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Aims: Around the world, the emergence of robotic technology has improved surgical precision and accuracy in total knee arthroplasty (TKA). This territory-wide study compares the results of various robotic TKA (R-TKA) systems with those of conventional TKA (C-TKA) and computer-navigated TKA (N-TKA)., Methods: This is a retrospective study utilizing territory-wide data from the Clinical Data Analysis and Reporting System (CDARS). All patients who underwent primary TKA in all 47 public hospitals in Hong Kong between January 2021 and December 2023 were analyzed. Primary outcomes were the percentage use of various robotic and navigation platforms. Secondary outcomes were: 1) mean length of stay (LOS); 2) 30-day emergency department (ED) attendance rate; 3) 90-day ED attendance rate; 4) 90-day reoperation rate; 5) 90-day mortality rate; and 6) surgical time., Results: A total of 8,492 knees from 7,746 patients were included in the study. Overall robotic use had risen to 20.4% (2023 Q3 to Q4: 355/1,738) by the end of 2023, with Mako being the most popular at 10.3% (179/1,738). R-TKA had the shortest mean LOS compared with N-TKA and C-TKA (5.5 vs 6.3 and 7.1 days, respectively; p < 0.001). Only Mako (9.7%) demonstrated reduced 90-day ED attendance compared to C-TKA (13.1%; p = 0.009), Cori/Navio (15.0%; p = 0.005), and Rosa (16.4%; p < 0.001). No differences in 90-day reoperation rate and mortality were observed between all groups. Mean surgical times were longer in R-TKA groups by 20.6 minutes (p < 0.001)., Conclusion: R-TKA use has increased in recent years, and has been shown to reduce hospital stay despite having a slightly longer surgical time, proving a promising candidate to alleviate the burden on healthcare systems. Individual differences between R-TKA systems contributed to variable clinical outcomes., Competing Interests: H. Fu reports consulting fees from Stryker, and payment or honoraria for lectures, presentations, speaker bureaus, manuscript writing or educational events from Stryker, Depuy Synthes, and Zimmer Biomet, unrelated to this study. H. Fu is also an international member of HKOA, AAHKS, and AAOS. K. Y. Chiu reports consulting fees and payment or honoraria for lectures, presentations, speaker bureaus, manuscript writing or educational events from Stryker, Depuy Synthes, Smith & Nephew, and Zimmer, unrelated to this study., (© 2025 Chan et al.)

The tissues of origin of plasma DNA can be revealed by methylation patterns. However, the relative DNA contributions from megakaryocytes and erythroblasts into plasma appeared inconsistent among studies. To shed light into this phenomenon, we developed droplet digital PCR (ddPCR) assays for the differential detection of contributions from these cell types in plasma based on megakaryocyte-specific and erythroblast-specific methylation markers. Megakaryocytic DNA and erythroid DNA contributed a median of 44.2% and 6.2% in healthy individuals, respectively. Patients with idiopathic thrombocytopenic purpura had a significantly higher proportion of megakaryocytic DNA in plasma compared to healthy controls (median: 59.9% versus 44.2%; P = 0.03). Similarly, patients with β-thalassemia were shown to have higher proportions of plasma erythroid DNA compared to healthy controls (median: 50.9% versus 6.2%) (P < 0.0001). Hence, the concurrent analysis of megakaryocytic and erythroid lineage-specific markers could facilitate the dissection of their relative contributions and provide information on patients with hematological disorders., (© 2024. The Author(s).)

Dysbiosis of the human oral microbiota has been reported to be associated with oral cavity squamous cell carcinoma (OSCC) while the host-microbiota interactions with respect to the potential impact of pathogenic bacteria on host genomic and epigenomic abnormalities remain poorly studied. In this study, the mucosal bacterial community, host genome-wide transcriptome and DNA CpG methylation were simultaneously profiled in tumors and their adjacent normal tissues of OSCC patients. Significant enrichment in the relative abundance of seven bacteria species (Fusobacterium nucleatum, Treponema medium, Peptostreptococcus stomatis, Gemella morbillorum, Catonella morbi, Peptoanaerobacter yurli and Peptococcus simiae) were observed in OSCC tumor microenvironment. These tumor-enriched bacteria formed 254 positive correlations with 206 up-regulated host genes, mainly involving signaling pathways related to cell adhesion, migration and proliferation. Integrative analysis of bacteria-transcriptome and bacteria-methylation correlations identified at least 20 dysregulated host genes with inverted CpG methylation in their promoter regions associated with enrichment of bacterial pathogens, implying a potential of pathogenic bacteria to regulate gene expression, in part, through epigenetic alterations. An in vitro model further confirmed that Fusobacterium nucleatum might contribute to cellular invasion via crosstalk with E-cadherin/β-catenin signaling, TNFα/NF-κB pathway and extracellular matrix remodeling by up-regulating SNAI2 gene, a key transcription factor of epithelial-mesenchymal transition (EMT). Our work using multi-omics approaches explored complex host-microbiota interactions and provided important insights into genetic and functional basis in OSCC tumorigenesis, which may serve as a precursor for hypothesis-driven study to better understand the causational relationship of pathogenic bacteria in this deadly cancer., (© 2024. The Author(s).)

Recent studies have revealed an unexplored population of long cell-free DNA (cfDNA) molecules in human plasma using long-read sequencing technologies. However, the biological properties of long cfDNA molecules (>500 bp) remain largely unknown. To this end, we have investigated the origins of long cfDNA molecules from different genomic elements. Analysis of plasma cfDNA using long-read sequencing reveals an uneven distribution of long molecules from across the genome. Long cfDNA molecules show overrepresentation in euchromatic regions of the genome, in sharp contrast to short DNA molecules. We observe a stronger relationship between the abundance of long molecules and mRNA gene expression levels, compared with short molecules (Pearson's r = 0.71 vs. -0.14). Moreover, long and short molecules show distinct fragmentation patterns surrounding CpG sites. Leveraging the cleavage preferences surrounding CpG sites, the combined cleavage ratios of long and short molecules can differentiate patients with hepatocellular carcinoma (HCC) from non-HCC subjects (AUC = 0.87). We also investigated knockout mice in which selected nuclease genes had been inactivated in comparison with wild-type mice. The proportion of long molecules originating from transcription start sites are lower in Dffb -deficient mice but higher in Dnase1l3 -deficient mice compared with that of wild-type mice. This work thus provides new insights into the biological properties and potential clinical applications of long cfDNA molecules., (© 2024 Che et al.; Published by Cold Spring Harbor Laboratory Press.)

Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5' 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as "founder" end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.

A meeting of experts was held in November 2021 to review and discuss available data on performance of Epstein-Barr virus (EBV)-based approaches to screen for early stage nasopharyngeal carcinoma (NPC) and methods for the investigation and management of screen-positive individuals. Serum EBV antibody and plasma EBV DNA testing methods were considered. Both approaches were found to have favorable performance characteristics and to be cost-effective in high-risk populations. In addition to endoscopy, use of magnetic resonance imaging (MRI) to investigate screen-positive individuals was found to increase the sensitivity of NPC detection with minimal impact on cost-effectiveness of the screening program., (© The Author(s) 2023. Published by Oxford University Press.)

Radiotherapy (RT) is the standard-of-care for Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), where the post-RT clearance of plasma EBV DNA is prognostic. Currently, it is not known whether the post-RT clearance of plasma EBV DNA is related to the presence of circulating T-cell subsets. Blood samples from NPC patients were used to assess the frequency of T-cell subsets relating to differentiation, co-signaling and chemotaxis. Patients with undetectable versus detectable plasma EBV DNA levels post-RT were categorized as clearers vs. non-clearers. Clearers had a lower frequency of PD1+CD8+ T cells as well as CXCR3+CD8+ T cells during RT compared to non-clearers. Clearers exclusively showed a temporal increase in chemo-attractant receptors CCR1, 4 and/or 5, expressing CD8+ T cells upon RT. The increase in CCR-expressing CD8+ T cells was accompanied by a drop in naïve CD8+ T cells and an increase in OX40+CD8+ T cells. Upon stratifying these patients based on clinical outcome, the dynamics of CCR-expressing CD8+ T cells were in concordance with the non-recurrence of NPC. In a second cohort, non-recurrence associated with higher quantities of circulating CCL14 and CCL15. Collectively, our findings relate plasma EBV DNA clearance post-RT to T-cell chemotaxis, which requires validation in larger cohorts.

Background: Recent studies using single molecule, real-time (SMRT) sequencing revealed a substantial population of analyzable long cell-free DNA (cfDNA) in plasma. Potential clinical utilities of such long cfDNA in pregnancy and cancer have been demonstrated. However, the performance of different long-read sequencing platforms for the analysis of long cfDNA remains unknown., Methods: Size biases of SMRT sequencing by Pacific Biosciences (PacBio) and nanopore sequencing by Oxford Nanopore Technologies (ONT) were evaluated using artificial mixtures of sonicated human and mouse DNA of different sizes. cfDNA from plasma samples of pregnant women at different trimesters, hepatitis B carriers, and patients with hepatocellular carcinoma were sequenced with the 2 platforms., Results: Both platforms showed biases to sequence longer (1500 bp vs 200 bp) DNA fragments, with PacBio showing a stronger bias (5-fold overrepresentation of long fragments vs 2-fold in ONT). Percentages of cfDNA fragments 500 bp were around 6-fold higher in PacBio compared with ONT. End motif profiles of cfDNA from PacBio and ONT were similar, yet exhibited platform-dependent patterns. Tissue-of-origin analysis based on single-molecule methylation patterns showed comparable performance on both platforms., Conclusions: SMRT sequencing generated data with higher percentages of long cfDNA compared with nanopore sequencing. Yet, a higher number of long cfDNA fragments eligible for the tissue-of-origin analysis could be obtained from nanopore sequencing due to its much higher throughput. When analyzing the size and end motif of cfDNA, one should be aware of the analytical characteristics and possible biases of the sequencing platforms being used., (© American Association for Clinical Chemistry 2022.)

Cell-free DNA (cfDNA) fragmentation patterns contain important molecular information linked to tissues of origin. We explored the possibility of using fragmentation patterns to predict cytosine-phosphate-guanine (CpG) methylation of cfDNA, obviating the use of bisulfite treatment and associated risks of DNA degradation. This study investigated the cfDNA cleavage profile surrounding a CpG (i.e., within an 11-nucleotide [nt] window) to analyze cfDNA methylation. The cfDNA cleavage proportion across positions within the window appeared nonrandom and exhibited correlation with methylation status. The mean cleavage proportion was ∼twofold higher at the cytosine of methylated CpGs than unmethylated ones in healthy controls. In contrast, the mean cleavage proportion rapidly decreased at the 1-nt position immediately preceding methylated CpGs. Such differential cleavages resulted in a characteristic change in relative presentations of CGN and NCG motifs at 5' ends, where N represented any nucleotide. CGN/NCG motif ratios were correlated with methylation levels at tissue-specific methylated CpGs (e.g., placenta or liver) (Pearson's absolute r > 0.86). cfDNA cleavage profiles were thus informative for cfDNA methylation and tissue-of-origin analyses. Using CG-containing end motifs, we achieved an area under a receiver operating characteristic curve (AUC) of 0.98 in differentiating patients with and without hepatocellular carcinoma and enhanced the positive predictive value of nasopharyngeal carcinoma screening (from 19.6 to 26.8%). Furthermore, we elucidated the feasibility of using cfDNA cleavage patterns to deduce CpG methylation at single CpG resolution using a deep learning algorithm and achieved an AUC of 0.93. FRAGmentomics-based Methylation Analysis (FRAGMA) presents many possibilities for noninvasive prenatal, cancer, and organ transplantation assessment.

Advances in Next Generation Sequencing (NGS) technologies have enabled the accurate detection and quantification of circulating tumor-derived (ct)DNA in most gastrointestinal (GI) cancers. The prognostic and predictive utility of ctDNA in patiets with different stages of colorectal (CRC), gastro-esophageal (GEC) and pancreaticobiliary cancers (PBC) are currently under active investigation. The most mature clinical data to date are derived from studies in the prognostic utility of personalized ctDNA-based NGS assays in the detection of minimal residual disease (MRD) and early recurrence after surgery in CRC and other GI cancers. These findings are being validated in several prospective studies which are designed to test if ctDNA could outperform conventional approaches in guiding adjuvant chemotherapy, and in post-operative surveillance in some GI cancers. Several adaptive studies using ctDNA as a screening platform are also being used to identify patients with actionable genomic alterations for clinical trials of targeted therapies. In the palliative setting, ctDNA monitoring during treatment has shown promise in the detection and tracking of clonal variants associated with acquired resistance to targeted therapies and immune-checkpoint inhibitors (ICI). Moreover, ctDNA may help to guide the therapeutic re-challenge of targeted therapies in patients who have prior exposure to such treatment. This review will examine the most updated research findings on ctDNA as a biomarker in CRC, GEC and PBCs. It aims to provide insights into how the unique strengths of this biomarker could be optimally leveraged in improving the management of these GI cancers., Competing Interests: KC is a director of Take2, DRA and Novostics. KC holds equities in Take2, DRA, Grail/Illumina. KC and WL were previous consultants to Grail. WL holds equity in Grail/Illumina. KC holds patents portfolio in molecular diagnostics and receive royalties from Take2, DRA, Grail, Illumina, Sequenome, Xcelom. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lam, Johnson, Lam, Li, Wong, Lam, Chan and Ma.)

Background: Analysis of circulating tumor DNA has become increasingly important as a tool for cancer care. However, the focus of previous studies has been on short fragments of DNA. Also, bisulfite sequencing, a conventional approach for methylation analysis, causes DNA degradation, which is not ideal for the assessment of long DNA properties and methylation patterns. This study attempted to overcome such obstacles by single-molecule sequencing., Methods: Single-molecule real-time (SMRT) sequencing was used to sequence plasma DNA. We performed fragment size and direct methylation analysis for each molecule. A methylation score concerning single-molecule methylation patterns was used for cancer detection., Results: A substantial proportion of plasma DNA was longer than 1 kb with a median of 16% in hepatocellular carcinoma (HCC) patients, hepatitis B virus carriers, and healthy individuals. The longest plasma DNA molecule in the HCC patients was 39.8 kb. Tumoral cell-free DNA (cfDNA) was generally shorter than nontumoral cfDNA. The longest tumoral cfDNA was 13.6 kb. Tumoral cfDNA had lower methylation levels compared with nontumoral cfDNA (median: 59.3% vs 76.9%). We developed and analyzed a metric reflecting single-molecule methylation patterns associated with cancer, named the HCC methylation score. HCC patients displayed significantly higher HCC methylation scores than those without HCC. Interestingly, compared to using short cfDNA (area under the receiver operating characteristic [ROC] curve, AUC: 0.75), the use of long cfDNA molecules greatly enhanced the discriminatory power (AUC: 0.91)., Conclusions: A previously unidentified long cfDNA population was revealed in cancer patients. The presence and direct methylation analysis of these molecules open new possibilities for cancer liquid biopsy., Competing Interests: Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:, (© American Association for Clinical Chemistry 2022.)

A 59-year-old female was admitted for non-ST-segment elevation myocardial infarction and Killip class 3 heart failure with a left ventricular ejection fraction of 30%. Coronary angiogram showed moderate to severe stenosis over the ostial-proximal left anterior descending artery with minor disease over the left circumflex artery and right coronary artery. We describe a complication encountered where a protruding stent was weakened and elongated during our attempts to remove it, risking possible breakage, stent embolization, and long stent protrusion inside the aorta. We then describe treatment with what we call the alpha-loop snaring technique, which, to our knowledge, is the first report describing this novel approach, which can salvage a failing snaring attempt of a completely deployed and dislodged coronary stent.

Background: Quantitative measurement of plasma Epstein-Barr virus (EBV) DNA by real-time PCR at the end of primary treatment is a robust prognostic marker for nasopharyngeal carcinoma (NPC) patients. However, up to 40% of patients who would later develop disease recurrence had undetectable post-treatment plasma EBV DNA. Targeted sequencing for the entire EBV genome potentially allows a more comprehensive and unbiased detection of plasma EBV DNA and enables the use of other parameters such as fragment size as biomarkers. Hence, we explored if plasma EBV DNA sequencing might allow more accurate prognostication of NPC patients., Patients and Methods: Plasma samples collected from 769 patients with stage IIB-IVB NPC at 6-8 weeks after radiotherapy were analysed using targeted sequencing for EBV DNA., Results: The sensitivities of the PCR-based analysis, at a cut-off of any detectable levels of plasma EBV DNA, for prediction of local and distant recurrences were 42.3% and 85.3%, respectively. The sequencing-based analysis (involving quantitation and size profiling) achieved better performance for both local and distant recurrences than PCR. Using a cut-off of the proportion of plasma EBV DNA deduced by sequencing at 0.01%, the sensitivities of the sequencing-based analysis for local and distant recurrences were 88.5% and 97.1%, with the resultant negative predictive values of 99.1% and 99.4%, respectively. Among patients with undetectable EBV DNA on quantitative PCR, sequencing could further define a subgroup that enjoyed superior survival outcomes based on the proportion of plasma EBV DNA, with a 5-year progression-free survival (PFS) approaching 90%. On multivariate analysis, sequencing-based quantitative level of plasma EBV DNA was the independent prognostic factor with the highest hazard ratio for prediction of overall survival and PFS., Conclusion: NPC prognostication using post-treatment plasma EBV DNA could be enhanced through sequencing., Competing Interests: Disclosure KCAC and YMDL hold equities in DRA, Take2 and Grail/Illumina; were consultants to Grail and previously received research funding from Grail. YMDL is a scientific cofounder of Grail. WKJL holds equities in Grail/Illumina. PJ holds equities in DRA and Grail/Illumina; is a consultant to Take2; is a Director of KingMed Future. DCTC, WKJL, KCAC and YMDL have filed patent applications based on the data generated from this work. EPH receives speaker’s honoraria from Merck Sharp & Dohme (MSD) and Merck Serono and serves as a consultant or in the advisory board from MSD. BBYM serves in the advisory board and receives speaker’s honorarium from Novartis, Bristol-Myers Squibb and MSD and receives research grant from Novartis and is a consultant to Viracta Therapeutics and Y-Biologics. ATCC receives research funding from Merck Serono, MSD, Novartis, Pfizer and Eli Lily, speaker's honorarium from Beigene, Springer and Pfizer, travel funding from BMS, MSD, Pfizer, Roche, Novartis and AstraZeneca, and is an advisory board member for MSD, Tessa Therapeutics. BZ is the founder and shareholder of Health View Bioanalytic Limited. QTL is a consultant to MSD and serves in the advisory boards of Nanobiotics, Roche and Coherus. Patent royalties are received from Grail, Illumina, Sequenom, DRA, Take2 and Xcelom. All other authors have declared no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Urinary cell-free DNA (ucfDNA) is a potential biomarker for bladder cancer detection. However, the biological characteristics of ucfDNA are not well understood. We explored the roles of deoxyribonuclease 1 (DNASE1) and deoxyribonuclease 1-like 3 (DNASE1L3) in the fragmentation of ucfDNA using mouse models. The deletion of Dnase1 in mice (Dnase1-/-) caused aberrations in ucfDNA fragmentation, including a 24-fold increase in DNA concentration, and a 3-fold enrichment of long DNA molecules, with a relative decrease of fragments with thymine ends and reduction of jaggedness (i.e., the presence of single-stranded protruding ends). In contrast, such changes were not observed in mice with Dnase1l3 deletion (Dnase1l3-/-). These results suggested that DNASE1 was an important nuclease contributing to the ucfDNA fragmentation. Western blot analysis revealed that the concentration of DNASE1 protein was higher in urine than DNASE1L3. The native-polyacrylamide gel electrophoresis zymogram showed that DNASE1 activity in urine was higher than that in plasma. Furthermore, the proportion of ucfDNA fragment ends within DNase I hypersensitive sites (DHSs) was significantly increased in Dnase1-deficient mice. In humans, patients with bladder cancer had lower proportions of ucfDNA fragment ends within the DHSs when compared with participants without bladder cancer. The area under the curve (AUC) for differentiating patients with and without bladder cancer was 0.83, suggesting the analysis of ucfDNA fragmentation in the DHSs may have potential for bladder cancer detection. This work revealed the intrinsic links between the nucleases in urine and ucfDNA fragmentomics., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: K.C.A.C., R.W.K.C., and Y.M.D.L. hold equities in DRA, Take2, and Grail/Illumina. K.C.A.C., R.W.K.C., and Y.M.D.L. were consultants to Grail/Illumina. K.C.A.C., R.W.K.C., and Y.M.D.L. received research funding from Grail/Illumina. Y.M.D.L. was a scientific co-founder of and served on the scientific advisory board of Grail/Illumina. W.K.J.L and P.J. holds equities in Grail/Illumina. P.J. is a consultant to Take2. P.J. is a Director of KingMed Future.

Cell-free extrachromosomal circular DNA (eccDNA) as a distinct topological form from linear DNA has recently gained increasing research interest, with possible clinical applications as a class of biomarkers. In this study, we aimed to explore the relationship between nucleases and eccDNA characteristics in plasma. By using knockout mouse models with deficiencies in deoxyribonuclease 1 (DNASE1) or deoxyribonuclease 1 like 3 (DNASE1L3), we found that cell-free eccDNA in Dnase1l3-/- mice exhibited larger size distributions than that in wild-type mice. Such size alterations were not found in tissue eccDNA of either Dnase1-/- or Dnase1l3-/- mice, suggesting that DNASE1L3 could digest eccDNA extracellularly but did not seem to affect intracellular eccDNA. Using a mouse pregnancy model, we observed that in Dnase1l3-/- mice pregnant with Dnase1l3+/- fetuses, the eccDNA in the maternal plasma was shorter compared with that of Dnase1l3-/- mice carrying Dnase1l3-/- fetuses, highlighting the systemic effects of circulating fetal DNASE1L3 degrading the maternal eccDNA extracellularly. Furthermore, plasma eccDNA in patients with DNASE1L3 mutations also exhibited longer size distributions than that in healthy controls. Taken together, this study provided a hitherto missing link between nuclease activity and the biological manifestations of eccDNA in plasma, paving the way for future biomarker development of this special form of DNA molecules.

Single-stranded ends of double-stranded DNA (jagged ends) are more abundant in urinary DNA than in plasma DNA. However, the lengths of jagged ends in urinary DNA remained undetermined, as a previous method used for urinary DNA jagged end sequencing analysis (Jag-seq) relied on unmethylation at CpG sites, limiting the resolution. Here, we performed high-resolution Jag-seq analysis using methylation at non-CpG cytosine sites, allowing determination of exact length of jagged ends. The urinary DNA bore longer jagged ends (~26-nt) than plasma DNA (~17-nt). The jagged end length distribution displayed 10-nt periodicities in urinary DNA, which were much less observable in plasma DNA. Amplitude of the 10-nt periodicities increased in patients with renal cell carcinoma. Heparin treatment of urine diminished the 10-nt periodicities. The urinary DNA jagged ends often extended into nucleosomal cores, suggesting potential interactions with histones. This study has thus advanced our knowledge of jagged ends in urine DNA., (© 2022. The Author(s).)

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5' fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5' 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia., Competing Interests: Competing interest statement: A patent application on the described technology has been filed by S.C.Y.Y., P.J., W.P., S.H.C., Y.T.T.C., K.C.A.C., R.W.K.C., and Y.M.D.L. and licensed to Take2 Holdings Limited founded by K.C.A.C., R.W.K.C., and Y.M.D.L., (Copyright © 2021 the Author(s). Published by PNAS.)

The effects of DNASE1L3 or DNASE1 deficiency on cell-free DNA (cfDNA) methylation were explored in plasma of mice deficient in these nucleases and in DNASE1L3-deficient humans. Compared to wild-type cfDNA, cfDNA in DNASE1L3-deficient mice was significantly hypomethylated, while cfDNA in DNASE1-deficient mice was hypermethylated. The cfDNA hypomethylation in DNASE1L3-deficient mice was due to increased fragmentation and representation from open chromatin regions (OCRs) and CpG islands (CGIs). These findings were absent in DNASE1-deficient mice, demonstrating the preference of DNASE1 to cleave in hypomethylated OCRs and CGIs. We also observed a substantial decrease of fragment ends at methylated CpGs in the absence of DNASE1L3, thereby demonstrating that DNASE1L3 prefers to cleave at methylated CpGs. Furthermore, we found that methylation levels of cfDNA varied by fragment size in a periodic pattern, with cfDNA of specific sizes being more hypomethylated and enriched for OCRs and CGIs. These findings were confirmed in DNASE1L3-deficient human cfDNA. Thus, we have found that nuclease-mediated cfDNA fragmentation markedly affects cfDNA methylation level on a genome-wide scale. This work provides a foundational understanding of the relationship between methylation, nuclease biology, and cfDNA fragmentation., (© 2021 Han et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Human plasma contains RNA transcripts released by multiple cell types within the body. Single-cell transcriptomic analysis allows the cellular origin of circulating RNA molecules to be elucidated at high resolution and has been successfully utilized in the pregnancy context. We explored the application of a similar approach to develop plasma RNA markers for cancer detection., Methods: Single-cell RNA sequencing was performed to decipher transcriptomic profiles of single cells from hepatocellular carcinoma (HCC) samples. Cell-type-specific transcripts were identified and used for deducing the cell-type-specific gene signature (CELSIG) scores of plasma RNA from patients with and without HCC., Results: Six major cell clusters were identified, including hepatocyte-like, cholangiocyte-like, myofibroblast, endothelial, lymphoid, and myeloid cell clusters based on 4 HCC tumor tissues as well as their paired adjacent nontumoral tissues. The CELSIG score of hepatocyte-like cells was significantly increased in preoperative plasma RNA samples of patients with HCC (n = 14) compared with non-HCC participants (n = 49). The CELSIG score of hepatocyte-like cells declined in plasma RNA samples of patients with HCC within 3 days after tumor resection. Compared with the discriminating power between patients with and without HCC using the abundance of ALB transcript in plasma [area under curve (AUC) 0.72)], an improved performance (AUC: 0.84) was observed using the CELSIG score. The hepatocyte-specific transcript markers in plasma RNA were further validated by ddPCR assays. The CELSIG scores of hepatocyte-like cell and cholangiocyte trended with patients' survival., Conclusions: The combination of single-cell transcriptomic analysis and plasma RNA sequencing represents an approach for the development of new noninvasive cancer markers., (© American Association for Clinical Chemistry 2021.)

Objective: To evaluate the dynamics of the oral microbiome and associated patient outcomes following treatment of head and neck squamous cell carcinoma (HNSCC)., Materials and Methods: This was a prospective cohort study at a tertiary academic center in Hong Kong SAR of patients with head and neck squamous cell carcinoma evaluating the oral microbiome in pre- and postsurgery oral rinses (at 1, 3, and 6 months) with 16S rRNA gene V3-V4 amplicon sequencing., Results: In total, 76 HNSCC patients were evaluated. There was a significantly depressed alpha diversities of oral microbial communities observed in HNSCC oral rinse samples within the first 6 months post-surgery when compared to presurgery or healthy controls. Distant clustering between pre- and postsurgery was also observed ( p < 0.022). Following treatment, eight oral bacterial genera showed a trend towards the restoration in the relative abundances that approximate healthy persons. In evaluating patient outcomes, the decreased relative abundance of three periodontal bacteria ( Capnocytophaga , Prevotella 7 , and Leptotrichia ) and the increased relative abundance of two commensal bacteria ( Streptococcus and Rothia ) at 6 months postsurgery compared to presurgery showed a better 3-year disease-specific survival (a cutoff of Kaplan-Meier survival curve test p < 0.3 at 36 months). In particular, the postsurgery restoration of Prevotella 7 was statistically significant in the surveyed patients (survival rate of 84% vs. 56% at 36 months, p = 0.0065)., Conclusions: Oral microbiome dysbiosis associated with HNSCC is dynamic. These dynamics of the oral microbiome postsurgery are also associated with patient treatment and outcomes and may serve as potential biomarkers for patient management in HNSCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chan, Ng, Lan, Fung, Li, Cai, Lei, Mou, Meehan, Lau, Yeung, Chan, Wong, Chan and Chen.)

Purpose: To study the dynamic changes in plasma Epstein-Barr virus (pEBV) DNA after radiotherapy in nasopharyngeal cancer (NPC)., Experimental Design: We conducted a randomized controlled trial of adjuvant chemotherapy versus observation in patients with NPC who had detectable pEBV DNA at 6 weeks post-radiotherapy. Randomized patients had a second pEBV DNA checked at 6 months post-randomization. The primary endpoint was progression-free survival (PFS)., Results: We prospectively enrolled 789 patients. Baseline post-radiotherapy pEBV DNA was undetectable in 573 (72.6%) patients, and detectable in 216 (27.4%) patients, of whom 104 (13.2%) patients were eligible for randomization to adjuvant chemotherapy ( n = 52) versus observation ( n = 52). The first post-radiotherapy pEBV DNA had a sensitivity of 0.48, specificity of 0.81, area under receiver-operator characteristics curve (AUC) of 0.65, false positive (FP) rate of 13.8%, and false negative (FN) rate of 14.4% for disease progression. The second post-radiotherapy pEBV DNA had improved sensitivity of 0.81, specificity of 0.75, AUC of 0.78, FP rate of 14.3%, and FN rate of 8.1%. Patients with complete clearance of post-radiotherapy pEBV DNA (51%) had survival superior to that of patients without post-radiotherapy pEBV DNA clearance (5-year PFS, 85.5% vs. 23.3%; HR, 9.6; P < 0.0001), comparable with patients with initially undetectable post-radiotherapy pEBV DNA (5-year PFS, 77.1%), irrespective of adjuvant chemotherapy or observation., Conclusions: Patients with NPC with detectable post-radiotherapy pEBV DNA who experienced subsequent pEBV DNA clearance had superior survival comparable with patients with initially undetectable post-radiotherapy pEBV DNA. Post-radiotherapy pEBV DNA clearance may serve as an early surrogate endpoint for long-term survival in NPC., (©2021 American Association for Cancer Research.)

Background: Although the characterization of cell-free extrachromosomal circular DNA (eccDNA) has gained much research interest, the methylation status of these molecules is yet to be elucidated. We set out to compare the methylation densities of plasma eccDNA of maternal and fetal origins, and between small and large molecules. The clearance of fetal eccDNA from maternal circulation was also investigated., Methods: We developed a sequencing protocol for eccDNA methylation analysis using tagmentation and enzymatic conversion approaches. A restriction enzyme-based approach was applied to verify the tagmentation results. The efficiency of cell-free fetal eccDNA clearance was investigated by fetal eccDNA fraction evaluations at various postpartum time points., Results: The methylation densities of fetal eccDNA (median: 56.3%; range: 40.5-67.6%) were lower than the maternal eccDNA (median: 66.7%; range: 56.5-75.7%) (P = 0.02, paired t-test). In addition, eccDNA molecules from the smaller peak cluster (180-230 bp) were of lower methylation levels than those from the larger peak cluster (300-450 bp). Both of these findings were confirmed using the restriction enzyme approach. We also observed comparable methylation densities between linear and eccDNA of both maternal and fetal origins. The average half-lives of fetal linear and eccDNA in the maternal blood were 30.2 and 29.7 min, respectively., Conclusions: We found that fetal eccDNA in plasma was relatively hypomethylated compared to the maternal eccDNA. The methylation densities of eccDNA were positively correlated with their sizes. In addition, fetal eccDNA was found to be rapidly cleared from the maternal blood after delivery, similar to fetal linear DNA., (© American Association for Clinical Chemistry 2021.)

Background: Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends., Methods: Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing., Results: The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83., Conclusions: Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection., (© American Association for Clinical Chemistry 2021.)

5-Methylcytosine (5mC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine 5mC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.SssI-treated DNA (methylated) (M.SssI being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide 5mC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human-mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq ( r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses., Competing Interests: Competing interest statement: A patent application on the described technology has been filed and licensed to Take2 Holdings Limited, founded by the research team., (Copyright © 2021 the Author(s). Published by PNAS.)

Background and Purpose: T1ρ imaging is a new quantitative MR imaging pulse sequence with the potential to discriminate between malignant and benign tissue. In this study, we evaluated the capability of T1ρ imaging to characterize tissue by applying T1ρ imaging to malignant and benign tissue in the nasopharynx and to normal tissue in the head and neck., Materials and Methods: Participants with undifferentiated nasopharyngeal carcinoma and benign hyperplasia of the nasopharynx prospectively underwent T1ρ imaging. T1ρ measurements obtained from the histogram analysis for nasopharyngeal carcinoma in 43 participants were compared with those for benign hyperplasia and for normal tissue (brain, muscle, and parotid glands) in 41 participants using the Mann-Whitney U test. The area under the curve of significant T1ρ measurements was calculated and compared using receiver operating characteristic analysis and the Delong test, respectively. A P  < .   05 indicated statistical significance., Results: There were significant differences in T1ρ measurements between nasopharyngeal carcinoma and benign hyperplasia and between nasopharyngeal carcinoma and normal tissue (all, P  < .   05). Compared with benign hyperplasia, nasopharyngeal carcinoma showed a lower T1ρ mean (62.14 versus 65.45 × ms), SD (12.60 versus 17.73 × ms), and skewness (0.61 versus 0.76) (all P  < .05), but no difference in kurtosis ( P  = .   18). The T1ρ SD showed the highest area under the curve of 0.95 compared with the T1ρ mean (area under the curve  = 0.72) and T1ρ skewness (area under the curve  = 0.72) for discriminating nasopharyngeal carcinoma and benign hyperplasia (all, P  < .05)., Conclusions: Quantitative T1ρ imaging has the potential to discriminate malignant from benign and normal tissue in the head and neck., (© 2020 by American Journal of Neuroradiology.)

The role of oral microbiota in head and neck squamous cell carcinoma (HNSCC) is poorly understood. Here we sought to evaluate the association of the bacterial microbiome with host gene methylation and patient outcomes, and to explore its potential as a biomarker for early detection or intervention. Here we performed 16S rRNA gene amplicon sequencing in sixty-eight HNSCC patients across both tissue and oral rinse samples to identify oral bacteria with differential abundance between HNSCC and controls. A subset of thirty-one pairs of HNSCC tumor tissues and the adjacent normal tissues were characterized for host gene methylation profile using bisulfite capture sequencing. We observed significant enrichments of Fusobacterium and Peptostreptococcus in HNSCC tumor tissues when compared to the adjacent normal tissues, and in HNSCC oral rinses when compared to healthy subjects, while ten other bacterial genera were largely depleted. These HNSCC-related bacteria were discriminative for HNSCC and controls with area under the receiver operating curves (AUCs) of 0.84 and 0.86 in tissue and oral rinse samples, respectively. Moreover, Fusobacterium nucleatum abundance in HNSCC cases was strongly associated with non-smokers, lower tumor stage, lower rate of recurrence, and improved disease-specific survival. An integrative analysis identified that enrichment of F. nucleatum was associated with host gene promoter methylation, including hypermethylation of tumor suppressor genes LXN and SMARCA2 , for which gene expressions were downregulated in the HNSCC cohort from The Cancer Genome Atlas. In conclusion, we identified a taxonomically defined microbial consortium associated with HNSCC that may have clinical potential regarding biomarkers for early detection or intervention. Host-microbe interactions between F. nucleatum enrichment and clinical outcomes or host gene methylation imply a potential role of F. nucleatum as a pro-inflammatory driver in initiating HNSCC without traditional risk factors, which warrants further investigation for the underlying mechanisms.

Plasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a "CC" end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cell-free DNA in plasma has been used for noninvasive prenatal testing and cancer liquid biopsy. The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have attracted much recent interest, leading to the emerging field of cell-free DNA fragmentomics. However, one aspect of plasma DNA fragmentomics as to whether double-stranded plasma molecules might carry single-stranded ends, termed a jagged end in this study, remains underexplored. We have developed two approaches for investigating the presence of jagged ends in a plasma DNA pool. These approaches utilized DNA end repair to introduce differential methylation signals between the original sequence and the jagged ends, depending on whether unmethylated or methylated cytosines were used in the DNA end-repair procedure. The majority of plasma DNA molecules (87.8%) were found to bear jagged ends. The jaggedness varied according to plasma DNA fragment sizes and appeared to be in association with nucleosomal patterns. In the plasma of pregnant women, the jaggedness of fetal DNA molecules was higher than that of the maternal counterparts. The jaggedness of plasma DNA correlated with the fetal DNA fraction. Similarly, in the plasma of cancer patients, tumor-derived DNA molecules in patients with hepatocellular carcinoma showed an elevated jaggedness compared with nontumoral DNA. In mouse models, knocking out of the Dnase1 gene reduced jaggedness, whereas knocking out of the Dnase1l3 gene enhanced jaggedness. Hence, plasma DNA jagged ends represent an intrinsic property of plasma DNA and provide a link between nuclease activities and the fragmentation of plasma DNA., (© 2020 Jiang et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: After curative radiotherapy (RT) or chemoradiation (CRT), there is no validated tool to accurately identify patients for adjuvant therapy in nasopharyngeal carcinoma (NPC). Post-RT circulating plasma Epstein-Barr virus (EBV) DNA can detect minimal residual disease and is associated with recurrence and survival independent of TNM (tumor-lymph node-metastasis) stage. We aimed to develop and validate a risk model for stratification of NPC patients after completion of RT/CRT to observation or adjuvant therapy., Patients and Methods: The prospective multicenter 0502 EBV DNA screening cohort (Hong Kong NPC Study Group 0502 trial) enrolled from 2006 to 2015 (n = 745) was used for model development. For internal validation, we pooled independent patient cohorts from prospective clinical studies enrolled from 1997 to 2006 (n = 340). For external validation, we used retrospective cohort of NPC patients treated at Sun Yat-sen University Cancer Center from 2009 to 2012 (n = 837). Eligible patients had histologically confirmed NPC of Union for International Cancer Control (UICC) 7th Edition stage II-IVB who completed curative RT/CRT with or without neoadjuvant chemotherapy, had post-RT EBV DNA tested within 120 days after RT and received no adjuvant therapy. The primary end point was overall survival (OS). We used recursive-partitioning analysis (RPA) to classify patients into groups of low, intermediate, and high risk of death., Results: Combining post-RT EBV DNA level (0, 1-49, 50-499, and ≥500 copies/ml) and TNM stage (II, III, IVAB), RPA model classified patients into low-, intermediate-, and high-risk groups with 5-year OS of 89.4%, 78.5% and 37.2%, respectively. The RPA low-risk group had comparable OS to TNM stage II (5-year OS 88.5%) but identified more patients (64.8% versus stage II 28.1%) that could potentially be spared adjuvant therapy toxicity. The RPA model (c-index 0.712) showed better risk discrimination than either the TNM stage (0.604) or post-RT EBV DNA alone (0.675) with improved calibration and consistence. These results were validated in both internal and external cohorts., Conclusion: Combining post-RT EBV DNA and TNM stage improved risk stratification in NPC., Competing Interests: Disclosure EPH, speaker's honoraria from Merck Sharp & Dohme (MSD), Merck Serono; consultant/advisory board from MSD. BBYM, advisory board and speaker's honorarium from Novartis, Bristol-Myers Squibb, MSD; research grant from Novartis, Boehringer Ingelheim; WKJL holds equity in Grail; KCAC, grant support from the Research Grants Council of Hong Kong, Kadoorie Charitable Foundation and Cirina/Grail; royalties from Sequenom, Illumina, Grail/Cirina, Xcelom, DRA and Take2; holds equities of DRA and Take2; YMDL, received sponsored research agreement from Grail; research grant from Hong Kong Research Grants Council; endowed professorship: Li Ka Shing Foundation; shareholding, membership of scientific advisory board, sponsored research agreement, licensing, royalties from Grail; shareholding, licensing, royalties from Take2; consultancy to Decheng Capital: licensing, royalties from Sequenom, DRA Limited and Illumina; ATCC, received research and travel grants: MSD, Pfizer, Roche. All remaining authors have declared no conflicts of interest., (Copyright © 2020 European Society for Medical Oncology. Published by Elsevier Ltd. All rights reserved.)

Background: Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. Plasma EBV DNA is a validated screening tool for NPC. In screening, there are some individuals who do not have NPC but carry EBV DNA in plasma. Currently it is not known from screening if there may be any genotypic differences in EBV isolates from NPC and non-NPC subjects. Also, low concentrations of EBV DNA in plasma could pose challenge to such EBV genotypic analysis through plasma DNA sequencing., Methods: In a training dataset comprised of plasma DNA sequencing data of NPC and non-NPC subjects, we studied the difference in the EBV single nucleotide variant (SNV) profiles between the two groups. The most differentiating SNVs across the EBV genome were identified. We proposed an NPC risk score to be derived from the genotypic patterns over these SNV sites. We subsequently analyzed the NPC risk scores in a testing set., Results: A total of 661 significant SNVs across the EBV genome were identified from the training set. In the testing set, NPC plasma samples were shown to have high NPC risk scores, which suggested the presence of NPC-associated EBV SNV profiles. Among the non-NPC samples, there was a wide range of NPC risk scores. These results support the presence of diverse SNV profiles of EBV isolates from non-NPC subjects., Conclusion: EBV genotypic analysis is feasible through plasma DNA sequencing. The NPC risk score may be used to inform the cancer risk based on the EBV genome-wide SNV profile., (© American Association for Clinical Chemistry 2020.)

Background and Purpose: We evaluated modifications to our contrast-enhanced MR imaging grading system for symptomatic patients with suspected nasopharyngeal carcinoma, aimed at improving discrimination of early-stage cancer and benign hyperplasia. We evaluated a second non-contrast-enhanced MR imaging grading system for asymptomatic patients from nasopharyngeal carcinoma plasma screening programs., Materials and Methods: Dedicated nasopharyngeal MR imaging before (plain scan system) and after intravenous contrast administration (current and modified systems) was reviewed in patients from a nasopharyngeal carcinoma-endemic region, comprising 383 patients with suspected disease without nasopharyngeal carcinoma and 383 patients with nasopharyngeal carcinoma. The modified and plain scan systems refined primary tumor criteria, added a nodal assessment, and expanded the system from 4 to 5 grades. The overall combined sensitivity and specificity of the 3 systems were compared using the extended McNemar test (a χ 2 value [Formula: see text]> 5.99 indicates significance)., Results: The current, modified, and plain scan MR imaging systems yielded sensitivities of 99.74%, 97.91%, and 97.65%, respectively, and specificities of 63.45%, 89.56% and 86.42%, respectively. The modified system yielded significantly better performance than the current ([Formula: see text] = 122) and plain scan ([Formula: see text] = 6.1) systems. The percentages of patients with nasopharyngeal carcinoma in grades 1-2, grade 3, and grades 4-5 for the modified and plain scan MR imaging systems were 0.42% and 0.44%; 6.31% and 6.96%; and 90.36% and 87.79%, respectively. No additional cancers were detected after contrast administration in cases of a plain scan graded 1-2., Conclusions: We propose a modified MR imaging grading system that improves diagnostic performance for nasopharyngeal carcinoma detection. Contrast was not valuable for low MR imaging grades, and the plain scan shows potential for use in screening programs., (© 2020 by American Journal of Neuroradiology.)

We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5'-untranslated regions (5'-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications., Competing Interests: Competing interest statement: S.T.K.S., P.J., J.D., L.J., K.C.A.C., R.W.K.C. and Y.M.D.L. have filed a patent application based on the data generated from this work., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: Cellular mitochondrial DNA (mtDNA) is organized as circular, covalently closed and double-stranded DNA. Studies have demonstrated the presence of short mtDNA fragments in plasma. It is not known whether circular mtDNA might concurrently exist with linear mtDNA in plasma., Methods: We elucidated the topology of plasma mtDNA using restriction enzyme Bfa I cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying Bfa I cleavage signatures were defined as circular-derived mtDNA, whereas those with no cleavage signature or with 1 cleavage signature were defined as linear-derived mtDNA. An independent assay using exonuclease V to remove linear DNA followed by restriction enzyme Msp I digestion was used for confirming the conclusions based on Bfa I cleavage analysis. We analyzed the presence of Bfa I cleavage signatures on plasma DNA ends in nonhematopoietically and hematopoietically derived DNA molecules by sequencing plasma DNA of patients with liver transplantation and bone marrow transplantation., Results: Both linear and circular mtDNA coexisted in plasma. In patients with liver transplantation, donor-derived (i.e., liver) mtDNA molecules were mainly linear (median fraction, 91%; range, 75%-97%), whereas recipient-derived (i.e., hematopoietic) mtDNA molecules were mainly circular (median fraction, 88%; range, 77%-93%). The proportion of linear mtDNA was well correlated with liver DNA contribution in the plasma DNA pool ( r = 0.83; P value = 0.0008). Consistent data were obtained from a bone marrow transplantation recipient in whom the donor-derived (i.e., hematopoietic) mtDNA molecules were predominantly circular., Conclusions: Linear and circular mtDNA molecules coexist in plasma and may have different tissue origins., (© 2019 American Association for Clinical Chemistry.)

Epstein-Barr virus (EBV) is associated with a number of diseases, including malignancies. Currently, it is not known whether patients with different EBV-associated diseases have different methylation profiles of circulating EBV DNA. Through whole-genome methylation analysis of plasma samples from patients with nasopharyngeal carcinoma (NPC), EBV-associated lymphoma and infectious mononucleosis, we demonstrate that EBV DNA methylation profiles exhibit a disease-associated pattern. This observation implies a significant potential for the development of methylation analysis of plasma EBV DNA for NPC diagnostics. We further analyse the plasma EBV DNA methylome of NPC and non-NPC subjects from a prospective screening cohort. Plasma EBV DNA fragments demonstrate differential methylation patterns between NPC and non-NPC subjects. Combining such differential methylation patterns with the fractional concentration (count) and size of plasma EBV DNA, population screening of NPC is performed with an improved positive predictive value of 35.1%, compared to a count- and size-based only protocol.

Background: The current diagnosis and monitoring of bladder cancer are heavily reliant on cystoscopy, an invasive and costly procedure. Previous efforts in urine-based detection of bladder cancer focused on targeted approaches that are predicated on the tumor expressing specific aberrations. We aimed to noninvasively detect bladder cancer by the genome-wide assessment of methylomic and copy number aberrations (CNAs). We also investigated the size of tumor cell-free (cf)DNA fragments., Methods: Shallow-depth paired-end genome-wide bisulfite sequencing of urinary cfDNA was done for 46 bladder cancer patients and 39 cancer-free controls with hematuria. We assessed ( a ) proportional contribution from different tissues by methylation deconvolution, ( b ) global hypomethylation, ( c ) CNA, and ( d ) cfDNA size profile., Results: Methylomic and copy number approaches were synergistically combined to detect bladder cancer with a sensitivity of 93.5% (84.2% for low-grade nonmuscle-invasive disease) and a specificity of 95.8%. The prevalence of methylomic and CNAs reflected disease stage and tumor size. Sampling over multiple time points could assess residual disease and changes in tumor load. Muscle-invasive bladder cancer was associated with a higher proportion of long cfDNA, as well as longer cfDNA fragments originating from genomic regions enriched for tumor DNA., Conclusions: Bladder cancer can be detected noninvasively in urinary cfDNA by methylomic and copy number analysis without previous knowledge or assumptions of specific aberrations. Such analysis could be used as a liquid biopsy to aid diagnosis and for potential longitudinal monitoring of tumor load. Further understanding of the differential size and fragmentation of cfDNA could improve the detection of bladder cancer., (© 2019 American Association for Clinical Chemistry.)

Background: Early-stage nasopharyngeal carcinoma (NPC) evades detection when the primary tumor is hidden from view on endoscopic examination. Therefore, in a prospective study of subjects being screened for NPC using plasma Epstein-Barr virus (EBV) DNA, we conducted a study to investigate whether magnetic resonance imaging (MRI) could detect endoscopically occult NPC., Patients and Methods: Participants with persistently positive EBV DNA underwent endoscopic examination and biopsy when suspicious for NPC, followed by MRI blinded to the endoscopic findings. Participants with a negative endoscopic examination and positive MRI were recalled for biopsy or surveillance. Diagnostic performance was assessed by calculating sensitivity, specificity and accuracy, based on the histologic confirmation of NPC in the initial study or in a follow-up period of at least two years., Results: Endoscopic examination and MRI were performed on 275 participants, 34 had NPC, 2 had other cancers and 239 without cancer were followed-up for a median of 36 months (24-60 months). Sensitivity, specificity and accuracy were 76.5%, 97.5% and 94.9%, respectively, for endoscopic examination and 91.2%, 97.5% and 96.7%, respectively, for MRI. NPC was detected only by endoscopic examination in 1/34 (2.9%) participants (a participant with stage I disease), and only by MRI in 6/34 (17.6%) participants (stage I = 4, II = 1, III = 1), two of whom had stage I disease and follow-up showing slow growth on MRI but no change on endoscopic examination for 36 months., Conclusion: MRI has a complementary role to play in NPC detection and can enable the earlier detection of endoscopically occult NPC., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cell-free DNA (cfDNA) in human plasma is a class of biomarkers with many current and potential future diagnostic applications. Recent studies have shown that cfDNA molecules are not randomly fragmented and possess information related to their tissues of origin. Pathologies causing death of cells from particular tissues result in perturbations in the relative distribution of DNA from the affected tissues. Such tissue-of-origin analysis is particularly useful in the development of liquid biopsies for cancer. It is therefore of value to accurately determine the relative contributions of the tissues to the plasma DNA pool in a simultaneous manner. In this work, we report that in open chromatin regions, cfDNA molecules show characteristic fragmentation patterns reflected by sequencing coverage imbalance and differentially phased fragment end signals. The latter refers to differences in the read densities of sequences corresponding to the orientation of the upstream and downstream ends of cfDNA molecules in relation to the reference genome. Such cfDNA fragmentation patterns preferentially occur in tissue-specific open chromatin regions where the corresponding tissues contributed DNA into the plasma. Quantitative analyses of such signals allow measurement of the relative contributions of various tissues toward the plasma DNA pool. These findings were validated by plasma DNA sequencing data obtained from pregnant women, organ transplantation recipients, and cancer patients. Orientation-aware plasma DNA fragmentation analysis therefore has potential diagnostic applications in noninvasive prenatal testing, organ transplantation monitoring, and cancer liquid biopsy., (© 2019 Sun et al.; Published by Cold Spring Harbor Laboratory Press.)

Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3 -/- mice carrying Dnase1l3 +/- fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3 -proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3 Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity., Competing Interests: Conflict of interest statement: Y.M.D.L. is a scientific cofounder and a member of the scientific advisory board for Grail. Y.M.D.L., R.W.K.C., and K.C.A.C. hold equity in Grail and receive research funding from Grail/Cirina for other projects. Y.M.D.L., R.W.K.C., K.C.A.C., and P.J. are consultants to Grail. Y.M.D.L., R.W.K.C., and K.C.A.C. are cofounders and board members of DRA Company Limited. Y.M.D.L., R.W.Y.C., R.W.K.C., K.C.A.C., P.J., L.S., and B.R. plan to file a patent application based on the data generated from this work., (Copyright © 2019 the Author(s). Published by PNAS.)

Background: Folate level was proposed to be a predictor for fluoropyrimidine-related toxicity. We conducted a prospective study to determine the association between serum and red-cell folate and capecitabine-related toxicity in patients with colorectal cancers., Materials and Methods: Eligibility criteria included diagnosis of colorectal cancers; eligible patients who were scheduled to undergo capecitabine monotherapy or capecitabine-oxaliplatin (CAPOX) for adjuvant or palliative purposes. Exclusion criteria included concomitant radiotherapy or chemotherapy other than capecitabine or CAPOX and creatinine clearance <30 mL/min. Fasting serum and red-cell folate were measured prior to chemotherapy. Capecitabine was administered at 2,500 mg/m 2 per day (monotherapy) or 2,000 mg/m 2 per day (CAPOX) for 14 days every 3 weeks. The toxicity of the first four cycles was documented by clinical investigators who were blinded to folate levels., Results: A total of 144 patients were recruited, of whom 126 were eligible; 40 patients had capecitabine alone, and 86 patients received CAPOX. The rates of grade 2 and grade 3 toxicity were 63.5% and 14.3%, respectively. Nausea and vomiting were the most common grade ≥2 adverse event (47.7%), followed by hand-foot syndrome (25.4%), diarrhea (23.1%), and neutropenia (22.3%). Combination with oxaliplatin (odds ratio [OR], 2.77; p  = .043) and serum folate (OR, 10.33; p  = .002) were independent predictors of grade ≥2 toxicity. Red-cell folate was not predictive of toxicity. For every 10 nmol/L increment in serum folate, the risk of grade ≥2 toxicity increased by 9%., Conclusion: Serum folate level, but not red-cell folate, was associated with higher rate of grade ≥2 toxicity during capecitabine-based treatment. Excessive folate intake may be avoided before and during capecitabine-based chemotherapy., Implications for Practice: This is the first prospective study to evaluate the association between serum folate level and capecitabine-related toxicity in patients with colon cancers. It shows that higher serum folate level is associated with increased risks of moderate to severe toxicity during capecitabine-based treatment. Excessive folate intake should be avoided before and during capecitabine-based chemotherapy., Competing Interests: Disclosures of potential conflicts of interest may be found at the end of this article., (© AlphaMed Press 2018.)

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment., Competing Interests: Conflict of interest statement: The authors declare a conflict of interest. K.C.A.C., R.W.K.C., and Y.M.D.L. hold equities in Grail. P.J., K.C.A.C., R.W.K.C., and Y.M.D.L. are consultants to Grail. K.C.A.C., R.W.K.C., and Y.M.D.L. receive research funding from Grail. Y.M.D.L. is a scientific cofounder of and serves on the scientific advisory board of Grail. P.J., K.C.A.C., R.W.K.C., and Y.M.D.L. have filed patent applications based on the data generated from this work. Patent royalties are received from Grail, Illumina, Sequenom, and Xcelom., (Copyright © 2018 the Author(s). Published by PNAS.)

Background: Measurement of DNA derived from different tissues in the circulating DNA pool can provide important information regarding the presence of many pathological conditions. However, existing methods involving genome-wide bisulfite sequencing are relatively expensive and may present challenges for large-scale analysis., Methods: Through identifying differentially methylated regions in the liver and colon compared with other tissues, we identified 2 markers and developed corresponding droplet digital PCR assays. Plasma concentrations of liver-derived and colon-derived DNA were measured for 13 liver transplant recipients, 40 liver cancer patients, and 62 colorectal cancer (CRC) patients (27 with and 35 without liver metastases)., Results: In liver transplant recipients, the fractional concentration of liver-derived DNA measured using the liver-specific methylation marker and donor-specific alleles showed good correlation (Pearson R = 0.99). In liver cancer patients, the concentration of liver-derived DNA correlated positively with the maximal dimension of the tumor (Spearman R = 0.74). In CRC patients with and without liver metastasis, the plasma concentrations of colon-derived DNA (median, 138 copies/mL and 4 copies/mL, respectively) were increased compared with the 30 healthy controls (26 had undetectable concentrations). The absolute concentration of liver-derived DNA provided a better differentiation between CRC patients with and without liver metastasis compared with the fractional concentration (area under ROC curve, 0.85 vs 0.75)., Conclusions: Quantitative analysis of plasma DNA with tissue-specific methylation patterns using droplet digital PCR is applicable for the investigation of cancers and assessing organ transplantation. This approach is useful for differentiating patients with and without metastases to other organs., (© 2018 American Association for Clinical Chemistry.)

Cell-free DNA in human plasma is nonrandomly fragmented and reflects genomewide nucleosomal organization. Previous studies had demonstrated tissue-specific preferred end sites in plasma DNA of pregnant women. In this study, we performed integrative analysis of preferred end sites with the size characteristics of plasma DNA fragments. We mined the preferred end sites in short and long plasma DNA molecules separately and found that these "size-tagged" ends showed improved accuracy in fetal DNA fraction estimation and enhanced noninvasive fetal trisomy 21 testing. Further analysis revealed that the fetal and maternal preferred ends were generated from different locations within the nucleosomal structure. Hence, fetal DNA was frequently cut within the nucleosome core while maternal DNA was mostly cut within the linker region. We further demonstrated that the nucleosome accessibility in placental cells was higher than that for white blood cells, which might explain the difference in the cutting positions and the shortness of fetal DNA in maternal plasma. Interestingly, short and long size-tagged ends were also observable in the plasma of nonpregnant healthy subjects and demonstrated size differences similar to those in the pregnant samples. Because the nonpregnant samples did not contain fetal DNA, the data suggested that the interrelationship of preferred DNA ends, chromatin accessibility, and plasma DNA size profile is likely a general one, extending beyond the context of pregnancy. Plasma DNA fragment end patterns have thus shed light on production mechanisms and show utility in future developments in plasma DNA-based noninvasive molecular diagnostics., Competing Interests: Conflict of interest statement: K.C.A.C., R.W.K.C., and Y.M.D.L. hold equities in DRA and Grail, are consultants to Grail, and receive research funding from Grail/Cirina. P.J. is a consultant to Xcelom and Grail. Y.M.D.L. is a scientific cofounder and a member of the scientific advisory board for Grail., (Copyright © 2018 the Author(s). Published by PNAS.)

Circulating tumor-derived DNA testing for cancer screening has recently been demonstrated in a prospective study on identification of nasopharyngeal carcinoma (NPC) among 20,174 asymptomatic individuals. Plasma EBV DNA, a marker for NPC, was detected using real-time PCR. While plasma EBV DNA was persistently detectable in 97.1% of the NPCs identified, ∼5% of the general population had transiently detectable plasma EBV DNA. We hypothesized that EBV DNA in plasma of subjects with or without NPC may have different molecular characteristics. We performed target-capture sequencing of plasma EBV DNA and identified differences in the abundance and size profiles of EBV DNA molecules within plasma of NPC and non-NPC subjects. NPC patients had significantly higher amounts of plasma EBV DNA, which showed longer fragment lengths. Cutoff values were established from an exploratory dataset and tested in a validation sample set. Adopting an algorithm that required a sample to concurrently pass cutoffs for EBV DNA counting and size measurements, NPCs were detected at a positive predictive value (PPV) of 19.6%. This represented superior performance compared with the PPV of 11.0% in the prospective screening study, which required participants with an initially detectable plasma EBV DNA result to be retested within 4 weeks. The observed differences in the molecular nature of EBV DNA molecules in plasma of subjects with or without NPC were successfully translated into a sequencing-based test that had a high PPV for NPC screening and achievable through single time-point testing., Competing Interests: Conflict of interest statement: Y.M.D.L. is a scientific cofounder and member of the scientific advisory board for Grail. Y.M.D.L., R.W.K.C., and K.C.A.C. hold equity in Grail and receive research funding from Grail/Cirina. P.J. is a consultant to Xcelom and Grail. W.K.J.L. is a consultant to Grail. E.P.H. receives fees for serving on an advisory board for Bristol-Myers Squibb and Merck Sharp & Dohme. Y.M.D.L., R.W.K.C., K.C.A.C., P.J., and W.K.J.L. have filed patent applications based on the data generated from this work., (Copyright © 2018 the Author(s). Published by PNAS.)

Background: Plasma Epstein-Barr virus (pEBV) DNA and fluorodeoxyglucose positron emission (PET) reflect tumour burden in advanced NPC. This study hypothesised that a dual endpoint based on assessing pEBV DNA clearance and PET response could predict early drug response., Methods: Eligible patients underwent a computed tomography (CT) scan and dual PET-CT at baseline, a PET-CT at 4 weeks, and then a CT scan at 10 weeks after starting palliative or induction chemotherapy. Plasma EBV DNA clearance was determined., Results: Fifty-eight out of 70 enrolled patients completed all imaging and 50/58 had falling pEBV DNA level, which allowed calculation of the clearance. At a median follow-up of 29.1 months, the dual endpoint (pEBV DNA clearance ≤ 10 days and > 50% drop in sum of SUVmax of target lesions) was an independent indicator of overall survival (hazard ratio (HR) = 0.135, 95% CI = 0.039 to 0.466, p = 0.0015) and progression-free survival (HR = 0.136, 95% CI = 0.048 to 0.385, p = 0002). This dual endpoint could predict subsequent response by Response Evaluation Criteria In Solid Tumours (RECIST) criteria at 10 weeks after chemotherapy., Conclusions: Early PET-CT response and pEBV DNA clearance could predict survival and subsequent response. This dual endpoint is an innovative tool for assessing early drug response.

Background and Purpose: MR imaging can detect nasopharyngeal carcinoma that is hidden from endoscopic view, but for accurate detection carcinoma confined within the nasopharynx (stage T1) must be distinguished from benign hyperplasia of the nasopharynx. This study aimed to document the MR imaging features of stage T1 nasopharyngeal carcinoma and to attempt to identify features distinguishing it from benign hyperplasia., Materials and Methods: MR images of 189 patients with nasopharyngeal carcinoma confined to the nasopharynx and those of 144 patients with benign hyperplasia were reviewed and compared in this retrospective study. The center, volume, size asymmetry (maximum percentage difference in area between the right and left nasopharyngeal halves), signal intensity asymmetry, deep mucosal white line (greater contrast enhancement along the deep tumor margin), and absence/distortion of the adenoidal septa were evaluated. Differences were assessed with logistic regression and the χ 2 test., Results: The nasopharyngeal carcinoma center was lateral, central, or diffuse in 134/189 (70.9%), 25/189 (13.2%), and 30/189 (15.9%) cases, respectively. Nasopharyngeal carcinomas involving the walls showed that a deep mucosal white line was present in 180/183 (98.4%), with a focal loss of this line in 153/180 (85%) cases. Adenoidal septa were absent or distorted in 111/111 (100%) nasopharyngeal carcinomas involving the adenoid. Compared with benign hyperplasia, nasopharyngeal carcinoma had a significantly greater volume, size asymmetry, signal asymmetry, focal loss of the deep mucosal white line, and absence/distortion of the adenoidal septa ( P < .001). Although size asymmetry was the most accurate criterion (89.5%) for nasopharyngeal carcinoma detection, use of this parameter alone would have missed 11.9% of early-stage T1 nasopharyngeal carcinomas., Conclusions: MR imaging features can help distinguish stage T1 nasopharyngeal carcinoma from benign hyperplasia in most cases., (© 2018 by American Journal of Neuroradiology.)