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Background: The inadequate early detection strategies makes hepatocellular carcinoma (HCC) patients with poor prognisis. Therefore, more effective detection methods are urgently needed for early detection and early intervention of HCC.Methods: 17 cases of suspected HCC patients and 11 cases of HBV-related decompensated cirrhosis (HBV-DeCi) patients were enrolled. For each patient, 5 ml blood sample was separated into circulating tumor cells (CTCs) and plasma, CTCs were stained with Diff staining for counting. Plasma was used for extracting cell free DNA (cfDNA) and then analyzed by qMSP assay. Ct values were recorded for GNB4 and Riplet as target genes and β-actin as an endogenous reference gene. Finally, clinical efficacy of CTC count combined with GNB4/Riplet methylation detection for early diagnosis of HCC was analyzed.Results: The CTC of HCC patients has pleomorphic characteristics, but it is difficult to distinguish from other blood cells with non-obviously pleomorphic of CTC. Although a small number of CTCs can also be detected in HBV-DeCi patients (control group), the number is significantly lower than that in HCC patients, the sensitivity and specificity of CTC for HCC detection were 70.6% and 90.9% (AUC = 0.81). The Ct values of GNB4 and Riplet methylation were significantly different between HCC patients and control group patients. When CTC combined with two genes, the AUC value was significantly increased to 0.98, the sensitivity was 88.2%, and the specificity was 100%.Conclusion: Our study has developed a novel test that CTC count combined with GNB4/Riplet methylation detection and showed its high performance for early diagnosis of HCC.

Abstract Background Nucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.Q™ H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay. A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU. Results Dogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas. Conclusions The Nu.Q™ H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines.

Abstract Background Nucleosomes consist of DNA wrapped around a histone octamer core like thread on a spool to condense DNA as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death, chromatin fragmentation and release of nucleosomes into the blood. The Nu.Q™ platform measures circulating nucleosomes in the blood of humans that result from disease and has been used to detect and identify cancer even at early stages. The objectives of this study are to quantify and better characterize nucleosomes in dogs with various stages of hemangiosarcoma (HSA) using this ELISA-based assay. Samples from 77 dogs with a confirmed diagnosis of hemangiosarcoma and 134 healthy controls were utilized for this study. The HSA samples were recruited from the Texas A&M University Small Animal Clinic (TAMU-SAC) or purchased from biobanks. All control samples were recruited from the TAMU-SAC. Results Dogs with hemangiosarcoma had a 6.6-fold increase in their median plasma nucleosome concentrations compared to controls (AUC 92.9 %). Elevated nucleosome concentrations were seen at all stages of disease and nucleosome concentrations increased with the stage of the disease. Conclusions Plasma nucleosome concentrations are a reliable way to differentiate dogs with hemangiosarcoma from healthy dogs. Further testing is underway to better characterize cancer associated HSA circulating nucleosomes and optimize future diagnostics for canine HSA detection.

This review focuses on recent findings in the preimplantation genetic testing (PGT) of embryos. Different preimplantation genetic tests are presented along with different genetic materials and their analysis. Original material concerning preimplantation genetic testing for aneuploidy (PGT-A) was sourced by searching the PubMed and ScienceDirect databases in October and November 2021. The searches comprised keywords such as 'preimplantation', 'cfDNA'; 'miRNA', 'PGT-A', 'niPGT-A', 'aneuploidy', 'mosaicism', 'blastocyst biopsy', 'blastocentesis', 'blastocoel fluid', 'NGS', 'FISH', and 'aCGH'. Non-invasive PGT-A (niPGT-A) is a novel approach to the genetic analysis of embryos. The premise is that the genetic material in the spent embryo culture media (SECM) corresponds to the genetic material in the embryo cells. The limitations of niPGT-A are a lower quantity and lesser quality of the cell-free genetic material, and its unknown origin. The concordance rate varies when compared to invasive PGT-A. Some authors have also hypothesized that mosaicism and aneuploid cells are preferentially excluded from the embryo during early development. Cell-free genetic material is readily available in the spent embryo culture media, which provides an easier, more economic, and safer extraction of genetic material for analysis. The sampling of the SECM and DNA extraction and amplification must be optimized. The origin of the cell-free media, the percentage of apoptotic events, and the levels of DNA contamination are currently unknown; these topics need to be further investigated. [ABSTRACT FROM AUTHOR]

Objective: The aim of the objective was to compare the detection rate for trisomy 21 of universal cell free DNA (cfDNA) screening with contingent screening.Methods: Retrospective study was carried out at 3 German centers. The study included euploid and trisomy 21 pregnancies where cfDNA and first trimester (FT) screening assessment was carried out. The FT risk for trisomy 21 was computed based on combined screening and stratified into the following classes: high risk ≥1:10, intermediate risk 1:11-1,000, low risk ≤1,001. For universal cfDNA screening, the cfDNA test results were examined. For the contingent screening model, the result of the cfDNA test was taken into account in case of an intermediate FT risk. Different strategies combining maternal age, nuchal translucency, nasal bone, beta-hCG, and PAPP-A were evaluated. Screen positivity was defined as either a high risk after FT screening or a cfDNA test indicating a high-risk result. An inconclusive cfDNA test was also considered as screen positive.Results: The search of the database identified 2,255 euploid and 163 affected pregnancies. All affected fetuses were identified by universal cfDNA screening. 1.3% of the euploid fetuses were classified as screen positive due to final inconclusive cfDNA test result. The detection and false-positive rate of a contingent approach that is based on combined screening and cfDNA screening in the intermediate group would be 98.4% and 0.7%, respectively. With this approach, cfDNA screening would be necessary in only about 27% of all pregnancies.Conclusion: This study demonstrates that a contingent approach provides similar detection rates for trisomy 21 as universal cfDNA screening, by a reduction of 73% the number of cfDNA tests. [ABSTRACT FROM AUTHOR]

Squamous cell carcinomas of the esophagus (ESCC) and of the head and neck (HNSCC) are two neoplasms that share common risk factors and have the same embryological origin, but a very different prognosis, the 5-year survival of HNSCC being almost double (40–50%) compared to the 5-year survival of ESCC (20%). Current guidelines emphasize the importance of screening for ESCC in patients diagnosed with head and neck cancers. A liquid biopsy is a novel tool for diagnosis, prognostic stratification, and personalized therapy. Liquid biopsy biomarkers for these two malignancies could help both their early detection, facilitate residual disease identification, and provide prognosis information. The present systematic review of the literature was aimed at describing the liquid biopsy biomarkers present in these two malignancies, with an emphasis on potential clinical applications.

Currently the clinical management of breast cancer relies on relatively few prognostic/predictive clinical markers (estrogen receptor, progesterone receptor, HER2), based on primary tumor biology. Circulating biomarkers, such as circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs) may enhance our treatment options by focusing on the very cells that are the direct precursors of distant metastatic disease, and probably inherently different than the primary tumor's biology. To shift the current clinical paradigm, assessing tumor biology in real time by molecularly profiling CTCs or ctDNA may serve to discover therapeutic targets, detect minimal residual disease and predict response to treatment. This review serves to elucidate the detection, characterization, and clinical application of CTCs and ctDNA with the goal of precision treatment of breast cancer.

Blood‐based molecular diagnostics have emerged as diagnostic tools applicable to a broad spectrum of tumor types from early diagnosis to treatment monitoring and the detection of recurrent disease. Here, we discuss emerging data on HPV‐specific liquid biopsies and their utility as diagnostic tests for HPV‐associated oropharyngeal squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Background: The introduction of cell-free DNA into clinical practice has changed the screening approach. Healthcare professionals and future parents tend to overestimate NIPT (noninvasive prenatal testing) capabilities despite its relatively high cost and limited information.Objective: In this review, our aim was to survey how various countries have introduced contingent screening models and to discuss the advantages and disadvantages of the combined screening test and the use of NIPT.Data Source: The Web of Science, PubMed database and institutional websites were searched for information regarding screening approaches and the implementation in different countries.Results: There are nine countries and regions that have already approved contingent screening test, while others (e.g. Australia) are discussing the implementation of contingent screening versus universal use of NIPT. There are several recent meta-analyses debating whether to use NIPT for universal screening for trisomies and other fetal conditions.Conclusions: NIPT is a reasonable option as an advanced screening test for trisomy 21, 18 and 13 only. Introducing screening by NIPT instead of a first-trimester screening will cause the loss of other valuable information including accurate dating of pregnancy, diagnosing major structural fetal abnormalities and multiple pregnancies at an early gestational age. Additionally, the opportunity to screen for early preeclampsia will be lost. Currently, the price for NIPT is still high adding extra strain on publicly funded health systems. [ABSTRACT FROM AUTHOR]

Abstract Objectives Recent years have witnessed a shift from invasive methods of prenatal screening to non-invasive strategies. Accordingly, non-invasive prenatal testing (NIPT) using cell-free fetal DNA in maternal plasma has gained a considerable deal of interest from both geneticists and obstetricians. Efficacy of this method in identification of common aneuploidies has been extensively assessed in singleton pregnancies. However, a limited number of studies have addressed the twin pregnancies. In this context, the present study is aimed at identification of the efficacy of NIPT in twin pregnancies. Methods NIPT was performed on twin pregnancies to screen trisomies 13, 18 and 21. Pregnant women referring to Nilou Clinical Laboratory between March 2016 and December 2018 were included in this research. Results In the current study, a total 356 twin pregnancies were screened in search for trisomies 13, 18 and 21. 6 cases exhibited positive NIPT results in which the presence of trisomies 13, 18 and 21 was confirmed by fetal karyotype in 1, 2 and 2 cases, respectively. One twin pregnancy showed normal karyotype. The combined false-positive rate for these trisomies was 0.28%. No false negative case was observed. The combined sensitivity and specificity of NIPT in twin pregnancies were 100 and 99.7%, respectively. Conclusion The results of the current study verify the feasibility, sensitivity and specificity of NIPT in twin pregnancies.

Abstract Background Liquid biopsies of blood plasma cell free DNA can be used to monitor treatment response and potentially detect mutations that are present in resistant clones in metastatic cancer patients. Case presentation In our non-interventional liquid biopsy study, a male patient in his fifties diagnosed with stage IV colorectal cancer and polytope liver metastases rapidly progressed after completing chemotherapy and deceased 8 months after diagnosis. Retrospective cell free DNA testing showed that the APC/TP53/KRAS major clone responded quickly after 3 cycles of FOLFIRI + Bevacizumab. Retrospective exome sequencing of pre-chemotherapy and post-chemotherapy tissue samples including metastases confirmed that the APC/TP53/KRAS and other major clonal mutations (GPR50, SLC5A, ZIC3, SF3A1 and others) were present in all samples. After the last chemotherapy cycle, CT imaging, CEA and CA19–9 markers validated the cfDNA findings of treatment response. However, 5 weeks later, the tumour had rapidly progressed. Conclusion As FOLFIRI+Bevacizumab has recently also been associated with sustained complete remission in a APC/TP53/KRAS triple-mutated patient, these driver genes should be tested and monitored in a more in-depth manner in future patients. Patients with metastatic disease should be monitored more closely during and after chemotherapy, ideally using cfDNA.

Early detection of cancer is vital for increasing patient survivability chances. The three major techniques used to diagnose cancers are instrumental examination, tissue biopsy, and tumor biomarker detection. Circulating tumor DNA (ctDNA) has gained much attention in recent years due to advantages over traditional technology, such as high sensitivity, high specificity, and noninvasive nature. Through the mechanism of apoptosis, necrosis, and circulating exosome release in tumor cells, ctDNA can spread throughout the circulatory system and carry modifications such as methylations, mutations, gene rearrangements, and microsatellite instability. Traditional gene-detection technology struggles to achieve real-time, low-cost, and portable ctDNA measurement, whereas electrochemical biosensors offer low cost, high specificity alongside sensitivity, and portability for the detection of ctDNA. Therefore, this review focuses on describing the recent advancements in ctDNA biomarkers for various cancer types and biosensor developments for real-time, noninvasive, and rapid ctDNA detection. Further in the review, ctDNA sensors are also discussed in regards to their selections of probes for receptors based on the electrode surface recognition elements.

Gene overexpression by transient transfection of in vitro cultured model cell lines with plasmid DNA is a commonly used method for studying molecular aspects of human biology and pathobiology. However, there is accumulating evidence suggesting that human cells may actively secrete fragments of DNA and the implications of this phenomenon for in vitro cultured cells transiently transfected with foreign nucleic acids has been overlooked. Therefore, in the current study we investigated whether a cell-to-cell transmission of acquired plasmid DNA takes place in a commonly used human cell line model. We transiently transfected HEK293 cells with EGFP encoding plasmids to serve as donor cells and either co-cultured these with stably mCherry expressing recipient cells in different set-ups or transferred their culture medium to the recipient cells. We found that recipient cells produced EGFP after being co-cultured with donor cells but not when they were exposed to their culture medium. The employment of different co-culture set-ups excluded that the observed effect stemmed from technical artefacts and provided evidence that an intercellular plasmid transfer takes place requiring physical proximity between living cells. This phenomenon could represent a significant biological artefact for certain studies such as those addressing protein transmissions in prion diseases., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Inc.)

Background and Objectives: The role of cell-free DNA (cfDNA) in operable nonsmall cell lung cancer (NSCLC) is unclear. This study was aimed to evaluate the feasibility for identification of cfDNA in pleural lavage fluid and its correlation with plasma in resectable NSCLCs., Methods: Consecutively resected NSCLCs were evaluated for cfDNA levels in preoperative plasma (PLS1), intraoperative pleural-lavage (PLV) and postoperative (at 1 month) plasma sample (PLS2). CfDNA was isolated and measured quantitatively by qPCR in a TaqMan probe-detection approach using the human β-actin gene as the amplifying target., Results: All (n = 34) except one were negative for malignant cells in PLV cytology. CfDNA could be isolated from all the three samples (PLS1, PLV, and PLS2) successfully in each patient. The median cfDNA levels in PLS1, PLV and PLS2 were 118 ng/mL (IQR 61-158), 167 ng/mL (IQR 59.9-179.9) and 103 ng/mL (IQR 66.5-125.4) respectively. The median follow-up was 34.1 months (IQR 25.2-41.6). A significant overall-survival (OS) and disease-free survival (DFS) were recorded for patients with cfDNA level cut-offs at 125, 170, and 100 ng/mL, respectively for PLS1, PLV, and PLS2. Patients with raised cfDNA in PLS1 (>125 ng/mL) and PLV (>170 ng/mL) had significantly poorer 2-year OS, p = 0.005 and p = 0.012, respectively. The hazards (OS) were also higher for those with raised cfDNA in PLV (HR = 5.779, 95% CI = 1.162-28.745, p = 0.032). PLV (>170 ng/mL) had increased pleural recurrences (p = 0.021) and correlated significantly with poorer DFS at 2-years (p = 0.001) with increased hazards (HR = 9.767, 95% CI = 2.098-45.451, p = 0.004). Multivariable analysis suggested higher cfDNA in PLV as a poor prognostic factor for both OS and DFS., Conclusions: Among patients with operable NSCLC, it is feasible to identify cfDNA in pleural lavage and correlate PLV cfDNA with pleural recurrences and outcomes., (© 2024 Wiley Periodicals LLC.)

Background: Relative haplotype dosage (RHDO) approach has been applied in noninvasive prenatal diagnosis (NIPD) of Duchenne muscular dystrophy (DMD). However, the RHDO procedure is relatively complicated and the parental haplotypes need to be constructed. Furthermore, it is not suitable for the diagnosis of de novo mutations or mosaicism in germ cells. Here, we investigated NIPD of DMD using a relative mutation dosage (RMD)-based approach—cell-free DNA Barcode-Enabled Single-Molecule Test (cfBEST), which has not previously been applied in the diagnosis of exon deletion. Methods: Five DMD families caused by DMD gene point mutations or exon deletion were recruited for this study. After the breakpoints of exon deletion were precisely mapped with multiple PCR, the genotypes of the fetuses from the five DMD families were inferred using cfBEST, and were further validated by invasive prenatal diagnosis. Results: The cfBEST results of the five families indicated that one fetus was female and did not carry the familial molecular alteration, three fetuses were carriers and one was male without the familial mutation. The invasive prenatal diagnosis results were consistent with those of the cfBEST procedure. Conclusion: This is the first report of NIPD of DMD using the RMD-based approach. We extended the application of cfBEST from point mutation to exon deletion mutation. The results showed that cfBEST would be suitable for NIPD of DMD caused by different kinds of mutation types. [ABSTRACT FROM AUTHOR]

The use of Next Generation Sequencing (NGS) to interrogate cell-free DNA (cfDNA) as a transplant diagnostic provides a crucial step in improving the accuracy of post-transplant monitoring of allograft health. cfDNA interrogation provides a powerful, yet minimally invasive, biomarker for disease and tissue injury. cfDNA can be isolated from a variety of body fluids and analyzed using bioinformatics to unlock its origins. Furthermore, cfDNA characteristics can reveal the mechanisms and conditions under which it was generated and released. In transplantation, donor-derived cfDNA monitoring provides a tool for identifying active allograft injury at the time of transplant, infection, and rejection. Multiple detection and interrogation methods for cfDNA detection are now being evaluated for clinical validity and hold the promise to provide minimally invasive, quantitative, and reproducible measures of allograft injury across organ types. [ABSTRACT FROM AUTHOR]

Most ovarian cancers, despite improvement in management of cancer, are still diagnosed at an advanced stage. Early detection plays an essential role in reducing ovarian cancer mortality and, therefore, is critically needed. Liquid biopsies-based approaches hold significant promise for cancer detection. The present study investigates a panel of epigenetic biomarkers for the detection of epithelial ovarian cancer. A qPCR assay has been developed based on the assessment of DNA methylation markers in circulating cell-free DNA as a minimally invasive tool. Herein, the promoter methylation of seven ovarian cancer-specific genes (RASSF1A, DAPK1, SOX1, HOXA9, HIC1, SPARC, and SFRP1) was analyzed quantitatively in 120 tissue samples by MethyLight assay. The best-performing genes were further evaluated for their methylation status in 70 matched serum cell-free DNA of cancerous and non-cancerous samples. Additionally, DNA methylation patterns of these best-performing genes were validated by clonal bisulfite sequencing. The ROC (Receiver-operator characteristic) curves were constructed to evaluate the diagnostic performances of both individual and combined gene panels. The seven candidate genes displayed a methylation frequency of 61.0–88.0% in tissue samples. The promoter methylation frequencies for all the seven candidate genes were significantly higher in cancer samples than in normal matched controls. In tissue samples, the multiplex MethyLight assay for HOXA9, HIC1, and SOX1 were the best performing gene panels in terms of sensitivity and specificity. The three best-performing genes exhibited individual frequencies of 53.0–71.0% in serum CFDNA, and the multiplex assay for these genes were identified to discriminate serum from cancer patients and healthy individuals (area under the curve: HOXA9+HIC1 = 0.95, HIC1+SOX1 = 0.93 and HOXA9+SOX1 = 0.85). The results of MethyLight showed high concordance with clonal bisulfite sequencing results. Individual genes and combined panel exhibited better discriminatory efficiencies to identify ovarian cancer at various stages of disease when analyzed in tissue and serum cell-free DNA. We report a qPCR-based non-invasive epigenetic biomarker assay with high sensitivity and specificity for OC screening. Our findings also reveal the potential utility of methylation-based detection of circulating cell-free tumor DNA in the clinical management of ovarian cancer.

Purpose: Circulating tumor DNA (ctDNA) is released by many tumors into the plasma. Its analysis has minimal procedural risk and, in many cancers, has the potential for clinical applications. In retinoblastoma, the clinical correlations of ctDNA in eyes treated without enucleation have not been studied. This purpose of this study was to determine how the ctDNA RB1 variant allele frequency (VAF) changes in patients with unilateral retinoblastoma after intra-arterial chemotherapy (IAC) treatment. Variant allele frequency is a proxy for tumor fraction. Design: Case series from a single tertiary cancer referral center. Participants: Five patients with retinoblastoma with at least 1 measurable ctDNA plasma specimen both at the time of active intraocular retinoblastoma before IAC and after at least 1 IAC cycle. Methods: Circulating tumor DNA RB1 was detected and VAF was measured before and after IAC treatment. Clinical correlations were made using clinical examination, fundus photography, ultrasound, and OCT. Main Outcome Measures: Comparison of ctDNA RB1 VAF before and after IAC treatment for retinoblastoma and concordance of ctDNA RB1 detectability with activity of intraocular disease. Results: Twenty-three ctDNA specimens were included from 5 patients. The 5 baseline RB1 VAFs ranged from 0.27% to 4.23%. In all patients, the subsequent post–intra-arterial RB1 VAF was lower than baseline (0.0%–0.17%). At 4 months (2 months after IAC completion), the ctDNA consistently was negative in the patients who demonstrated clinically inactive intraocular disease. Conclusions: In this small cohort, a decremental decrease in ctDNA RB1 VAF was found after IAC, suggesting that relative VAF changes could be a biomarker of treatment response.

BackgroundTraumatic brain injury (TBI) is a major cause of death and disability. In this study a new method to measure cell free DNA (CFD) for the management of TBI is tested. Our hypothesis was that CFD concentrations correlate to the magnitude of brain damage, and may predict the outcome of injured patients.MethodsTwenty eight patients with isolated head injury were enrolled. Their demographic and clinical data were recorded. CFD levels were determined in patients' sera samples by a direct fluorescence method developed in our laboratory.ResultsMean admission CFD values were lower in patients with mild TBI compared to severe injury (760 ± 340 ng/ml vs. 1600 ± 2100 ng/ml, p = 0.03), and in patients with complete recovery upon discharge compared to patients with disabilities (680 ± 260 ng/ml vs. 2000 ± 2300 ng/ml, p = 0.003). Patients with high CFD values had a relative risk to require surgery of 1.5 (95% CI 0.83 to 2.9) a relative risk to have impaired outcome on discharge of 2.8 (95% CI 0.75 - 10), and a longer length of stay (12 ± 13 days vs. 3.4 ± 4.8 days, p = 0.02). CFD values did not correlate with CT scan based grading.ConclusionsCFD levels may be used as a marker to assess the severity of TBI and to predict the prognosis. Its use should be considered as an additional tool along with currently used methods or as a surrogate for them in limited resources environment.

Background: Exosomes are ubiquitous extracellular nanovesicles secreted from almost all living cells that are thought to be involved in several important cellular processes, including cell–cell communication and signaling. Exosomes serve as a liquid biopsy tool for clinical and translational research. Although many techniques have been used to isolate exosomes, including ultracentrigation, size-exclusion chromatography, and immunocapturing-based techniques, these techniques are not convenient, they require expensive instrumentation, and they are unhandy for clinical samples. Precipitation techniques from available commercial kits that contain polyethelene glycol (PEG) are now widely used, but these kits are expensive, especially if a large number of biological samples are to be processed. Objective: the purpose of this study is to compare and optimize the efficacy of different concentrations of PEG with two commercial kits ExoQuick (SBI) and Total Exosome Isolation (TEI) from Invitrogen in human plasma. Methods and Materials: we determined exosome quantity, size distribution, marker expression, and downstream application. Results: among the precipitation methods, we found the size of particles and concentrations with 10–20% PEG are similar to ExoQuick and better than TEI. Interestingly, we detected cfDNA with ExoQuick and 10–20% PEG but not TEI and 5% PEG. Moreover, 10% PEG detection of miR-122 and miR-16 expression was superior to ExoQuick and TEI. Furthermore, in proteomics results it also found the identified proteins better than commercial kits but there was a high level of contamination of other proteins in serum. Conclusions: together, these findings show that an optimal concentration of 10% PEG serves as a guide for use with clinical samples in exosome isolation for downstream applications.

Background: Nucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.Q™ H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay. A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU. Results: Dogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas. Conclusions: The Nu.Q™ H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines. [ABSTRACT FROM AUTHOR]

Background: Anaplastic thyroid carcinoma (ATC) is an aggressive thyroid cancer that requires a rapid diagnosis and treatment to achieve disease control. Gene mutation profiling of circulating cell-free DNA (cfDNA) in peripheral blood may help to facilitate early diagnosis and treatment selection. The relatively rapid turnaround time compared with conventional tumor mutation testing is a major advantage. The objectives of this study were to examine the concordance of ATC-related mutations detected in cfDNA with those detected in the corresponding tumor tissue, and to determine the prognostic significance of cfDNA mutations in ATC patients. Methods: The ATC patients who were diagnosed and treated at The University of Texas MD Anderson Cancer Center between January 2015 and February 2018 and who had cfDNA testing were included in this study. cfDNA was collected by blood draw and was analyzed by next-generation sequencing (NGS) using the Guardant360-73 gene platform. Results: A total of 87 patients were included in the study. The most frequently mutated genes detected in cfDNA were TP53, BRAF, and PIK3CA. In 28 treatment naive ATC patients, the concordance rate of detected mutations in TP53, BRAFV600E, and PIK3CA between cfDNA and matched tissue NGS was 82.1%, 92.9%, and 92.9%, respectively. Patients with a PIK3CA mutation detected on cfDNA had worse overall survival (OS) (p = 0.03). This association was observed across various treatment modalities, including surgery, cytotoxic chemotherapy, radiation, and BRAF inhibitor (BRAFi) therapy. With regard to treatment, BRAFi therapy significantly improved ATC OS (p = 0.003). Conclusions: cfDNA is a valuable tool to evaluate a tumor's molecular profile in ATC patients. We identified high concordance rates between the gene mutations identified via cfDNA analysis and those identified from the NGS of the corresponding tumor tissue sequencing. Identified mutations in cfDNA can potentially provide timely information to guide treatment selection and evaluate the prognosis in patients with ATC. [ABSTRACT FROM AUTHOR]

Circulating tumor DNA (ctDNA) is utilized for molecular profiling of cancers, and is under investigation for a growing number of applications based on the assumption that ctDNA levels faithfully reflect disease burden. Our objective was to investigate whether patient and tumor characteristics may impact ctDNA detection or levels and the prognostic significance of ctDNA levels or mutations. We performed a retrospective cohort analysis of a comprehensively annotated cohort of 561 patients at a National Cancer Institute–designated comprehensive cancer center with advanced solid cancers who underwent ctDNA testing using a commercial targeted next-generation sequencing assay. ctDNA detection in advanced cancers was associated with older age, non-obese body mass index, and diabetes, but not with tumor diameter, volume, lesion number, or other pathological features. Regression models indicate that no more than 14.3% of the variance in ctDNA levels between patients was explained by known clinical factors and disease burden. Even after adjusting for established prognostic factors and tumor burden, ctDNA levels were associated with worse survival among patients without prior systemic therapy, while ctDNA mutations were associated with survival among patients who previously received systemic treatment. These findings uncover clinical factors that affect ctDNA detection in patients with advanced cancers and challenge the convention that ctDNA is a surrogate for tumor burden. Our study also indicates that the prognostic value of ctDNA levels and mutations are independent of tumor burden and dependent on treatment context. [ABSTRACT FROM AUTHOR]

Background: Nucleosomes consist of DNA wrapped around a histone octamer core like thread on a spool to condense DNA as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death, chromatin fragmentation and release of nucleosomes into the blood. The Nu.Q™ platform measures circulating nucleosomes in the blood of humans that result from disease and has been used to detect and identify cancer even at early stages. The objectives of this study are to quantify and better characterize nucleosomes in dogs with various stages of hemangiosarcoma (HSA) using this ELISA-based assay. Samples from 77 dogs with a confirmed diagnosis of hemangiosarcoma and 134 healthy controls were utilized for this study. The HSA samples were recruited from the Texas A&M University Small Animal Clinic (TAMU-SAC) or purchased from biobanks. All control samples were recruited from the TAMU-SAC. Results: Dogs with hemangiosarcoma had a 6.6-fold increase in their median plasma nucleosome concentrations compared to controls (AUC 92.9 %). Elevated nucleosome concentrations were seen at all stages of disease and nucleosome concentrations increased with the stage of the disease. Conclusions: Plasma nucleosome concentrations are a reliable way to differentiate dogs with hemangiosarcoma from healthy dogs. Further testing is underway to better characterize cancer associated HSA circulating nucleosomes and optimize future diagnostics for canine HSA detection. [ABSTRACT FROM AUTHOR]

Summary: Circulating tumour DNA (ctDNA) is a highly versatile analyte and an emerging biomarker for detection of tumour‐specific sequences in lymphoid malignancies. Since ctDNA is derived from tumour cells throughout the body, it overcomes fundamental limitations of tissue biopsies by capturing the complete molecular profile of tumours, including those from inaccessible anatomic locations. Assays for ctDNA are minimally invasive and serial sampling monitors the effectiveness of therapy and identifies minimal residual disease below the detection limit of standard imaging scans. Dynamic changes in ctDNA levels measure real‐time tumour kinetics, and early reductions in ctDNA during treatment correlate with clinical outcomes in multiple B‐cell lymphomas. After therapy, ctDNA can effectively discriminate between patients who achieved a complete molecular remission from those with residual treatment‐resistant disease. Serial monitoring of ctDNA after therapy can detect early molecular relapse and identify drug‐resistant clones that harbour targetable mutations. In order for ctDNA to reach its full potential, the standardization and harmonization of the optimal pre‐analytical and analytical techniques for B‐cell lymphomas is a critically necessary requirement. Prospective validation of ctDNA within clinical studies is also required to determine its clinical utility as an adjunctive decision‐making tool. [ABSTRACT FROM AUTHOR]

It is imperative for proper evidence triage that forensic biologists understand what kind of results to expect from certain evidence types submitted for DNA analysis. The persistence of trace DNA has been insufficiently investigated and there is little data available pertaining to the persistence of DNA in different environmental conditions and on different materials. The goal of this study is to increase the available data on this topic which would, in turn, help forensic biologists manage expectations when submitting specific evidence types for DNA testing. The work presented herein is a large-scale persistence project aimed to identify trends in the persistence of trace DNA and indicate how different environmental storage conditions and target surface characteristics influence the persistence of cellular and cell free DNA (cfDNA) over time. To eliminate variation within the experiment we used a proxy DNA deposit consisting of a synthetic fingerprint solution, cellular DNA, and/or cfDNA. Samples were collected and analysed from 7 metals over the course of 1 year (27 time points) under 3 different environmental storage conditions. The results of this experiment show that metal type greatly influences DNA persistence. For instance, copper exhibited an expected poor DNA persistence (up to 4 h) which a purification step did not help increase the DNA yield. Alternatively, DNA can persist for up to a year on lead at levels potentially high enough to allow for forensic DNA testing. Additionally, this study showed that the sample storage environment had no impact on DNA persistence in most cases. When considering DNA type, cfDNA was shown to persist for longer than cellular DNA and persistence as a whole appears to be better when DNA is deposited as mixtures over when deposited alone. Unsurprisingly, it can be expected that DNA recovery rates from trace deposits will decrease over time. However, DNA decay is highly dependent on the metal surface and extremely variable at short time points but slightly less variable as time since deposition increases. This data is intended to add to our understanding of DNA persistence and the factors which affect it. • DNA has the capacity to persist on lead for up to one year. • Poor persistence of DNA on copper is likely due to DNA damage not PCR inhibition. • Cell free DNA persists for longer than cellular DNA on metals. • Rate of DNA loss is highly metal dependent. • In many instances, environmental conditions did not influence DNA persistence. [ABSTRACT FROM AUTHOR]

Stress is a pervasive issue in modern life, affecting both physical and mental health. Identifying biomarkers like cell-free DNA (cfDNA) could provide insights into stress response and help detect individuals at risk for stress-related disorders. The aim of this study is to investigate the potential use of cfDNA as a diagnostic biomarker in individuals experiencing stress. A case-control analysis was conducted using convenient sampling on university participants (N = 285 cases, N = 500 controls) aged 18–24. The study assessed haematological and lipid profile parameters using the Sysmex XP-300TM automated analyzer and an automated biochemistry analyzer, and cfDNA was extracted using a standardized in house developed Phenol-Chloroform protocol and estimated using Agarose Gel Electrophoresis and Nanodrop. Statistical analysis was performed using SPSS ver. 21.0. The results indicated a significant difference between stressed individuals and healthy controls in demographic, haematological and biochemical parameters. Specifically, stressed cases had significantly higher levels of cholesterol, LDL cholesterol, triglycerides, glucose, VLDL cholesterol, and lower levels of HDL compared to healthy controls. Stressed cases also showed significantly elevated levels of circulating cfDNA relative to healthy controls. These findings suggest that cfDNA may have potential as a diagnostic biomarker for stress. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the knowledge and attitude of pregnant women before undergoing cell-free DNA screening at King Chulalongkorn Memorial Hospital (KCMH). Materials and Methods: A cross-sectional study recruited 400 singleton pregnant women who underwent cell-free DNA screening at KCMH from December 2016 to August 2017. Self-administered questionnaires were used to evaluate participants’ knowledge and attitude. Results: Four-hundred pregnant women answered the questionnaires and 344 responses were considered valid. The maternal age ranged from 23 to 46 years and mean age was 34.8 ± 3.6 years. Almost all of the participants answered correctly about the test’s ability to detect trisomy 21, trisomy 18, trisomy 13, and fetal sex (96.8, 83.7, 84.8, and 93.9% respectively). Eighty-nine percent of the women answered correctly about the time to start cell-free DNA testing, and 68% answered correctly about detection rate of trisomy 21. Some participants had misconceptions about the test’s ability such as false negative rate, thalassemia screening, fetal malignancy detection, autistic detection, cleft lip-cleft palate detection, and the option of termination of pregnancy if the screening was positive. Seventy-nine percent were aware of the possibility of re-sampling. Additional data showed that participants had positive attitude towards cell-free DNA screening, and preferred to use it again for future pregnancy. Conclusion: Our study showed that the majority of the participants had good knowledge of the test’s ability to detect trisomy, and a possibility of re-sampling. However, almost half of the participants misunderstood that it could detect all genetic abnormalities. This study showed the magnitude of expectations and misunderstandings about cell-free DNA screening.

We hypothesize that the anti - apoptotic response in mesenchymal stem cells (MSC) under low doses of radiation may develop through the participation of an extracellular mediator of signal transmission between cells - fragments of oxidized cell - free DNA (cfDNA).Aim. To investigate the effect of radiation in low doses (10 sGy) and oxidized fragments of cfDNA on the activation of genes in MSCs signalling cascades that regulate the cellular response to DNA damage. Materials and methods. We investigated the effect of radiation in low doses (10 cGy) and fragments of oxidized cfDNA (concentration 50 ng/ml) on the activation of signalling gene cascades in MSC that regulate the cellular response to DNA damage.Results. We have shown, that in MSC under small doses of radiation for 15-40 minutes the transcriptional activity of pro - apoptotic genes is activated, the level of apoptosis increases and due to the death of part of the cells in the culture of MSC, fragments of oxidized cfDNA are formed. 3-72 hours later the expression of genes ING2, HUWE1, TP53, BBC3, NOXA, P53AIP1, BAX, BAK1, MMP7, ENDOG decreases 1.5-3 times ( p

Abstract Objective DNA methylation analysis via real-time PCR or other analytical techniques requires purified bisulfite converted DNA. We report on an automated high throughput solution for DNA extraction, bisulfite-conversion, and purification of 96 samples with an input volume of up to 3.5 mL of plasma or urine, using reagents from the commercially available Epi BisKit. Results Magnetic bead-based DNA extraction, bisulfite conversion at high temperature, and efficient DNA purification was conducted on a customized commercially available liquid-handling platform. A highly interlaced 4 × 24 sample protocol was implemented for DNA extraction, elution in a 96-well plate, efficient bisulfite-conversion and extensive purification. The resulting bisulfite-converted DNA was stored in a 96-well format, ready for PCR set-up or other down-stream applications. The automated method is a walk-away solution for processing 96 samples in 7 h 30 min. Performance of the method was validated by comparison with the standard manual method of the Epi BiSKit using technical and biological samples. Overall DNA yield was assessed with a standardized β-actin assay. The automated workflow demonstrated equivalent performance to the manual method for technical, plasma and urine samples. It may provide a new standard for effective high-throughput preparation of bisulfite-converted DNA from a variety of high volume liquid biopsy specimens.

Tumor-derived circulating tumor DNA (ctDNA) has demonstrated its excellent potential for cancer diagnosis by DNA methylome; therefore, this study aimed to identify the retinoblastoma (RB) specific methylated CpG loci as the RB diagnostic biomarkers and design a methylation specific assay to detect these biomarker from aqueous humor of RB patients. Through a genome-wide methylation profiling of tissue samples from patients with RB, normal retina and other retinal diseases, we shortlisted two CpG loci were only methylated in RB but not in normal retina or other retinal diseases. Both of these two CpG loci were located in the genome of TFAP2A. Through the screening, a primer and probe set for the two CpG loci were tested in fully methylated standards and RB tissues with a significant differentiation of RB. Our results of this assay tested in aqueous humor from RB revealed an accuracy of 92.7% for RB diagnosis. These results suggested our assay targeting the TFAP2A ctDNA methylation can be utilized for RB diagnosis and cancer monitoring.

Context: Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare bone and endocrine disorder resulting in fractures, pain, and disability. There are no targeted or effective therapies to alter the disease course. Disease arises from somatic gain-of-function variants at the R201 codon in GNAS, replacing arginine by either cysteine or histidine. The relative pathogenicity of these variants is not fully understood.Objective: This work aimed 1) to determine whether the most common GNAS variants (R201C and R201H) are associated with a specific clinical phenotype, and 2) to determine the prevalence of the most common GNAS variants in a large patient cohort.Methods: This retrospective cross-sectional analysis measured the correlation between genotype and phenotype characterized by clinical, biochemical, and radiographic data.Results: Sixty-one individuals were genotyped using DNA extracted from tissue or circulating cell-free DNA. Twenty-two patients (36.1%) had the R201C variant, and 39 (63.9%) had the R201H variant. FD skeletal disease burden, hypophosphatemia prevalence, fracture incidence, and ambulation status were similar between the 2 groups. There was no difference in the prevalence of endocrinopathies, ultrasonographic gonadal or thyroid abnormalities, or pancreatic involvement. There was a nonsignificant association of cancer with the R201H variant.Conclusion: There is no clear genotype-phenotype correlation in patients with the most common FD/MAS pathogenic variants. The predominance of the R201H variant observed in our cohort and reported in the literature indicates it is likely responsible for a larger burden of disease in the overall population of patients with FD/MAS, which may have important implications for the future development of targeted therapies. [ABSTRACT FROM AUTHOR]

BackgroundCell-free DNA (cfDNA) has arisen as an alternative target for evaluating somatic mutations in cancer. KRAS mutation status is critical for targeted therapy in colorectal adenocarcinoma (CRAC). We evaluated KRASG12/G13 mutations in cfDNA extracted from serum and compared the results with KRASG12/G13 mutations detected in tissue samples. We assessed the clinical significance of KRASG12/G13 mutation in serum in regard to recurrence and metastasis of CRAC.MethodsA total of 146 CRAC patients were enrolled, and KRASG12/G13 mutations were evaluated in 146 pairs of serum and tissue samples. In addition, 35 pairs of primary and metastatic CRAC tissue samples were evaluated for KRASG12/G13 mutational status.ResultsDetection of KRASG12/13 mutation from serum and tissue had a 55% concordance rate, and serum detection had a sensitivity of 39.8%. Detection of the KRASG12/13 mutation yielded a 14% discordance rate between primary and metastatic tissue. CRAC patients with mutant KRASG12/13 mutation in serum but wild-type KRASG12/13 in tissue had concurrent KRASG12/13-mutant metastatic tumors, indicating spatial genetic heterogeneity. Changes in serum KRASG12/G13 mutation status during postoperative follow-up were associated with recurrence. Conclusion: Although serum detection of the KRASG12/13 mutation cannot substitute for detection in tissue, serum testing can support the interpretation of a CRAC patient’s status in regard to concurrent metastasis. Dynamic changes in serum KRASG12/13 mutation status during follow-up indicated that cfDNA from serum represents a potential source for monitoring recurrence in CRAC patients.

BackgroundMolecular profiling of advanced EGFR mutated NSCLC has recently demonstrated the co-existence of multiple genetic alterations. Specifically, co-existing KRAS-mutations in EGFR NSCLCs have been described, despite their prevalence at progression and their role in the response to EGFR tyrosine kinase inhibitors (TKIs) remain marginally explored. Aim of our study was to investigate the prevalence of co-existing KRAS mutations at the time of progressive disease and explore their impact on clinical outcome.Materials and MethodsWe retrospectively analyzed by digital droplet PCR prevalence of KRAS co-mutations in 106 plasma samples of EGFR mutated NSCLC patients, in progressive disease after EGFR TKI treatment as first-line therapy.ResultsKRAS co-mutations (codon 12 and 13) were identified in 3 patients (2.8% of analyzed samples), with low allelic frequency (

BackgroundEGFR-positive Non-small Cell Lung Cancer (NSCLC) is a dynamic entity and tumor progression and resistance to tyrosine kinase inhibitors (TKIs) arise from the accumulation, over time and across different disease sites, of subclonal genetic mutations. For instance, the occurrence of EGFR T790M is associated with resistance to gefitinib, erlotinib, and afatinib, while EGFR C797S causes osimertinib to lose activity. Sensitive technologies as radiomics and liquid biopsy have great potential to monitor tumor heterogeneity since they are both minimally invasive, easy to perform, and can be repeated over patient’s follow-up, enabling the extraction of valuable information. Yet, to date, there are no reported cases associating liquid biopsy and radiomics during treatment.Case presentationIn this case series, seven patients with metastatic EGFR-positive NSCLC have been monitored during target therapy. Plasma-derived cell free DNA (cfDNA) was analyzed by a digital droplet PCR (ddPCR), while radiomic analyses were performed using the validated LifeX® software on computed tomography (CT)-images. The dynamics of EGFR mutations in cfDNA was compared with that of radiomic features. Then, for each EGFR mutation, a radiomic signature was defines as the sum of the most predictive features, weighted by their corresponding regression coefficients for the least absolute shrinkage and selection operator (LASSO) model. The receiver operating characteristic (ROC) curves were computed to estimate their diagnostic performance. The signatures achieved promising performance on predicting the presence of EGFR mutations (R2 = 0.447, p

This review focuses on recent findings in the preimplantation genetic testing (PGT) of embryos. Different preimplantation genetic tests are presented along with different genetic materials and their analysis. Original material concerning preimplantation genetic testing for aneuploidy (PGT-A) was sourced by searching the PubMed and ScienceDirect databases in October and November 2021. The searches comprised keywords such as ‘preimplantation’, ‘cfDNA’; ‘miRNA’, ‘PGT-A’, ‘niPGT-A’, ‘aneuploidy’, ‘mosaicism’, ‘blastocyst biopsy’, ‘blastocentesis’, ‘blastocoel fluid’, ‘NGS’, ‘FISH’, and ‘aCGH’. Non-invasive PGT-A (niPGT-A) is a novel approach to the genetic analysis of embryos. The premise is that the genetic material in the spent embryo culture media (SECM) corresponds to the genetic material in the embryo cells. The limitations of niPGT-A are a lower quantity and lesser quality of the cell-free genetic material, and its unknown origin. The concordance rate varies when compared to invasive PGT-A. Some authors have also hypothesized that mosaicism and aneuploid cells are preferentially excluded from the embryo during early development. Cell-free genetic material is readily available in the spent embryo culture media, which provides an easier, more economic, and safer extraction of genetic material for analysis. The sampling of the SECM and DNA extraction and amplification must be optimized. The origin of the cell-free media, the percentage of apoptotic events, and the levels of DNA contamination are currently unknown; these topics need to be further investigated.

Background: Molecular profiling of advanced EGFR mutated NSCLC has recently demonstrated the co-existence of multiple genetic alterations. Specifically, co-existing KRAS -mutations in EGFR NSCLCs have been described, despite their prevalence at progression and their role in the response to EGFR tyrosine kinase inhibitors (TKIs) remain marginally explored. Aim of our study was to investigate the prevalence of co-existing KRAS mutations at the time of progressive disease and explore their impact on clinical outcome. Materials and Methods: We retrospectively analyzed by digital droplet PCR prevalence of KRAS co-mutations in 106 plasma samples of EGFR mutated NSCLC patients, in progressive disease after EGFR TKI treatment as first-line therapy. Results: KRAS co-mutations (codon 12 and 13) were identified in 3 patients (2.8% of analyzed samples), with low allelic frequency (<0.2%), and had a negative impact on clinical outcome to first-line EGFR TKI. Conclusion: Detection of KRAS mutations in cell-free DNA of EGFR mutant NSCLC patients at progression after first or second generation EGFR TKI is a rare event. Due to their low abundance, the negative impact of KRAS mutations on the response to EGFR TKI remains to be confirmed in larger studies. [ABSTRACT FROM AUTHOR]

Background: Cell-free DNA (cfDNA) has arisen as an alternative target for evaluating somatic mutations in cancer. KRAS mutation status is critical for targeted therapy in colorectal adenocarcinoma (CRAC). We evaluated KRASG12/G13 mutations in cfDNA extracted from serum and compared the results with KRASG12/G13 mutations detected in tissue samples. We assessed the clinical significance of KRASG12/G13 mutation in serum in regard to recurrence and metastasis of CRAC. Methods: A total of 146 CRAC patients were enrolled, and KRASG12/G13 mutations were evaluated in 146 pairs of serum and tissue samples. In addition, 35 pairs of primary and metastatic CRAC tissue samples were evaluated for KRASG12/G13 mutational status. Results: Detection of KRASG12/13 mutation from serum and tissue had a 55% concordance rate, and serum detection had a sensitivity of 39.8%. Detection of the KRASG12/13 mutation yielded a 14% discordance rate between primary and metastatic tissue. CRAC patients with mutant KRASG12/13 mutation in serum but wild-type KRASG12/13 in tissue had concurrent KRASG12/13-mutant metastatic tumors, indicating spatial genetic heterogeneity. Changes in serum KRASG12/G13 mutation status during postoperative follow-up were associated with recurrence. Conclusion: Although serum detection of the KRASG12/13 mutation cannot substitute for detection in tissue, serum testing can support the interpretation of a CRAC patient's status in regard to concurrent metastasis. Dynamic changes in serum KRASG12/13 mutation status during follow-up indicated that cfDNA from serum represents a potential source for monitoring recurrence in CRAC patients. [ABSTRACT FROM AUTHOR]

Introduction: Cell-free DNA (cfDNA) methylation is an important molecular biomarker, which provides information about the regulation of gene expression in the tissue of origin. There is an inverse correlation between SOST gene methylation and expression levels. Methods: We analyzed SOST promoter methylation in cfDNA from serum, and compared it with DNA from blood and bone cells from patients undergoing hip replacement surgery. We also measured cfDNA methylation in 28 osteoporotic patients at baseline and after 6 months of antiosteoporotic therapy (alendronate, teriparatide, or denosumab). Results:SOST gene promoter methylation levels in serum cfDNA were very similar to those of bone-derived DNA (79% ± 12% and 82% ± 7%, respectively), but lower than methylation levels in blood cell DNA (87% ± 10%). Furthermore, there was a positive correlation between an individual's SOST DNA methylation values in serum and bone. No differences in either serum sclerostin levels or SOST methylation were found after 6-months of therapy with antiosteoporotic drugs. Conclusions: Our results suggest that serum cfDNA does not originate from blood cells, but rather from bone. However, since we did not confirm changes in this marker after therapy with bone-active drugs, further studies examining the correlation between bone changes of SOST expression and SOST methylation in cfDNA are needed to confirm its potential role as a bone biomarker. [ABSTRACT FROM AUTHOR]

Background: Esophageal cancer (EC) is a prevalent malignancy characterized by a low 5-year survival rate, primarily attributed to delayed diagnosis and limited therapeutic options. Currently, early detection of EC heavily relies on endoscopy and pathological examination, which pose challenges due to their invasiveness and high costs, leading to low patient compliance. The detection of DNA methylation offers a non-endoscopic, cost-effective, and secure approach that holds promising prospects for early EC detection., Methods: To identify improved methylation markers for early EC detection, we conducted a comprehensive review of relevant literature, summarized the performance of DNA methylation markers based on different input samples and analytical methods in EC early detection and screening., Findings: This review reveals that blood cell free DNA methylation-based method is an effective non-invasive method for early detection of EC, although there is still a need to improve its sensitivity and specificity. Another highly sensitive and specific non-endoscopic approach for early detection of EC is the esophageal exfoliated cells based-DNA methylation analysis. However, while there are substantial studies in esophageal adenocarcinoma, further more validation is required in esophageal squamous cell carcinoma., Conclusion: In conclusion, DNA methylation detection holds significant potential as an early detection and screening technology for EC., Competing Interests: All authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xu, Wang, Pei, Wang, Xue and Zhao.)

Head and neck squamous cell carcinoma (HNSCC) is the seventh most commonly diagnosed cancer globally. HNSCC develops from the mucosa of the oral cavity, pharynx and larynx. Methylation levels of septin 9 ( SEPT9 ) and short stature homeobox 2 ( SHOX2 ) genes in circulating cell‑free DNA (ccfDNA) are considered epigenetic biomarkers and have shown predictive value in preliminary reports in HNSCC. Liquid biopsy is a non‑invasive procedure that collects tumor‑derived molecules, including ccfDNA. In the present study, a droplet digital PCR (ddPCR)‑based assay was developed to detect DNA methylation levels of circulating SEPT9 and SHOX2 in the plasma of patients with HNSCC. The assay was first set up using commercial methylated and unmethylated DNA. The dynamic changes in the methylation levels of SEPT9 and SHOX2 were then quantified in 20 patients with HNSCC during follow‑up. The results highlighted: i) The ability of the ddPCR‑based assay to detect very low copies of methylated molecules; ii) the significant decrease in SEPT9 and SHOX2 methylation levels in the plasma of patients with HNSCC at the first time points of follow‑up with respect to T 0 ; iii) a different trend of longitudinally DNA methylation variations in small groups of stratified patients. The absolute and precise quantification of SEPT9 and SHOX2 methylation levels in HNSCC may be useful for studies with translational potential.

Background: EGFR-positive Non-small Cell Lung Cancer (NSCLC) is a dynamic entity and tumor progression and resistance to tyrosine kinase inhibitors (TKIs) arise from the accumulation, over time and across different disease sites, of subclonal genetic mutations. For instance, the occurrence of EGFR T790M is associated with resistance to gefitinib, erlotinib, and afatinib, while EGFR C797S causes osimertinib to lose activity. Sensitive technologies as radiomics and liquid biopsy have great potential to monitor tumor heterogeneity since they are both minimally invasive, easy to perform, and can be repeated over patient's follow-up, enabling the extraction of valuable information. Yet, to date, there are no reported cases associating liquid biopsy and radiomics during treatment. Case presentation: In this case series, seven patients with metastatic EGFR-positive NSCLC have been monitored during target therapy. Plasma-derived cell free DNA (cfDNA) was analyzed by a digital droplet PCR (ddPCR), while radiomic analyses were performed using the validated LifeX® software on computed tomography (CT)-images. The dynamics of EGFR mutations in cfDNA was compared with that of radiomic features. Then, for each EGFR mutation, a radiomic signature was defines as the sum of the most predictive features, weighted by their corresponding regression coefficients for the least absolute shrinkage and selection operator (LASSO) model. The receiver operating characteristic (ROC) curves were computed to estimate their diagnostic performance. The signatures achieved promising performance on predicting the presence of EGFR mutations (R2 = 0.447, p <0.001 EGFR activating mutations R2 = 0.301, p = 0.003 for T790M; and R2 = 0.354, p = 0.001 for activating plus resistance mutations), confirmed by ROC analysis. Conclusion: To our knowledge, these are the first cases to highlight a potentially promising strategy to detect clonal heterogeneity and ultimately identify patients at risk of progression during treatment. Together, radiomics and liquid biopsy could detect the appearance of new mutations and therefore suggest new therapeutic management. [ABSTRACT FROM AUTHOR]

Introduction: Plasma androgen receptor (AR) copy number (CN) status identifies castration-resistant prostate cancer (CRPC) patients with worse outcome on abiraterone/enzalutamide. However, the impact of AR CN changes on clinical outcome in CRPC is unknown.Materials and Methods: Plasma samples from 73 patients treated with abiraterone or enzalutamide were collected at baseline and at the time of progression disease (PD). Droplet digital polymerase chain reaction was used to assess AR CN status.Results: We showed that 11 patients (15.1%) changed AR CN status from baseline to PD (9 patients from normal to gain, 2 from gain to normal). Patients changing AR CN status from normal at baseline to gain at PD had intermediate median overall survival (OS) of 20.5 months (95% CI = 8.0–44.2) between those who remained AR CN normal from baseline to PD (27.3 months [95% CI = 21.9–34.4]) and those who remained AR CN gain from baseline to PD (9.1 months [95% CI = 3.8–14.5], p < 0.0001). Patients changing AR CN from normal at baseline to gain at PD had a median progression-free survival (PFS) of 9.2 months (95% CI = 2.0–14.7), patients who remained AR CN normal had a median PFS of 9.1 months (95% CI = 7.2–10.1), and patients who remained AR CN gain had a median PFS of 5.4 (95% CI = 3.6–6.5, p = 0.0005). Both OS and PFS were not significantly different between patients with AR CN that changes from normal to gain and patients with stable AR CN normal.Conclusions: We showed that CRPC patients changing AR CN status from baseline to progression time point had intermediate OS and we suggested that AR CN evaluation at baseline could be the most informative for clinical outcome of CRPC patients treated with abiraterone or enzalutamide. Larger prospective studies are warranted.

Precision medicine stands for 4Ps – precise, preventive, participatory, and personal; in which “precision” is important because the current modern medicine starts from “trial and error,” and “one does not fit all”. Current targeted therapies for cancer have changed treatment approaches and led the precision medicine; however, clinical use of liquid biopsy, using blood or other liquid specimens to characterize circulating tumor cells (CTC) or tumor genes instead of biopsies of tumor tissues, still awaits availability of more information regarding non-invasive cancer detection and characterization, prediction of treatment response, monitoring the disease course and relapse possibilities, identification of mechanisms of drug resistance, and newer pathogenesis. In this review, we will introduce the basic concept of CTC, circulating cell free DNA, and exosomes and their possible application for gastric cancer relevant with Helicobacter pylori infection.

Purpose Several blood markers have been evaluated in stroke patients, but their role remains limited in clinical practice. This study was designed to evaluate the utility of cell free DNA (cf DNA) in stroke patients undergoing therapeutic intervention in the form of mechanical thrombectomy in acute ischemic stroke patients. Materials and Methods Twenty-six patients with ischemic stroke who were managed with interventions like intravenous thrombolysis (IVT) and mechanical thrombectomy were recruited consecutively in this study. The cf DNA was extracted by using circulating nucleic acid kit and measured by real-time quantitative PCR assay for β-globin gene. The neurological outcome was measured by modified Rankin scale (mRS) score at three months after the onset of symptoms. Results Cf DNA levels correlated with severity of stroke at the time of admission (r=0.421, P=0.032) and poor outcome at three months (r=0.606, P=0.001). Therapeutic intervention in the form of mechanical thrombectomy or IVT was associated with improved outcome in patients with cf DNA

Background/Aims: Colonoscopy screening has been accepted broadly to evaluate the risk and incidence of colorectal cancer (CRC) during health examination in outpatients. However, the intrusiveness, complexity and discomfort of colonoscopy may limit its application and the compliance of patients. Thus, more reliable and convenient diagnostic methods are necessary for CRC screening. Genome instability, especially copy-number variation (CNV), is a hallmark of cancer and has been proved to have potential in clinical application. Methods: We determined the diagnostic potential of chromosomal CNV at the arm level by whole-genome sequencing of CRC plasma samples (n = 32) and healthy controls (n = 38). Arm level CNV was determined and the consistence of arm-level CNV between plasma and tissue was further analyzed. Two methods including regular z score and trained Support Vector Machine (SVM) classifier were applied for detection of colorectal cancer. Results: In plasma samples of CRC patients, the most frequent deletions were detected on chromosomes 6, 8p, 14q and 1p, and the most frequent amplifications occurred on chromosome 19, 5, 2, 9p and 20p. These arm-level alterations detected in plasma were also observed in tumor tissues. We showed that the specificity of regular z score analysis for the detection of colorectal cancer was 86.8% (33/38), whereas its sensitivity was only 56.3% (18/32). Applying a trained SVM classifier (n = 40 in trained group) as the standard to detect colorectal cancer relevance ratio in the test samples (n = 30), a sensitivity of 91.7% (11/12) and a specificity 88.9% (16/18) were finally reached. Furthermore, all five early CRC patients in stages I and II were successfully detected. Conclusion: Trained SVM classifier based on arm-level CNVs can be used as a promising method to screen early-stage CRC.

Extracellular DNA (ecDNA) is a potential marker and predictor in several inflammatory diseases. Periodontitis, a chronic inflammatory disease, is associated with epithelial cell death and could lead to release of DNA. Our aim was to analyze salivary DNA concentration and deoxyribonuclease (DNase) activity in periodontitis patients. We hypothesized that salivary ecDNA will be higher than in controls and could serve as a marker of periodontitis severity. Samples of saliva were collected from 25 patients with chronic periodontitis and 29 age-matched controls. DNA was quantified fluorometrically in whole saliva, as well as in supernatants after centrifugation (depletion of cells at 1600× g) and in double-centrifuged supernatants (depletion of cell debris at 1600× g and 16,000× g). The subcellular origin of ecDNA was assessed using real-time PCR. In comparison to controls, patients with periodontitis had twofold higher salivary DNA (p < 0.01), higher mitochondrial DNA in centrifuged supernatants (p < 0.05) and lower nuclear ecDNA in double-centrifuged samples (p < 0.05). No correlations were found between salivary DNA and oral health status, but mitochondrial DNA positively correlated with papillary bleeding index in centrifuged samples. Salivary DNase activity was comparable between the groups. In conclusion, we proved that salivary DNA is higher in periodontitis. The source of the higher mitochondrial DNA in cell-free saliva and the causes of lower nuclear ecDNA remain to be elucidated. Further studies should focus on the role of mitochondrial DNA as a potential driver of inflammation in periodontitis. [ABSTRACT FROM AUTHOR]