Plasma DNA Research Articles

The adverse impact of cytomegalovirus infection on intensive care units outcomes in critically ill COVID-19 patients: a single-center prospective observational study.

To assess the incidence and clinical impact of CMV infection in critically ill COVID-19 patients, examining ICU and hospital mortality, and length of hospital stay. In this single-center, prospective observational study (March 2020 - September 2022), 431 patients with COVID-19 pneumonia and moderate to severe ARDS were included. An active CMV surveillance protocol was implemented, analyzing CMV DNA in plasma and bronchoalveolar lavage (BAL). Clinical characteristics and outcomes were compared between CMV-COVID co-infected patients and those without CMV reactivation. CMV-COVID co-infection was detected in 14.8% (64/431) of the cohort. Patients with CMV-COVID co-infection exhibited significantly higher ICU mortality (43.8% vs. 13.6%; p < 0.001) and hospital mortality (48.4% vs. 13.6%; p < 0.001) compared to patients without CMV. CMV infection was an independent predictor of hospital mortality (OR 4.91; 95% CI 2.76-8.75; p = 0.019). Earlier CMV reactivation was associated with an increased risk of hospital mortality (HR = 0.94; 95% CI: 0.90-0.98; p = 0.003). Additionally, CMV-COVID patients had a higher incidence of ICU-acquired infections and a prolonged hospital stay. In critically ill patients with SARS-CoV-2 pneumonia, CMV infection was frequently observed, and associated with increased ICU and hospital mortality. CMV co-infection correlated with a higher incidence of ICU-acquired bacterial and fungal infections and prolonged hospital stays. This emphasizes the importance of early CMV monitoring upon ICU admission, as timely detection and intervention could potentially mitigate its impact on patient outcomes.

The Cryoprotectant Effects of Safflower Polysaccharides on the Quality of Frozen–Thawed Boar Sperm

The low resistance of boar sperm to cryopreservation dictates that adding antioxidants and energetic substances to the diluent to improve sperm quality is necessary. This study is designed to assess the impact of various concentrations of safflower polysaccharides (SPSs; 0, 0.5, 1.0, 1.5, and 2.0 g/L) on the quality of boar sperm following freezing and thawing. The results of the study showed that the supplementation of 1.5 g/L SPS significantly enhanced the motility, average path velocity, straight-line velocity, curvilinear velocity, beat cross frequency, acrosome integrity, plasma membrane integrity, mitochondrial activity, and DNA integrity compared with the control group (p &lt; 0.05). In addition, the supplementation of 1.5 g/L SPS significantly enhanced the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, and catalase activity while significantly decreasing malondialdehyde and hydrogen peroxide content (p &lt; 0.05). Therefore, the supplement SPS has potentially positive implications for improving the quality of cryopreserved boar sperm, and the recommended concentration is 1.5 g/L SPS.

Enrichment of Microbial DNA in Plasma to Improve Pathogen Detection in Sepsis: A Pilot Study.

Diagnosis of sepsis and timely identification of pathogens in critically ill patients remains challenging. Plasma metagenomic sequencing to detect microbial cell-free DNA (mDNA) has shown promise, but low abundance of mDNA in plasma limits sensitivity and necessitates high sequencing depth. mDNA is shorter and more fragmented than human cell-free DNA. Here, we evaluated whether combining single-stranded DNA (ssDNA) sequencing library preparation and size selection can enrich mDNA and improve pathogen detection. We prospectively enrolled 48 trauma patients and collected daily blood samples during the first 10 days of intensive care unit (ICU) admission. For patients with culture-proven infections, we extracted plasma DNA, prepared double-stranded DNA (dsDNA) and ssDNA sequencing libraries, and applied size selection to exclude fragments >110 bp. Following sequencing, we performed taxonomic classification, and evaluated differences in mDNA fractions and in sensitivity for pathogen detection (compared to background noise). We analyzed 46 plasma samples from 5 patients who developed culture-proven infections, including 17 samples coincident with positive microbial cultures. Size-selected ssDNA libraries showed the total mDNA fraction 204-fold higher on average than conventional dsDNA libraries (P < 0.0001). However, for pathogen-specific DNA (at the genus level), the highest sensitivity was observed in size-selected dsDNA (82%), compared to dsDNA (41%), ssDNA (71%), and size-selected ssDNA (35%) library preparations. Our results demonstrate that combining ssDNA library preparation together with fragment size selection improves mDNA yield, potentially reducing sequencing requirements. However, at the genus level, this combination also increases background noise, which limits sensitivity for pathogen detection.

Genomic and fragmentomic landscapes of cell-free DNA for early cancer detection.

Genomic analyses of cell-free DNA (cfDNA) in plasma are enabling noninvasive blood-based biomarker approaches to cancer detection and disease monitoring. Current approaches for identification of circulating tumour DNA typically use targeted tumour-specific mutations or methylation analyses. An emerging approach is based on the recognition of altered genome-wide cfDNA fragmentation in patients with cancer. Recent studies have revealed a multitude of characteristics that can affect the compendium of cfDNA fragments across the genome, collectively called the 'cfDNA fragmentome'. These changes result from genomic, epigenomic, transcriptomic and chromatin states of an individual and affect the size, position, coverage, mutation, structuraland methylation characteristics of cfDNA. Identifying and monitoring these changes has the potential to improve early detection of cancer, especially using highly sensitive multi-feature machine learning approaches that would be amenable to broad use in populations at increased risk. This Review highlights the rapidly evolving field of genome-wide analyses of cfDNA characteristics, their comparison to existing cfDNA methods, and recent related innovations at the intersection of large-scale sequencing and artificial intelligence. As the breadth of clinical applications of cfDNA fragmentome methods have enormous public health implications for cancer screening and personalized approaches for clinical management of patients with cancer, we outline the challenges and opportunities ahead.

An interference-based covalent organic framework biosensor for the detection of specific circulating tumor DNA in plasma

An interference-based covalent organic framework biosensor for the detection of specific circulating tumor DNA in plasma

HPV Biomarkers in Oral and Blood-Derived Body Fluids in Head and Neck Cancer Patients.

Oral HPV DNA and circulating tumor (ct) HPV DNA in plasma were evaluated as potential biomarkers for HPV-associated head and neck cancer (HNC). Samples from HNC patients (n = 132), including 23 oropharyngeal cancers (OPC), and non-HNC controls (n = 10) were analyzed. HPV status was determined using a multiplex bead-based test (E7-MPG) applied to formalin-fixed paraffin-embedded (FFPE) tissues (n = 90), plasma (n = 141), gargle samples (n = 141), and oral swabs (n = 142). HPV DNA was detected in 25.8% of HNC tissues, 12% of plasma samples, 20.6% of gargles and 7% of oral swabs with HPV16 as the most prevalent genotype. Among OPC cases, HPV16 DNA was found in 71.4% of FFPE samples. High concordance was observed between paired OPC tissues and plasma (91.3%) or gargles (95.2%), with moderate concordance for oral swabs (59.1%). Gargle samples alone demonstrated a 100% detection rate for HPV16-positive OPC, regardless of the cT stage, outperforming plasma (86.7%). Combined oral gargle and plasma analyses detected all HPV-positive OPC cases (7/7) at the early cT1 stage. These findings highlight the limited involvement of HPV in non-oropharyngeal HNC compared to OPC, and support gargle and plasma samples as minimally invasive diagnostic tools for detecting HPV-associated OPC.

Acute Human Bocavirus 1 Infection in Children Hospitalized for Acute Bronchiolitis: A 2-Year Prospective Study.

The objective of this prospective study was to assess the proportion and clinical consequences of human bocavirus 1 (HBoV1) replication in children hospitalized for acute bronchiolitis (AB) with HBoV1 DNA in the nasopharynx (NP). For this purpose, we detected HBoV1 DNA and mRNA (evidence of viral replication and viable virus) in NP in cases and healthy control children. This research allowed us to distinguish active HBoV1 infections from inactive ones. HBoV1 DNA was detected in the NP of 37 of 307 patients with AB (12.1%) and 9 of 150 children in a healthy control group (6%) with a high codetection rate with other respiratory viruses in AB patients, 28 of 37 (75.7%). Only 9 of 37 HBoV1 DNA-positive NP swabs (24.3%) with high DNA load were also HBoV1 mRNA positive, moreover, HBoV1 DNA was also detected in the plasma of these patients. Based on the results of our study, we can conclude that children with AB acute HBoV1 infection has a high HBoV1 DNA load in NP samples together with detected HBoV1 mRNA and detected HBoV1 DNA in plasma.

Equex-STM Paste-Added Freezing Medium and Straw Volume Influence Hampshire Crossbred Boar Semen Quality Following Cryopreservation.

Cryopreservation of boar semen is essential for maintaining genetic diversity and improving reproductive efficiency. However, optimizing semen extenders and packaging methods is crucial to enhancing sperm quality and cryotolerance. Equex-STM is commonly used as a surfactant to enhance cryoprotective properties by stabilizing sperm membranes during freezing and thawing. A total of 24 ejaculates (n = 24), six from each of four Hampshire crossbred boars, aged between 18 and 24 months were used in the present study. Semen was collected twice weekly by gloved hand technique. The sperm-rich fraction showing more than 70% initial motility was considered for further processing and freezing. The fresh semen was diluted in Beltsville thawing solution (BTS) at a 1:1 ratio and further processed with the addition of Fraction I and Fraction II extenders supplemented with and without 1.5% Equex-STM paste {Control (C)-no Equex; Treatment (T-1.5% Equex-STM supplementation}. The extended semen was packaged in 0.25 and 0.5 mL straws to check the effect of straw size (T1-0.25 mL straw and T2-0.50 mL straw). Straws were cryopreserved in liquid nitrogen (LN2) for post-thaw sperm quality evaluation. Results showed that the sperm motility, viability, plasma membrane integrity (PMI), DNA integrity, and mitochondrial membrane potential (MMP) were significantly (p < 0.001) higher in the T group in comparison with control (C). Sperm motility, PMI, and DNA integrity were highly significant (p < 0.001) and sperm viability and MMP were significant (p < 0.05) in group T2 in comparison with T1.

Genomic alterations and transcriptional phenotypes in circulating free DNA and matched metastatic tumor

BackgroundProfiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), due to the aggressive clinical course of this cancer, which makes obtaining tumor biopsies exceedingly challenging.MethodsIn this study, we analyzed a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC. We conducted cfDNA low-pass whole genome sequencing (0.1X coverage), comparing it with time-point matched tumor characterized using whole-exome (130X) and transcriptome sequencing.ResultsA direct comparison of cfDNA and tumor biopsy revealed that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not detected in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Sequencing coverage of plasma DNA fragments around transcription start sites showed distinct treatment-related changes and captured the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors. This allowed for the prediction of SCLC neuroendocrine phenotypes and treatment responses.ConclusionscfDNA captures a comprehensive view of tumor heterogeneity and evolution. These findings have significant implications for the non-invasive stratification of SCLC, a disease currently treated as a single entity.

Adrenocortical activity, measured via feather corticosterone, affects semen quality but not blood plasma prolactin in the capercaillie (Tetrao urogallus L.)

Capercaillie populations are experiencing worrying decline in several European countries, better knowledge of the factors affecting their reproductive success is therefore much needed. Chronic stress is a major cause of infertility in wild animals used in ex situ conservation programmes. In birds, the main hormonal stress response is an increase in corticosterone (CORT) secretion. Prolactin (PRL) is also involved, but its role in sperm functionality in birds is not well known. This work reports the relationship between CORT and PRL, and their influence on capercaillie semen quality. Feather CORT concentrations were determined by ELISA, along with blood plasma PRL by radioimmunoassay, in 14 sub-adult (1–2 years old) and 5 adult (> 3 years old) capercaillies. Sperm concentration was determined using a Neubauer chamber. Nigrosin-eosin dye was used to examine sperm viability; DNA integrity was assessed via the TUNEL assay. A significant, negative correlation was detected between CORT and sperm concentration (rs = 0.30, p = 0.02), while a positive correlation was detected between plasma PRL and DNA integrity (rs = 0.78, p = 0.007). No clear association was found between CORT and PRL (rs = 0.49, p = 0.21). These results suggest that the CORT concentration provides a suitable biomarker of stress in capercaillies, a factor known to affect reproductive function in males of this species.

Plasma Microbial Cell-free DNA Sequencing for the Detection of Kingella kingae Pediatric Spinal Infections.

Diagnosis of Kingella kingae skeletal system infections is made challenging by the microbe's fastidious nature. Detection and quantification of circulating microbial cell-free DNA (mcfDNA) in plasma by the Karius Test, a commercial metagenomic sequencing test, may offer promise in diagnosing pediatric spinal infections caused by difficult-to-culture organisms such as K. kingae. Plasma mcfDNA sequencing detections of K. kingae from April 2018 to December 2020 were reviewed to identify pediatric (age <18 years) patients. Medical charts of those with spinal infections were reviewed, and mcfDNA sequencing diagnostic performance was compared with usual care tests (ie, cultures, polymerase chain reaction). Ten children with K. kingae spinal infections were identified across 7 institutions. The median age was 16.5 months (range 11-23 months). All case-patients had vertebral osteomyelitis with 9 having spondylodiscitis. Compared with usual care tests, mcfDNA sequencing was significantly more sensitive (McNemar's test 6.25, 2-tailed P = 0.0133). It was the only method of microbiological diagnosis in 9 patients, providing results in a median of 2.5 days (range 2-5 days) from sample collection. K. kingae mcfDNA was detected despite antibiotic pretreatment in 5/5 case-patients. Pathogen-tailoring of antimicrobial coverage was undertaken in 9 children. Plasma mcfDNA sequencing offers a rapid, noninvasive method of detecting K. kingae causing pediatric spinal infections. This culture-independent approach may facilitate diagnosis, despite antibiotic pretreatment and subsequently targeted therapy and potentially obviate the need for biopsy.

Application of Next-Generation Sequencing to Realize Principles of Precision Therapy in Management of Cancer Patients.

All cancers are diseases of the genome, since the cancer cell genome typically consists of 10,000s of passenger alterations, 5-10 biologically relevant alterations, and 1-2 "actionable" alterations. Therefore, somatic mutations in cancer cells can have diagnostic, prognostic, and predictive value. Traditional methods are widely used for testing, such as immunohistochemistry, Sanger sequencing, and allele-specific PCR. However, due to the low throughput, these methods are focused exclusively on testing the most common mutations in target genes. The modern next generation sequencing (NGS) is a technology that enables precision oncology in its current form. ESCAT and ESMO Guidelines defined NGS for routine use in patients with advanced cancers such as non-squamous non-small cell lung cancer, prostate cancer, ovarian cancer, and cholangiocarcinoma. The high sensitivity of the NGS method allows it to be used to search for specific mutations in circulating tumor DNA in blood plasma and other body fluids. NGS testing has evolved from hotspot panels, actionable gene panels, and disease-specific panels to more comprehensive panels. The exome and whole genome sequencing approaches are just beginning to emerge, that is why panel-based testing remains most optimal in oncology practice. NGS is also widely used to identify new and rare mutations in cancer genes and detect inherited cancer mutations.

Whole-genome bisulfite sequencing of cell-free DNA unveils age-dependent and ALS-associated methylation alterations

BackgroundCell-free DNA (cfDNA) in plasma carries epigenetic signatures specific to tissue or cell of origin. Aberrant methylation patterns in circulating cfDNA have emerged as valuable tools for noninvasive cancer detection, prenatal diagnostics, and organ transplant assessment. Such epigenetic changes also hold significant promise for the diagnosis of neurodegenerative diseases, which often progresses slowly and has a lengthy asymptomatic period. However, genome-wide cfDNA methylation changes in neurodegenerative diseases remain poorly understood.ResultsWe used whole-genome bisulfite sequencing (WGBS) to profile age-dependent and ALS-associated methylation signatures in cfDNA from 30 individuals, including young and middle-aged controls, as well as ALS patients with matched controls. We identified 5,223 age-related differentially methylated loci (DMLs) (FDR < 0.05), with 51.6% showing hypomethylation in older individuals. Our results significantly overlapped with age-associated CpGs identified in a large blood-based epigenome-wide association study (EWAS). Comparing ALS patients to controls, we detected 1,045 differentially methylated regions (DMRs) in gene bodies, promoters, and intergenic regions. Notably, these DMRs were linked to key ALS-associated pathways, including endocytosis and cell adhesion. Integration with spinal cord transcriptomics revealed that 31% of DMR-associated genes exhibited differential expression in ALS patients compared to controls, with over 20 genes significantly correlating with disease duration. Furthermore, comparison with published single-nucleus RNA sequencing (snRNA-Seq) data of ALS demonstrated that cfDNA methylation changes reflects cell-type-specific gene dysregulation in the brain of ALS patients, particularly in excitatory neurons and astrocytes. Deconvolution of cfDNA methylation profiles suggested altered proportions of immune and liver-derived cfDNA in ALS patients.ConclusionscfDNA methylation is a powerful tool for assessing age-related changes and ALS-specific molecular dysregulation by revealing perturbed locus, genes, and the proportional contributions of different tissues/cells to the plasma. This technique holds promise for clinical application in biomarker discovery across a broad spectrum of neurodegenerative disorders.

Early circulating tumor DNA changes predict outcomes in head and neck cancer patients under re-radiotherapy.

Local recurrence after radiotherapy is common in locally advanced head and neck cancer (HNC) patients. Re-irradiation can improve local disease control, but disease progression remains frequent. Hence, predictive biomarkers are needed to adapt treatment intensity to the patient's individual risk. We quantified circulating tumor DNA (ctDNA) in sequential plasma samples and correlated ctDNA levels with disease outcome. Ninety four longitudinal plasma samples from 16 locally advanced HNC patients and 57 healthy donors were collected at re-radiotherapy baseline, after 5 and 10 radiation fractions, at irradiation end, and at routine follow-up visits. Plasma DNA was subjected to low coverage whole genome sequencing for copy number variation (CNV) profiling to quantify ctDNA burden. CNV-based ctDNA burden was detected in 8/16 patients and 25/94 plasma samples. Ten additional ctDNA-positive samples were identified by tracking patient-specific CNVs found in earlier sequential plasma samples. ctDNA-positivity after 5 and 10 radiation fractions (both: log-rank, p = .050) as well as at the end of irradiation correlated with short progression-free survival (log-rank, p = .006). Moreover, a pronounced decrease of ctDNA toward re-radiotherapy termination was associated with worse treatment outcome (log-rank, p = .005). Dynamic ctDNA tracking in serial plasma beyond re-radiotherapy reflected treatment response and imminent disease progression. In five patients, molecular progression was detected prior to tumor progression based on clinical imaging. Our findings emphasize that quantifying ctDNA during re-radiotherapy may contribute to disease monitoring and personalization of adjuvant treatment, follow-up intervals, and dose prescription.

Feasibility of enriched amplicon circulating tumor DNA sequencing to detect minimal residual disease (MRD) after prostatectomy in localized prostate cancer.

402 Background: Nearly one third of men with prostate cancer experience relapse after radical prostatectomy, often despite achieving undetectable PSA nadir. An assay to detect minimal residual disease would be valuable to identify patients who could benefit from adjuvant therapy. Methods: Eligible patients had biopsy proven prostate cancer and planned to undergo prostatectomy for definitive therapy. Blood was drawn at pre-specified timepoints (TP) including prior to prostatectomy (TP1), on the day after surgery or at the 1-week post-op catheter removal visit (TP2), and at 1-month post-op with no interval treatment (TP3). We performed tumor-guided plasma DNA analysis using a novel method based on single-stranded adapter ligation, incorporation of unique molecular identifiers (UMIs), targeted PCR, normalization of amplicons using hybrid-capture, and targeted sequencing. Using tumor and germline exome sequencing, we designed and validated multiplexed assays to target somatic founder mutations. We prepared targeted sequencing libraries using a median of 6.7 ng input plasma DNA. Results: 11 patients were enrolled after IRB approval. 1 (9.09%) had grade group 2, 1 (9.09%) grade group 3, 5 (45.45%) grade group 4, 4 (36.36%) grade group 5 disease. Median PSA pre-op was 9.3 ng/mL (range: 1.7 – 21.47). Tumor DNA sequencing was successful and targeted assays were generated for all patients (11/11). Median number of targets tested was 20 (range 4 to 88). We analyzed 31 plasma DNA samples obtained from 11 patients. Median on-target rate in plasma DNA was 90.5% (range 76.0% to 96.3%). Prior to surgery (TP1), 5/11 (45%) patients had detectable ctDNA at a median tumor fraction of 0.077% (range: 0.006% to 3.4%). At post-op TP (TP2), the ctDNA was detectable in 7/11 patients (64%) at a median tumor fraction of 0.024% (range: 0.004% to 3.1%). Detectable ctDNA was observed at 1 month (TP3) in 5/8 patients (62.5%) at a median tumor fraction of 0.022% (range 0.010% to 0.037%). For an exploratory analysis, we examined whether persistent ctDNA detection at the 1 month TP3 was associated with later relapse. Of the 5 patients with PSA relapse, 3 (60%) of patients had detectable residual ctDNA at TP3 with median follow up time of 61.75 months. Conclusions: Localized prostate cancer has been noted to have low tumor shed, limiting meaningful ctDNA detection rates with prior assays (Hennigan 2019). Our results show promising feasibility of a novel tumor-informed ctDNA assay in this setting. In addition, there was preliminary evidence of residual ctDNA detection predicting relapse.

Correlates of immunotherapy response in metastatic clear cell renal cell carcinoma from circulating cell-free epigenomes.

593 Background: Immune checkpoint inhibitors (ICIs) are a mainstay of treatment for metastatic clear cell renal cell carcinoma (mccRCC). With a growing number of treatment options for mccRCC, biomarkers are needed to guide treatment selection by predicting benefit from ICIs. Cell-free DNA (cfDNA)-based assays that measure tumor DNA in plasma are promising biomarkers for treatment response in several cancers, but their role in RCC remains unclear. Here, we tested whether epigenomic profiling of cell-free DNA can identify or predict radiographic responses to immunotherapy in mccRCC. Methods: Plasma samples were obtained from 180 patients (400 total samples collected, up to 4 samples per patient) with mccRCC enrolled in the DOD-funded Kidney Cancer Research Consortium ctDNA study (NCT04883827). Patients received an immune checkpoint inhibitor and were categorized as non-responders or responders based on CT scan results obtained at 3- or 6-months following treatment initiation. Cell-free chromatin immunoprecipitation sequencing (cfChIP-seq) was performed on patient plasma to profile H3K4me3, a histone modification associated with active gene promoters. DNA methylation was profiled with cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq). H3K4me3 signal at the promoters of ccRCC-associated genes variation between pre-treatment and on-treatment timepoints was assessed for non-responders and responders. We calculated changes in H3K4me3 signal for on- and pre-treatment plasma draws. We then compared these changes between non-responders and responders using the Wilcoxon sum-rank test. The immune cell types contributing to cfDNA were inferred from cfMeDIP-seq data. Results: Comparing pre- and on-treatment timepoints drawn between 1-5 months after starting therapy, cfDNA promoter H3K4me3 at RCC-associated genes was concordant with radiographic response in 10 of 11 patients (91%), with signal increasing from pre- to on-treatment samples in 6 of 7 non-responders (86%) and decreasing in 4 of 4 responders (100%). The median change in H3K4me3 differed significantly for non-responders and responders (p = 0.02). Analysis of cfDNA methylation profiles identified a higher proportion of memory CD8 T cell-derived cfDNA in non-responders compared to responders in pre-treatment samples (p = 0.003) Conclusions: This pilot study suggests that epigenomic profiling of cfDNA may identify pre-treatment and on-treatment biomarkers of ICI response. Profiling of larger cohorts is underway to test the generalizability of these findings.

Extracellular DNA in patients with urothelial carcinoma of the bladder.

834 Background: Higher concentration of extracellular DNA (ecDNA) in plasma of subjects with various cancers is associated with worse prognosis. The association of ecDNA and its subcellular origin – nuclear DNA (ncDNA) and mitochondrial DNA (mtDNA) with urothelial carcinoma of the bladder (BC) is unexplored. Deoxyribonuclease (DNase) can cleave ecDNA and modulate the observed association. The objective of this study was to identify the potential differences in ecDNA, ncDNA, mtDNA and DNase between patients with non-muscle-infiltrating BC (NMIBC) and muscle-infiltrating BC (MIBC) compared to controls. Methods: We analyzed a population of 62 individuals with BC (44 males, median age 67.5 years), 20 had Ta stage, 26 T1 stage (NMIBC) and 16 T2–T3 stage (MIBC). The control group consisted of 15 males and 6 females without history of cancer, median age 67.3 years. EcDNA was quantified fluorometrically after isolation from double centrifuged plasma. The subcellular origin of ecDNA was analyzed using quantitative real time PCR targeting specific nuclear and mitochondrial sequences. DNase activity was assessed using the single radial enzyme diffusion method. For statistical analysis, basic nonparametric tests were used. Results: In patients with BC compared to controls, we identified significantly higher ecDNA (median 15.0 v 11.7 ng/ml, P=.021) and ncDNA (median 4707 v 2136 GE/ml, P=.0046), but not mtDNA and DNase. Moreover, we determined a higher activity of DNase (median 2.3 vs 1.9 ng/ml, P=.0084) in subjects with NMIBC (Ta+T1) v MIBC. By mutual comparisons of all subgroups, following significant differences were recognized: ecDNA and ncDNA in MIBC v controls (P=.0065 and P=.0127, respectively) and DNase for Ta v MIBC (P=.0177). In addition, we detected a significant correlation on a stage of BC and rising ecDNA concentrations (P=.0076). Conclusions: In this study, we showed higher plasma ecDNA and ncDNA in patients with MIBC than controls and lower DNase activity in MIBC compared to NMIBC. The ecDNA concentrations increased with rising tumor stage. These results suggest a possible diagnostic and a likely prognostic value of ecDNA and DNase in BC. Further studies will focus on a potential association between plasma ecDNA and urinary ecDNA, which are assessed non-invasively and more frequently. This study was supported by the Ministry of Education, Science, Research and Sport of the Slovak Republic (grant number : VEGA 1/0090/22), the Slovak Research and Development Agency (grant number: APVV-22-0231) and OncoReSearch. Key Words: Bladder Cancer. Extracellular DNA. Nuclear DNA. Mitochondrial DNA. Nuclease.

Evaluation of the Alinity m CMV assay for detecting and quantifying cytomegalovirus DNA in non-plasma samples.

Evaluation of the Alinity m CMV assay for detecting and quantifying cytomegalovirus DNA in non-plasma samples.

Whole genome sequencing-powered ctDNA sequencing for breast cancer detection.

Circulating tumour DNA (ctDNA)-based detection of molecular residual disease (MRD) presents a strategy to identify patients at high risk of relapse. In this article, we profile early breast cancer patients with an ultrasensitive, whole genome sequencing (WGS)-based, tumour-informed ctDNA platform. We analysed 617 plasma samples (median 8, range 2-14) from 78 patients (23 triple-negative breast cancer, 35 human epidermal growth factor receptor 2-positive, 18 hormone receptor-positive, and 2 unknown). Samples were collected at diagnosis before therapy, cycle 2 of neoadjuvant chemotherapy, post-surgery after neoad'juvant therapy if administered, every 3 months during the first year, and every 6 months thereafter. Plasma DNA was analysed using the NeXT Personal MRD platform, a tumour-informed WGS approach to produce personalized ctDNA sequencing panels tracking a median of 1451 variants per patient. MRD detection was correlated with clinical outcomes. ctDNA was detected at levels ranging from 2.19 parts per million (PPM) to 204 900 PPM (median 405 PPM), with 39% of all ctDNA detections in the ultra-low range <100 PPM. Of patients with samples at diagnosis, 98% (49/50) had ctDNA detected before treatment. At a median follow-up of 76 months (range 5-118 months), detection of ctDNA was associated with high risk of future relapse (P < 0.0001; log-rank test) and shortened overall survival (P < 0.0001) with a median lead time from ctDNA detection to clinical relapse of 15 months (range 0.9-61.5 months). MRD was identified in 100% (11/11) of patients who relapsed, with a median level of ctDNA at first MRD detection of 13.1 PPM. No ctDNA-undetected patients relapsed throughout follow-up (64/64). Comparison with exome-powered MRD detection assays showed improved sensitivity and lead time. A whole genome-powered MRD assay detected breast cancer relapse with a long lead time over clinical relapse, and was strongly associated with relapse-free survival. Rates of ctDNA detection at diagnosis were higher than those reported with exome-based tumour-informed assays.

Fetal Fraction of Cell-Free DNA in Noninvasive Prenatal Testing and Adverse Pregnancy Outcomes: A Nationwide Retrospective Cohort Study of 56,110 Pregnant Women

(Abstracted from Am J Obstet Gynecol 2024;231:244.e1–244.e18) Noninvasive prenatal testing (NIPT) for Down, Edwards, and Patau syndromes is widely available. Its accuracy depends on a sufficient amount of fetal cell-free DNA (cfDNA) in maternal plasma.