Multi-centre analytical performance verification of an IVD assay to quantify donor-derived cell-free DNA in solid organ transplant recipients.

Donor-derived cell-free DNA (dd-cfDNA) has been widely studied as biomarker for non-invasive allograft rejection monitoring. Earlier rejection detection enables more prompt diagnosis and intervention, ultimately improving patient treatment and outcomes. This multi-centre study aims to verify analytical performance of a next-generation sequencing-based dd-cfDNA assay at end-user environments. Three independent laboratories received the same experimental design and 16 blinded samples to perform cfDNA extraction and the dd-cfDNA assay workflow. dd-cfDNA results were compared between sites and against manufacturer validation to evaluate concordance, reproducibility, repeatability and verify analytical performance. A total of 247 sample libraries were generated across 18 runs, with completion time of <24 h. A 96.0% first pass rate highlighted minimal failures. Overall observed versus expected dd-cfDNA results demonstrated good concordance and a strong positive correlation with linear least squares regression r2 = 0.9989, and high repeatability and reproducibility within and between sites, respectively (p > 0.05). Manufacturer validation established limit of blank 0.18%, limit of detection 0.23% and limit of quantification 0.23%, and results from independent sites verified those limits. Parallel analyses illustrated no significant difference (p = 0.951) between dd-cfDNA results with or without recipient genotype. The dd-cfDNA assay evaluated here has been verified as a reliable method for efficient, reproducible dd-cfDNA quantification in plasma from solid organ transplant recipients without requiring genotyping. Implementation of onsite dd-cfDNA testing at clinical laboratories could facilitate earlier detection of allograft injury, bearing great potential for patient care.

Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer.

OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

Predicting Preterm Birth Using Cell-Free Ribonucleic Acid.

Spontaneous preterm birth (sPTB) is a complex and clinically heterogeneous condition that remains incompletely understood, leading to insufficient interventions to effectively prevent it from occurring. Cell-free ribonucleic acid signatures in the maternal circulation have the potential to identify biologically relevant subtypes of sPTB. These could one day be used to predict and prevent sPTB in asymptomatic individuals, and to aid in prognosis and management for individuals presenting with threatened preterm labor and preterm prelabor rupture of membranes.

Multimodal analysis of cfDNA methylomes for early detecting esophageal squamous cell carcinoma and precancerous lesions.

Detecting early-stage esophageal squamous cell carcinoma (ESCC) and precancerous lesions is critical for improving survival. Here, we conduct whole-genome bisulfite sequencing (WGBS) on 460 cfDNA samples from patients with non-metastatic ESCC or precancerous lesions and matched healthy controls. We develop an expanded multimodal analysis (EMMA) framework to simultaneously identify cfDNA methylation, copy number variants (CNVs), and fragmentation markers in cfDNA WGBS data. cfDNA methylation markers are the earliest and most sensitive, detectable in 70% of ESCCs and 50% of precancerous lesions, and associated with molecular subtypes and tumor microenvironments. CNVs and fragmentation features show high specificity but are linked to late-stage disease. EMMA significantly improves detection rates, increasing AUCs from 0.90 to 0.99, and detects 87% of ESCCs and 62% of precancerous lesions with >95% specificity in validation cohorts. Our findings demonstrate the potential of multimodal analysis of cfDNA methylome for early detection and monitoring of molecular characteristics in ESCC.

Role of mitochondrial DNA level in epidural-related maternal fever: a single-centre, observational, pilot study.

INTRODUCTION: Epidural analgesia has been associated with intrapartum maternal fever development. Epidural-related maternal fever (ERMF) is believed to be based on a non-infectious inflammatory reaction. Circulating cell-free mitochondrial deoxyribonucleic acid (mtDNA) is one of the possible triggers of sterile inflammatory processes; however, a connection has not been investigated so far. Therefore, this study aimed to investigate cell-free mtDNA alterations in women in labour with ERMF in comparison with non-febrile women. MATERIAL AND METHODS: A total of 60 women in labour were assessed for maternal temperature every 4 h and blood samples were obtained at the beginning and after delivery. Depending on the analgesia and the development of fever (axillary temperature ≥ 37.5 °C), the women were allocated either to the group of no epidural analgesia (n = 17), to epidural analgesia no fever (n = 34) or to ERMF (n = 9). Circulating cell-free mtDNA was analysed in the maternal plasma for the primary outcome whereas secondary outcomes include the evaluation of inflammatory cytokine release, as well as placental inflammatory signs. RESULTS: Of the women with epidural analgesia, 20% (n = 9) developed ERMF and demonstrated a decrease of circulating mtDNA levels during labour (p = 0.04), but a trend towards higher free nuclear DNA. Furthermore, women with maternal pyrexia showed a 1.5 fold increased level of Interleukin-6 during labour. A correlation was found between premature rupture of membranes and ERMF. CONCLUSIONS: The pilot trial revealed an evident obstetric anaesthesia phenomenon of maternal fever due to epidural analgesia in 20% of women in labour, demonstrating counterregulated free mtDNA and nDNA. Further work is urgently required to understand the connections between the ERMF occurrence and circulating cell-free mtDNA as a potential source of sterile inflammation. TRIAL REGISTRATION: NCT0405223 on clinicaltrials.gov (registered on 25/07/2019).

5-Hydroxymethylcytosine in Cell-Free DNA Predicts Immunotherapy Response in Lung Cancer.

Immune checkpoint inhibitors (ICIs) drastically improve therapeutic outcomes for lung cancer, but accurately predicting individual patient responses to ICIs remains a challenge. We performed the genome-wide profiling of 5-hydroxymethylcytosine (5hmC) in 85 plasma cell-free DNA (cfDNA) samples from lung cancer patients and developed a 5hmC signature that was significantly associated with progression-free survival (PFS). We built a 5hmC predictive model to quantify the 5hmC level and validated the model in the validation, test, and control sets. Low weighted predictive scores (wp-scores) were significantly associated with a longer PFS compared to high wp-scores in the validation [median 7.6 versus 1.8 months; p = 0.0012; hazard ratio (HR) 0.12; 95% confidence interval (CI), 0.03-0.54] and test (median 14.9 versus 3.3 months; p = 0.00074; HR 0.10; 95% CI, 0.02-0.50) sets. Objective response rates in patients with a low or high wp-score were 75.0% (95% CI, 42.8-94.5%) versus 0.0% (95% CI, 0.0-60.2%) in the validation set (p = 0.019) and 80.0% (95% CI, 44.4-97.5%) versus 0.0% (95% CI, 0.0-36.9%) in the test set (p = 0.0011). The wp-scores were also significantly associated with PFS in patients receiving single-agent ICI treatment (p < 0.05). In addition, the 5hmC predictive signature demonstrated superior predictive capability to tumor programmed death-ligand 1 and specificity to ICI treatment response prediction. Moreover, we identified novel 5hmC-associated genes and signaling pathways integral to ICI treatment response in lung cancer. This study provides proof-of-concept evidence that the cfDNA 5hmC signature is a robust biomarker for predicting ICI treatment response in lung cancer.

Dynamics and prognostic value of plasma cell-free DNA PCR in patients with invasive aspergillosis and mucormycosis.

Aspergillus species and Mucorales agents are the primary etiologies of invasive fungal disease (IFD). Biomarkers that predict outcomes are needed to improve care. Patients diagnosed with invasive aspergillosis and mucormycosis using plasma cell-free DNA (cfDNA) PCR were retested weekly for 4 weeks. The primary outcome included all-cause mortality at 6 weeks and 6 months based on baseline cycle threshold (CT) values and results of follow-up cfDNA PCR testing. Forty-five patients with Aspergillus and 30 with invasive Mucorales infection were retested weekly for a total of 197 tests. Using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSG) criteria, 30.7% (23/75), 25.3% (19/75), and 38.7% (29/75) had proven, probable, and possible IFD, respectively. In addition, 97.3% (73/75) were immunocompromised. Baseline CT increased significantly starting at week 1 for Mucorales and week 2 for Aspergillus. Aspergillosis and mucormycosis patients with higher baseline CT (CT >40 and >35, respectively) had a nonsignificantly higher survival rate at 6 weeks, compared with patients with lower baseline CT. Mucormycosis patients with higher baseline CT had a significantly higher survival rate at 6 months. Mucormycosis, but not aspergillosis patients, with repeat positive cfDNA PCR results had a nonsignificantly lower survival rate at 6 weeks and 6 months compared with patients who reverted to negative. Aspergillosis patients with baseline serum Aspergillus galactomannan index <0.5 and <1.0 had significantly higher survival rates at 6 weeks when compared with those with index ≥0.5 and ≥1.0, respectively. Baseline plasma cfDNA PCR CT can potentially be used to prognosticate survival in patients with invasive Aspergillus and Mucorales infections. IMPORTANCE: We show that Aspergillus and Mucorales plasma cell-free DNA PCR can be used not only to noninvasively diagnose patients with invasive fungal disease but also to correlate the baseline cycle threshold with survival outcomes, thus potentially allowing the identification of patients at risk for poor outcomes, who may benefit from more targeted therapies.

Circulating tumor DNA in liquid biopsy: Current diagnostic limitation.

With the rapid development of science and technology, cell-free DNA (cfDNA) is rapidly becoming an important biomarker for tumor diagnosis, monitoring and prognosis, and this cfDNA-based liquid biopsy technology has great potential to become an important part of precision medicine. cfDNA is the total amount of free DNA in the systemic circulation, including DNA fragments derived from tumor cells and all other somatic cells. Tumor cells release fragments of DNA into the bloodstream, and this source of cfDNA is called circulating tumor DNA (ctDNA). cfDNA detection has become a major focus in the field of tumor research in recent years, which provides a new opportunity for non-invasive diagnosis and prognosis of cancer. In this paper, we discuss the limitations of the study on the origin and dynamics analysis of ctDNA, and how to solve these problems in the future. Although the future faces major challenges, it also contains great potential.

Mitochondrial Fraction of Circulating Cell-Free DNA as an Indicator of Human Pathology.

Circulating cell-free DNA (ccfDNA) of mitochondrial origin (ccf-mtDNA) consists of a minor fraction of total ccfDNA in blood or in other biological fluids. Aberrant levels of ccf-mtDNA have been observed in many pathologies. Here, we introduce a simple and effective standardized Taqman probe-based dual-qPCR assay for the simultaneous detection and relative quantification of nuclear and mitochondrial fragments of ccfDNA. Three pathologies of major burden, one malignancy (Breast Cancer, BrCa), one inflammatory (Osteoarthritis, OA) and one metabolic (Type 2 Diabetes, T2D), were studied. Higher levels of ccf-mtDNA were detected both in BrCa and T2D in relation to health, but not in OA. In BrCa, hormonal receptor status was associated with ccf-mtDNA levels. Machine learning analysis of ccf-mtDNA datasets was used to build biosignatures of clinical relevance. (A) a three-feature biosignature discriminating between health and BrCa (AUC: 0.887) and a five-feature biosignature for predicting the overall survival of BrCa patients (Concordance Index: 0.756). (B) a five-feature biosignature stratifying among T2D, prediabetes and health (AUC: 0.772); a five-feature biosignature discriminating between T2D and health (AUC: 0.797); and a four-feature biosignature identifying prediabetes from health (AUC: 0.795). (C) a biosignature including total plasma ccfDNA with very high performance in discriminating OA from health (AUC: 0.934). Aberrant ccf-mtDNA levels could have diagnostic/prognostic potential in BrCa and Diabetes, while the developed multiparameter biosignatures can add value to their clinical management.

Diagnostic and Prognostic Value of Circulating DNA Fragments in Glioblastoma Multiforme Patients.

Novel blood-circulating molecules, as potential biomarkers for glioblastoma multiforme (GBM) diagnosis and monitoring, are attracting particular attention due to limitations of imaging modalities and invasive tissue biopsy procedures. This study aims to assess the diagnostic and prognostic values of circulating cell-free DNA (cfDNA) in relation to inflammatory status in GBM patients and to determine the concentration and average size of DNA fragments typical of tumour-derived DNA fractions. Preoperative plasma samples from 40 patients (GBM 65.0 ± 11.3 years) and 40 healthy controls (HC 70.4 ± 5.4 years) were compared. The cfDNA concentrations and lengths were measured using the electrophoresis platform, and inflammatory indices (NLR, PLR, LMR, and SII) were calculated from complete blood cell analysis. More fragmented cfDNA and 4-fold higher 50-700 bp cfDNA concentrations were detected in GBM patients than in healthy controls. The average cfDNA size in the GBM group was significantly longer (median 336 bp) than in the HC group (median 271 bp). Optimal threshold values were 1265 pg/µL for 50-700 bp cfDNA (AUC = 0.857) and 290 bp for average cfDNA size (AUC = 0.814). A Kaplan-Meier survival curves analysis also demonstrated a higher mortality risk in the GBM group with a cut-off >303 bp cfDNA. This study is the first to have revealed glioblastoma association with high levels of cfDNA > 1000 pg/µL of 50-700 bp in length, which can be aggravated by immunoinflammatory reactivity.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

Technical Advances in Circulating Cell-Free DNA Detection and Analysis for Personalized Medicine in Patients' Care.

Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in the recent literature, several challenges remain, represented by a low abundance, a need for highly sensitive assays, and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it allows us to monitor patients' conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a diagnostic marker to be further validated, and very few centers are implementing its analysis in routine diagnostics. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients' quality of life.

Novel Blood Biomarkers for Response Prediction and Monitoring of Stereotactic Ablative Radiotherapy and Immunotherapy in Metastatic Oligoprogressive Lung Cancer.

Up to 80% of patients under immune checkpoint inhibitors (ICI) face resistance. In this context, stereotactic ablative radiotherapy (SABR) can induce an immune or abscopal response. However, its molecular determinants remain unknown. We present early results of a translational study assessing biomarkers of response to combined ICI and SABR (I-SABR) in liquid biopsy from oligoprogressive patients in a prospective observational multicenter study. Cohort A includes metastatic patients in oligoprogression to ICI maintaining the same ICI due to clinical benefit and who receive concomitant SABR. B is a comparative group of oligometastatic patients receiving only SABR. Blood samples are extracted at baseline (T1), after the first (T2) and last (T3) fraction, two months post-SABR (T4) and at further progression (TP). Response is evaluated by iRECIST and defined by the objective response rate (ORR)-complete and partial responses. We assess peripheral blood mononuclear cells (PBMCs), circulating cell-free DNA (cfDNA) and small RNA from extracellular vesicles. Twenty-seven patients could be analyzed (cohort A: n = 19; B: n = 8). Most were males with non-small cell lung cancer and one progressing lesion. With a median follow-up of 6 months, the last ORR was 63% (26% complete and 37% partial response). A decrease in cfDNA from T2 to T3 correlated with a good response. At T2, CD8+PD1+ and CD8+PDL1+ cells were increased in non-responders and responders, respectively. At T2, 27 microRNAs were differentially expressed. These are potential biomarkers of response to I-SABR in oligoprogressive disease.

A Cell-free DNA Blood-Based Test for Colorectal Cancer Screening.

BACKGROUND: Colorectal cancer is the third most diagnosed cancer in adults in the United States. Early detection could prevent more than 90% of colorectal cancer-related deaths, yet more than one third of the screening-eligible population is not up to date with screening despite multiple available tests. A blood-based test has the potential to improve screening adherence, detect colorectal cancer earlier, and reduce colorectal cancer-related mortality. METHODS: We assessed the performance characteristics of a cell-free DNA (cfDNA) blood-based test in a population eligible for colorectal cancer screening. The coprimary outcomes were sensitivity for colorectal cancer and specificity for advanced neoplasia (colorectal cancer or advanced precancerous lesions) relative to screening colonoscopy. The secondary outcome was sensitivity to detect advanced precancerous lesions. RESULTS: The clinical validation cohort included 10,258 persons, 7861 of whom met eligibility criteria and were evaluable. A total of 83.1% of the participants with colorectal cancer detected by colonoscopy had a positive cfDNA test and 16.9% had a negative test, which indicates a sensitivity of the cfDNA test for detection of colorectal cancer of 83.1% (95% confidence interval [CI], 72.2 to 90.3). Sensitivity for stage I, II, or III colorectal cancer was 87.5% (95% CI, 75.3 to 94.1), and sensitivity for advanced precancerous lesions was 13.2% (95% CI, 11.3 to 15.3). A total of 89.6% of the participants without any advanced colorectal neoplasia (colorectal cancer or advanced precancerous lesions) identified on colonoscopy had a negative cfDNA blood-based test, whereas 10.4% had a positive cfDNA blood-based test, which indicates a specificity for any advanced neoplasia of 89.6% (95% CI, 88.8 to 90.3). Specificity for negative colonoscopy (no colorectal cancer, advanced precancerous lesions, or nonadvanced precancerous lesions) was 89.9% (95% CI, 89.0 to 90.7). CONCLUSIONS: In an average-risk screening population, this cfDNA blood-based test had 83% sensitivity for colorectal cancer, 90% specificity for advanced neoplasia, and 13% sensitivity for advanced precancerous lesions. (Funded by Guardant Health; ECLIPSE ClinicalTrials.gov number, NCT04136002.).

Fragmentation patterns of cell-free DNA and somatic mutations in the urine of metastatic breast cancer patients.

BACKGROUND: Urinary cell-free deoxyribonucleic acid (DNA) (ucfDNA) holds promise as a biomarker; however, its potential remains largely unexplored. We examined the fragmentation pattern of ucfDNA and identified somatic mutations within urine samples from metastatic breast cancer (MBC) patients. METHODS: Urine and blood specimens were collected before treatment from 45 MBC patients and posttreatment urine samples from 16 of the 45 patients at the China National Cancer Center. Somatic mutations and tumor mutational burden (TMB) in the urine and plasma of 10 patients were analyzed by next-generation sequencing (NGS). Fragmentation patterns of cfDNA were displayed using electropherograms. Differences in the extracted amount of cfDNA, length of cfDNA fragments, and TMB between urine and plasma were compared using a Wilcoxon test. RESULTS: The fragmentation patterns of ucfDNA were categorized as follows: (1) profile A (n = 26) containing a short peak (100-200 bp) and a long peak (>1500 bp); (2) profile B (n = 8) containing only a long peak; and (3) profile C (n = 11) containing flat pattern. For profile A patients, the short-peaked ucfDNA circulating in the bloodstream was much shorter compared with plasma cfDNA (149 vs. 171 bp, Wilcoxon test, P = 0.023). The fragmentation patterns in lung metastasis patients exhibited a higher propensity toward profile C ( P = 0.002). After treatment, 87.5% of the patients exhibited consistent fragmentation patterns. The concordance rate for somatic mutations in the plasma and urine was 30%, and the median TMB of urine and plasma was not significantly different. CONCLUSIONS: This study established a fragmentation pattern for ucfDNA and detected somatic mutations in the urine of MBC patients. These results suggest the potential application of ucfDNA as a biomarker for MBC.

Performance of cell-free DNA testing for common fetal trisomies in triplet pregnancies.

OBJECTIVE: In singleton pregnancies, the use of cell-free DNA (cfDNA) analysis as a screening test for common fetal trisomies has spread worldwide though we still lack sufficient data for its use in triplet pregnancies. The objective of this study is to assess the performance of cfDNA testing in detecting fetal aneuploidies in triplet pregnancies as a first-tier test. METHOD: We performed a retrospective cohort study including data from pregnant women with a triplet pregnancy who underwent cfDNA testing between May 1, 2017, and January 15, 2020. cfDNA was obtained by massive parallel sequencing (VeriSeq NIPT solution; Illumina®). The objectives of the study were to assess the diagnostic performance of cfDNA testing for trisomy 21 (T21) (primary outcome), trisomy 18 (T18) and 13 (secondary outcomes). RESULTS: During the study period, cfDNA testing was performed in 255 women with triplet pregnancy, of which 165 (64.7%) had a neonatal outcome available. Three tests were positive for T21, one of which was confirmed by an antenatal karyotype, and the other was confirmed at birth. The third case did not undergo an invasive procedure and was not confirmed at birth (false positive). In one case, cfDNA testing was positive for T18 and was confirmed by an antenatal karyotype. There were no cases of trisomy 13 in the cohort. The no-call rate was 2.4% at first sampling. Fifty-eight (22.7%) women had embryo reduction, which in 40 (69%) of whom was performed after the cfDNA test result. CONCLUSION: cfDNA testing could be offered as primary screening for main fetal aneuploidies in triplet pregnancies after provision of appropriate patient information.

Eliminating first trimester combined testing: Consequences for early detection of significant fetal anomalies.

OBJECTIVE: To determine whether implementation of cell-free DNA (cfDNA) testing for aneuploidy as a first-tier test and subsequent abolition of first trimester combined testing (FCT) affected the first trimester detection (<14 weeks) of certain fetal anomalies. METHODS: We performed a geographical cohort study in two Fetal Medicine Units between 2011 and 2020, including 705 fetuses with prenatally detected severe brain, abdominal wall and congenital heart defects. Cases were divided into two groups: before (n = 396) and after (n = 309) cfDNA introduction. The primary outcome was the first trimester detection rate (<14 weeks) overall and for non-chromosomal anomalies solely. RESULTS: Overall, gastroschisis, AVSD and HLHS were detected more often in the first trimester in the before group compared to the after group, respectively 54.5% versus 18.5% (p = 0.004), 45.9% versus 26.9% (p = 0.008) and 30% versus 3.4% (p = 0.005). After exclusion of chromosomal anomalies identifiable through cfDNA testing, the detection of AVSD remained higher in the before group (43.3% vs. 9.5%, p = 0.02), leading to a possible earlier gestation at termination. The termination of pregnancy (TOP) rate did not differ among the groups. In the after group, referrals for suspected anomalies following a dating scan between 11 and 14 weeks significantly increased from 17.4% to 29.1% (p < 0.001). CONCLUSION: This study underscores the value of a scan dedicated to fetal anatomy in the first trimester as we observed a decline in the early detection of certain fetal anomalies (detectable in the first trimester) subsequent to the abolition of FCT.

Evaluating the effectiveness of pre-operative diagnosis of ovarian cancer using minimally invasive liquid biopsies by combining serum human epididymis protein 4 and cell-free DNA in patients with an ovarian mass.

OBJECTIVE: To assess the feasibility of scalable, objective, and minimally invasive liquid biopsy-derived biomarkers such as cell-free DNA copy number profiles, human epididymis protein 4 (HE4), and cancer antigen 125 (CA125) for pre-operative risk assessment of early-stage ovarian cancer in a clinically representative and diagnostically challenging population and to compare the performance of these biomarkers with the Risk of Malignancy Index (RMI). METHODS: In this case-control study, we included 100 patients with an ovarian mass clinically suspected to be early-stage ovarian cancer. Of these 100 patients, 50 were confirmed to have a malignant mass (cases) and 50 had a benign mass (controls). Using WisecondorX, an algorithm used extensively in non-invasive prenatal testing, we calculated the benign-calibrated copy number profile abnormality score. This score represents how different a sample is from benign controls based on copy number profiles. We combined this score with HE4 serum concentration to separate cases and controls. RESULTS: Combining the benign-calibrated copy number profile abnormality score with HE4, we obtained a model with a significantly higher sensitivity (42% vs 0%; p<0.002) at 99% specificity as compared with the RMI that is currently employed in clinical practice. Investigating performance in subgroups, we observed especially large differences in the advanced stage and non-high-grade serous ovarian cancer groups. CONCLUSION: This study demonstrates that cell-free DNA can be successfully employed to perform pre-operative risk of malignancy assessment for ovarian masses; however, results warrant validation in a more extensive clinical study.

Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.