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BACKGROUND: Plasma cell-free DNA (cfDNA) is derived from cellular death in various tissues. Investigating the tissue origin of cfDNA through cell type deconvolution, we can detect changes in tissue homeostasis that occur during disease progression or in response to treatment. Consequently, cfDNA has emerged as a valuable noninvasive biomarker for disease detection and treatment monitoring. Although there are many methylation-based methods for cfDNA cell type deconvolution, a comprehensive and systematic evaluation of these methods has yet to be conducted. RESULTS: In this study, we benchmark five methods: MethAtlas, cfNOMe toolkit, CelFiE, CelFEER, and UXM. Utilizing deep whole-genome bisulfite sequencing data from 35 human cell types, we generate in silico cfDNA samples with ground truth cell type proportions to assess the deconvolution performance of the five methods under multiple scenarios. Our findings indicate that multiple factors, including reference marker selection, sequencing depth, and reference atlas completeness, jointly influence the deconvolution performance. Notably, an incomplete reference with missing markers or cell types leads to suboptimal results. We observe performance differences among methods under varying conditions, underscoring the importance of tailoring cfDNA deconvolution analyses. To increase the clinical relevance of our findings, we further evaluate each methods performance in potential clinical applications using real-world datasets. CONCLUSIONS: Based on the benchmark results, we propose general guidelines to choose the suitable methods based on sequencing depth of the cfDNA data and completeness of the reference atlas to maximize the performance of methylation-based cfDNA cell type deconvolution.

Non-invasive prenatal testing (NIPT) employs ultra-low-pass sequencing of maternal plasma cell-free DNA to detect fetal trisomy. Its global adoption has established NIPT as a large human genetic resource for exploring genetic variations and their associations with phenotypes. Here, we present methods for analyzing large-scale, low-depth NIPT data, including customized algorithms and software for genetic variant detection, genotype imputation, family relatedness, population structure inference, and genome-wide association analysis of maternal genomes. Our results demonstrate accurate allele frequency estimation and high genotype imputation accuracy (R2>0.84) for NIPT sequencing depths from 0.1× to 0.3×. We also achieve effective classification of duplicates and first-degree relatives, along with robust principal-component analysis. Additionally, we obtain an R2>0.81 for estimating genetic effect sizes across genotyping and sequencing platforms with adequate sample sizes. These methods offer a robust theoretical and practical foundation for utilizing NIPT data in medical genetic research.

A large U.S. clinical validation study of a commercially available noninvasive prenatal cell-free DNA test for fetal RHD shows high test performance. OBJECTIVE: To present a large U.S. clinical validation of a next-generation sequencing–based, noninvasive prenatal cell-free DNA test for fetal RHD. METHODS: This clinical validation study assessed the performance of a commercially available, next-generation sequencing–based cell-free DNA test for fetal RHD status. Samples that passed quality metrics were included if the patient had a previously reported cell-free DNA result for fetal aneuploidy, maternal RhD-negative serology, newborn RhD serology, and maternal RHD deletion or RHD-CE-D hybrid(r's) genotype. Dizygotic twin pregnancies were excluded. Maternal and fetal RHD genotypes were evaluated with prospective cell-free DNA next-generation sequencing analysis. At the time of analysis, investigators were blinded to fetal RhD status. RESULTS: The cohort consisted of 655 pregnant patients with serologic results for RhD antigen. Patient demographics included a representative distribution of race and ethnicities in the RhD-negative U.S. population (74.0% White, 13.7% Hispanic, 7.0% Black, and 2.1% Asian). Cell-free DNA fetal RHD was not reported in two cases. There were zero false-negative cases; 356 of 356 fetuses were correctly identified as fetal RhD positive (sensitivity 100%, 95% CI, 98.9–100%). Of the 297 RhD-negative fetuses, 295 were correctly identified as RhD negative (specificity 99.3%, 95% CI, 97.6–99.8%). Of the fetuses with a negative RhD phenotype, the cell-free DNA test accurately identified three with the fetal RHD pseudogene (RHDΨ) genotype. CONCLUSION: Validation of this test in this large U.S. cohort of RhD-negative patients provides data on early and accurate noninvasive prenatal identification of fetal RHD genotype at 9 weeks of gestation or more. This test has the potential to assist patients and clinicians in the prevention and management of RhD alloimmunization. [ABSTRACT FROM AUTHOR]

Hemangiosarcoma is a highly malignant tumor commonly affecting canines, originating from endothelial cells that line blood vessels, underscoring the importance of early detection. This canine cancer is analogous to human angiosarcoma, and the development of liquid biopsies leveraging cell-free DNA (cfDNA) represents a promising step forward in early cancer diagnosis. In this study, we utilized Whole Genome Sequencing (WGS) to analyze fragment sizes and copy number alterations (CNAs) in cfDNA from 21 hemangiosarcoma-affected and 36 healthy dogs, aiming to enhance early cancer detection accuracy through machine learning models. Our findings reveal that similar to trends in human oncology, hemangiosarcoma samples exhibited shorter DNA fragment sizes compared to healthy controls, with a notable leftward shift in the primary peak. Interestingly, canine hemangiosarcoma DNA fragment sizes demonstrated eight distinct periodic patterns diverging from those typically observed in human angiosarcoma. Additionally, we identified seven novel genomic gains and nine losses in the hemangiosarcoma samples. Applying machine learning to the cfDNA fragment size distribution, we achieved an impressive average Area Under the Curve (AUC) of 0.93 in 10-fold cross-validation, underscoring the potential of this approach for precise early-stage cancer classification. This study confirms distinctive cfDNA fragment size and CNA patterns in hemangiosarcoma-affected vs. healthy dogs and demonstrates the promise of these biomarkers in canine cancer screening, early detection, and monitoring via liquid biopsies. These findings establish a foundation for broader research on cfDNA analysis in various canine cancers, integrating methodologies from human oncology to enhance early detection and diagnostic precision in veterinary medicine. [ABSTRACT FROM AUTHOR]

The management of early-stage colon cancer involves surgical resection of the primary tumor with or without chemotherapy, depending on pathological staging. The benefit of adjuvant chemotherapy for stage II and III colon cancer is approximately 5% and 15%, indicating the need for optimization for risk stratification and patient selection. Several studies have revealed that current clinicopathological factors lack precision. Circulating tumor DNA (ctDNA) is cell-free DNA originating from cancer cells and can be detected even in the absence of radiologically detectable disease among patients with colon cancer. Recent cohort studies revealed that ctDNA is one of the most significant prognostic factors for patients with early-stage colon cancer, surpassing pathological and clinical risk factors. Prospective cohort studies also suggest there may be a predictive role for ctDNA on the decision for consideration of adjuvant therapy. Currently, randomized clinical trials are enrolling to better define this role. In this review article, we review recent literature on ctDNA and its role in patients with colon cancer. We also elaborate on the future clinical utility of ctDNA in clinical practice and the unmet need for research to optimize currently available ctDNA assays. [ABSTRACT FROM AUTHOR]

Second Primary Cancers (SPCs) are defined as cancers that develop either simultaneously or metachronously in the same individual who has been diagnosed with and survived one primary cancer. SPCs exhibit a high incidence rate and represent the primary cause of mortality among survivors of first primary cancers. There is growing concern about the dangers of SPCs. This review summarizes recent studies on the mechanisms of SPCs, including the roles of genomic changes after first primary cancer (FPC) treatments, stromal cell phenotypic and metabolic changes, hormone levels and receptor expression, immunosuppression, aberrant gene methylation, EGFR signaling, and cell-free DNA in SPC development. This comprehensive analysis contributes to elucidating current research trends in SPC mechanisms and enhances our understanding of the underlying pathophysiology. Furthermore, potential applications of intratumoral microbes, single-cell multi-omics, and metabolomics in investigating SPC mechanisms are also discussed, providing new ideas for follow-up studies. [ABSTRACT FROM AUTHOR]

Background: Noninvasive prenatal testing (NIPT) is increasingly used to screen for fetal chromosomal aneuploidy by analyzing cell-free DNA (cfDNA) in peripheral maternal blood. The method provides an opportunity for early detection of large genetic abnormalities without an increased risk of miscarriage due to invasive procedures. Commercial applications for use at clinical laboratories often take advantage of DNA sequencing technologies and include the bioinformatic workup of the sequence data. The interpretation of the test results and the clinical report writing, however, remains the responsibility of the diagnostic laboratory. In order to facilitate this step, we developed NIPTviewer, a web-based application to visualize and guide the interpretation of NIPT data results. Results: NIPTviewer has a database functionality to store the NIPT results and a web interface for user interaction and visualization. The application has been implemented as part of a novel analysis pipeline for NIPT in a diagnostic laboratory at Uppsala University Hospital. The validation data set included 84 previously analyzed plasma samples with known results regarding chromosomes 13, 18, 21, X and Y. They were sequenced in six different experiments, uploaded to NIPTviewer and assigned to a clinical laboratory geneticist for interpretation. The results of all previously analyzed samples were replicated. Conclusion: NIPTviewer facilitates NIPT results interpretation and has been implemented as part of a NIPT analysis routine that was accredited by the national accreditation body for Sweden (Swedac). [ABSTRACT FROM AUTHOR]

Cell-free DNA (cfDNA) has attracted increasing attention as a promising biomarker in liquid biopsy due to its crucial role in disease diagnosis. However, previous cfDNA detection methods are commonly based on the development of target-specific primers and integrated signal amplification strategies, which may induce false-positive results. This paper presents a sensitive yet accurate method for cfDNA detection that combines phosphorothioated-terminal hairpin creation with a self-priming extension process. This approach initiates a self-priming mediated chain extension-based signal cycle following the trans-cleavage of H0@MBs when the CRISPR-Cas12a complex is activated by target cfDNA, resulting in the production of a substantial quantity of pyrophosphate. A pyrophosphate sensing probe (pp probe) was utilized, facilitating both high-efficiency and stable colorimetric signaling. This innovative technique for colorimetric detection of target cfDNA demonstrated exceptional sensitivity with a low limit of detection of 1.04 fM and greatly enhanced selectivity, with the complete detection process taking around 60 min. In addition, this technique is capable of detecting cfDNA from the culture medium of HEK293 cells, indicating its clinical application potential. Compared with the previous CRISPR-Cas system-based cfDNA method that necessitates an amplification step before detection, Cas12a was directly used to identify a target sequence that can avoid false target amplification. This technique is simple, accurate, and rapid, engineered to identify cancer-associated cfDNA via a highly sensitive colorimetric change, which is expected to be beneficial for applications requiring point-of-care cancer detection. [ABSTRACT FROM AUTHOR]

Enzyme engineering is limited by the challenge of rapidly generating and using large datasets of sequence-function relationships for predictive design. To address this challenge, we develop a machine learning (ML)-guided platform that integrates cell-free DNA assembly, cell-free gene expression, and functional assays to rapidly map fitness landscapes across protein sequence space and optimize enzymes for multiple, distinct chemical reactions. We apply this platform to engineer amide synthetases by evaluating substrate preference for 1217 enzyme variants in 10,953 unique reactions. We use these data to build augmented ridge regression ML models for predicting amide synthetase variants capable of making 9 small molecule pharmaceuticals. Over these nine compounds, ML-predicted enzyme variants demonstrate 1.6- to 42-fold improved activity relative to the parent. Our ML-guided, cell-free framework promises to accelerate enzyme engineering by enabling iterative exploration of protein sequence space to build specialized biocatalysts in parallel. While machine learning shows promise in expanding protein engineering efforts, its potential is limited by the challenge of gathering large datasets of sequence-function relationships. Here, authors introduce a platform that integrates cell-free DNA assembly and gene expression to accelerate enzyme engineering. [ABSTRACT FROM AUTHOR]

The emergence of liquid biopsy technologies holds great promise in the cancer setting, including in pediatric central nervous system (CNS) tumors. In contrast to broad lower-depth sequencing, commonly referred to as low pass whole genome sequencing (WGS), targeted platforms with a higher depth of coverage have also been established. Here, we review targeted liquid biopsy techniques with applicability to pediatric CNS tumors. These include polymerase chain reaction (PCR), both droplet digital PCR and reverse transcription-based PCR, Sanger sequencing, and next-generation sequencing approaches that incorporate amplicon- and hybrid capture-based methods. The goal of this paper is to facilitate an understanding of these targeted techniques and provide a context for clinical relevance within disease categories, as well as a discussion on optimizing real-world implementation for pediatric CNS tumors. [ABSTRACT FROM AUTHOR]

The treatment landscape for advanced melanoma has transformed significantly with the advent of BRAF and MEK inhibitors (BRAF/MEKi) targeting BRAFV600 mutations, as well as immune checkpoint inhibitors (ICI) like anti-PD-1 monotherapy or its combinations with anti-CTLA-4 or anti-LAG-3. Despite that, many patients still do not benefit from these treatments at all or develop resistance mechanisms. Therefore, prognostic and predictive biomarkers are needed to identify patients who should switch or escalate their treatment strategies or initiate an intensive follow-up. In melanoma, liquid biopsy has shown promising results, with a potential role in predicting relapse in resected high-risk patients or in disease monitoring during the treatment of advanced disease. Several components in peripheral blood have been analyzed, such as circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulant tumoral DNA (ctDNA), which have turned out to be particularly promising. To analyze ctDNA in blood, different techniques have proven to be useful, including digital droplet polymerase chain reaction (ddPCR) to detect specific mutations and, more recently, next-generation sequencing (NGS) techniques, which allow analyzing a broader repertoire of the mutation landscape of each patient. In this review, our goal is to update the current understanding of liquid biopsy, focusing on the use of ctDNA as a biological material in the daily clinical management of melanoma patients, in particular those with advanced disease treated with ICI. [ABSTRACT FROM AUTHOR]

The progression of colorectal cancer is strongly influenced by environmental and genetic conditions. One of the key factors is tumor heterogeneity which is extensively studied by cfDNA and bulk sequencing methods; however, we lack knowledge regarding its effects at the single-cell level. Motivated by this, we aimed to employ an end-to-end single-cell sequencing workflow from tissue-derived sample isolation to exome sequencing. Our main goal was to investigate the heterogeneity patterns by laser microdissecting samples from different locations of a tissue slide. Moreover, by studying healthy colon control, tumor-associated normal, and colorectal cancer tissues, we explored tissue-specific heterogeneity motifs. For completeness, we also compared the performance of the whole-exome bulk, cfDNA, and single-cell sequencing methods based on variation at the level of a single nucleotide. [ABSTRACT FROM AUTHOR]

Non-invasive prenatal testing (NIPT) has been widely adopted for the screening of chromosomal abnormalities; however, its adoption for monogenic disorders, such as β-thalassaemia, has proven challenging. Haemoglobinopathies are the most common monogenic disorders globally, with β-thalassaemia being particularly prevalent in Cyprus. This study introduces a non-invasive prenatal haplotyping (NIPH) assay for β-thalassaemia, utilizing cell-free DNA (cfDNA) from maternal plasma. The assay determines paternal inheritance by analyzing highly heterozygous single-nucleotide variants (SNVs) in the β-globin gene cluster. To identify highly heterozygous SNVs in the population, 96 randomly selected samples were processed using Illumina DNA-prep NGS chemistry. A custom, high-density NGS genotyping panel, named HAPLONID, was designed with 169 SNVs, including 15 common pathogenic ones. The AmpliSeq for Illumina assay was then applied to cfDNA to evaluate the panel's efficiency in performing NIPT for β-thalassaemia. Analysis revealed 219 highly polymorphic SNVs, and the sequencing of 17 families confirmed successful paternal allele determination. The NIPH assay demonstrated 100% success in diagnostic interpretation. This study achieved the advancement of an integrated NGS-NIPT assay for β-thalassaemia, bringing it one step closer to being a diagnostic assay and thereby enabling a reduction in the number of risky invasive prenatal sampling procedures in Cyprus and elsewhere. [ABSTRACT FROM AUTHOR]

Purpose: Renal cell carcinoma (RCC) is a common malignancy, with patients frequently diagnosed at an advanced stage due to the absence of sufficiently sensitive detection technologies, significantly compromising patient survival and quality of life. Advances in cell-free DNA (cfDNA) methylation profiling using liquid biopsies offer a promising non-invasive diagnostic option, but robust biomarkers for early detection are current not available. This study aimed to identify methylation biomarkers for RCC and establish a DNA methylation signature-based prognostic model for this disease. Methods: High-throughput methylation sequencing was performed on peripheral blood samples obtained from 49 primarily Stage I RCC patients and 44 healthy controls. Comparative analysis and Least Absolute Shrinkage and Selection Operator (LASSO) regression methods were employed to identify RCC methylation signatures.Subsequently, methylation markers-based diagnostic and prognostic models for RCC were independently trained and validated using random forest and Cox regression methodologies, respectively. Results: Comparative analysis revealed 864 differentially methylated CpG islands (DMCGIs), 96.3% of which were hypermethylated. Using a training set from The Cancer Genome Atlas (TCGA) dataset of 443 early-stage RCC tumors and matched normal tissues, we applied LASSO regression and identified 23 methylation signatures. We then constructed a random forest-based diagnostic model for early-stage RCC and validated the model using two independent datasets: a TCGA set of 460 RCC tumors and controls, and a blood sample set from our study of 15 RCC cases and 29 healthy controls. For Stage I RCC tissue, the model showed excellent discrimination (AUC-ROC: 0.999, sensitivity: 98.5%, specificity: 100%). Blood sample validation also yielded commendable results (AUC-ROC: 0.852, sensitivity: 73.9%, specificity: 89.7%). Further analysis using Cox regression identified 7 of the 23 DMCGIs as prognostic markers for RCC, allowing the development of a prognostic model with strong predictive power for 1-, 3-, and 5-year survival (AUC-ROC > 0.7). Conclusions: Our findings highlight the critical role of hypermethylation in RCC etiology and progression, and present these identified biomarkers as promising candidates for diagnostic and prognostic applications. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) lacks a specific biomarker, but is defined by relatively selective toxicity to motor neurons (MN). As others have highlighted, this offers an opportunity to develop a sensitive and specific biomarker based on detection of DNA released from dying MN within accessible biofluids. Here we have performed whole genome bisulfite sequencing (WGBS) of iPSC-derived MN from neurologically normal individuals. By comparing MN methylation with an atlas of tissue methylation we have derived a MN-specific signature of hypomethylated genomic regions, which accords with genes important for MN function. Through simulation we have optimised the selection of regions for biomarker detection in plasma and CSF cell-free DNA (cfDNA). However, we show that MN-derived DNA is not detectable via WGBS in plasma cfDNA. In support of our experimental finding, we show theoretically that the relative sparsity of lower MN sets a limit on the proportion of plasma cfDNA derived from MN which is below the threshold for detection via WGBS. Our findings are important for the ongoing development of ALS biomarkers. The MN-specific hypomethylated genomic regions we have derived could be usefully combined with more sensitive detection methods and perhaps with study of CSF instead of plasma. Indeed we demonstrate that neuronal-derived DNA is detectable in CSF. Our work is relevant for all diseases featuring death of rare cell-types. [ABSTRACT FROM AUTHOR]

Introduction: Placental DNA methylation differences have been associated with timing in gestation and pregnancy complications. Maternal cell-free DNA (cfDNA) partly originates from the placenta and could enable the minimally invasive study of placental DNA methylation dynamics. We will for the first time longitudinally investigate cfDNA methylation during pregnancy by using Methylated DNA Sequencing (MeD-seq), which is compatible with low cfDNA levels and has an extensive genome-wide coverage. We aim to investigate DNA methylation in placental tissues and cfDNA during different trimesters in uncomplicated pregnancies, and in pregnancies with placental-related complications, including preeclampsia and fetal growth restriction. Identified gestational-age and disease-specific differentially methylated regions (DMRs) could lead to numerous applications including biomarker development. Methods and analysis: Our study design involves three sub-studies. Sub-study 1 is a single-centre prospective, observational subcohort embedded within the Rotterdam Periconception cohort (Predict study). We will longitudinally collect maternal plasma in each trimester and during delivery, and sample postpartum placentas (n = 300). In sub-study 2, we will prospectively collect first and second trimester placental tissues (n = 10 per trimester). In sub-study 3 we will retrospectively collect plasma after non-invasive prenatal testing (NIPT) in an independent validation case-control cohort (n = 30–60). A methylation-dependent restriction enzyme (LpnPI) will be used to generate DNA fragments followed by sequencing on the Illumina NextSeq2000 platform. DMRs will be identified in placental tissues and cell types, and in cfDNA related to gestational-age or placental-related complications. (Paired) placental methylation profiles will be correlated to DMRs in cfDNA to aid tissue-of-origin analysis. We will establish a methylation score to predict associated diseases. Discussion: This study will provide insights in placental DNA methylation dynamics in health and disease, and could lead to clinical relevant biomarkers. [ABSTRACT FROM AUTHOR]

Plasma cell‐free DNA (cfDNA) analysis to track estrogen receptor 1 (ESR1) mutations is highly beneficial for the identification of tumor molecular dynamics and the improvement of personalized treatments for patients with metastatic breast cancer (MBC). Plasma‐cfDNA is, up to now, the most frequent liquid biopsy analyte used to evaluate ESR1 mutational status. Circulating tumor cell (CTC) enumeration and molecular characterization analysis provides important clinical information in patients with MBC. In this study, we investigated whether analysis of CTCs and circulating tumor DNA (ctDNA) provide similar or complementary information for the analysis of ESR1 mutations. We analyzed both plasma‐cfDNA (n = 90) and paired CTC‐derived genomic DNA (gDNA; n = 42) from 90 MBC patients for seven ESR1 mutations. Eight out of 90 (8.9%) plasma‐cfDNA samples tested using the ddPLEX Mutation Detection Assay (Bio‐Rad, Hercules, CA, USA), were found positive for one ESR1 mutation, whereas 11/42 (26.2%) CTC‐derived gDNA samples were found positive for at least one ESR1 mutation. Direct comparison of paired samples (n = 42) revealed that the ESR1 mutation rate was higher in CTC‐derived gDNA (11/42, 26.2%) than in plasma‐cfDNA (6/42, 14.3%) samples. Our results, using this highly sensitive ddPLEX assay, reveal a higher percentage of mutations in CTC‐derived gDNAs than in paired ctDNA in patients with MBC. CTC‐derived gDNA analysis should be further evaluated as an important and complementary tool to ctDNA for identifying patients with ESR1 mutations and for guiding individualized therapy. [ABSTRACT FROM AUTHOR]

Background: Cell-free DNA (cfDNA) is present in healthy individuals but is elevated in those undergoing physical exertion, trauma, sepsis, and certain cancers. Maintaining cfDNA concentrations is vital for immune homeostasis and preventing inflammatory responses. Understanding cfDNA release and clearance is essential for using cfDNA as a biomarker in clinical diagnostics. We focused on the fragment size of cfDNA and investigated cfDNA dynamics and half-life, particularly the 100–250 base pair fragments. Methods: Healthy, adult men (n = 5; age 40 ± 4.1 years) were subjected to a 30 min treadmill exercise. Blood samples were collected at 0, 5, 10, 15, 30, and 60 min post-exercise using PAXgene® Blood ccfDNA tubes to stabilize and prevent nuclease-mediated cfDNA degradation and minimize genomic DNA contamination risk. The cfDNA concentration was measured using an electrophoresis-based technique (4150 TapeStation system) to quantify the concentration based on cfDNA fragment size. Results: The results showed a cfDNA half-life of 24.2 min, with a transient increase in 100–250 base pair cfDNA fragments post-exercise, likely due to nuclease activity. These levels rapidly reverted to the baseline within an hour. Conclusions: The rapid clearance of cfDNA underscores its potential as a biomarker for real-time disease monitoring and the evaluation of treatment efficacy. This study is expected to standardize cfDNA investigations, enhancing diagnosis and treatment monitoring across various disease conditions. [ABSTRACT FROM AUTHOR]

Background: Given the poor prognosis of metastatic pancreatic adenocarcinoma (mPDAC), closer disease monitoring through liquid biopsy, most frequently based on serial measurements of cell-free mutated KRAS (KRASmut cfDNA), has become a highly active research focus, aimed at improving patients' long-term outcomes. However, most of the available data show only a limited predictive and prognostic value of single-parameter-based methods. We hypothesized that a combined longitudinal analysis of KRASmut cfDNA and novel protein biomarkers could improve risk stratification and molecular monitoring of patients with mPDAC. Methods: We prospectively collected 160 plasma samples from 47 patients with mPDAC at our institution. Highly sensitive single-target ddPCR assays were employed to detect and quantify KRASmut cfDNA. Additionally, analysis of ten protein biomarkers was performed through Enzyme-linked Immunosorbent Assay (ELISA), and Carbohydrate-Antigen 19-9 (CA 19-9) dynamics were registered. Results: KRASmut cfDNA was detectable in 37/47 (78.7%) patients throughout the course of study, and CA 19-9 levels were elevated in 40 out of 47 (85.1%) patients. KRASmut cfDNA increase at the time of the first follow-up could predict inferior progression-free survival (PFS) (Hazard ratio (HR) = 3.40, p = 0.0003) and overall survival (OS) (HR = 4.91, p < 0.0001). In contrast to CA 19-9 kinetics, which were not predictive of outcome, integrated analysis of KRASmut cfDNA combined with six evaluated circulating protein biomarkers allowed basal risk stratification at the time of the first follow-up (HR = 10.2, p = 0.0014). Conclusions: A combined longitudinal analysis of KRASmut cfDNA with selected protein biomarkers offers significantly improved prognostic value for patients with mPDAC compared to single-parameter methods. This innovative approach is a step forward in the molecular monitoring of mPDAC and should be validated in further prospective studies. [ABSTRACT FROM AUTHOR]

Endometriosis is a chronic, estrogen-dependent disorder associated with the presence of endometrial cells mainly in the pelvic cavity, causing systemic immune inflammation, infertility, epigenetic dysregulation of differential DNA methylation, coelomic metaplasia, and pain. It affects approximately 10–12% of women. Despite decades of research, full pathophysiology, a diagnostic roadmap, and clinical management strategies for endometriosis are not yet fully elucidated. Cell-free DNA (Cf-DNA) in the peripheral blood of diseased and healthy individuals was discovered in the 1950s. Quantifying peripheral Cf-DNA and the specific differential methylation of a group of genes have been proposed as potential non-invasive diagnostic biomarkers for somatic and constitutional genetics and for various other pathological disorders. In this study, we investigated the Cf-DNA levels of 78 young women, 38 of whom had endometriosis confirmed via laparoscopy and 40 of whom were healthy. We found a significant difference between the two groups when Cf-DNA was quantified, with 3.9 times more Cf-DNA in the serum of women with endometriosis. We also identified nine target genes potentially involved in the pathogenesis of endometriosis, with a different methylation profile between the two groups. Our data suggest that the combination of cell-free DNA quantification and the assessment of the epigenetic signature of differential methylation of nine genes can be proposed as a non-invasive predictive and diagnostic test for endometriosis. [ABSTRACT FROM AUTHOR]

PURPOSE: Circulating tumor DNA (ctDNA) analysis is an alternative to tissue biopsy for genotyping in various cancers. We aimed to establish a plasma ctDNA sequencing assay, then evaluate its clinical utility in advanced urothelial cancer (UC). MATERIALS AND METHODS: This study included 82 patients with muscle-invasive or metastatic UC. A total of 158 blood samples and 73 tumor tissue samples were sequenced using our designed panel covering 53 UC-relevant genes and TERT promoter. We investigated the association between the proportion of ctDNA in total cell-free DNA (ctDNA fraction) and response to pembrolizumab treatment. ctDNA dynamics were explored during systemic therapy. RESULTS: In paired tissue-ctDNA samples with estimated tumor fraction, half (50.2%) of the somatic mutations were shared between both sources, while some (17.6%) mutations were found only in ctDNA. In the metastatic setting, on-treatment increase in ctDNA fraction during pembrolizumab treatment was significantly associated with a poor response rate (18.7% v 76.1%; P =.001) and progression-free survival (median 2.8 v 9.8 months; P <.001). A comparison of cancer cell fraction, the fraction of tumor cells carrying somatic mutations, between pembrolizumab initiation and on-treatment showed a strong correlation among patients where ctDNA fraction increased during treatment (r = 0.73). By contrast, no correlation was observed in patients without ctDNA fraction increase (r = 0.09). Most mutations newly detected at pembrolizumab resistance have already been identified in tumor tissues at earlier stages. CONCLUSION: Our newly established assay is suitable for assessing the genomic profile of ctDNA in patients with advanced UC. Our data may support future analyses of prognostic or predictive biomarkers for UC. [ABSTRACT FROM AUTHOR]

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

Objective Low fetal fraction (FF) on cell-free DNA (cfDNA)-based noninvasive prenatal screening (NIPS) is a common etiology for indeterminate results. As maternal Black race is implicated as a risk factor for low FF and more indeterminate results, we sought to evaluate this association. Study Design This was a single-institution, retrospective cohort study of cfDNA-based NIPS performed between May 2017 and May 2022 with complete clinical data abstraction. We compared FF, indeterminate rates, and total cfDNA concentration among self-reported Black, White, and Other groups from NIPS results from 2017 to 2022 with full clinical data abstraction. Using linear regression and interaction testing, we evaluated associations between self-reported race, FF, indeterminate rate, and total cfDNA concentration. Results In total, 1,591 participants met the inclusion criteria; 70.8% (n = 1,126) self-identified as White, 6.9% (n = 110) as Black, and 22.3% (n = 355) self-identified with another race. Mean FF was not different between the White, Black, or Other groups (11.8 vs. 11.2 vs. 11.7%, respectively, p = 0.52). This remained true after adjusting for body mass index (BMI), gestational age (GA) at draw, and fetal sex (all p > 0.17). Interaction testing for FF and total cfDNA by race with BMI, GA at draw, and fetal sex demonstrated no effect modification. Conclusion In our population, maternal self-identified race, particularly Black race, does not affect FF. Biological plausibility for race-based differences on clinical tests requires ongoing thoughtful consideration. Key Points NIPS is widely used to screen for fetal aneuploidy. FF is an important test metric, and low FF is associated with adverse outcomes, like aneuploidy. In existing studies, Black race is implicated as a risk factor for lower FF. Our study found no differences in FF between groups by self-reported race. Biological plausibility for race-based differences on clinical tests requires ongoing consideration. [ABSTRACT FROM AUTHOR]

Background: A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma. Questions/purposes: Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes? Methods: This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software. Results: This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable. Conclusion: In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes. Clinical Relevance: These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose. [ABSTRACT FROM AUTHOR]

Blood cell-free DNA (cfDNA) can be a new reliable tool for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. However, the currently reported cfDNA assays have a limited role in detecting drug-resistant mutations due to their deficiencies in sensitivity, stability, or mutation detection rate. We developed an Archaeoglobus fulgidus-derived flap endonuclease (Afu FEN)-based DNA-enhanced amplification system of mutated cfDNA by designing a pair of hairpin probes to anneal with wild-type cfDNA to form two 5′-flaps, allowing for the specific cleavage of wild-type cfDNA by Afu FEN. When the dominant wild-type somatic cfDNA fragments were cleaved by structure-recognition-specific Afu FEN, the proportion of mutated cfDNA in the reaction system was greatly enriched. As the amount of mutated cfDNA in the system was further increased by PCR amplification, the mutation status could be easily detected through first-generation sequencing. In a mixture of synthetic wild-type and T790M EGFR DNA fragments, our new assay still could detect T790M mutation at the fg level with remarkably high sensitivity. We also tested its performance in detecting low variant allele frequency (VAF) mutations in clinical samples from NSCLC patients. The plasma cfDNA samples with low VAF (0.1 and 0.5 %) could be easily detected by DNA-enhanced amplification. This system with enhanced amplification of mutated cfDNA is an effective tool used for the early screening and individualized targeted therapy of NSCLC by providing a rapid, sensitive, and economical way for the detection of drug-resistant mutations in tumors. [ABSTRACT FROM AUTHOR]

Objective: To report the diagnostic accuracy of cell‐free fetal DNA (cfDNA) in detecting fetal chromosomal anomalies in women experiencing miscarriage. Methods: PubMed, MEDLINE, EMBASE and Cochrane databases were searched from inception to June 2024. The inclusion criteria were women experiencing miscarriage (defined as pregnancy loss before 20 weeks of gestation) who underwent cfDNA screening for trisomies 21, 18 and 13, other autosomal aneuploidies, sex‐chromosome aneuploidies and/or copy‐number variants (CNVs). The index test was cfDNA screening for each of the chromosomal anomalies listed. The reference standard was cytogenetic analysis of pregnancy tissue. The quality of the studies was assessed using the revised tool for the quality assessment of diagnostic accuracy studies. Summary estimates of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio were computed using the hierarchical summary receiver‐operating‐characteristics model. Results: Seven studies (887 women) were included in the systematic review and meta‐analysis. cfDNA had a sensitivity and specificity of 100% (95% CI, 81.5–100%) and 100% (95% CI, 99.1–100%), respectively, for trisomy 21, 100% (95% CI, 54.1–100%) and 100% (95% CI, 99.0–100%), respectively, for trisomy 18, and 88.9% (95% CI, 51.8–99.7%) and 100% (95% CI, 99.1–100%), respectively, for trisomy 13. The respective values for other autosomal trisomies were 75.8% (95% CI, 65.7–84.2%) and 99.4% (95% CI, 97.9–99.9%), while those for CNVs were 60.0% (95% CI, 14.7–94.7%) and 100% (95% CI, 97.4–100%). Failure of cfDNA testing was reported in 7.3% (95% CI, 5.7–9.2%) of cases. Conclusion: cfDNA has high diagnostic accuracy in detecting fetal trisomies 21, 18 and 13 in women experiencing miscarriage, while its accuracy for other autosomal aneuploidies and CNVs is only moderate. © 2024 International Society of Ultrasound in Obstetrics and Gynecology. [ABSTRACT FROM AUTHOR]

Background/Aim: Cholangiocarcinoma (CCA) is an aggressive hepatobiliary malignancy characterized by genomic heterogeneity. KRAS mutations play a significant role in influencing patient prognosis and guiding therapeutic decision-making. This study aimed to determine the prevalence and prognostic significance of KRAS mutations in CCA, asses the detection of KRAS G12/G13 mutations in plasma cell-free DNA (cfDNA), and evaluate the prognostic value of KRAS G12/G13 mutant allele frequency (MAF) in cfDNA in relation to clinicopathological data and patient survival. Materials and Methods: A retrospective analysis of 937 CCA patients was performed using data from cBioPortal to examine KRAS mutation profiles and their association with survival. Plasma from 101 CCA patients was analyzed for KRAS G12/G13 mutations in the cfDNA using droplet digital PCR, and the results were compared with tissuebased sequencing from 78 matched samples. Results: KRAS driver mutations were found in 15.6% of patients, with common variants being G12D (37.0%), G12V (24.0%) and Q61H (8.2%). Patients harboring KRAS mutations exhibited decreased overall and recurrence-free survival. KRAS G12/G13 mutations were detected in 14.9% of cfDNA samples, showing moderate concordance with tissue sequencing, and achieving 80% sensitivity and 93% specificity. Elevated KRAS G12/G13 MAF in cfDNA, combined with high CA19-9 levels, correlated with poorer survival outcomes. Conclusion: The presence of KRAS mutations was associated with poor survival in CCA, underscoring the importance of KRAS mutations as prognostic markers. The detection of KRAS mutations in cfDNA demonstrated potential as a promising non-invasive alternative for mutation detection and, when combined with CA19-9 levels, may improve prognostic efficacy in CCA. [ABSTRACT FROM AUTHOR]

Heart failure (HF) remains a major global health challenge, and more effective and comprehensive plasma biomarkers are needed to effectively treat HF patients. Multiomics studies have shown that DNA fragments, noncoding RNAs, proteins, and metabolites may be potential plasma biomarkers for HF. However, comprehensive reviews that focus on research on plasma biomarkers for HF from an omics perspective are lacking. This review summarizes the applications of various omics approaches in the exploration of biomarkers related to the risk assessment, diagnosis, subtype classification, medical management, and prognosis prediction of HF. Moreover, as heart transplantation and left ventricular assistant device (LVAD) implantation are terminal therapies for end-stage HF patients, this review also discusses the role of cell-free DNA as a biomarker for cardiac transplant rejection and omics studies of plasma biomarkers in patients who respond to LVAD therapy. Our findings suggest that future omics research on HF biomarkers should employ integrated multiomics methods and expand the sample size to increase the robustness of the results and that the identified biomarkers should be further validated in large cohorts. [ABSTRACT FROM AUTHOR]

The autonomous and active Long-Interspersed Element-1 (LINE-1, L1) and the non-autonomous Alu retrotransposon elements, contributing to 30% of the human genome, are the most abundant repeated sequences. With more than 90% of their sequences being methylated in normal cells, these elements undeniably contribute to the global DNA methylation level and constitute a major part of circulating-cell-free DNA (cfDNA). So far, the hypomethylation status of LINE-1 and Alu in cellular and extracellular DNA has long been considered a prevailing hallmark of ageing-related diseases and cancer. This study demonstrated that errors in LINE-1 and Alu methylation level measurements were caused by an excessive input quantity of genomic DNA used for bisulfite conversion. Using the minuscule DNA amount of 0.5 ng, much less than what has been used and recommended so far (500 ng-2 μg) or 1 μL of cfDNA extracted from 1 mL of blood, we revealed hypermethylation of LINE-1 and Alu in 407 tumour samples of primary breast, colon and lung cancers when compared with the corresponding pair-matched adjacent normal tissue samples (P < 0.05–0.001), and in cfDNA from 296 samples of lung cancers as compared with 477 samples from healthy controls (P < 0.0001). More importantly, LINE-1 hypermethylation in cfDNA is associated with healthy ageing. Our results have not only contributed to the standardized bisulfite-based protocols for DNA methylation assays, particularly in applications on repeated sequences but also provided another perspective for other repetitive sequences whose epigenetic properties may have crucial impacts on genome architecture and human health. [ABSTRACT FROM AUTHOR]

Donor-derived cell-free DNA (dd-cfDNA) is an emerging non-invasive biomarker for allograft injury detection. This study aimed to evaluate a new, decentralized dd-cfDNA testing kit against a centralized dd-cfDNA testing service broadly utilized in the United States. Kidney transplant recipients with decentralized and centralized dd-cfDNA measurements and concomitant kidney allograft biopsies were included in the study. 580 kidney allograft recipients from 3 referral centers were included for 603 total evaluations. Correlation between assays was evaluated using r-squared (r 2) and Spearman's rank correlation test, and associations with rejection using logistic regression analyses and discrimination using area under the curve. Mean dd-cfDNA levels from decentralized and centralized tests were 0.51% ± 0.81% and 0.43% ± 0.78%, respectively. The assays were highly correlated, with r 2 = 0.95 and Spearman's rank correlation 0.88 (p < 0.0001). Both tests showed significant association with allograft rejection (p < 0.0001) and good and similar discriminations to predict rejection (AUC: 0.758 for the decentralized and AUC: 0.760 for the centralized dd-cfDNA; p = 0.8466). Consistency between the assays was also confirmed across clinical scenarios including post-transplant timepoint, allograft stability, and allograft rejection subcategories. This decentralized dd-cfDNA assessment demonstrates high accuracy and value to non-invasively monitor kidney recipients. [ABSTRACT FROM AUTHOR]

Metastasis in patients with oral squamous cell carcinoma has been associated with a poor prognosis. However, sensitive and reliable tests for monitoring their occurrence are unavailable, with the exception of PET-CT. Circulating tumor cells and cell-free DNA have emerged as promising biomarkers for determining treatment efficacy and as prognostic predictors in solid tumors such as breast cancer and colorectal cancer. Hence, this study aimed to determine the potential role of liquid biopsy, circulating tumor cells, and cell-free DNA as biomarkers of oral squamous cell carcinoma. Thirteen patients with primary oral squamous cell carcinoma who visited our hospital between 2022 and 2023 were recruited, and plasma samples were collected from each patient preoperatively and postoperatively. We examined the relationship between the prognosis, the number of circulating tumor cells per four milliliters of peripheral blood, and the amount of cell-free DNA per milliliter of serum or the gene mutation in cell-free DNA. We observed no correlation between the number of preoperative circulating tumor cells and metastatic events. However, the number of circulating tumor cell clusters or the amount of preoperative cell-free DNA in metastatic cases was higher than that in non-metastatic cases. In oral squamous cell carcinoma, circulating tumor cell clusters or cell-free DNA levels may help inform management decisions regarding metastasis. However, further studies are required to provide a possible window for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Esophageal adenocarcinoma (EAC) is an aggressive cancer characterized by a high risk of relapse post-surgery. Current follow-up methods (serum carcinoembryonic antigen detection and PET-CT) lack sensitivity and reliability, necessitating a novel approach. Analyzing cell-free DNA (cfDNA) from blood plasma emerges as a promising avenue. This study aims to evaluate the cost-effective and genome-wide cell-free reduced representation bisulfite sequencing (cfRRBS) method combined with computational deconvolution for effective disease monitoring in EAC patients. cfDNA methylation profiling with cfRRBS was performed on 162 blood plasma samples from 33 EAC cancer patients and 28 blood plasma samples from 20 healthy donors. The estimated tumor fraction for EAC patients at the time of diagnosis was significantly different from the healthy donor plasma samples (one-sided Wilcoxon rank-sum test: p-value = 0.032). Tumor fractions above 15% and focal gains/amplifications in MYC (chr8), KRAS (chr12), EGFR (chr7) and NOTCH2 (chr1) were observed in four samples of distinct patients at the time metastatic disease was detected. This study showed feasibility to estimate tumor fractions in blood plasma of EAC patients based on cfDNA methylation using cfRRBS and computational deconvolution. Nevertheless, in this study only cancer patients with evidence of metastatic disease show high tumor fractions and copy number alterations. [ABSTRACT FROM AUTHOR]

Background: Esophageal cancer (EC) is a highly lethal disease lacking early detection approaches. We previously identified that OTOP2 and KCNA3 were specifically hypermethylated in circulating cell-free DNA from patients with EC. We then developed a blood-based methylation assay targeting OTOP2 and KCNA3 (named "IEsohunter") for esophageal cancer noninvasive detection. This double-blinded, multicenter, prospective study aimed to comprehensively evaluate its clinical diagnostic performance. Methods: Participants with EC, high-grade intraepithelial neoplasia (HGIN), other malignancies, benign gastrointestinal lesions, or no abnormalities were prospectively enrolled from 5 tertiary referral centers across China. Peripheral blood samples were collected, followed by plasma cell-free DNA methylation analysis using the IEsohunter test based on multiplex quantitative polymerase chain reaction adopting an algorithm-free interpretation strategy. The primary outcome was the diagnostic accuracy of IEsohunter test for EC. Results: We prospectively enrolled 1116 participants, including 334 patients with EC, 71 with HGIN, and 711 controls. The areas under the receiver operating characteristic curves of the IEsohunter test for detecting EC and HGIN were 0.903 (95% CI 0.880–0.927) and 0.727 (95% CI 0.653–0.801), respectively. IEsohunter test showed sensitivities of 78.5% (95% CI 69.1–85.6), 87.3% (95% CI 79.4–92.4), 92.5% (95% CI 85.9–96.2), and 96.9% (95% CI 84.3–99.8) for stage I-IV EC, respectively, with an overall sensitivity of 87.4% (95% CI 83.4–90.6) and specificity of 93.3% (95% CI 91.2–94.9) for EC detection. The IEsohunter test status turned negative (100.0%, 47/47) after surgical resection of EC. Conclusions: The IEsohunter test showed high diagnostic accuracy for EC detection, indicating that it could potentially serve as a tool for noninvasive early detection and surveillance of EC. [ABSTRACT FROM AUTHOR]

The advent of non-invasive prenatal testing (NIPT) in the screening of fetal abnormalities has optimized prenatal care and decreased the rate of invasive diagnostic tests. In this retrospective descriptive study, we began with 1874 singleton pregnancies. After exclusion of some cases, the study cohort ended up with 1674 cases. We analyzed the performance of NIPT based on the results of first trimester screening (FTS) using serum screening combined with NT. The cases were also compared to diagnostic testing/pregnancy outcomes. Notably, in the subgroup with FTS risk < 1000, NIPT was reported to be normal in all cases with no false negative results. In the risk group of 1/300-1/1000, NIPT could detect all trisomy 21 cases with one false positive result. Moreover, in the risk group of 1/11 − 1/300, NIPT could detect all cases of trisomy 21, 13 and 18 with low false positive rate. However, the false positive rate for sex chromosomal abnormalities was high. Taken together, the current study confirms the applicability of NIPT as a tool for detection of fetal trisomies with high sensitivity and specificity. Yet, the high rate of false positive results for sex chromosome abnormalities should be considered in the interpretation of the results. [ABSTRACT FROM AUTHOR]

Glioma is the most common primary malignant brain tumor. Despite significant advances in the past decade in understanding the molecular pathogenesis of this tumor and exploring therapeutic strategies, the prognosis of patients with glioma remains poor. Accurate diagnosis of glioma is very important for the treatment and prognosis. Although the gold-standard method for the diagnosis and prognosis prediction of patients with glioma is tissue biopsy, it still has many limitations. Liquid biopsy can provide information on the auxiliary diagnosis and prognosis of gliomas. In this review, we summarized the application of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) in the auxiliary diagnosis and prognosis of glioma. The common methods used to detect ctDNA in gliomas using samples including blood and cerebrospinal fluid (CSF) and the detection techniques for ctDNA, including droplet digital PCR (ddPCR) and next-generation sequencing (NGS), were discussed. Detection of ctDNA from plasma of patients with brain tumors remains challenging because of the blood–brain barrier (BBB). CSF has been proposed as a medium for ctDNA analysis in brain tumors, and mutation detection using plasma ctDNA was less sensitive than CSF ctDNA sequencing. Moreover, ongoing relevant clinical studies were summarized. Finally, we discussed the challenges, and future directions for the studies on ctDNA in glioma. [ABSTRACT FROM AUTHOR]