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Knowledge of the composition of proteins that interact with plasma DNA will provide a better understanding of the homeostasis of circulating nucleic acids and the various modes of interaction with target cells, which may be useful in the development of gene targeted therapy approaches. The goal of the present study is to shed light on the composition and architecture of histone-containing nucleoprotein complexes (NPCs) from the blood plasma of healthy females (HFs) and breast cancer patients (BCPs) and to explore the relationship of proteins with crucial steps of tumor progression: epithelial-mesenchymal transition (EMT), cell proliferation, invasion, cell migration, stimulation of angiogenesis, and immune response. MALDI-TOF mass spectrometric analysis of NPCs isolated from blood samples using affine chromatography was performed. Bioinformatics analysis showed that the shares of DNA-binding proteins in the compositions of NPCs in normal and cancer patients are comparable and amount to 40% and 33%, respectively; in total, we identified 38 types of DNA-binding motifs. Functional enrichment analysis using FunRich 3.13 showed that, in BCP blood, the share of DNA-binding proteins involved in nucleic acid metabolism increased, while the proportion of proteins involved in intercellular communication and signal transduction decreased. The representation of NPC passenger proteins in breast cancer also changes: the proportion of proteins involved in transport increases and the share of proteins involved in energy biological pathways decreases. Moreover, in the HF blood, proteins involved in the processes of apoptosis were more represented in the composition of NPCs and in the BCP blood-in the processes of active secretion. For the first time, bioinformatics approaches were used to visualize the architecture of circulating NPCs in the blood and to show that breast cancer has an increased representation of passenger proteins involved in EMT, cell proliferation, invasion, cell migration, and immune response. Using breast cancer protein data from the Human Protein Atlas (HPA) and DEPC, we found that 86% of NPC proteins in the blood of BCPs were not previously annotated in these databases. The obtained data may indirectly indicate directed protein sorting in NPCs, which, along with extracellular vesicles, can not only be diagnostically significant molecules for liquid biopsy, but can also carry out the directed transfer of genetic material from donor cells to recipient cells.
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Neoplasias da Mama , Proteínas de Ligação a DNA , Humanos , Feminino , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Carcinogênese/metabolismo , Proliferação de Células , DNA/metabolismo , DNA/sangue , Biologia Computacional/métodos , Nucleoproteínas/metabolismo , Nucleoproteínas/sangue , Movimento CelularRESUMO
BACKGROUND: Cell-free DNA (cfDNA) fragmentomic characteristics are promising analytes with abundant physiological signals for non-invasive disease diagnosis and monitoring. Previous studies on plasma cfDNA fragmentomics commonly employed a two-step centrifugation process for removing cell debris, involving a low-speed centrifugation followed by a high-speed centrifugation. However, the effects of centrifugation conditions on the analysis of cfDNA fragmentome remain uncertain. METHODS: We collected blood samples from 10 healthy individuals and divided each sample into two aliquots for plasma preparation with one- and two-step centrifugation processes. We performed whole genome sequencing (WGS) of the plasma cfDNA in the two groups and comprehensively compared the cfDNA fragmentomic features. Additionally, we reanalyzed the fragmentomic features of cfDNA from 16 healthy individuals and 16 COVID-19 patients, processed through one- and two-step centrifugation in our previous study, to investigate the impact of centrifugation on disease signals. RESULTS: Our results showed that there were no significant differences observed in the characteristics of nuclear cfDNA, including size, motif diversity score (MDS) of end motifs, and genome distribution, between plasma samples treated with one- and two-step centrifugation. The cfDNA size shortening in COVID-19 patients was observed in plasma samples with one- and two-step centrifugation methods. However, we observed a significantly higher relative abundance and longer size of cell-free mitochondrial DNA (mtDNA) in the one-step samples compared to the two-step samples. This difference in mtDNA caused by the one- and two-step centrifugation methods surpasses the pathological difference between COVID-19 patients and healthy individuals. CONCLUSIONS: Our findings indicate that one-step low-speed centrifugation is a simple and potentially suitable method for analyzing nuclear cfDNA fragmentation characteristics. These results offer valuable guidance for cfDNA research in various clinical scenarios.
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COVID-19 , Ácidos Nucleicos Livres , Centrifugação , SARS-CoV-2 , Humanos , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/isolamento & purificação , Ácidos Nucleicos Livres/genética , COVID-19/sangue , COVID-19/diagnóstico , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Coleta de Amostras Sanguíneas , Masculino , Feminino , Sequenciamento Completo do Genoma , AdultoRESUMO
BACKGROUND: Cell-free DNA (cfDNA) is a promising analyte for non-invasive liquid biopsy, carrying abundant signatures for disease diagnosis and monitoring. In infectious disease researches, blood plasma samples are routinely heat-inactivated before proceeding with downstream analyses. However, the effects of heat inactivation on cfDNA fragmentomic analysis remain largely unclear, potentially introducing biases or altering the characteristics of cfDNA. METHODS: We performed a comprehensive investigation of cfDNA concentrations and fragmentomics in 21 plasma samples from 7 healthy individuals, by comparing the sample group without the heat inactivation to those exposed to once or twice heat-inactivation at 56 °C for 30 min and following freeze-thaw. RESULTS: Plasma samples with once and twice heat inactivation displayed no significant deviations in primary characteristics, including cfDNA concentrations, size profiles, end motif features, and genome-wide distributions, compared to samples without heat treatment. CONCLUSIONS: Heat-inactivated cfDNA can be utilized for liquid biopsy in infectious disease researches, without substantial impact on cfDNA concentrations and fragmentomic properties. This study provides essential insights into the effects of heat inactivation on cfDNA properties and will contribute to the development of reliable non-invasive biomarkers for infectious disease.
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Ácidos Nucleicos Livres , Temperatura Alta , Humanos , Biomarcadores , Biópsia Líquida , Biomarcadores Tumorais/genéticaRESUMO
BACKGROUND: Glial fibrillary acidic protein (GFAP) in plasma is an established biomarker of traumatic brain injury (TBI) in humans. Plasma extracellular DNA (ecDNA) is a very sensitive, although nonspecific marker of tissue damage including TBI. Whether plasma GFAP or ecDNA could be used as an early non-invasive biomarker in the mouse model of closed head injury is unknown. The aim of this paper was to describe the early dynamics of plasma GFAP and ecDNA in the animal model of closed head TBI. METHODS: Closed head TBI was induced using the weight-drop method in 40 adult CD1 mice and blood was collected in different time points (1, 2 or 3h) after TBI in different groups of mice. Plasma GFAP and ecDNA and ecDNA fragmentation from the experimental groups were compared to healthy controls. In the surviving mice, a static rods test was performed 30 days after TBI to assess the neurological outcome of TBI. RESULTS: Despite a trend of higher plasma GFAP after TBI the differences between the groups were not statistically significant. Plasma ecDNA was higher by 50% after 1h (P<0.05) and 2h (P<0.05) after TBI and was highly variable after 3h. Plasma ecDNA, but not GFAP, was partially predictive of the neurological impairment of the mice. CONCLUSION: In this study, we have described the early dynamics of plasma GFAP and ecDNA after TBI in mice. According to our results, ecDNA in plasma is a more sensitive early marker of TBI than GFAP. Analysis of tissue-specific ecDNA might improve its predictive value regarding the survival and neurobehavioral outcome.
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Lesões Encefálicas Traumáticas , DNA , Proteína Glial Fibrilar Ácida , Animais , Camundongos , Biomarcadores/sangue , Lesões Encefálicas Traumáticas/diagnóstico , DNA/sangue , Proteína Glial Fibrilar Ácida/sangueRESUMO
Objective: Extrachromosomal circular DNA elements (eccDNAs) are known for their broad existence in cells and plasma, which may potentially play important roles in many biological processes. Our aim was to identify potentially functional or marked eccDNAs in gout patients. Methods: The Circle-Seq approach was applied for eccDNA detection from plasma in acute gout patients and healthy controls. Further analysis was performed on the distribution of genomic elements and eccDNA gene annotations in two groups. Results: We detected 57,216 and 109,683 eccDNAs from the acute gout and healthy control plasma, respectively. EccDNAs were mapped to the reference genome to identify diverse classes of genomic elements and there was no significant difference of eccDNAs on genomic element annotation between gout and control group. A total of 256 eccDNA-associated genes were detected as gout unique eccDNA genes, including COL1A1 and EPB42, which potentially contribute to hyperuricemia and gout, and a couple of genes involved in inflammation or immune response. Enrichment analysis showed that these eccDNA genes were highly correlated with defense response, stress response, and immune and inflammatory responses, including T cell receptor signaling pathway, Fc epsilon RI signaling pathway, and JAK-STAT signaling pathway. Conclusion: Our discovery reveals the novel potential biological roles of plasma eccDNAs in gouty arthritis.
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Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in regions of equatorial Africa where P. falciparum malaria is holoendemic. The tumor is consistently associated with Epstein-Barr virus (EBV). Screening for EBV DNA in plasma in a high-risk population in Hong Kong has been shown to be useful in facilitating the early diagnosis of nasopharyngeal carcinoma, another EBV-associated tumor. Here, we investigate plasma EBV as a diagnostic marker for eBL in children in Uganda. We studied plasma specimens from 25 children with eBL and 25 controls matched for age (<3-16 years), gender and geography, including many with asymptomatic P. falciparum infection. These specimens were previously collected under the auspices of the EMBLEM (Epidemiology of Burkitt lymphoma in East African children and minors) study. After cell-free DNA isolation, plasma EBV DNA was measured using a quantitative PCR assay that amplifies the large internal repeats of the EBV genome. All children with eBL had measurable plasma EBV, as compared to 84% of control children. The median plasma EBV DNA level was 5.23 log10 copies/mL (interquartile range 3.54-6.08 log10 copies/mL) in children with eBL. In contrast, the median plasma EBV DNA level was 0.37 log10 copies/mL (interquartile range 0.18-1.05 log10 copies/mL) in children without lymphoma. An EBV threshold of 2.52 log10 copies/mL yielded a sensitivity of.88 and a specificity of 1. The estimated AUC was 0.936 (95% CI: 0.8496 - 1.00) for the corresponding ROC curve. Plasma EBV copy number did not depend on age, gender, or malaria screening status. However, two control children with asymptomatic P. falciparum infection and parasitemia also had high plasma EBV copy number. Our analysis suggests that measurements of EBV copy number in plasma may be useful in identifying children with eBL versus control children. A promising area for future research is the differentiation of high copy number associated with tumor versus high copy number associated with asymptomatic parasitemia.
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BACKGROUND: Recent research highlighted the potential role of circulating cell-free DNA (cfDNA), resulted by apoptosis or cell necrosis, as a prognostic marker in the setting of different clinical conditions. Cardiorenal syndrome type 1 (CRS type 1) is characterized by a rapid worsening of cardiac function leading to acute kidney injury (AKI). Apoptosis of renal epithelial cells is proposed as a mechanism involved in CRS type 1. In this study, we investigated cfDNA levels in patients with acute heart failure (AHF) and CRS type 1 and the possible correlation between cfDNA levels and inflammatory and apoptotic parameters. METHODS: We enrolled 17 AHF patients and 15 CRS type 1 who exhibited AKI at the time of admission (caused by AHF) or developed AKI during the first 48 h from admission. cfDNA was extracted from plasma and quantified by real-time polymerase chain reaction. Plasma levels of NGAL, tumor necrosis factor-α, interleukin (IL)-6, IL-18, and caspase-3 were measured. RESULTS: We observed significantly higher levels of cfDNA in patients with CRS type 1 than patients with AHF. Caspase-3, IL-6, IL-18, and NGAL levels resulted significantly increased in patients with CRS type 1. Moreover, a positive correlation between cfDNA levels and caspase-3 levels was found, as well as between cfDNA levels and IL-6 and renal parameters. CONCLUSION: Our study explores the premise of cfDNA as a marker for apoptosis and inflammation in CRS type 1 patients. cfDNA could potentially serve as an index for noninvasive monitoring of tissue damage and apoptosis in patients with AKI induced by AHF.
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Injúria Renal Aguda , Síndrome Cardiorrenal , Ácidos Nucleicos Livres , Insuficiência Cardíaca , DNA , HumanosRESUMO
Cell-free DNA circulates in plasma at low levels as a normal by-product of cellular apoptosis. Multiple clinical pathologies, as well as environmental stressors can lead to increased circulating cell-free DNA (ccfDNA) levels. Plasma DNA studies frequently employ targeted amplicon deep sequencing platforms due to limited concentrations (ng/ml) of ccfDNA in the blood. Here, we report whole genome sequencing (WGS) and read distribution across chromosomes of ccfDNA extracted from two human plasma samples from normal, healthy subjects, representative of limited clinical samples at <1 ml. Amplification was sufficiently robust with ~90% of the reference genome (GRCh38.p2) exhibiting 10X coverage. Chromosome read coverage was uniform and directly proportional to the number of reads for each chromosome across both samples. Almost 99% of the identified genomic sequence variants were known annotated dbSNP variants in the hg38 reference genome. A high prevalence of C>T and T>C mutations was present along with a strong concordance of variants shared between the germline genome databases; gnomAD (81.1%) and the 1000 Genome Project (93.6%). This study demonstrates isolation and amplification procedures from low input ccfDNA samples that can detect sequence variants across the whole genome from amplified human plasma ccfDNA that can translate to multiple clinical research disciplines.
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Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Cromossomos Humanos/genética , Genoma Humano , Mutação , Sequenciamento Completo do Genoma/métodos , HumanosRESUMO
Symptoms of coronavirus disease 2019 (COVID-19) range from asymptomatic to severe pneumonia and death. A deep understanding of the variation of biological characteristics in severe COVID-19 patients is crucial for the detection of individuals at high risk of critical condition for the clinical management of the disease. Herein, by profiling the gene expression spectrum deduced from DNA coverage in regions surrounding transcriptional start site in plasma cell-free DNA (cfDNA) of COVID-19 patients, we deciphered the altered biological processes in the severe cases and demonstrated the feasibility of cfDNA in measuring the COVID-19 progression. The up- and downregulated genes in the plasma of severe patient were found to be closely related to the biological processes and functions affected by COVID-19 progression. More importantly, with the analysis of transcriptome data of blood cells and lung cells from control group and cases with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection, we revealed that the upregulated genes were predominantly involved in the viral and antiviral activity in blood cells, reflecting the intense viral replication and the active reaction of immune system in the severe patients. Pathway analysis of downregulated genes in plasma DNA and lung cells also demonstrated the diminished adenosine triphosphate synthesis function in lung cells, which was evidenced to correlate with the severe COVID-19 symptoms, such as a cytokine storm and acute respiratory distress. Overall, this study revealed tissue involvement, provided insights into the mechanism of COVID-19 progression, and highlighted the utility of cfDNA as a noninvasive biomarker for disease severity inspections.
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BACKGROUND: Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth. METHODS: Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES). RESULTS: cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches. CONCLUSIONS: cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.
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Biomarcadores Tumorais , DNA Tumoral Circulante , Biópsia Líquida/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Variações do Número de Cópias de DNA , Genômica/métodos , Humanos , Mutação , Curva ROC , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Plasma AR status has been identified as a potential biomarker of response in metastatic castration-resistant prostate cancer (mCRPC) patients receiving docetaxel or AR-targeted therapies. However, the relevance of plasma AR in the overall management of CRPC patients receiving different docetaxel doses is unknown. PATIENTS AND METHODS: This was a multi-institution study of associations between baseline plasma AR copy number status, assessed by droplet digital PCR, and outcome in 325 mCRPC patients receiving docetaxel at standard or adapted regimen at the discretion of the treating physician. Upon analysis, patients were assigned randomly to either a training (n = 217) or validation (n = 108) cohort. RESULTS: In the training cohort, AR-gained patients treated with adapted docetaxel regimen had a significantly worse median progression-free survival (PFS) (3.8 vs 6.3 months, hazard ratio [HR] 2.58, 95% confidence interval [CI] 1.34-4.95, p < 0.0001), median overall survival (10.8 vs 20.6 months, HR 1.98, 95% CI 1.09-3.62, p = 0.0064) and PSA response (PSA > -50%: odds ratio 4.88 95%CI 1.55-14.32, p = 0.013) as compared to plasma AR normal patients. These findings were all confirmed in the validation cohort. However, in patients treated with standard docetaxel regimen, these differences were not seen. The interaction between AR CN status and dose reduction of docetaxel was considered as independent factor for PFS in both the training and validation cohort (HR 2.84, 95% CI 1.41-5.73, p = 0.003, and HR 4.79, 95% CI 1.79-12.82, p = 0.002). CONCLUSION: Despite the retrospective non-randomised design of this study, our hypothesis-generating findings could suggest plasma AR as a potential biomarker for optimal docetaxel timing and dose in mCRPC patients. Prospective trials are warranted.
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Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Biomarcadores Tumorais/sangue , Docetaxel/administração & dosagem , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores Androgênicos/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Relação Dose-Resposta a Droga , Esquema de Medicação , Monitoramento de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos , Humanos , Masculino , Pessoa de Meia-Idade , Prednisona/administração & dosagem , Intervalo Livre de Progressão , Estudos Prospectivos , Neoplasias de Próstata Resistentes à Castração/sangueRESUMO
BACKGROUND: Plasma tumor DNA fraction is prognostic in metastatic cancers. This could improve risk stratification before commencing a new treatment. We hypothesized that a second sample collected after one cycle of treatment could refine outcome prediction of patients identified as poor prognosis based on plasma DNA collected pre-treatment. PATIENTS AND METHODS: Plasma DNA [128 pre-treatment, 134 cycle 2 day 1 (C2D1), and 49 progression] from 151 chemotherapy-naive metastatic castration-resistant prostate cancer (mCRPC) patients in a phase II study of abiraterone acetate (NCT01867710) were subjected to custom targeted next-generation sequencing covering exons of these genes: TP53, AR, RB1, PTEN, PIK3CA, BRCA1, BRCA2, ATM, CDK12, CHEK2, FANCA HDAC2 and PALB2. We also captured 1500 pan-genome regions enriched for single nucleotide polymorphisms to allow detection of tumor DNA using the rolling B-allele method. We tested associations with overall survival (OS) and progression-free survival (PFS). RESULTS: Plasma tumor DNA detection was associated with shorter OS [hazard ratio (HR): 2.89, 95% confidence intervals (CI): 1.77-4.73, P ≤ 0.0001] and PFS (HR: 2.05; 95% CI: 1.36-3.11, P < 0.001). Using a multivariable model including plasma tumor DNA, patients who had a TP53, RB1 or PTEN gene alteration pre-treatment and at C2D1 had a significantly shorter OS than patients with no alteration at either time point (TP53: HR 7.13, 95% CI 2.37-21.47, P < 0.001; RB1: HR 6.24, 95% CI 1.97-19.73, P = 0.002; PTEN: HR 11.9, 95% CI 3.6-39.34, P < 0.001). Patients who were positive pre-treatment and converted to undetectable had no evidence of a difference in survival compared with those who were undetectable pre-treatment (P = 0.48, P = 0.43, P = 0.5, respectively). Progression samples harbored AR gain in all patients who had gain pre-treatment (9/49) and de novo AR somatic point mutations were detected in 8/49 patients. CONCLUSIONS: Plasma gene testing after one cycle treatment refines prognostication and could provide an early indication of treatment benefit.
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Neoplasias de Próstata Resistentes à Castração , Acetato de Abiraterona , Biomarcadores Tumorais/genética , Conversão Gênica , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Resultado do TratamentoRESUMO
We developed genetic-epigenetic tissue mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung transplant recipients, we showed that, at 72 hr after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles, and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring, and cancer screening.
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DNA/sangue , Epigenômica/métodos , Neoplasias/genética , Transplante de Órgãos/métodos , Diagnóstico Pré-Natal/métodos , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , DNA/genética , Metilação de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Epigênese Genética , Feminino , Feto/metabolismo , Variação Genética , Humanos , Neoplasias Hepáticas/genética , Linfoma/genética , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Placenta/metabolismo , Gravidez , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND: The identification of effective prognosis biomarkers for nasopharyngeal carcinoma (NPC) is crucial to improve treatment and patient outcomes. In the present study, we have attempted to evaluate the correlation between pre-treatment plasmatic Epstein-Barr virus (EBV) DNA load and the conventional prognostic factors in Moroccan patients with NPC. METHODS: The present study was conducted on 121 histologically confirmed NPC patients, recruited from January 2017 to December 2018. Circulating levels of EBV DNA were measured before therapy initiation using real-time quantitative PCR. RESULTS: Overall, undifferentiated non-keratinizingcarcinoma type was the most common histological type (90.1 %), and 61.8 % of patients were diagnosed at an advanced disease stage (IV). Results of pre-treatment plasma EBV load showed that 90.9 % of patients had detectable EBV DNA, with a median plasmatic viral load of 7710 IU/ml. The correlation between pre-treatment EBV DNA load and the conventional prognostic factors showed a significant association with patients' age (p = 0.01), tumor classification (p = 0.01), lymph node status (p = 0.003), metastasis status (p = 0.00) and overall cancer stage (p = 0.01). Unexpectedly, a significant higher level of pre-treatment EBV DNA was also found in plasma of NPC patients with a family history of cancer (p = 0.04). The risk of NPC mortality in patients with high pretreatment EBVDNA levels was significantly higher than that of those with low pre-treatment plasma EBV-DNA levels (p < 0.05). Furthermore, patients with high pre-treatment EBV-DNA levels (≥ 2000, ≥ 4000) had a significant low overall survival (OS) rates (p < 0.05). Interestingly, lymph node involvement, metastasis status and OS were found to be the most important factors influencing the EBV DNA load in NPC patients. CONCLUSIONS: The results of the present study clearly showed a high association between pre-treatment EBV DNA load, the crucial classical prognostic factors (T, N, M and disease stage) of NPC and OS, suggesting that pre-treatment EBV DNA can be a useful prognostic biomarker in clinical decision-making and improving NPC treatment in Morocco.
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Neurofibromatosis type 1 (NF1) is an autosomal dominant syndrome whose characteristic manifestations include benign neurofibromas, yet NF1 is also associated with a high risk of cancer. Measurements of circulating free plasma DNA (cfDNA) are gaining wider applicability in cancer diagnostics, targeting of therapy, and monitoring of therapeutic response. Individuals with NF1 are likely to be followed up using this method, but the effects of NF1 and neurofibromas on cfDNA levels are not known. We studied peripheral blood samples from 19 adults with NF1 and 12 healthy controls. The cfDNA was isolated from plasma with QIAamp Circulating Nucleic Acid Kit and quantified using the Qubit 2.0 Fluorometer. The cfDNA concentration of each sample was normalized relative to the plasma protein concentration. The normalized median concentration of cfDNA in plasma was 19.3 ng/ml (range 6.6-78.6) among individuals with NF1 and 15.9 ng/ml (range 4.8-47.0) among controls (p = .369). Individuals with NF1 who also had plexiform neurofibroma (pNF) showed non-significantly elevated cfDNA concentration compared to individuals with NF1 and without known pNF (median 25.4 vs. 18.8 ng/ml, p = .122). The effect of NF1 on cfDNA seems to be relatively small and NF1 is therefore unlikely to hamper the use of cfDNA-based assays.
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Ácidos Nucleicos Livres/sangue , Neurofibroma/sangue , Neurofibromatose 1/sangue , Neurofibromina 1/sangue , Adolescente , Adulto , Ácidos Nucleicos Livres/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/genética , Neoplasias/patologia , Neurofibroma/genética , Neurofibroma/patologia , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Adulto JovemRESUMO
Analysis of androgen receptor (AR) status, particularly AR copy number, in plasma DNA is a minimally invasive method with the potential to identify treatment resistance in patients with castration-resistant prostate cancer (CRPC) starting enzalutamide or abiraterone. Patients with elevated plasma AR do not have worse outcomes than patients with normal plasma AR when treated with taxanes. Consequently, circulating AR may improve clinical decision-making between AR-directed therapies versus taxanes and probably also between adapted versus standard taxane regimens. The evidence indicates that circulating AR could have a role in overall CRPC management. Promising clinical implications of plasma AR testing are measurement in earlier stages of prostate cancer, disease monitoring, and within the context of a multiplex biomarker strategy to improve treatment selection for CRPC patients. PATIENT SUMMARY: Measurement of the copy number of androgen receptor genes in plasma is a promising tool for guiding personalised treatment in patients with castration-resistant prostate cancer. However, prospective trials to validate these findings are needed.
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Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos , Humanos , Masculino , Seleção de Pacientes , Prognóstico , Neoplasias de Próstata Resistentes à Castração/diagnóstico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genéticaRESUMO
Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of â¼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.
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Núcleo Celular/genética , Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , Ensaios de Triagem em Larga Escala/métodos , Mitocôndrias/genética , Automação , Ácidos Nucleicos Livres/isolamento & purificação , Ácidos Nucleicos Livres/metabolismo , DNA Mitocondrial/isolamento & purificação , DNA Mitocondrial/metabolismo , Endopeptidase K/metabolismo , Humanos , Magnetismo , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real , TemperaturaRESUMO
We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at â¼202 and â¼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5'-untranslated regions (5'-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.
Assuntos
Ácidos Nucleicos Livres/isolamento & purificação , DNA Circular/isolamento & purificação , Plasma/química , Ácidos Nucleicos Livres/química , Ácidos Nucleicos Livres/genética , DNA Circular/química , DNA Circular/genética , Feminino , Genoma Humano , Hong Kong , Humanos , Teste Pré-Natal não Invasivo , GravidezRESUMO
Extracellular DNA is studied as a diagnostic biomarker, but also as a factor involved in the pathophysiology of several diseases due to its pro-inflammatory properties. Extracellular DNA can be extracted from plasma, urine, saliva or other biofluids using standard DNA isolation procedures and specialized commercial kits. Sample preparation for isolation is important, freezing and thawing may affect the amount of extracellular DNA extracted. Subsequent centrifugations remove cells and cell debris from the samples to obtain true extracellular DNA. Small volume of samples especially from animal experiments is often an issue and it affects the DNA yield. Very short fragments ( 100 bp) can be lost during isolation and are difficult to quantify using PCR. Fluorometric methods asses all stained DNA fragments. Selecting the method for quantification of extracellular DNA is crucial and combination of at least two methods is ideal. Standardization of procedures or at least their reporting in research papers is of utmost importance for comparison of results.
RESUMO
The therapeutic landscape of prostate cancer has expanded rapidly over the past 10 years, and there is now an even greater need to understand the biological mechanisms of resistance and to develop noninvasive biomarkers to guide treatment. The androgen receptor (AR) is known to be involved in the pathogenesis and progression of prostate cancer. Recently, highly sensitive next-generation sequencing and PCR-based methods for analyzing androgen receptor gene (AR) copy numbers (CN) and mutations in plasma were established in cell-free DNA (cfDNA) of patients with castration-resistant prostate cancer (CRPC) treated with different drugs. The study of cfDNA holds great promise for improving treatment in CRPC, especially in the advanced stage of the disease. Recent findings showed the significant association of plasma AR aberrations with clinical outcome in CRPC patients treated with AR-directed therapies, whereas no association was observed in patients treated with taxanes. This suggests the potential for using plasma AR as a biomarker for selecting treatment, i.e., hormone therapy or chemotherapy, and the possibility of modulating taxane dose. In recent years, plasma AR status has also been investigated in association with novel agents, such as 177Lu-PSMA radioligand therapy and PARP inhibitors. This review will focus on AR testing in plasma that may have clinical utility for treatment selection in advanced prostate cancer.