Relationship Between Plasma Cell-Free DNA Changes and Lysyl Oxidase During the Treatment and Prognosis of Canine Transmissible Venereal Tumor

2021 ◽  
Author(s):  
Mona MohammadZahri ◽  
Hadi Cheraghi ◽  
Dariush Shirani ◽  
Ali Hatamkhani
Keyword(s):  
Oxidase Activity ◽  
Roc Analysis ◽  
Lysyl Oxidase ◽  
Quantitative Polymerase Chain Reaction ◽  
High Sensitivity ◽  
Cell Free Dna ◽  
Diagnosis And Prognosis ◽  
Free Dna ◽  
Integrity Index ◽  
Polymerase Chain

Abstract BackgroundTransmissible Venereal Tumor (TVT) is a wide tumor of canine, there are no effective markers to monitor the therapeutic response in real-time. Circulating biomarkers may be valuable for the early diagnosis and prognosis of cancers, so in this study, we aimed to investigate the significance of the cell-free DNA (cfDNA) and cfDNA integrity index to monitor the response of TVT to vincristine and compare them with lysyl oxidase activity. Plasma and sera were collected before drug administration within four weeks from fifteen male dogs. The analytical method was mainly based on quantitative polymerase chain reaction for short and long cfDNA, and lysyl oxidase activity was measured in serum.ResultsThe results of cfDNA integrity index showed significant (p<0.05) difference in a baseline to 2nd and 3rd week (with cut-off value 1.118 and 93.33% specificity). We found that the cfDNA integrity index was increased during weeks due to the reduction of shorts cfDNA in the 1st week after treatment. lysyl Oxidase activity was increased during the 4th week (p<0.001) but there were no significant differences in the other weeks compared to baseline. ROC analysis of lysyl Oxidase revealed high sensitivity (100%) and specificity (93%) 2nd, 3rd weeks on comparison between Baseline. Multivariate analysis between cfDNA integrity index and lysyl Oxidase showed significant correlation (p<0.05) only baseline results. ConclusionsTaken together, we propose short cfDNA, cfDNA integrity index, and lysyl Oxidase activity as a diagnostic biomarker and a putative prognostic candidate in TVT patients. These biomarkers could be used simultaneously for quickly diagnose TVT in combination with cytology.

scholarly journals Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant

Tumor Biology ◽  
2019 ◽  
Vol 41 (8) ◽  
pp. 101042831986636 ◽  
Author(s):  
Abel Jacobus Bronkhorst ◽  
Vida Ungerer ◽  
Stefan Holdenrieder
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Culture Supernatant ◽  
Quantitative Polymerase Chain Reaction ◽  
High Sensitivity ◽  
Cell Types ◽  
Cell Culture Supernatant ◽  
Cell Free Dna ◽  
Free Dna

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA–based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.

scholarly journals Enhanced specificity of clinical high-sensitivity tumor mutation profiling in cell-free DNA via paired normal sequencing using MSK-ACCESS

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
A. Rose Brannon ◽  
Gowtham Jayakumaran ◽  
Monica Diosdado ◽  
Juber Patel ◽  
Anna Razumova ◽  
...  
Keyword(s):  
Cancer Patients ◽  
De Novo ◽  
Low Frequency ◽  
High Sensitivity ◽  
Clinical Analysis ◽  
De Novo Mutation ◽  
Cell Free Dna ◽  
Free Dna ◽  
Somatic Alterations ◽  
Mutation Profiling

AbstractCirculating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.

scholarly journals Tissue and Cell-Free DNA-Based Epigenomic Approaches for Cancer Detection

2019 ◽  
Vol 66 (1) ◽  
pp. 105-116 ◽  
Author(s):  
Alessandro Leal ◽  
David Sidransky ◽  
Mariana Brait
Keyword(s):  
Cancer Detection ◽  
Cancer Diagnosis ◽  
Malignant Tumors ◽  
Unmet Need ◽  
Clinical Routine ◽  
Commercial Development ◽  
Cell Free Dna ◽  
Cancer Management ◽  
Diagnosis And Prognosis ◽  
Free Dna

Abstract BACKGROUND Over 9 million people die of cancer each year worldwide, reflecting the unmet need for effective biomarkers for both cancer diagnosis and prognosis. Cancer diagnosis is complex because the majority of malignant tumors present with long periods of latency and lack of clinical presentation at early stages. During carcinogenesis, premalignant cells experience changes in their epigenetic landscapes, such as differential DNA methylation, histone modifications, nucleosome positioning, and higher orders of chromatin changes that confer growth advantage and contribute to determining the biologic phenotype of human cancers. CONTENT Recent progress in microarray platforms and next-generation sequencing approaches has allowed the characterization of abnormal epigenetic patterns genome wide in a large number of cancer cases. The sizable amount of processed data also comes with challenges regarding data management and assessment for effective biomarker exploration to be further applied in prospective clinical trials. Epigenetics-based single or panel tests of genes are being explored for clinical management to fulfill unmet needs in oncology. The advance of these tests to the clinical routine will depend on rigorous, extensive, and independent validation in well-annotated cohort of patients and commercial development of clinical routine–friendly and adequate procedures. SUMMARY In this review we discuss the analytic validation of tissue and cell-free DNA-based epigenomic approaches for early cancer detection, diagnosis, and treatment monitoring and the clinical utility of candidate epigenetic alterations applied to colorectal, glioblastoma, breast, prostate, bladder, and lung cancer management.

scholarly journals Comparison of Four Commercial Kits for Isolation of Urinary Cell-Free DNA and Sample Storage Conditions

Diagnostics ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 234 ◽  
Author(s):  
Eun Young Lee ◽  
Eun-Ju Lee ◽  
Hana Yoon ◽  
Dong Hyeon Lee ◽  
Kwang Hyun Kim
Keyword(s):  
Cost Efficiency ◽  
High Sensitivity ◽  
Storage Conditions ◽  
Sample Storage ◽  
Sample Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Zymo Research ◽  
Free Circulating Dna ◽  
Commercial Kits

Urinary cell-free DNA (cfDNA) is an attractive body fluid for liquid biopsy. In this study, we compared the efficiencies of four commercial kits for urinary cell-free DNA (cfDNA) isolation and of various sample storage conditions. Urinary cfDNA was isolated from 10 healthy individuals using four commercial kits: QIAamp Circulating Nucleic Acid Kit (QC; Qiagen), MagMAX™ Cell-Free DNA Isolation Kit (MM; Applied Biosystems), Urine Cell-Free Circulating DNA Purification Midi Kit (NU; Norgen Biotek), and Quick-DNA™ Urine Kit (ZQ; Zymo Research). To assess the isolation efficiency, an Agilent 2100 Bioanalyzer with High Sensitivity DNA chips was used, and cfDNA yield was defined as the amount of cfDNA obtained from 1 mL of urine. MM and QC provided the highest cfDNA yield in the 50–300 bp range, and MM and NU gave the highest cfDNA yield in the 50–100 bp range. In particular, the NU kit was efficient for isolation of more fragmented cfDNA in the range of 50–100 bp with the lowest cellular genomic DNA contamination. ZQ had the best cost-efficiency for isolating the same amount of urinary cfDNA. Samples stored at −70 °C with the addition of 10 mM EDTA resulted in the highest cfDNA yield 3 months after sample collection.

Dynamics of Pneumococcal Carriage in Adults: A New Look at an Old Paradigm

2020 ◽  
Author(s):  
Sónia T Almeida ◽  
Ana Cristina Paulo ◽  
Filipe Froes ◽  
Hermínia de Lencastre ◽  
Raquel Sá-Leão
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
High Sensitivity ◽  
Carrier State ◽  
Healthy Adults ◽  
Limited Information ◽  
Pneumococcal Carriage ◽  
The Social ◽  
Polymerase Chain ◽  
Pneumococcal Colonization ◽  
Regular Contact

Abstract Background Limited information is available on pneumococcal colonization among adults. We studied pneumococcal carriage dynamics in healthy adults using high-sensitivity approaches. Methods Eighty-seven adults (25–50 years old) were followed for 6 months in Portugal. Nasopharyngeal, oropharyngeal, and saliva samples were obtained monthly; pneumococcal carriers were also sampled weekly. Carriage was investigated by quantitative polymerase chain reaction (targeting lytA and piaB) and culture. Positive samples were serotyped. Results Approximately 20% of the adults were intermittent carriers; 10% were persistent carriers (&gt;4 months). Pneumococcal acquisition and clearance rates were 16.5 (95% confidence interval [CI], 11.2–24.2) and 95.9 (95% CI, 62.3–145.0) cases/1000 person-weeks, respectively. Living with children increased pneumococcal acquisition (hazard ratio, 9.7 [95% CI, 2.6–20.5]; P &lt; .001). Median duration of carriage was 7 weeks and did not depend on regular contact with children. Conclusions The pneumococcal carrier state in healthy adults is more dynamic than generally assumed: Acquisition is frequent and duration of carriage is often long. This suggests that some adults may act as reservoirs of pneumococci and hence, depending on the social structure of a community, the magnitude of herd effects potentially attainable through children vaccination may vary. These findings are important when designing strategies to prevent pneumococcal disease in adults.

scholarly journals Quantification of plasma cell-free DNA levels in dogs with various tumors

2019 ◽  
Vol 31 (6) ◽  
pp. 836-843 ◽  
Author(s):  
Michihito Tagawa ◽  
Genya Shimbo ◽  
Hisashi Inokuma ◽  
Kazuro Miyahara
Keyword(s):  
Disease Progression ◽  
Distant Metastasis ◽  
Malignant Tumors ◽  
Clinical Stage ◽  
Extracellular Dna ◽  
Dna Integrity ◽  
Healthy Controls ◽  
Cell Free Dna ◽  
Free Dna ◽  
Integrity Index

Circulating cell-free DNA (cfDNA) is extracellular DNA released into the bloodstream by apoptotic or necrotic tumor cells, with cfDNA determination proposed as a noninvasive, sensitive marker for the diagnosis of human cancer. We evaluated cfDNA quantification as a diagnostic and prognostic tool in dogs with various tumors. We quantified plasma cfDNA concentration by absolute real-time PCR of long interspersed nuclear elements in 50 dogs with malignant tumors, 13 dogs with benign tumors or nodules, and 11 healthy controls. Six patients with malignant tumors were followed-up, and plasma cfDNA was quantified throughout disease progression. We found that plasma cfDNA concentrations were significantly elevated in dogs with malignant tumors compared with dogs with benign nodules or healthy controls. The DNA integrity index (the ratio between long and short cfDNA fragments) was significantly lower in dogs with malignant tumors compared to healthy controls. Significantly higher cfDNA levels and a lower DNA integrity index were observed in dogs with lymphoma or leukemia, hemangiosarcoma, and distant metastasis; cfDNA levels correlated well with clinical stage and tended to increase during or before periods of disease progression, suggesting potential efficacy of cfDNA for the detection of distant metastasis and to monitor the clinical stage of neoplasia.

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