Predicting gestational diabetes mellitus risk at 11-13 weeks' gestation: the role of extrachromosomal circular DNA.

BACKGROUND: Gestational diabetes mellitus (GDM) significantly impacts maternal and infant health both immediately and over the long term, yet effective early diagnostic biomarkers are currently lacking. Thus, it is essential to identify early diagnostic biomarkers for GDM risk screening. Extrachromosomal circular DNA (eccDNA), being more stable than linear DNA and involved in disease pathologies, is a viable biomarker candidate for diverse conditions. In this study, eccDNA biomarkers identified for early diagnosis and assessment of GDM risk were explored. METHODS: Using Circle-seq, we identified plasma eccDNA profiles in five pregnant women who later developed GDM and five matched healthy controls at 11-13 weeks of gestation. These profiles were subsequently analyzed through bioinformatics and validated through outward PCR combined with Sanger sequencing. Furthermore, candidate eccDNA was validated by quantitative PCR (qPCR) in a larger cohort of 70 women who developed GDM and 70 normal glucose-tolerant (NGT) subjects. A ROC curve assessed the eccDNA's diagnostic potential for GDM. RESULTS: 2217 eccDNAs were differentially detected between future GDM patients and controls, with 1289 increased and 928 decreased in abundance. KEGG analysis linked eccDNA genes mainly to GDM-related pathways such as Rap1, MAPK, and PI3K-Akt, and Insulin resistance, among others. Validation confirmed a significant decrease in eccDNA PRDM16circle in the plasma of 70 women who developed GDM compared to 70 NGT women, consistent with the eccDNA-seq results. PRDM16circle showed significant diagnostic value in 11-13 weeks of gestation (AUC = 0.941, p < 0.001). CONCLUSIONS: Our study first demonstrats that eccDNAs are aberrantly produced in women who develop GDM, including PRDM16circle, which can predict GDM at an early stage of pregnancy, indicating its potential as a biomarker. TRIAL REGISTRATION: ChiCTR2300075971, http://www.chictr.org.cn . Registered 20 September 2023.

Differential HBV replicative markers and covalently closed circular DNA transcription in immune-active chronic hepatitis B with and without HBeAg.

BACKGROUND AND AIMS: Molecular processes driving immune-active chronic hepatitis B (CHB) with and without hepatitis B e antigen (HBeAg) remain incompletely understood. This study aimed to investigate expression profiles of serum and intrahepatic HBV markers and replicative activity of HBV in CHB patients with or without HBeAg. METHODS: This study recruited 111 untreated immune-active CHB (60 HBeAg-positive and 51 HBeAg-negative) patients and quantified intrahepatic covalently closed circular DNA (cccDNA), pre-genomic RNA (pgRNA), total HBV DNA (tDNA), and replicative intermediates as well as serum HBV markers (HBV DNA, hepatitis B surface antigen, hepatitis B core-related antigen). Correlations between HBV markers and clinico-virological factors influencing expression levels of HBV markers were analysed. RESULTS: Levels of all serum markers and intrahepatic cccDNA/tDNA as well as cccDNA transcriptional activity and virion productivity were significantly reduced in HBeAg-negative patients compared to those in HBeAg-positive patients. Additionally, correlations between intrahepatic cccDNA/pgRNA and serum markers were impaired in HBeAg-negative individuals. Aminotransferase levels were positively correlated with cccDNA transcriptional activity in HBeAg-positive patients, but not in HBeAg-negative patients. Notably, among HBeAg-positive patients, there was a progressive decline in pgRNA level, transcriptional activity, and serum HBV markers as liver fibrosis advanced, which was not observed in HBeAg-negative patients. CONCLUSIONS: HBeAg loss is correlated with diminished intrahepatic HBV reservoirs and cccDNA transcription, leading to decreased serum HBV marker levels. Circulating HBV markers are not reliable indicators of intrahepatic HBV replicative activity for HBeAg-negative patients. Our findings reveal distinct disease phenotypes between immune-active CHB with and without HBeAg, highlighting the need to establish optimal surrogate biomarkers that can accurately mirror intrahepatic viral activity to aid in decision-making for antiviral therapy for immune-active CHB.

One-Step Reverse Transcriptase qPCR Method for Serum Hepatitis B Virus RNA Quantification.

In recent years, serum hepatitis B virus (HBV) RNA has been identified as a promising noninvasive surrogate biomarker of intrahepatic covalently closed circular DNA (cccDNA), detection of which requires an invasive liver biopsy in patients with chronic HBV infection. It is impractical to detect intrahepatic cccDNA as a routine diagnosis for chronic hepatitis B (CHB) patients in clinical management. Here, we describe a detailed protocol for serum HBV RNA quantification, which can reflect the activity of intrahepatic cccDNA. The procedure includes three major steps: (1) Simultaneous isolation of HBV DNA and RNA from patients' serum, (2) DNase I digestion for removing HBV DNA contamination, and (3) HBV RNA quantification by one-step reverse transcription qPCR.

Characterization of extrachromosomal circular DNAs in plasma of patients with clear cell renal cell carcinoma.

BACKGROUND AND PURPOSE: Extrachromosomal circular DNAs (eccDNAs) have been recognized for their significant involvement in numerous biological processes. Nonetheless, the existence and molecular characteristics of eccDNA in the peripheral blood of patients diagnosed with clear cell renal cell carcinoma (ccRCC) have not yet been reported. Our aim was to identify potentially marked plasma eccDNAs in ccRCC patients. METHODS AND MATERIALS: The detection of plasma eccDNA in ccRCC patients and healthy controls was performed using the Tn5-tagmentation and next-generation sequencing (NGS) method. Comparisons were made between ccRCC patients and healthy controls regarding the distribution of length, gene annotation, pattern of junctional nucleotide motif, and expression pattern of plasma eccDNA. RESULTS: We found 8,568 and 8,150 plasma eccDNAs in ccRCC patients and healthy controls, respectively. There were no statistical differences in the length distribution, gene annotation, and motif signature of plasma eccDNAs between the two groups. A total of 701 differentially expressed plasma eccDNAs were identified, and 25 plasma eccDNAs with potential diagnostic value for ccRCC have been successfully screened. These up-regulated plasma eccDNAs also be indicated to originate from the genomic region of the tumor-associated genes. CONCLUSION: This work demonstrates the characterization of plasma eccDNAs in ccRCC and suggests that the up-regulated plasma eccDNAs could be considered as a promising non-invasive biomarker in ccRCC.

Categorizing Extrachromosomal Circular DNA as Biomarkers in Serum of Cancer.

Extrachromosomal circular DNA (eccDNA), a double-stranded circular DNA molecule found in multiple organisms, has garnered an increasing amount of attention in recent years due to its close association with the initiation, malignant progression, and heterogeneous evolution of cancer. The presence of eccDNA in serum assists in non-invasive tumor diagnosis as a biomarker that can be assessed via liquid biopsies. Furthermore, the specific expression patterns of eccDNA provide new insights into personalized cancer therapy. EccDNA plays a pivotal role in tumorigenesis, development, diagnosis, and treatment. In this review, we comprehensively outline the research trajectory of eccDNA, discuss its role as a diagnostic and prognostic biomarker, and elucidate its regulatory mechanisms in cancer. In particular, we emphasize the potential application value of eccDNA in cancer diagnosis and treatment and anticipate the development of novel tumor diagnosis strategies based on serum eccDNA in the future.

Differential expression and analysis of extrachromosomal circular DNAs as serum biomarkers in pulmonary arterial hypertension.

BACKGROUND: Extrachromosomal circular DNAs (eccDNAs) have been reported to play a key role in the occurrence and development of various diseases. However, the characterization and role of eccDNAs in pulmonary arterial hypertension (PAH) remain unclear. METHODS: In the discovery cohort, we first explored eccDNA expression profiles by Circle-sequencing analysis. The candidate eccDNAs were validated by routine polymerase chain reaction (PCR), TOPO-TA cloning and Sanger sequencing. In the validation cohort, 30 patients with PAH and 10 healthy controls were recruited for qPCR amplification to detect the candidate eccDNAs. Datas at the baseline were collected, including clinical background, biochemical variables, echocardiography and hemodynamic factors. Receiver operating characteristic curve was used to investigate the diagnostic effect of the eccDNA. RESULTS: We identified a total of 21,741 eccDNAs in plasma samples of 3 IPAH patients and 3 individuals in good health, and the expression frequency, GC content, length distribution, and genome distribution of the eccDNAs were thoroughly characterized and analyzed. In the validation cohort, 687 eccDNAs were differentially expressed in patients with IPAH compared with healthy controls (screening threshold: |FC|≥2 and P < 0.05). Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the specific eccDNAs in IPAH were significantly enriched in calcium channel activity, the mitogen-activated protein kinase pathway, and the wnt signaling pathway. Verification queue found that the expression of eccDNA-chr2:131208878-131,424,362 in PAH was considerably higher than that in healthy controls and exhibited a high level of accuracy in predicting PAH with a sensitivity of 86.67% and a specificity of 90%. Furthermore, correlation analysis disclosed a significant association between serum eccDNA-chr2:131208878-131,424,362 and mean pulmonary artery pressure (mPAP) (r = 0.396, P = 0.03), 6 min walking distance (6MWD) (r = -0.399, P = 0.029), N-terminal pro-B-type natriuretic peptide (NT-proBNP) (r = 0.685, P < 0.001) and cardiac index (CI) (r = - 0.419, P = 0.021). CONCLUSIONS: This is the first study to identify and characterize eccDNAs in patients with PAH. We revealed that serum eccDNA-chr2:131208878-131,424,362 is significantly overexpressed and can be used in the diagnosis of PAH, indicating its potential as a novel non-invasive biomarker.

Differential expression and analysis of extrachromosomal circular DNAs as serum biomarkers in lung adenocarcinoma.

BACKGROUND: Extrachromosomal circular DNAs (eccDNAs) increase the number of proto-oncogenes by enhancing oncogene expression to promote tumorigenesis. However, there are limited reports on differential eccDNA expression and analysis in lung cancer, especially in lung adenocarcinoma (LAD). METHODS: Three LAD and three corresponding NT tissues samples were used for eccDNA next-generation sequencing analysis, and an additional 20 were used for quantitative PCR (qPCR) evaluations. We further performed qPCR amplification using serum samples from LAD patients and healthy medical examiners. RESULTS: eccDNAs from LAD samples were mainly 200-1000 bp in length. Gene annotation analysis revealed that most eccDNAs were derived from chromosomes 1 and 2. The top-ten increased and top-ten decreased eccDNAs in LAD tissues were CircD-ARPC1B, CircD-ARPC1A, CircD-FAM49B, CircD-SDK1, CircD-KCNG1, CircD-POLR2F, CircD-SS18L1, CircD-SLC16A3, CircD-CSNK1D, CircD-KCTD1, and CircD-TMIGD2, CircD-PDIA5, CircD-VAV2, CircD-GATAD2A, CircD-CAB39L, CircD-KHDC1, CircD-FOXN3, CircD-SULT2B1, CircD-DPP9, and CircD-CSNK1D. qPCR demonstrated that the expression of CircD-DZRN3 was higher in LAD tissues than in normal lung tissues, whereas CircD-LGR6 and CircD-UMODL1 expression levels were lower in LAD than in normal lung tissues. Furthermore, the serum CircD-PDZRN3 level increased, while CircD-LGR6 decreased in LAD. Receiver operating characteristic (ROC) analysis showed that area under curve (AUC) of serum CircD-PDZRN3 (0.991), CircD-LGR6 (0.916) was higher than that of serum carcinoembryonic antigen (CEA) (0.825), CY211 (cytokeratin 19 fragment) (0.842), SCCA(squamous cell carcinoma antigen) (0.857) for the diagnosis of LAD. CONCLUSIONS: Our study first showed that several eccDNAs were aberrantly expressed in LAD, among which CircD-PDZRN3 and CircD-LGR6 clearly distinguished LAD patients from healthy controls, indicating their potential as biomarkers.

Ultrasensitive Assay for Hepatitis B Core-Related Antigen Predicts Hepatocellular Carcinoma Incidences During Entecavir.

Serum hepatitis B core-related antigen (HBcrAg) and surface antigen (HBsAg) are surrogate markers of intrahepatic covalently closed circular DNA. The measurement range of the current HBcrAg assay is relatively narrow. Thus, we examined the potential of HBcrAg and HBsAg measured by ultrasensitive assays for predicting hepatocellular carcinoma (HCC) development in patients with chronic hepatitis B treated with entecavir (ETV). We conducted a retrospective cohort study of 180 patients who received ETV for >1 year. All patients had hepatitis B e-antigen negativity at baseline. Serum HBcrAg and HBsAg levels at baseline and year 1 were measured in all patients by ultrasensitive assays using immunoassay for total antigen including complex by pretreatment (iTACT) technology. During the median follow-up of 11.0 years, 22 patients developed HCC (11.8/1,000 person-years). Baseline HBsAg levels were not associated with HCC development during ETV treatment. However, high HBcrAg levels at baseline and at year 1 were significantly associated with HCC development (log-rank test; P < 0.001). In 110 patients (61.1%) with ≥4.0 log U/mL at baseline (high HBcrAg cohort), HBcrAg declined to ≤2.9 log U/mL at year 1 in 25 patients (22.7%). The adjusted hazard ratio for HCC incidence was significantly lower in patients with HBcrAg ≤2.9 log U/mL at year 1 than in those in the high HBcrAg cohort. Conclusion: Measurement of HBcrAg by ultrasensitive assay has better potential for predicting HCC during antiviral treatment than the current HBcrAg assay.

Analysis of HBsAg Immunocomplexes and cccDNA Activity During and Persisting After NAP-Based Therapy.

Therapy with nucleic acid polymers (NAPs), tenofovir disoproxil fumarate (TDF), and pegylated interferon (pegIFN) achieve high rates of HBsAg loss/seroconversion and functional cure in chronic hepatitis B virus (HBV) infection. The role of hepatitis B surface antigen (HBsAg) seroconversion and inactivation of covalently closed circular DNA (cccDNA) in establishing functional cure were examined. Archived serum from the REP 401 study was analyzed using the Abbott ARCHITECT HBsAg NEXT assay (Chicago, IL), Abbott research use-only assays for HBsAg immune complexes (HBsAg ICs), circulating HBV RNA, and the Fujirebio assay for hepatitis B core-related antigen (HBcrAg; Malvern, PA). HBsAg became < 0.005 IU/mL in 23 participants during NAP exposure, which persisted in all participants with functional cure. HBsAg IC declined during lead-in TDF monotherapy and correlated with minor declines in HBsAg. Following the addition of NAPs and pegIFN, minor HBsAg IC increases (n = 13) or flares (n = 2) during therapy were not correlated with HBsAg decline, hepatitis B surface antibody (anti-HBs) titers, or alanine aminotransferase. HBsAg IC universally declined during follow-up in participants with virologic control or functional cure. Universal declines in HBV RNA and HBcrAg during TDF monotherapy continued with NAP + pegIFN regardless of therapeutic outcome. At the end of therapy, HBV RNA was undetectable in only 5 of 14 participants with functional cure but became undetectable after removal of therapy in all participants with functional cure. Undetectable HBV RNA at the end of therapy in 5 participants was followed by relapse to virologic control or viral rebound. Conclusion: Anti-HBs-independent mechanisms contribute to HBsAg clearance during NAP therapy. Inactivation of cccDNA does not predict functional cure following NAP-based therapy; however, functional cure is accompanied by persistent inactivation of cccDNA. Persistent HBsAg loss with functional cure may also reflect reduction/clearance of integrated HBV DNA. Clinicaltrials.org number NCT02565719.

Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies.

Liquid biopsies that analyze cell-free DNA in blood plasma are used for noninvasive prenatal testing, oncology, and monitoring of organ transplant recipients. DNA molecules are released into the plasma from various bodily tissues. Physical and molecular features of cell-free DNA fragments and their distribution over the genome bear information about their tissues of origin. Moreover, patterns of DNA methylation of these molecules reflect those of their tissue sources. The nucleosomal organization and nuclease content of the tissue of origin affect the fragmentation profile of plasma DNA molecules, such as fragment size and end motifs. Besides double-stranded linear fragments, other topological forms of cell-free DNA also exist-namely circular and single-stranded molecules. Enhanced by these features, liquid biopsies hold promise for the noninvasive detection of tissue-specific pathologies with a range of clinical applications.

Factors and virological significance of hepatitis B virus pregenomic RNA status after 5 years of antiviral therapy.

OBJECTIVES: To investigate the factors and virological significance of serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) status after long-term antiviral therapy with nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB). METHODS: In total, 51 treatment-naïve patients with CHB were included in the study. Clinical data were collected at baseline, during 5 years and at year 10 of NA treatment. Serum HBV pgRNA status of 51 patients was determined at year 5. RESULTS: At year 5, 45% of the patients remained positive for HBV pgRNA. There were significant differences in baseline hepatitis B e antigen (HBeAg) status, HBV DNA load and hepatitis B surface antigen (HBsAg) levels between patients testing positive and negative for HBV pgRNA at year 5. Serum HBV pgRNA status and serum HBV DNA load were correlated after 5 years of NA treatment (r = 0.347, P = 0.013). Being HBV pgRNA positive at year 5 was an independent risk factor for sustainedly undetectable HBV DNA after 10 years of NA treatment (odds ratio 13.638, 95% confidence interval 1.32-140.81; P = 0.028). Furthermore, HBV pgRNA status at year 5 was associated with HBV DNA re-appearance at year 10 (P = 0.009). CONCLUSIONS: HBV pgRNA status at year 5 can reveal HBV covalently closed circular DNA (cccDNA) activity, and HBV pgRNA positivity after long-term antiviral therapy may indicate higher transcriptional activity of HBV cccDNA. Long-term dynamic monitoring of HBV pgRNA should be considered.

Evolution of a highly functional circular DNA aptamer in serum.

Circular DNA aptamers are powerful candidates for therapeutic applications given their dramatically enhanced biostability. Herein we report the first effort to evolve circular DNA aptamers that bind a human protein directly in serum, a complex biofluid. Targeting human thrombin, this strategy has led to the discovery of a circular aptamer, named CTBA4T-B1, that exhibits very high binding affinity (with a dissociation constant of 19 pM), excellent anticoagulation activity (with the half maximal inhibitory concentration of 90 pM) and high stability (with a half-life of 8 h) in human serum, highlighting the advantage of performing aptamer selection directly in the environment where the application is intended. CTBA4T-B1 is predicted to adopt a unique structural fold with a central two-tiered guanine quadruplex capped by two long stem-loops. This structural arrangement differs from all known thrombin binding linear DNA aptamers, demonstrating the added advantage of evolving aptamers from circular DNA libraries. The method described here permits the derivation of circular DNA aptamers directly in biological fluids and could potentially be adapted to generate other types of aptamers for therapeutic applications.

Establishing Simultaneous T Cell Receptor Excision Circles (TREC) and K-Deleting Recombination Excision Circles (KREC) Quantification Assays and Laboratory Reference Intervals in Healthy Individuals of Different Age Groups in Hong Kong.

The clinical experience gathered throughout the years has raised awareness of primary immunodeficiency diseases (PIDD). T cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) assays for thymic and bone marrow outputs measurement have been widely implemented in newborn screening (NBS) programs for Severe Combined Immunodeficiency. The potential applications of combined TREC and KREC assay in PIDD diagnosis and immune reconstitution monitoring in non-neonatal patients have been suggested. Given that ethnicity, gender, and age can contribute to variations in immunity, defining the reference intervals of TREC and KREC levels in the local population is crucial for setting up cut-offs for PIDD diagnosis. In this retrospective study, 479 healthy Chinese sibling donors (240 males and 239 females; age range: 1 month-74 years) from Hong Kong were tested for TREC and KREC levels using a simultaneous quantitative real-time PCR assay. Age-specific 5th-95th percentile reference intervals of TREC and KREC levels (expressed in copies per µL blood and copies per 106 cells) were established in both pediatric and adult age groups. Significant inverse correlations between age and both TREC and KREC levels were observed in the pediatric age group. A significant higher KREC level was observed in females than males after 9-12 years of age but not for TREC. Low TREC or KREC levels were detected in patients diagnosed with mild or severe PIDD. This assay with the established local reference intervals would allow accurate diagnosis of PIDD, and potentially monitoring immune reconstitution following haematopoietic stem cell transplantation or highly active anti-retroviral therapy in the future.

Second-Tier Next Generation Sequencing Integrated in Nationwide Newborn Screening Provides Rapid Molecular Diagnostics of Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) and other T cell lymphopenias can be detected during newborn screening (NBS) by measuring T cell receptor excision circles (TRECs) in dried blood spot (DBS) DNA. Second tier next generation sequencing (NGS) with an amplicon based targeted gene panel using the same DBS DNA was introduced as part of our prospective pilot research project in 2015. With written parental consent, 21 000 newborns were TREC-tested in the pilot. Three newborns were identified with SCID, and disease-causing variants in IL2RG, RAG2, and RMRP were confirmed by NGS on the initial DBS DNA. The molecular findings directed follow-up and therapy: the IL2RG-SCID underwent early hematopoietic stem cell transplantation (HSCT) without any complications; the leaky RAG2-SCID received prophylactic antibiotics, antifungals, and immunoglobulin infusions, and underwent HSCT at 1 year of age. The child with RMRP-SCID had complete Hirschsprung disease and died at 1 month of age. Since January 2018, all newborns in Norway have been offered NBS for SCID using 1st tier TRECs and 2nd tier gene panel NGS on DBS DNA. During the first 20 months of nationwide SCID screening an additional 88 000 newborns were TREC tested, and four new SCID cases were identified. Disease-causing variants in DCLRE1C, JAK3, NBN, and IL2RG were molecularly confirmed on day 8, 15, 8 and 6, respectively after birth, using the initial NBS blood spot. Targeted gene panel NGS integrated into the NBS algorithm rapidly delineated the specific molecular diagnoses and provided information useful for management, targeted therapy and follow-up i.e., X rays and CT scans were avoided in the radiosensitive SCID. Second tier targeted NGS on the same DBS DNA as the TREC test provided instant confirmation or exclusion of SCID, and made it possible to use a less stringent TREC cut-off value. This allowed for the detection of leaky SCIDs, and simultaneously reduced the number of control samples, recalls and false positives. Mothers were instructed to stop breastfeeding until maternal cytomegalovirus (CMV) status was determined. Our limited data suggest that shorter time-interval from birth to intervention, may prevent breast milk transmitted CMV infection in classical SCID.

Expanding TREC and KREC Utility in Primary Immunodeficiency Diseases Diagnosis.

Primary immunodeficiency diseases (PID) area heterogeneous group of disorders caused by genetic defects of the immune system, which manifest clinically as recurrent infections, autoimmune diseases or malignancies. Early detection of PID remains a challenge, particularly in older children with milder and less specific symptoms. This study aimed to assess TREC and KREC diagnostic ability in PID. Data from children assessed by clinical immunologists at Speransky Children's Hospital, Moscow, Russia with suspected immunodeficiencies were analyzed between May 2013 and August 2016. Peripheral blood samples were sent for TREC/KREC, flow cytometry (CD3, CD4, CD8 and CD19), IgA and IgG analysis. A total of 434 children [189 healthy, 97 with group I and II PID (combined T and B cell immunodeficiencies & well-defined syndromes with immunodeficiency) and 148 group III PID (predominantly antibody deficiencies)] were included. Area under the curve (AUC) for TREC in PID groups I and II diagnosis reached 0.82 (CI = 0.75-0.90), with best model providing sensitivity of 65% and specificity of 92%. Neither TREC, nor KREC had added value in PID group III diagnosis. In this study, the predictive value of TREC and KREC in PID diagnosis was examined. We found that the TREC had some diagnostic utility for groups I and II PID. Possibly, addition of TREC measurements to existing clinical diagnostic algorithms may improve their predictive value. Further investigations on a larger cohort are needed to evaluate TREC/KREC abilities to be used as diagnostic tools on a wider scale.

Detection of somatic mutations in peritoneal lavages and plasma of endometrial cancer patients: A proof-of-concept study.

Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Although most patients are diagnosed at early stages, 15-20% will relapse despite local treatment. Presently, there are no reliable markers to identify patients with worse outcomes who may benefit from adjuvant treatments, such as chemotherapy, and liquid biopsies may be of use in this setting. Peritoneal lavages are systematically performed during endometrial surgery but little data are available about their potential as liquid biopsies. We analyzed KRAS and PIK3CA mutations in paired surgical biopsies, blood and cytology-negative peritoneal lavages in a cohort of 50 EC patients. Surgical biopsies were submitted to next-generation sequencing (NGS) while circulating-free DNA (cfDNA) purified from plasma and peritoneal lavages was analyzed for KRAS and PIK3CA hotspot mutations using a sensitive quantitative polymerase chain reaction (PCR) assay. NGS of biopsies revealed KRAS, PIK3CA or concomitant KRAS + PIK3CA mutations in 33/50 (66%) EC patients. Of those, 19 cases carried hotspot mutations. Quantitative PCR revealed KRAS and/or PIK3CA mutations in the lavages of 9/19 (47.4%) hotspot EC patients. In contrast, only 2/19 (10.5%) blood samples from hotspot EC patients were positive. Mutations found in cfDNA consistently matched those in paired biopsies. One of the two patients positive in plasma and lavage died in less than 6 months. In conclusion, mutational analysis in peritoneal lavages and blood from early stage EC is feasible. Further studies are warranted to determine if it might help to identify patients with worse prognosis. Human genes discussed: KRAS, KRAS proto-oncogene, GTPase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Diagnostic Value of Detection of Pregenomic RNA in Sera of Hepatitis B Virus-Infected Patients with Different Clinical Outcomes.

Pregenomic RNA (pgRNA) is a direct transcription product of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and it plays important roles in viral genome amplification and replication. This study was designed to investigate whether serum pgRNA is a strong alternative marker for reflecting HBV cccDNA levels and to analyze the correlation between serum pgRNA, serum HBV DNA, and hepatitis B surface antigen (HBsAg). A total of 400 HBV-infected patients who received nucleos(t)ide analog (NA) therapy with different clinical outcomes were involved in this research. Case groups included asymptomatic hepatitis B virus carrier (ASC), chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC) patients, with 100 patients in each group. The results showed that the levels of HBV pgRNA had significant differences between these 4 groups. Serum pgRNA levels correlated well with serum HBV DNA and HBsAg levels (HBV pgRNA levels versus HBV DNA levels, r = 0.58, P < 0.001; HBV pgRNA levels versus HBsAg levels, r = 0.47, P < 0.001). In addition, we focused on the 108 HBV-infected patients with HBV DNA levels of <500 IU/ml; it was surprising to find that in 17.57% (13/74) of cases, HBV pgRNA could be detected even when the HBV DNA level was below 20 IU/ml. In conclusion, HBV pgRNA levels in serum can be a surrogate marker for intrahepatic HBV cccDNA compared with serum HBV DNA and HBsAg. The detection of serum HBV pgRNA levels may provide a reference for clinical monitoring of cccDNA levels and the selection of appropriate timing for discontinuing antiviral therapy, especially when HBV DNA levels are below the detection limit.