Therefore, this article will comprehensively review the application and findings of gd-cfDNA in monitoring various solid organs, discussing the advantages, limitations, and some future research directions to aid in its clinical application. [ABSTRACT FROM AUTHOR]

We reported a performance during an implementation of prenatal cell-free (cf) DNA screening using single nucleotide polymorphism (SNP) approach in our accredited laboratory.Prospective audit with prompt intervention was set for the processing of 2,502 samples from singleton pregnancy from August 2017 to July 2019. Risks of trisomy 21 (T21), T18, T13, monosomy X (XO), and other sex chromosome aneuploidies (SCAs) were clarified by a proprietary bioinformatics algorithm.Laboratory failure occurred in 192 samples (7.7 %) as a result of inadequate sequencing (n=144), fundamental limitation of the testing (n=19), and obvious human error (n=29). Faulty setting of the calibration curve was the most common human error (n=22/29). After a redraw (n=110), 79 (71.8 %) were settled. Overall, 2,389/2,502 samples (95.5 %) were reportable. Thirty-five samples were high-risk for T21 (n=19), T18 (n=5), T13 (n=1), XO (n=3), and other SCAs (n=7), respectively. Positive predictive values calculated from either prenatal confirmatory tests or postnatal findings were 93.8 % (n=16), 100 % (n=4), 50 % (n=2), and 83.3 % (n=6) for T21, T18, XO, and other SCAs, respectively, with high sensitivity and specificity (>99.9 %). Vanishing twin was detected from 1 out of 4 samples with detected additional haplotypes.An overall performance of SNP-based prenatal cf-DNA screening during our initial implementation can be optimized with proactive approach. The technical know-how was a significant limiting factor for adopting the technology. The lessons learnt may be of interest to the academic laboratory considering adopting their own test instead of sending blood to a testing service of a supplier. [ABSTRACT FROM AUTHOR]

Background: Uveal melanoma (UM) is the most common primary intraocular tumour in adults, and approximately 50% of patients will develop metastasis. Epigenetic changes are a major factor in cancer progression. We aimed to determine whether methylation profiles could be altered using a DNA methyltransferase (DNMT) inhibitor in UM cell lines. Methods: Four primary and metastatic UM cell lines were treated with azacytidine and analysed for cell proliferation, colony formation, and BAP1 protein expression. Genomic and cell-free (cf)DNA methylation were compared. Results: In all cell lines, azacytidine treatment resulted in dose-dependent effects on proliferation, colony formation, and radiosensitivity. Methylation profiling revealed differences in methylation between cell lines according to BAP1 expression. Matched primary and metastatic cell lines showed very similar patterns. Alterations were seen in pathways known to be important in UM progression, such as PI3K/Akt and MAPK signaling, and in pathways involved in cancer progression, such as regulation of stemlike potential, cell motility, and invasion. These changes were maintained in genomic and cell-free DNA. Conclusions: This data suggests that DNMT inhibitors cause changes in UM cells that are maintained in cfDNA. The results suggest that targeting methylation in UM treatment and monitoring response to treatment using cfDNA methylation could be a valuable tool. [ABSTRACT FROM AUTHOR]

Aim: This study was aimed at comparing the positive predictive value of a high‐risk cell‐free fetal DNA test result for sex chromosome aneuploidies (45,X0, 47,XXX, 47,XXY, and 47,XYY) and autosomal trisomies (T21, T18, and T13) with confirmatory tests in singleton pregnancies. Additionally, we identify the main reason for discordant and inconclusive results. Methods: PubMed, Web of Science, and Scopus were searched from 2017 for primary research articles on cell‐free fetal DNA testing of autosomal trisomies and sex chromosome aneuploidies in singleton pregnancies. The methodological characteristics and the statistical results of the studies were collected, and the risk of bias was assessed. Results: Fourteen studies were included. Among the autosomal trisomies, T21 had the highest, whereas T13 showed the lowest positive predictive values. As for the sex chromosome aneuploidies, the lowest values were found with 45,X0. Although discordant and inconclusive results were reported inconsistently, false positives were mainly caused by mosaicism, and inconclusive results were mostly secondary to a low fetal DNA fraction. Conclusion: Cell‐free fetal DNA is a reliable screening tool for autosomal trisomies. It is also useful for sex chromosome aneuploidies, although the positive predictive values are lower. A positive screening result should be followed with a confirmatory test. [ABSTRACT FROM AUTHOR]

Circulating cell-free DNA (cfDNA) assays for monitoring individuals with cancer typically rely on prior identification of tumor-specific mutations. Here, we develop a tumor-independent and mutation-independent approach (DELFI-tumor fraction, DELFI-TF) using low-coverage whole genome sequencing to determine the cfDNA tumor fraction and validate the method in two independent cohorts of patients with colorectal or lung cancer. DELFI-TF scores strongly correlate with circulating tumor DNA levels (ctDNA) (r = 0.90, p < 0.0001, Pearson correlation) even in cases where mutations are undetectable. DELFI-TF scores prior to therapy initiation are associated with clinical response and are independent predictors of overall survival (HR = 9.84, 95% CI = 1.72-56.10, p < 0.0001). Patients with lower DELFI-TF scores during treatment have longer overall survival (62.8 vs 29.1 months, HR = 3.12, 95% CI 1.62-6.00, p < 0.001) and the approach predicts clinical outcomes more accurately than imaging. These results demonstrate the potential of using cfDNA fragmentomes to estimate tumor burden in cfDNA for treatment response monitoring and clinical outcome prediction. Circulating cell-free DNA (cfDNA) assays for monitoring individuals with cancer typically use tumor-specific mutations that may not be detected in many patients. Here, the authors develop a tumor-independent and mutation-independent approach (DELFI-TF) to estimate tumor burden in cfDNA for treatment response monitoring and clinical outcome prediction. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer is a public health issue and was the third leading cause of cancer-related death worldwide in 2022. Early diagnosis can improve prognosis, making screening a central part of colorectal cancer management. Blood-based screening, diagnosis and follow-up of colorectal cancer patients are possible with the study of cell-free circulating tumor DNA. This study aimed to identify novel DNA methylation biomarkers of colorectal cancer that can be used for the follow-up of patients with colorectal cancer. Methods: A DNA methylation profile was established in the Gene Expression Omnibus (GEO) database (n = 507) using bioinformatics analysis and subsequently confirmed using The Cancer Genome Atlas (TCGA) database (n = 348). The in silico profile was then validated on local tissue and cell-free DNA samples using methylation-specific digital PCR in colorectal cancer patients (n = 35) and healthy donors (n = 35). Results: The DNA methylation of COL25A1 and METAP1D was predicted to be a colorectal cancer biomarker by bioinformatics analysis (ROC AUC = 1, 95% CI [0.999–1]). The two biomarkers were confirmed with tissue samples, and the combination of COL25A1 and METAP1D yielded 49% sensitivity and 100% specificity for cell-free DNA. Conclusion: Bioinformatics analysis of public databases revealed COL25A1 and METAP1D DNA methylation as clinically applicable liquid biopsies DNA methylation biomarkers. The specificity implies an excellent positive predictive value for follow-up, and the high sensitivity and relative noninvasiveness of a blood-based test make these biomarkers compatible with colorectal cancer screening. However, the clinical impact of these biomarkers in colorectal cancer screening and follow-up needs to be established in further prospective studies. [ABSTRACT FROM AUTHOR]

High heterogeneity in clinical benefit characterizes the use of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC). We prospectively enrolled 113 advanced NSCLC patients treated with ICIs and performed liquid biopsy at the time of ICI start (T1), after 3 weeks (T2) and at the time of radiological evaluation (T3). Molecular variables were associated with outcome endpoints: cfDNA quantification, its dynamic change (∆T2–T1), variant allele frequency (VAF) of the gene with the highest frequency detected at baseline with NGS (maxVAF) and its dynamic change (∆T2–T1). At multivariate analysis, PD-L1 negativity, T1 cfDNA, cfDNA increase (∆T2–T1), and T2 VAF were significantly associated with shorter progression-free survival (PFS); PD-L1 negativity, squamous histology, T1 cfDNA, cfDNA (∆T2–T1) increase, and T2 maxVAF affected overall survival (OS). Among high PD-L1 expressing patients treated in first-line, elevated T2 maxVAF and cfDNA increase (∆T2–T1) correlated with worse PFS; higher T2 maxVAF and cfDNA increase (∆T2–T1) with worse OS. Derived integrated models were used to build nomograms and categorize different risk groups. [ABSTRACT FROM AUTHOR]

Background/Objectives: Currently, the prediction of disease recurrence after radical prostatectomy (RP) in localized prostate cancer (PCa) relies on clinicopathological parameters, which lack accuracy in predicting clinical outcomes. This study focused on evaluating the utility of cfDNA levels and fragmentation patterns as prognostic biomarkers in progressive prostate-specific antigen (PSA) patients, including those with persistent PSA and biochemical recurrence (BR), after primary treatment in localized PCa patients. Methods: Twenty-nine high-risk localized PCa patients were enrolled in the study between February 2022 and May 2023. Blood samples were obtained before robotic RP. cfDNA concentration and fragment size were quantified using the Quant-it PicoGreen dsDNA Assay kit and Agilent 2200 TapeStation System, respectively. Results: The mean PSA value at diagnosis was 9.4 ng/mL. Seven patients (24.1%) had stage pT2 and 22 (75.9%) pT3. Nine patients (31%) had detectable PSA at the first PSA control six weeks after surgery, and four patients (20%) had BR during a mean follow-up of 18.4 months. No associations were found between cfDNA levels or fragmentation patterns and clinicopathological data. Although not statistically significant, patients with detectable PSA levels post-surgery exhibited higher cfDNA levels and shorter fragments compared with those with undetectable PSA. Conclusions: Our study indicated a tendency toward more fragmented cfDNA levels in PCa patients with persistent PSA. Strikingly, biochemical recurrent PCa patients exhibited similar cfDNA levels and fragmentation patterns compared to non-recurrent patients. Further studies exploring liquid biopsy-derived biomarkers in localized PCa patients are needed to elucidate their clinical utility in predicting PSA persistence. [ABSTRACT FROM AUTHOR]

Background: The role of epigenetics in cardiovascular diseases has paved the way for innovative therapeutic approaches. Investigating epigenetic changes using cell-free DNA (cfDNA) holds substantial promise beyond mere diagnostics, especially for heart-related conditions like acute myocardial infarction (AMI), where obtaining tissue samples is a challenge. This study explores the methylation patterns of cfDNA in AMI patients and compares them with genomic DNA (gDNA) from the same individuals, aiming to evaluate the effectiveness of cfDNA as a valuable resource for studying heart-related diseases. Methodology: We generated global methylome profiles of cfDNA and gDNA from 25 AMI patients using EM-Seq. Tissue deconvolution analysis was performed to estimate tissue specificity based on the methylation patterns. Differentially methylated loci were identified and explored to understand AMI pathophysiology. Results: Comparative analysis of cfDNA and gDNA methylation patterns in AMI patients reveals cfDNA holds more significance than gDNA. Principal component analysis revealed distinct clusters for cfDNA and gDNA, indicating distinct methylome profiles. cfDNA originated from multiple sources, predominantly from neutrophils (~ 75%) and about 10% from the left atrium, highlighting cardiac-specific changes. In contrast, immune cells are the major source of gDNA, indicative of inflammatory responses. Gene set enrichment analysis (GSEA) associates cfDNA methylation patterns with pathways related to cardiac muscle contraction, inflammation, hypoxia, and lipid metabolism. The affected genes include G protein-coupled receptors (GHSR, FFAR2, HTR1A, and VIPR2) that are part of the cAMP signaling pathway. Conclusion: Epigenetic changes in cfDNA are more specific to cardiac tissue compared to those in gDNA, providing better insights into the molecular mechanisms involved in AMI. Genes that are differentially methylated in cfDNA and regulate core pathways, such as cAMP signaling, could be targeted for clinical applications, including the development of effective biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

Background Early diagnosis of invasive fungal disease is essential for optimizing management. Although the clinical utility of fungal polymerase chain reaction (PCR) testing on plasma and bronchoalveolar lavage (BAL) has been established, the role of follow-up testing remains unclear. Methods This was a retrospective single-center study. The yield of follow-up PCR for Aspergillus species, Mucorales agents, Fusarium species, Scedosporium species, dimorphic fungi, Pneumocystis jirovecii , and Candida species on plasma and/or BAL was measured at intervals of 1, 2, 3, and 4 weeks following a negative result. Results A total of 1389 follow-up tests on 406 plasma specimens from 264 patients and 983 BAL specimens from 431 patients were evaluated. Overall, the positivity rate at 1, 2, 3, and 4 weeks was 2.7% (4/148), 3.3% (4/123), 5.1% (4/78), and 3.5% (2/57), respectively, on plasma, and 0% (0/333), 0.3% (1/288), 0.4% (1/228), and 0.7% (1/134), respectively, on BAL. Conversions occurred with Aspergillus species, Mucorales agents, and Fusarium species PCR on plasma and Aspergillus species and P jirovecii PCR on BAL. All patients who converted were immunocompromised. Within 1 week of a prior negative test, 2 Aspergillus and 2 Mucorales PCRs were positive on plasma, and zero tests were positive on BAL. In week 1, only 1 Aspergillus species that was positive on day 7 was classified as probable fungal disease. Conclusions Fungal PCR follow-up testing on plasma and BAL within 4 weeks of a prior negative result was of low yield and rarely generated a positive result considered clinically significant in the first week. [ABSTRACT FROM AUTHOR]

Background: Recent studies have highlighted the importance of the cell-free DNA (cfDNA) methylation profile in detecting breast cancer (BC) and its different subtypes. We investigated whether plasma cfDNA methylation, using cell-free Methylated DNA Immunoprecipitation and High-Throughput Sequencing (cfMeDIP-seq), may be informative in characterizing breast cancer in patients with BRCA1/2 germline mutations for early cancer detection and response to therapy. Methods: We enrolled 23 BC patients with germline mutation of BRCA1 and BRCA2 genes, 19 healthy controls without BRCA1/2 mutation, and two healthy individuals who carried BRCA1/2 mutations. Blood samples were collected for all study subjects at the diagnosis, and plasma was isolated by centrifugation. Cell-free DNA was extracted from 1 mL of plasma, and cfMeDIP-seq was performed for each sample. Shallow whole genome sequencing was performed on the immuno-precipitated samples. Then, the differentially methylated 300-bp regions (DMRs) between 25 BRCA germline mutation carriers and 19 non-carriers were identified. DMRs were compared with tumor-specific regions from public datasets to perform an unbiased analysis. Finally, two statistical classifiers were trained based on the GLMnet and random forest model to evaluate if the identified DMRs could discriminate BRCA-positive from healthy samples. Results: We identified 7,095 hypermethylated and 212 hypomethylated regions in 25 BRCA germline mutation carriers compared to 19 controls. These regions discriminate tumors from healthy samples with high accuracy and sensitivity. We show that the circulating tumor DNA of BRCA1/2 mutant breast cancers is characterized by the hypomethylation of genes involved in DNA repair and cell cycle. We uncovered the TFs associated with these DRMs and identified that proteins of the Erythroblast Transformation Specific (ETS) family are particularly active in the hypermethylated regions. Finally, we assessed that these regions could discriminate between BRCA positives from healthy samples with an AUC of 0.95, a sensitivity of 88%, and a specificity of 94.74%. Conclusions: Our study emphasizes the importance of tumor cell-derived DNA methylation in BC, reporting a different methylation profile between patients carrying mutations in BRCA1, BRCA2, and wild-type controls. Our minimally invasive approach could allow early cancer diagnosis, assessment of minimal residual disease, and monitoring of response to therapy. [ABSTRACT FROM AUTHOR]

Background: Liquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release. Methods: We measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release. Results: Higher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding. Conclusions: Methylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays. [ABSTRACT FROM AUTHOR]

Inflammation plays a pivotal role in the pathogenesis of primary and post-essential thrombocythemia or post-polycythemia vera myelofibrosis (MF) in close cooperation with the underlying molecular drivers. This inflammatory state is induced by a dynamic spectrum of inflammatory cytokines, although recent evidence points to the participation of additional soluble inflammatory mediators. Damage-associated molecular patterns (DAMPs) represent endogenous signals released upon cell death or damage which trigger a potent innate immune response. We assessed the contribution of two prototypical DAMPs, HMGB1 and S100A8/A9, to MF inflammation. Circulating HMGB1 and S100A8/A9 were elevated in MF patients in parallel to the degree of systemic inflammation and levels increased progressively during advanced disease stages. Patients with elevated DAMPs had higher frequency of adverse clinical features, such as anemia, and inferior survival, suggesting their contribution to disease progression. Monocytes, which are key players inMF inflammation, were identified as a source of S100A8/A9 but not HMGB1 release, while both DAMPs correlated with cell death parameters, such as serumLDH and cell-free DNA, indicating that passive release is an additional mechanism leading to increased DAMPs. HMGB1 and S100A8/A9 promote inflammation through binding to Toll-like receptor (TLR) 4, whereas the former also binds TLR2. Monocytes from MF patients were shown to be hyperactivated at baseline, as reflected by higher CD11β and tissue factor exposure and increased expression levels of proinflammatory cytokines IL-1β and IL-6. Patient monocytes showed preserved TLR4 and TLR2 expression and were able to mount normal or even exacerbated functional responses and cytokine upregulation following stimulation of TLR4 and TLR2. Elevated levels of endogenous TLR ligands HMGB1 and S100A8/A9 coupled to the finding of preserved or hyperreactive TLR-triggered responses indicate that DAMPs may promote monocyte activation and cytokine production in MF, fueling inflammation. Plasma IL-1β and IL-6 were elevated in MF and correlated with DAMPs levels, raising the possibility that DAMPs could contribute to cytokine generation in vivo. In conclusion, this study highlights that, in cooperation with classic proinflammatory cytokines, DAMPs represent additional inflammatory mediators that may participate in the generation of MF inflammatory state, potentially providing novel biomarkers of disease progression and new therapeutic targets. [ABSTRACT FROM AUTHOR]

Background: A blood-based diagnostic test is a promising strategy for colorectal cancer (CRC). The MethyDT test (IColohunter), which detects methylation levels of NTMT1 and MAP3K14-AS1, exhibited potential in discriminating CRC, but its clinical performance needs to be validated in large-scale populations. Methods: A multicenter, double-blinded, cross-sectional study that enrolled 1194 participants was performed. Plasma samples were collected to detect methylation levels of NTMT1 and MAP3K14-AS1 using quantitative methylation-specific PCR with the MethyDT test, and the accuracy was further evaluated by Sanger sequencing. Results: The sensitivities of the MethyDT test for detecting CRC, early stages of CRC (I and II), advanced adenoma (AA), and high-grade intraepithelial neoplasia (HGIN) were 91.2% (95% confidence interval [CI], 88.4–94.0), 87.4% (95% CI, 82.5–92.2), 43.5% (95% CI, 35.7–51.4), and 72.7% (95% CI, 57.5–87.9), respectively. The specificities for participants with non-AA, interfering diseases (ID), and no evidence of disease (NED) were 85.0% (95% CI, 78.8–91.3), 93.7% (95% CI, 91.4–95.9) and 97.3% (95% CI, 90.5–99.7), respectively, and its overall specificity for all-controls was 92.4% (95% CI, 90.3–94.4). The consistency of the MethyDT test with pathology for CRC was high with a kappa value of 0.830 (95% CI, 0.795–0.865). Additionally, the MethyDT test was comparable to Sanger sequencing for detecting methylation with kappa values > 0.97. Conclusions: The MethyDT test demonstrates excellent sensitivity and specificity for CRC and high consistency with Sanger sequencing for methylation, suggesting it may serve as a potential noninvasive diagnostic tool for the detection of CRC. Trial registration: This clinical trial has been registered in ClinicalTrials.gov (NCT05508503). [ABSTRACT FROM AUTHOR]

Background: The mortality rate of COVID-19 patients with critical symptoms is reported to be 40.5%. Early identification of patients with poor progression in the critical cohort is essential to timely clinical intervention and reduction of mortality. Although older age, chronic diseases, have been recognized as risk factors for COVID-19 mortality, we still lack an accurate prediction method for every patient. This study aimed to delve into the cell-free DNA fragmentomics of critically ill patients, and develop new promising biomarkers for identifying the patients with high mortality risk. Methods: We utilized whole genome sequencing on the plasma cell-free DNA (cfDNA) from 33 COVID-19 patients with critical symptoms, whose outcomes were classified as survival (n = 16) and death (n = 17). Mitochondrial DNA (mtDNA) abundance and fragmentomic properties of cfDNA, including size profiles, ends motif and promoter coverages were interrogated and compared between survival and death groups. Results: Significantly decreased abundance (~ 76% reduction) and dramatically shorter fragment size of cell-free mtDNA were observed in deceased patients. Likewise, the deceased patients exhibited distinct end-motif patterns of cfDNA with an enhanced preference for "CC" started motifs, which are related to the activity of nuclease DNASE1L3. Several dysregulated genes involved in the COVID-19 progression-related pathways were further inferred from promoter coverages. These informative cfDNA features enabled a high PPV of 83.3% in predicting deceased patients in the critical cohort. Conclusion: The dysregulated biological processes observed in COVID-19 patients with fatal outcomes may contribute to abnormal release and modifications of plasma cfDNA. Our findings provided the feasibility of plasma cfDNA as a promising biomarker in the prognosis prediction in critically ill COVID-19 patients in clinical practice. [ABSTRACT FROM AUTHOR]

Cancer is a prominent global cause of mortality, primarily due to delayed detection leading to limited treatment options. Current screening methods are mostly invasive and involve complex lengthy processes with high costs. Moreover, each screening typically focuses on a single type of cancer. This imposes a growing need for innovative, precise, and minimally invasive methods for early cancer detection. With the current advances in assay technologies and data science, multi-cancer early detection (MCED) tests are gaining increased interest in the research community as they offer potential for earlier diagnosis and improved patient outcomes. Different approaches are followed for MCED, and multiple machine learning methods are considered. In this paper, we systematically explore various MCED studies and their applied machine learning (ML) models for different types of biomarker data. We discuss the strengths and limitations of different study designs and compare their performance. Future directions are proposed, emphasizing the importance of integrating multi-omics data, enhancing model transparency, and fostering collaborative efforts to develop robust, cost effective and clinically applicable MCED tools. [ABSTRACT FROM AUTHOR]

Background: Arising in the late 1990s, when a promising role in prenatal diagnostics was first delineated for circulating fetal DNA, non-invasive prenatal tests (NIPTs) have been increasingly used with more frequency and popularity. These exams have been used as a prenatal screening tests for genetic diseases. Initially, they were developed for the investigation of the main fetal chromosomal aneuploidies, but lately they have also been used to rule out genomic microrearrangements and monogenic conditions. However, along with great opportunities and potential, the tests can show inconclusive or unexpected results. Several studies have shown that the current pre-test counseling is often insufficient, and more oriented at providing pieces of information about the identifiable diseases rather than providing extensive information on all possible scenarios which may affect both the fetus and the pregnant mother, especially in the case of an invasive test for the pregnant mother. Methods and Results: We have gathered from the literature on NIPT the main pitfalls, imperfections, and particular cases associated with this innovative diagnostic procedure. Conclusions: In view of further improvements in the methods that can limit the inconclusive or unexpected results, this paper aims to reinforce the importance of more accurate pre-test counseling with comprehensive information about the above-mentioned questions, as well as ultrasound use and also the creation of an international consensus statement concerning these topics. [ABSTRACT FROM AUTHOR]

Objective: Cell‐free DNA (cfDNA) is used as a biomarker after transplantation to detect graft injury, relying on the donor fraction (DF). We have established a PCR‐based approach allowing us to separately quantify absolute values of dd‐cfDNA and recipient‐derived cfDNA (rd‐cfDNA). We aimed to present typical clinical scenarios after heart transplantation (HTx) to illustrate the advantages of absolute cfDNA values over DF. Methods: We used the cfDNA results of our cohort (509 samples of 52 patients followed during the first year after HTx) as background and determined the trajectories of cfDNA in specific clinical situations. We profiled an uncomplicated clinical course, viral and bacterial infections, acute and chronic rejection, and false‐negative and false‐positive rejections in six patients (five adults, one child). Results: There was a substantial discrepancy between relative (DF) and absolute cfDNA‐levels in several clinical situations. Rd‐ and dd‐cfDNA were independently elevated during episodes of rejection and infection and were better suited to depict treatment response than DF alone. Conclusions: Absolute quantification of cfDNA may offer clinically relevant information additive to DF in various situations after HTx and could be helpful for more accurate monitoring of diagnosis and treatment of rejection. [ABSTRACT FROM AUTHOR]

The article in the Clinical & Translational Medicine journal discusses the importance of detecting molecular residual disease (MRD) using circulating tumor DNA (ctDNA) in postoperative colorectal cancer patients. The study included 104 patients and found that positive MRD status was associated with a higher risk of recurrence. Genetic markers like KRAS, APC, and FGFR2 variants in ctDNA were linked to inferior disease-free survival. Integrating cfDNA_KRAS variants with MRD improved risk stratification precision, and mutation sites were found to be enriched in shorter cfDNA fragments. The study highlights the potential of ctDNA-based MRD monitoring for better disease control and survival rates in colorectal cancer patients. [Extracted from the article]

The article discusses the importance of adjusting performance parameters in evaluating diagnostic methods to account for sample size. Existing performance parameters like accuracy, sensitivity, and specificity may be misleading when sample size is not considered. Adjusted performance parameters, which incorporate the estimated performance and uncertainty, provide a more reliable evaluation of a method's performance, especially in studies with small sample sizes. The proposed adjustment allows for a better comparison of different methods and a more realistic assessment of method performance, particularly in cases of 100% accuracy. [Extracted from the article]

Background: Intense exercise leads to neutrophil extracellular traps (NETs) formation, which triggers cell disintegration. NET, as well as other processes of apoptosis, necrosis, and spontaneous secretion, result in increased levels of cell-free DNA (cf-DNA) in the circulation. An increment of cf-DNA is also observed in autoimmune diseases, such as type 1 diabetes mellitus (T1DM). Repeated exhaustive exercises are an impulse for physiological adaptation; therefore, in this case–control study, we compared the exercise-induced increase in cf-DNA in men with T1DM and healthy controls to determine the development of the tolerance to exercise. Methods: Volunteers performed a treadmill run to exhaustion at a speed matching 70% of their personal VO2 max at three consecutive visits, separated by a 72 h resting period. Blood was collected before and after exercise for the determination of plasma cell-free nuclear and mitochondrial DNA (cf n-DNA, cf mt-DNA) by real-time PCR, blood cell count and metabolic markers. Results: Each bout of exhaustive exercise induced a great elevation of cf n-DNA levels. An increase in cf mt-DNA was observed after each run. However, the significance of the increase was noted only after the second bout in T1DM participants (p < 0.02). Changes in cf-DNA concentration were transient and returned to baseline values during 72 h of resting. The exercise-induced increment in circulating cf n-DNA and cf mt-DNA was not significantly different between the studied groups (p > 0.05). Conclusions: Cf-DNA appears to be a sensitive marker of inflammation, with a lower post-exercise increase in individuals with T1DM than in healthy men. [ABSTRACT FROM AUTHOR]