Large language model produces high accurate diagnosis of cancer from end-motif profiles of cell-free DNA.

Instruction-tuned large language models (LLMs) demonstrate exceptional ability to align with human intentions. We present an LLM-based model-instruction-tuned LLM for assessment of cancer (iLLMAC)-that can detect cancer using cell-free deoxyribonucleic acid (cfDNA) end-motif profiles. Developed on plasma cfDNA sequencing data from 1135 cancer patients and 1106 controls across three datasets, iLLMAC achieved area under the receiver operating curve (AUROC) of 0.866 [95% confidence interval (CI), 0.773-0.959] for cancer diagnosis and 0.924 (95% CI, 0.841-1.0) for hepatocellular carcinoma (HCC) detection using 16 end-motifs. Performance increased with more motifs, reaching 0.886 (95% CI, 0.794-0.977) and 0.956 (95% CI, 0.89-1.0) for cancer diagnosis and HCC detection, respectively, with 64 end-motifs. On an external-testing set, iLLMAC achieved AUROC of 0.912 (95% CI, 0.849-0.976) for cancer diagnosis and 0.938 (95% CI, 0.885-0.992) for HCC detection with 64 end-motifs, significantly outperforming benchmarked methods. Furthermore, iLLMAC achieved high classification performance on datasets with bisulfite and 5-hydroxymethylcytosine sequencing. Our study highlights the effectiveness of LLM-based instruction-tuning for cfDNA-based cancer detection.

Biomarkers in Kidney Transplantation.

Currently in the United States, there are more than 250,000 patients with a functioning kidney allograft and over 100,000 waitlisted patients awaiting kidney transplant, with a burgeoning number added to the kidney transplant wait list every year. Although early post-transplant care is delivered at the transplant center, the increasing number of kidney transplant recipients requires general nephrologists to actively participate in the long-term care of these patients. Serum creatinine and proteinuria are imperfect traditional biomarkers of allograft dysfunction and lag behind subclinical allograft injury. This manuscript reviews the various clinically available biomarkers in the field of kidney transplantation for a general nephrologist with a focus on the utility of donor-derived cell-free DNA, as a marker of early allograft injury. Blood gene expression profiling, initially studied in the context of early identification of subclinical rejection, awaits validation in larger multicentric trials. Urinary cellular messenger ribonucleic acid and chemokine CXCL10 hold promising potential for early diagnosis of both subclinical and acute rejection. Torque tenovirus, a ubiquitous DNA virus is emerging as a biomarker of immunosuppression exposure as peripheral blood torque tenovirus copy numbers might mirror the intensity of host immunosuppression. Although high-quality evidence is still being generated, evidence and recommendations are provided to aid the general nephrologist in implementation of novel biomarkers in their clinical practice.

Cell-free and extrachromosomal DNA profiling of small cell lung cancer.

Small cell lung cancer (SCLC) is highly aggressive with poor prognosis. Despite a relative prevalence of circulating tumour DNA (ctDNA) in SCLC, liquid biopsies are not currently implemented, unlike non-SCLC where cell-free DNA (cfDNA) mutation profiling in the blood has utility for guiding targeted therapies and assessing minimal residual disease. cfDNA methylation profiling is highly sensitive for SCLC detection and holds promise for disease monitoring and molecular subtyping; cfDNA fragmentation profiling has also demonstrated clinical potential. Extrachromosomal DNA (ecDNA), that is often observed in SCLC, promotes tumour heterogeneity and chemotherapy resistance and can be detected in blood. We discuss how these cfDNA profiling modalities can be harnessed to expand the clinical applications of liquid biopsy in SCLC.

Chapter 2: Non-invasive prenatal diagnosis.

Non-invasive prenatal diagnosis of monogenic disorders is becoming integrated into routine clinical care for many indications. This is carried out by testing cell-free DNA extracted from the plasma portion of a maternal blood sample. The cell-free DNA is low in concentration, and consists of a mixture of maternal and fetally-derived DNA which are not easy to separate. Methods used therefore need to be rapid, sensitive and specific, including real-time PCR, digital PCR and next generation sequencing with complex algorithms. Testing may be required for pregnancies with an increased chance of a monogenic disorder due to family history or carrier status, or where there are specific abnormalities identified by ultrasound scan. In these situations, testing is considered to be diagnostic and therefore does not require confirmation by invasive testing. With increased access to genomic technologies, and more diagnoses for rare disease patients, future demand for NIPD and possibilities during pregnancy will continue.

Biomarkers for diagnosis and therapeutic options in hepatocellular carcinoma.

Liver cancer is a global health challenge, causing a significant social-economic burden. Hepatocellular carcinoma (HCC) is the predominant type of primary liver cancer, which is highly heterogeneous in terms of molecular and cellular signatures. Early-stage or small tumors are typically treated with surgery or ablation. Currently, chemotherapies and immunotherapies are the best treatments for unresectable tumors or advanced HCC. However, drug response and acquired resistance are not predictable with the existing systematic guidelines regarding mutation patterns and molecular biomarkers, resulting in sub-optimal treatment outcomes for many patients with atypical molecular profiles. With advanced technological platforms, valuable information such as tumor genetic alterations, epigenetic data, and tumor microenvironments can be obtained from liquid biopsy. The inter- and intra-tumoral heterogeneity of HCC are illustrated, and these collective data provide solid evidence in the decision-making process of treatment regimens. This article reviews the current understanding of HCC detection methods and aims to update the development of HCC surveillance using liquid biopsy. Recent critical findings on the molecular basis, epigenetic profiles, circulating tumor cells, circulating DNAs, and omics studies are elaborated for HCC diagnosis. Besides, biomarkers related to the choice of therapeutic options are discussed. Some notable recent clinical trials working on targeted therapies are also highlighted. Insights are provided to translate the knowledge into potential biomarkers for detection and diagnosis, prognosis, treatment response, and drug resistance indicators in clinical practice.

Evaluation and integration of cell-free DNA signatures for detection of lung cancer.

Cell-free DNA (cfDNA) analysis has shown potential in detecting early-stage lung cancer based on non-genetic features. To distinguish patients with lung cancer from healthy individuals, peripheral blood were collected from 926 lung cancer patients and 611 healthy individuals followed by cfDNA extraction. Low-pass whole genome sequencing and targeted methylation sequencing were conducted and various features of cfDNA were evaluated. With our customized algorithm using the most optimal features, the ensemble stacked model was constructed, called ESim-seq (Early Screening tech with Integrated Model). In the independent validation cohort, the ESim-seq model achieved an area under the curve (AUC) of 0.948 (95 % CI: 0.915-0.981), with a sensitivity of 79.3 % (95 % CI: 71.5-87.0 %) across all stages at a specificity of 96.0 % (95 % CI: 90.6-100.0 %). Specifically, the sensitivity of the ESim-seq model was 76.5 % (95 % CI: 67.3-85.8 %) in stage I patients, 100 % (95 % CI: 100.0-100.0 %) in stage II patients, 100 % (95 % CI: 100.0-100.0 %) in stage III patients and 87.5 % (95 % CI: 64.6%-100.0 %) in stage IV patients in the independent validation cohort. Besides, we constructed LCSC model (Lung Cancer Subtype multiple Classification), which was able to accurately distinguish patients with small cell lung cancer from those with non-small cell lung cancer, achieving an AUC of 0.961 (95 % CI: 0.949-0.957). The present study has established a framework for assessing cfDNA features and demonstrated the benefits of integrating multiple features for early detection of lung cancer.

Somatic Activating ESR1 Mutation in an Aggressive Prolactinoma.

CONTEXT AND OBJECTIVE: The genetic profile of prolactinomas remains poorly understood. Our objective is to identify somatic genetic alterations associated with prolactinomas and to report the identification of an activating ESR1 mutation (ESR1Y537S) in an aggressive prolactinoma. SETTING: Brigham and Women's Hospital. DESIGN: Massively parallel-sequencing panel (OncoPanel) was performed in a cohort of patients with prolactinomas to identify mutations and copy number variation (CNV). RESULTS: Twenty subjects (mean age 38.6 years; 12 women and 8 men) were included in this study. A somatic ESR1Y537S mutation was identified in an aggressive prolactinoma in a post-menopausal woman. No SF3B1 or other somatic mutations were identified. The median number of CNV events identified in our samples was 46; the prolactinoma with ESR1Y537S had the highest number with 233 events. In breast cancer, ESR1Y537S has been shown to activate estrogen receptor alpha independent of ligand binding. In patients with resistant breast cancer and ESR1Y537S, elacestrant, a second-line ER degrader, improves progression-free survival. Therefore, given the lack of response to multimodality therapies, elacestrant was initiated in this patient after the third cycle of radiotherapy. Elacestrant, along with radiotherapy, controlled tumor growth and significantly reduced prolactin levels. CONCLUSION: Molecular profiling allowed the identification of ESR1Y537S, in an aggressive prolactinoma. ESR1Y537S was not detected early in the course of the disease and is likely conferring tumor aggressiveness. This finding emphasizes the significance of estrogen receptor signaling in prolactinomas. It also allowed the use of targeted therapy with successful control of disease progression.

Effects of exogenous deoxyribonuclease I in collagen antibody-induced arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is associated with a high concentration of extracellular DNA (ecDNA). This could be a consequence of the inflammation, but the ecDNA could also be involved in the unknown etiopathogenesis of RA. Clearance of ecDNA is hypothesized to prevent the development of RA. This study aimed to analyze the effects of exogenous deoxyribonuclease I (DNase I) administration in an animal model of RA. METHODS: The collagen antibody-induced arthritis (CAIA) model of RA was induced in adult female DBA/1J mice. CAIA mice were treated with saline or DNase I (10 mg/kg) every 12 h for the whole duration of the experiment. Arthritic scores were assessed. Paw volume and temperature were assessed using a plethysmometer and a thermal camera, respectively. Plasma ecDNA and its subcellular origin were analyzed using fluorometry and real-time PCR. DNase activity was quantified with single radial enzyme diffusion method. RESULTS: The CAIA model was successfully induced as proved by a higher volume, temperature and the overall arthritis score in comparison to controls. The administration of DNase I resulted in a nearly two-fold increase in serum DNase activity. Still, it did affect neither plasma ecDNA, nor the arthritis score or other measures of joint inflammation. CONCLUSION: Our results suggest that exogenous DNase I does not prevent the development of CAIA in mice. Whether this is true for other animal models of arthritis or clinical RA requires further research. EcDNA does not seem to be involved in the pathogenesis of CAIA. Additional studies are also needed to elucidate the role of ecDNA in the development of RA, focusing especially on its origin and inhibition of ecDNA release.

Analysis of urine cell-free DNA in bladder cancer diagnosis by emerging bioactive technologies and materials.

Urinary cell-free DNA (UcfDNA) is gaining recognition as an important biomarker for diagnosing bladder cancer. UcfDNA contains tumor derived DNA sequences, making it a viable candidate for non-invasive early detection, diagnosis, and surveillance of bladder cancer. The quantification and qualification of UcfDNA have demonstrated high sensitivity and specificity in the molecular characterization of bladder cancer. However, precise analysis of UcfDNA for clinical bladder cancer diagnosis remains challenging. This review summarizes the history of UcfDNA discovery, its biological properties, and the quantitative and qualitative evaluations of UcfDNA for its clinical significance and utility in bladder cancer patients, emphasizing the critical role of UcfDNA in bladder cancer diagnosis. Emerging bioactive technologies and materials currently offer promising tools for multiple UcfDNA analysis, aiming to achieve more precise and efficient capture of UcfDNA, thereby significantly enhancing diagnostic accuracy. This review also highlights breakthroughs in detection technologies and substrates with the potential to revolutionize bladder cancer diagnosis in clinic.

Clinical utility of plasma cell-free DNA (cfDNA) in diffuse gliomas for the detection of IDH1 R132H mutation.

Liquid biopsy for CNS tumors is in its nascent phase, hindered by the low levels of circulating tumor DNA (ctDNA). Overcoming this challenge requires highly sensitive molecular techniques. DD-PCR emerges as a standout technique due to its ability to identify rare mutations, copy number variations, and circulating nucleic acids, making it one of the best methods for identifying somatic mutations in cell-free DNA (cfDNA). Despite promising results from various studies demonstrating the feasibility of obtaining informative ctDNA profiles from liquid biopsy samples, challenges persist, including the need to standardize sample collection, storage, and processing methods, define clear assay positivity thresholds, and address the overall low assay sensitivity. Our two-phase study began by assessing DD-PCR efficacy in FFPE tissues, revealing robust concordance with immunohistochemistry. In Phase 1 (85 cases), DD-PCR on FFPE tissues demonstrated 100 % sensitivity and specificity for IDH1 R132H mutations. In Phase 2 (100 cases), analysis extended to cfDNA, maintaining high specificity (100 %) with moderate sensitivity (44.2 %). Overall concordance with immunohistochemistry was 61 %, highlighting liquid biopsy's potential in glioma management. The findings emphasized DD-PCR's clinical utility in both tissue and liquid biopsy, underscoring its role in early detection, diagnosis, and therapeutic monitoring of diffuse gliomas.

Cell-Free DNA Hydroxymethylation in Cancer: Current and Emerging Detection Methods and Clinical Applications.

In the era of precision oncology, identifying abnormal genetic and epigenetic alterations has transformed the way cancer is diagnosed, managed, and treated. 5-hydroxymethylcytosine (5hmC) is an emerging epigenetic modification formed through the oxidation of 5-methylcytosine (5mC) by ten-eleven translocase (TET) enzymes. DNA hydroxymethylation exhibits tissue- and cancer-specific patterns and is essential in DNA demethylation and gene regulation. Recent advancements in 5hmC detection methods and the discovery of 5hmC in cell-free DNA (cfDNA) have highlighted the potential for cell-free 5hmC as a cancer biomarker. This review explores the current and emerging techniques and applications of DNA hydroxymethylation in cancer, particularly in the context of cfDNA.

Minimal residual disease testing for classical Hodgkin lymphoma: A comprehensive review.

Classical Hodgkin lymphoma (cHL) is a common lymphoma that affects young patients. Fortunately, the disease is highly curable as it is susceptible to the currently available treatment modalities. Disease monitoring with Positron Emission Tomography and Computed Tomography (PET/ CT) is an integral part of managing these patients. PET guided protocols are currently used to adjust treatment according to the response. The pivotal idea behind the use of response-adapted approaches is to preserve efficacy while decreasing the toxicity. It also helps to intensify therapy in patients in need because of suboptimal response. However, imaging techniques are limited by their sensitivity and specificity. Minimal Residual Disease (MRD) assessment is a newly emerging concept in many hematologic malignancies. It utilizes various molecular techniques such as polymerase chain reaction (PCR), and next-generation sequencing (NGS) as well as flow cytometry, to detect disease traces. This review looks into MRD detection techniques, its current applications, and the evidence in the literature for its use in cHL.

Cerebrospinal fluid liquid biopsy by low-pass whole genome sequencing for clinical disease monitoring in pediatric embryonal tumors.

Background: Liquid biopsy assays that detect cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) are a promising tool for disease monitoring in pediatric patients with primary central nervous system (CNS) tumors. As a compliment to tissue-derived molecular analyses, CSF liquid biopsy has the potential to transform risk stratification, prognostication, and precision medicine approaches. Methods: In this pilot study, we evaluated a clinical pipeline to determine feasibility and sensitivity of low-pass whole genome sequencing (LP-WGS) of CSF-derived cfDNA from patients with CNS embryonal tumors. Thirty-two longitudinal CSF samples collected from 17 patients with molecularly characterized medulloblastoma (12), embryonal tumor with multilayered rosettes (2), CNS embryonal tumor, not elsewhere classified (NEC) (2), and atypical teratoid/rhabdoid tumor (1) were analyzed. Results: Adequate CSF-derived cfDNA for LP-WGS analysis was obtained in 94% of samples (30/32). Copy number variants compatible with neoplasia were detected in 90% (27/30) and included key alterations, such as isodicentric ch17, monosomy 6, and MYCN amplification, among others. Compared to tissue specimens, LP-WGS detected additional aberrations in CSF not previously identified in corresponding primary tumor specimens, suggesting a more comprehensive profile of tumor heterogeneity or evolution of cfDNA profiles over time. Among the 12 CSF samples obtained at initial staging, only 2 (17%) were cytologically positive, compared to 11 (92%) that were copy number positive by LP-WGS. Conclusions: LP-WGS of CSF-derived cfDNA is feasible using a clinical platform, with greater sensitivity for tumor detection compared to conventional CSF cytologic analysis at initial staging. Large prospective studies are needed to further evaluate LP-WGS as a predictive biomarker.

Liquid Biopsy in Neuropsychiatric Disorders: A Step Closer to Precision Medicine.

Psychiatric disorders are among the leading causes of disease burden worldwide. Despite their significant impact, their diagnosis remains challenging due to symptom heterogeneity, psychiatric comorbidity, and the lack of objective diagnostic tests and well-defined biomarkers. Leveraging genomic, epigenomic, and fragmentomic technologies, circulating cell-free DNA (ccfDNA)-based liquid biopsies have emerged as a potential non-invasive diagnosis and disease-monitoring tool. ccfDNA is a DNA species released into circulation from all types of cells through passive and active mechanisms and can serve as a biomarker for various diseases, namely, cancer. Despite their potential, the application of ccfDNA in neuropsychiatry remains underdeveloped. In this review, we provide an overview of liquid biopsies and their components, with a particular focus on ccfDNA. With a summary of pre-analytical practices and current ccfDNA technologies, we highlight the current state of research regarding the use of ccfDNA as a biomarker for neuropsychiatric disorders. Finally, we discuss future steps to unlock ccfDNA's potential in clinical practice.

Plasma Cell-Free DNA Concentration and Fragmentomes Predict Neoadjuvant Chemotherapy Response in Cervical Cancer Patients.

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

GWAS shows the genetics behind cell-free DNA and highlights the importance of p.Arg206Cys in DNASE1L3 for non-invasive testing.

The properties of cell-free DNA (cfDNA) are intensely studied for their potential as non-invasive biomarkers. We explored the effect of common genetic variants on the concentration and fragmentation properties of cfDNA using a genome-wide association study (GWAS) based on low-coverage whole-genome sequencing data of 140,000 Dutch non-invasive prenatal tests (NIPTs). Our GWAS detects many genome-wide significant loci, functional enrichments for phagocytes, liver, adipose tissue, and macrophages, and genetic correlations with autoimmune and cardiovascular disease. A common (7%) missense variant in DNASE1L3 (p.Arg206Cys) strongly affects all cfDNA properties. It increases the size of fragments, lowers cfDNA concentrations, affects the distribution of cleave-site motifs, and increases the fraction of circulating fetal DNA during pregnancy. For the application of NIPT, and potentially other cfDNA-based tests, this variant has direct clinical consequences, as it increases the odds of inconclusive results and impairs the sensitivity of NIPT by causing predictors to overestimate the fetal fraction.

Donor-derived post-transplant lymphoproliferative disease detection by donor-derived cell free DNA.

Post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication of organ transplantation, commonly diagnosed after patients present with nonspecific constitutional symptoms and/or transplant organ dysfunction. Here we report a case of a kidney transplant recipient who was found to have highly elevated circulating donor-derived cell free DNA (dd-cfDNA) levels on routine serum surveillance for allograft rejection, initially without organ dysfunction or evidence of allograft rejection on biopsy. Later for cause imaging revealed retroperitoneal lymphadenopathy and an allograft hilar mass, which was biopsied to show post-transplant lymphoproliferative disorder/diffuse large B-cell lymphoma (DLBCL). The elevated circulating dd-cfDNA levels in this patient prompted targeted next-generation sequencing of the same 266 single-nucleotide polymorphisms used to detect dd-cfDNA on the DLBCL, which identified it as donor-derived. The patient achieved complete remission with retained allograft kidney function after reduced immunosuppression and 6 cycles of immunochemotherapy. This case suggests that dd-cfDNA may be an early detection tool in rare but potentially life-threatening cases of donor-derived malignancy, such as donor-derived PTLD.

Detection of circulating tumor DNA by tumor-informed whole-genome sequencing enables prediction of recurrence in stage III colorectal cancer patients.

INTRODUCTION: Circulating tumor (ctDNA) can be used to detect residual disease after cancer treatment. Detecting low-level ctDNA is challenging, due to the limited number of recoverable ctDNA fragments at any target loci. In response, we applied tumor-informed whole-genome sequencing (WGS), leveraging thousands of mutations for ctDNA detection. METHODS: Performance was evaluated in serial plasma samples (n = 1283) from 144 stage III colorectal cancer patients. Tumor/normal WGS was used to establish a patient-specific mutational fingerprint, which was searched for in 20x WGS plasma profiles. For reproducibility, paired aliquots of 172 plasma samples were analyzed in two independent laboratories. De novo variant calling was performed for serial plasma samples with a ctDNA level > 10 % (n = 17) to explore genomic evolution. RESULTS: WGS-based ctDNA detection was prognostic of recurrence: post-operation (Hazard ratio [HR] 6.75, 95 %CI 3.18-14.3, p < 0.001), post-adjuvant chemotherapy (HR 28.9, 95 %CI 10.1-82.8; p < 0.001), and during surveillance (HR 22.8, 95 %CI 13.7-37.9, p < 0.0001). The 3-year cumulative incidence of ctDNA detection in recurrence patients was 95 %. ctDNA was detected a median of 8.7 months before radiological recurrence. The independently analyzed plasma aliquots showed excellent agreement (Cohens Kappa=0.9, r = 0.99). Genomic characterization of serial plasma revealed significant evolution in mutations and copy number alterations, and the timing of mutational processes, such as 5-fluorouracil-induced mutations. CONCLUSION: Our study supports the use of WGS for sensitive ctDNA detection and demonstrates that post-treatment ctDNA detection is highly prognostic of recurrence. Furthermore, plasma WGS can identify genomic differences distinguishing the primary tumor and relapsing metastasis, and monitor treatment-induced genomic changes.

Analytical and Diagnostic Performance of a Dual-Target Blood Detection Test for Hepatocellular Carcinoma.

BACKGROUND: Surveillance approaches with high sensitivity and specificity for hepatocellular carcinoma (HCC) are still urgently needed. Previous studies have shown that methylation of GNB4 and Riplet can effectively diagnose HCC. AIMS: This study plan to analyze the performance of a blood test for detecting HCC using GNB4 and Riplet methylation. METHODS AND RESULTS: This study mainly investigated the analytical performance of the dual-target HCC blood test (DT-HBT), including cut-off value, limit of detection (LOD), precision, analytical specificity, and coincidence rate. In addition, the detection performance for HCC was validated in 1030 clinical plasma samples (214 HCC and 816 non-HCC). Plasma samples from 25 HCC patients after hepatectomy were collected to assess the feasibility of the kit for postoperative recurrence monitoring. All analytical performance of the DT-HBT met prespecified requirements. The LOD for GNB4, Riplet, and ß-actin was 1% methylation/100 copies/µL with cut-offs of 43, 43, and 35, respectively. The DT-HBT showed excellent precision, within 5% CV. It had a specificity of 91.5% for detecting other cancers, and 100% for breast, lung, and bladder cancer. No cross-reactions were observed with 9 potential interfering substances. The DT-HBT achieved a 100% coincidence rate in detecting reference and clinical samples. The clinical performance study found that the kit showed a sensitivity of 81.7% for stage I HCC, and an overall sensitivity and specificity of 87.4% and 92.3%, respectively. The detection sensitivity for postoperative recurrent patients was 95.8%, with a specificity of 100%. CONCLUSION: The analytical performance of the DT-HBT met prespecified criteria. It provided HCC patients with a reliable and high-performing new blood test for the HCC diagnosis and surveillance. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT05685524.

Role of DNase I in DNA degradation and cell-free DNA generation after acetaminophen-induced hepatic injury.

Cell-free DNA (cfDNA), the DNA in the blood circulation, is a useful marker for diagnosing hereditary diseases and tumors. However, the mechanisms underlying the generation of cfDNA are not completely understood. We previously studied DNases [Caspase-activated DNase (CAD), DNase1L3, and DNase I] and reported that in acetaminophen-induced liver necrosis, DNase1L3 was the main endonuclease generating cfDNA, with CAD playing a supporting role. In this study, we generated triple-gene knockout (TKO) mice, Cad -/-DNase1L3 -/-DNase1 -/-, and found that DNase I also contributed to cfDNA generation. Given that a defect in DNase1L3 or DNase I is involved in autoimmune diseases, TKO mice would be useful as a disease model and tool for identifying the in vivo roles of endonucleases.