OBJECTIVE: To evaluate the diagnostic value of 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA) for nasopharyngeal carcinoma (NPC) and to develop a diagnostic model.
METHODS: Genome-wide 5hmC profiles in cfDNA from 174 NPC patients and 146 non-cancer individuals were analyzed using the 5hmC-Seal technique. A cfDNA 5hmC-based diagnostic model to identify NPC patients was developed using least absolute shrinkage and selection operator (LASSO) logistic regression, and performance was evaluated with receiver operating characteristic (ROC) curves and confusion matrices.
RESULTS: The 5hmC-Seal data from patients with NPC showed a different genome-wide distribution than non-tumor samples. Our initial analysis revealed a 12-gene-based 5hmC marker panel to be an accurate diagnostic model effectively distinguishing between NPC samples and non-cancerous samples (training set: area under curve (AUC)= 0.97 [95 % CI: 0.94-0.99]; and test set: AUC= 0.93 [95 % CI: 0.88-0.98]) superior to EBV DNA testing. The diagnostic score performed well in differentiating the non-cancer subjects from early-stage NPC (training set: AUC=0.99 [95 % CI: 0.98-1]; test set: AUC=0.98 [95 % CI: 0.95-1]), and advanced-stage NPC (training set: AUC=0.96 [95 % CI: 0.93-0.99]; test set: AUC=0.93 [95 % CI: 0.88-0.98]). Notably, in EBV-negative patients, the diagnostic scores showed excellent capacity for distinguishing EBV-negative patients with NPC from non-cancer subjects in both the training set (AUC= 0.94 [95 % CI: 0.88-1]) and test set (AUC=0.91 [95 % CI: 0.81-1]).
CONCLUSIONS: 5hmC modifications in cfDNA are promising noninvasive biomarkers for NPC, offering high sensitivity and specificity, particularly for early-stage and EBV-negative NPC.

This study was conducted with the primary objective of assessing the performance of cfDNA methylation in the detection of colorectal cancer (CRC). Five tumor tissue, 20 peripheral blood leucocyte, and 169 cfDNA samples were collected for whole-genome bisulfite sequencing (WGBS) analysis. Bioinformatic analysis was conducted to identify differentially methylated regions (DMRs) and their functional characteristics. Quantitative methylation-specific PCR (qMSP) was used to validate the methylation levels of DMRs in the tissues and leucocytes. cfDNA samples from CRC patients and healthy controls were used to evaluate the performance of the DMR analysis. WGBS analysis revealed a decrease in DNA methylation levels in the CpG context in CRC tumor tissues compared with adjacent normal tissues. A total of 132 DMRs in cfDNA were identified as potential markers for diagnosing CRC. In a cohort of 95 CRC patients and 74 healthy controls, a combination of the three DMRs (DAB1, PPP2R5C, and FAM19A5) yielded an AUC of 0.763, achieving 64.21% sensitivity and 78.38% specificity in discriminating CRC patients from healthy controls. This study provides insights into DNA methylation patterns in CRC and identifies a set of DMRs in cfDNA with potential diagnostic value for CRC. These DMRs hold promise as biomarkers for CRC detection, offering promise for non-invasive CRC diagnosis. Further research is warranted to validate these findings in larger cohorts.

OBJECTIVE: Uterine fibroids are monoclonal tumors, which are often genetically abnormal and associated with false-positive genome-wide cell-free DNA (cfDNA) screening results, particularly when large. It is plausible that fibroids may also increase the risk of cfDNA failure by affecting fetal fraction or due to their genetic anomalies confounding cfDNA algorithms. We aimed to investigate a possible association between fibroids and cfDNA non-informative results.
METHODS: This was a retrospective cohort study of women undergoing cfDNA screening for fetal chromosomal abnormalities between 2013 and 2020, comparing pregnancies with vs without uterine fibroids recorded on any obstetric ultrasound before 24 weeks\' gestation. Univariable and multivariable logistic regression models were used to investigate the association between fibroids and cfDNA failure, adjusting for gestational age, maternal age, weight and height at blood sampling, mode of conception, multiple gestation and test platform (chromosome-selective or genome-wide). Analyses were stratified according to the number of fibroids and total fibroid volume. The impact of fibroids on fetal fraction was assessed using linear regression, adjusting for the same covariates.
RESULTS: Among 19 818 pregnancies undergoing cfDNA screening, fibroids were reported in 2038 (10.28%) and cfDNA failure at the first screening attempt occurred in 228 (1.15%) pregnancies. Non-informative results occurred in 1.96% of pregnancies with fibroids and 1.06% of pregnancies without fibroids (adjusted odds ratio (aOR), 2.40 (95% CI, 1.65-3.48)). The risk of failure in the first screening attempt increased progressively with the number of fibroids (aOR, 5.05 (95% CI, 2.29-11.13) in women with four or more fibroids) and total fibroid volume, with greater than a 5-fold and 14-fold increase in risk among women with fibroid volumes of 100.1-400 mL (aOR, 5.52 (95% CI, 2.30-13.25)) and > 400 mL (aOR, 14.80 (95% CI, 4.50-48.69)), respectively. Although test failure was more common with chromosome-selective than genome-wide screening, fibroids similarly increased the risk of failure of both screening platforms. Compared to pregnancies without fibroids, those with fibroids had a fetal fraction on average 0.61% lower (adjusted mean difference, -0.61% (95% CI, -0.77% to -0.45%)).
CONCLUSIONS: Uterine fibroids are associated with lower fetal fraction and an increased risk of cfDNA screening failure. The strength of this association increases with increasing fibroid number and volume. © 2024 The Author(s). Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

UNASSIGNED: The purpose of this study was to investigate the potential of plasma cfDNA methylation patterns in reflecting tumour methylation changes, focusing on three candidate sites, cg02469161, cg11528914, and cg20131654. These sites were selected for verification, with a particular emphasis on their association with breast cancer.
UNASSIGNED: We conducted a comprehensive analysis of 850k whole-methylation sequencing data to identify potential markers for breast cancer detection. Subsequently, we investigated the methylation status of the genes Ran-binding protein 3 (RANBP3), Lymphocyte cytoplasmic protein 2 (LCP2), and GRB2 related adaptor protein 2 (GRAP2), situated at the specified sites, using cancer and canceradjacent tissues from 17 breast cancer patients. We also examined the methylation patterns in different molecular subtypes and pathological grades of breast cancer. Additionally, we compared the methylation levels of these genes in plasma cfDNA to their performance in tissues.
UNASSIGNED: Our analysis revealed that RANBP3, LCP2, and GRAP2 genes exhibited significant methylation differences between cancer and cancer-adjacent tissues. In breast cancer, these genes displayed diagnostic efficiencies of 91.0%, 90.6%, and 92.2%, respectively. Notably, RANBP3 showed a tendency towards lower methylation in HR+ breast cancer, and LCP2 methylation was correlated with tumour malignancy. Importantly, the methylation levels of these three genes in plasma cfDNA closely mirrored their tissue counterparts, with diagnostic efficiencies of 83.3%, 83.9%, and 77.6% for RANBP3, LCP2, and GRAP2, respectively.
UNASSIGNED: Our findings propose that the genes RANBP3, LCP2, and GRAP2, located at the identified methylation sites, hold significant potential as molecular markers in blood for the supplementary diagnosis of breast cancer. This study lays the groundwork for a more in-depth investigation into the changes in gene methylation patterns in circulating free DNA (cfDNA) for the early detection not only of breast cancer but also for various other types of cancer.
UNASSIGNED: Svrha ove studije je bila da se istraži potencijal obrazaca metilacije cfDNK plazme u odrazu promena metilacije tumora, fokusirajući se na tri kandidata, cg02469161, cg11528914 i cg20131654. Ove lokacije su odabrane za verifikaciju, sa posebnim naglaskom na njihovu povezanost sa rakom dojke.
UNASSIGNED: Sproveli smo sveobuhvatnu analizu 850.000 podataka sekvenciranja cele metilacije da bismo identifikovali potencijalne markere za otkrivanje raka dojke. Nakon toga, istražili smo status metilacije gena Ran-vezujući protein 3 (RANBP3), limfocitni citoplazmatski protein 2 (LCP2) i GRB2 povezan adapter protein 2 (GRAP2), koji se nalaze na navedenim mestima, koristeći kancer i susedna tkiva raka od 17 pacijenata obolelih od raka dojke. Takođe smo ispitali obrasce metilacije kod različitih molekularnih podtipova i patoloških stepena raka dojke. Pored toga, uporedili smo nivoe metilacije ovih gena u cfDNK plazme sa njihovim performansama u tkivima.
UNASSIGNED: Naša analiza je otkrila da geni RANBP3, LCP2 i GRAP2 pokazuju značajne razlike u metilaciji između raka i tkiva u blizini raka. Kod raka dojke, ovi geni su pokazali dijagnostičku efikasnost od 91,0%, 90,6% i 92,2%, respektivno. Značajno je da je RANBP3 pokazao tendenciju ka nižoj metilaciji kod HR+ raka dojke, a metilacija LCP2 bila je u korelaciji sa malignitetom tumora. Važno je da su nivoi metilacije ova tri gena u cfDNK u plazmi blisko odraz njihovih kolega u tkivu, sa dijagnostičkom efikasnošću od 83,3%, 83,9% i 77,6% za RANBP3, LCP2 i GRAP2, respektivno.
UNASSIGNED: Naši nalazi sugerišu da geni RANBP3, LCP2 i GRAP2, locirani na identifikovanim mestima metilacije, imaju značajan potencijal kao molekularni markeri u krvi za dodatnu dijagnozu raka dojke. Ova studija postavlja temelje za dublje istraživanje promena u obrascima metilacije gena u cirkulišućoj slobodnoj DNK (cfDNK) za rano otkrivanje ne samo raka dojke već i raznih drugih vrsta raka.

UNASSIGNED: Inflammatory bowel disease (IBD) is associated with increased circulating damage-associated molecular patterns, in particular, the highly pro-inflammatory mitochondrial DNA (mtDNA). Here, we study the importance of blood neutrophils in mtDNA release via neutrophil extracellular trap (NET) formation and mitochondrial NETosis, where neutrophils specifically expulse mtDNA as potential targetable biological pathways.
UNASSIGNED: We investigated the roles of A23187 (a known NET stimulant), granulocyte macrophage stimulating factor, lipopolysaccharide (LPS), and human IBD plasma in their ability to induce NET formation, mitochondrial NETosis, mtDNA, and total DNA release from human blood neutrophils; and the evidence for increased NET formation in IBD.
UNASSIGNED: We demonstrated that NET formation resulted in significant DNA (P < .0001) and mtDNA release (P < .0001) with long DNA fragments (>1000 base pairs) with NETs containing high levels of mtDNA. Using previously described in vitro conditions for mitochondrial NETosis, granulocyte macrophage stimulating factor + LPS triggered neutrophil mtDNA release at lower levels but not NETosis. LPS alone can trigger neutrophilic DNA release without NET formation. Heterologous coculture with plasma from patients with active IBD (vs remission [n = 6/group]) were not associated with significantly higher levels of NETs and mtDNA release. During coculture with active IBD plasma (vs remission), citrullinated histone 3 (CitH3) (a NETs biomarker) levels were significantly lower (P < .001). Similarly, CitH3 levels were lower in stool supernatants of patients with active IBD vs remission (n = 19/12, P = .0001). Stool CitH3 negatively correlates with stool calprotectin, a biomarker for gut inflammation (r = -0.47, P = .03).
UNASSIGNED: Hence, although blood neutrophils remain an important source of circulating mtDNA with defined mechanisms for release via NET formation and during neutrophil activation, our data do not support excessive systemic NET formation as a dominant underpinning pathobiological process in IBD.

A 35-year-old male presented with recurrent respiratory papillomatosis. Human papillomavirus type 11 was detected from all sites of tumor tissue DNA by PCR. The pre-surgery cell-free DNA (cfDNA) viral load (3.33 × 103 copies/ng DNA) fell below the post-surgical detection limits on achieving remission, suggesting cfDNA\'s potential as a biomarker.

UNASSIGNED: Colorectal cancer is a worldwide leading cause of death accounting for high-rate mortality. Mutations in the epidermal growth factor receptor and RAS/MAPK pathways, as well as altered methylation genes profiles, have been described as molecular mechanisms promoting and sustaining tumour development and progression. Aberrant methylation is a well-known epigenetic mechanism involved in gene regulation; particularly several genes were reported as hypermethylated in CRC. Recently, it was shown that epigenetic alterations in genes such as neuropeptide y, proenkephalin and Wnt inhibitory factor 1 can be used as promising disease biomarkers. Almost all methods developed for the DNA methylation analysis combined next generation sequencing, conventional qRT-PCR or ddPCR with the prior DNA modification with sodium bisulfite.
UNASSIGNED: We implemented a ddPCR method to assess the methylation status of Wnt inhibitory factor 1 and neuropeptide y using the methylation sensitive restriction enzyme approach that does not impact on DNA quality and guarantees the discrimination of DNA methylation independent of bisulfite conversion.
UNASSIGNED: We showed that this method is robust and sensitive also allowing the monitoring of CRC disease progression when applied to circulating free DNA samples from liquid biopsies, proving to be a fast and easy to implement assay to be used for the monitoring of the methylation pattern of clinically relevant target genes.

Epithelial ovarian cancer (EOC) is the deadliest women\'s cancer and has a poor prognosis. Early detection is the key for improving survival (a 5-year survival rate in stage I/II is over 70% compared to that of 25% in stage III/IV) and can be achieved through methylation markers from circulating cell-free DNA (cfDNA) using a liquid biopsy. In this study, we first identify top 500 EOC markers differentiating EOC from healthy female controls from 3.3 million methylome-wide CpG sites and validated them in 1,800 independent cfDNA samples. We then utilize a pretrained AI transformer system called MethylBERT to develop an EOC diagnostic model which achieves 80% sensitivity and 95% specificity in early-stage EOC diagnosis. We next develop a simple digital droplet PCR (ddPCR) assay which archives good performance, facilitating early EOC detection.

BACKGROUND: Uterine corpus endometrial carcinoma (UCEC) is a prevalent gynecologic malignancy with a favorable prognosis if detected early. However, there is a lack of accurate and reliable early detection tests for UCEC. This study aims to develop a precise and non-invasive diagnostic method for UCEC using circulating cell-free DNA (cfDNA) fragmentomics.
METHODS: Peripheral blood samples were collected from all participants, and cfDNA was extracted for analysis. Low-coverage whole-genome sequencing was performed to obtain cfDNA fragmentomics data. A robust machine learning model was developed using these features to differentiate between UCEC and healthy conditions.
RESULTS: The cfDNA fragmentomics-based model showed high predictive power for UCEC detection in training (n = 133; AUC 0.991) and validation cohorts (n = 89; AUC 0.994). The model manifested a specificity of 95.5% and a sensitivity of 98.5% in the training cohort, and a specificity of 95.5% and a sensitivity of 97.8% in the validation cohort. Physiological variables and preanalytical procedures had no significant impact on the classifier\'s outcomes. In terms of clinical benefit, our model would identify 99% of Chinese UCEC patients at stage I, compared to 21% under standard care, potentially raising the 5-year survival rate from 84 to 95%.
CONCLUSIONS: This study presents a novel approach for the early detection of UCEC using cfDNA fragmentomics and machine learning showing promising sensitivity and specificity. Using this model in clinical practice could significantly improve UCEC management and control, enabling early intervention and better patient outcomes. Further optimization and validation of this approach are warranted to establish its clinical utility.

We present a case of a man immunocompromised due to myelodysplastic syndrome with Candida krusei fungemia who had a rising cell-free DNA (cfDNA) giant magnetoresistance (GMR) signal when tested daily using plasma blood samples. With the rise in GMR signal paralleling the development of skin lesions in this patient, we conclude that cfDNA can be used to indicate uncontrolled infection and thus help monitor response to therapy. This index patient provides evidence that an invasive fungal infection requires both direct antifungal therapy and an intact immune system to control the infection. This biosensing platform has been simplified to potentially serve as a point-of-care test, setting it apart by overcoming the three common barriers of cfDNA testing: complexity, cost, and time.