Value of circulating DNA concentration and integrity as a screening test for detection of cancer in an Egyptian cohort

Zaher, Ebtsam R.; Anwar, Medhat Mohamed; Kohail, H.; El-Zoghby, S.M.; Aboeleneen, Marwa S.

doi:10.1016/j.ajme.2012.03.003

Cited by 6 publications

(5 citation statements)

References 32 publications

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“…The size distribution of ccfDNA was evaluated using two previously reported methods: two qPCR assays used to amplify the APP gene (67 and 180 bp) [10,11] and two semiquantitative PCR amplifications (400 and 800 bp) of the TP53 gene, which were subsequently analyzed by gel electrophoresis [12]. An integrity index of ccfDNA was calculated using C P180 /C P67 (C P =cycle threshold of the qPCR assay) for APP and I 800 /I 400 (I=intensity of the gel band) for TP53.…”

Section: Integrity Indexmentioning

confidence: 99%

“…Conditions for the amplification of the TP53 gene were as follows: 1× HotStar Taq buffer supplemented with 1.6 mM MgCl 2 , 0.4 mM of each dNTP, 0.08 U of HotStar Taq polymerase, 0.2 mM of forward and reverse primers, and 0.250 ng of ccfDNA in 25 μL [12]. PCR cycling conditions included an initial denaturation step for 15 min at 95°C, followed by 50 cycles of 30 s at 95°C, 30 s at 60°C, and 15 s at 72°C and a final step for 5 min at 72°C.…”

Section: Integrity Indexmentioning

confidence: 99%

“…The isolated ccfDNA was quantified using two newly developed quantitative real-time PCR (qPCR) assays targeting repetitive genomic elements (Kpn, DHFRP2) as well as a fluorometric assay. Finally, the integrity index was calculated using either qPCR assays to amplify the APP gene or gel electrophoresis to amplify products of the TP53 gene [10][11][12].…”

Section: Introductionmentioning

confidence: 99%

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Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma

et al. 2015

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Circulating cell-free DNA (ccfDNA) has great potential for non-invasive diagnostics, and prediction and monitoring of treatment response, but its amount is usually limited. Therefore, the choice of methods to extract and characterize ccfDNA is crucial. In the current study, we performed the most comprehensive comparison of methods for ccfDNA extraction (11 methods), quantification (3 methods), and estimation of the integrity index (2 methods) from small quantities of different kinds of plasma. The QIAamp® Circulating Nucleic Acid Kit and the Norgen Plasma/Serum Circulating DNA Purification Mini Kit showed the best accuracy and reproducibility, but the Norgen kit allowed to extract a higher amount of ccfDNA. This workflow provides a reliable protocol for the multiple applications of ccfDNA in biomedicine.

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Section: Integrity Indexmentioning

confidence: 99%

Section: Integrity Indexmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma

et al. 2015

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“…Eight studies included patients with benign breast disease, however, only four of them [ 23 – 24 , 27 – 28 ] had diagnostic data available for pooled quantitative analyses. For these four studies, the pooled sensitivity and specificity were 0.86 (95% CI, 78~92%; Q = 9.51, I 2 = 68.54%, P = 0.02) and 85% (95% CI, 65~95%; Q = 21.60, I 2 = 86.11%, P < 0.01), respectively (Figure 3A ).…”

Section: Resultsmentioning

confidence: 99%

Quantitation of cell-free DNA in blood is a potential screening and diagnostic maker of breast cancer: a meta-analysis

et al. 2017

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IntroductionIncreased cell-free DNA (cfDNA) levels in circulating blood have been associated with higher possibility of breast cancer, however, researchers have not reached an agreement on its analysis.Materials and MethodsWe conducted a meta-analysis of 12 retrospective studies to clarify the value of cfDNA quantification in screening and diagnosis of breast cancer. PubMed, EMBASE, Web of Science and Cochrane library were searched from January, 2000 to October, 2016. Pooled analyses were estimated using a random effects model.ResultsIn total, 1003 primary breast cancer patients, 283 cases with benign breast disease and 575 healthy individuals were included. Pooled diagnostic odds ratio (DOR) was 27.63 (95% confidence interval [CI]: 10.96~69.61, I2 = 86.2%, P < 0.001) in discriminating between breast cancer and healthy controls; the area under the summary receiver operating characteristic (SROC) curve measured 0.91 (95% CI: 0.17~1.00). Analysis of available data in distinguishing breast cancer and benign breast disease showed a pooled DOR of 35.30 (95% CI: 7.58~164.39, I2 = 79.9%, P = 0.002) with an area under SROC of 0.91 (95% CI: 0.89~0.93). Ethnic group distribution based geographical factors suggested by meta-regression and subgroup analyses explained most of the heterogeneity.ConclusionsQuantification of cfDNA is a promising test in screening and diagnostic of breast cancer, but population-based standardization of test methods require completion prior to clinical use.

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“…With the development of PCR and quantitative PCR techniques, understanding of cfDNA is rapidly growing. However, radioimmunoassay [18] , DNA Dip Stick-TM Kit, Pico Green assay [19] , direct nick translation DNA labelling [20] , and spectrophotometry [21] have also reported significantly increased concentration of cfDNA in plasma/serum of cancer patients. Since cfDNA analysis may serve as a liquid biopsy in several malignancies, selecting a consistent and efficient serum/plasma cfDNA quantification method would be important before estimating cfDNA in cancer patients.…”

Section: Introductionmentioning

confidence: 99%

Comparative Evaluation of Diagnostic Performance of Circulating free DNA by Conventional vs. Real Time PCR in Gallbladder Cancer

Kumari

Mishra

Agarwal

et al. 2022

ijirms

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Background: Circulating free DNA (cfDNA) in serum/plasma has been studied as a promising biomarker in various pathologies, including cancer. However, there is no standardized method for the isolation and quantification of serum cfDNA. An effective and reliable method for isolation and quantification is of utmost importance before any clinical decision. The current study compares the conventional and real-time PCR methods to find any differences and concordance in cfDNA levels between the two methods and the diagnostic accuracy of cfDNA by each method. Methods: Serum sample was collected from 67 subjects, including 17 normal healthy individuals (control, n=17), 19 disease controls (cholecystitis, n=19), and 31 Gallbladder cancer patients (cancer, n=31) before any treatment for cfDNA quantification. Results: The cfDNA level did not differ significantly between two methods in both control and cholecystitis groups. In cancer group, cfDNA level was significantly (P < 0.001) different and higher in real time PCR as compared to conventional PCR. There was no significant correlation between two methods in control (r=0.02, P = 0.937), cholecystitis (r=0.10, P = 0.697), cancer (r=-0.08, P = 0.657) and total cases (control + cholecystitis + cancer) (r=0.06, P = 0.622). The diagnostic accuracy of two methods was found similar (P > 0.05) when assessed between control vs. cholecystitis (Z=0.85, P = 0.397), and control vs. total cases (Z=1.35, P = 0.177). However, the diagnostic accuracy of real time PCR was found significantly different and higher as compared to conventional PCR when assessed between control vs. cancer (Z=2.98, P = 0.003), and cholecystitis vs. cancer (Z=4.41, P < 0.001). Conclusion: Quantitative real-time PCR method is of high accuracy, reproducibility, and time-effectiveness. The diagnostic accuracy of real-time PCR was higher compared to conventional PCR.

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Value of circulating DNA concentration and integrity as a screening test for detection of cancer in an Egyptian cohort

Cited by 6 publications

References 32 publications

Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma

Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma

Quantitation of cell-free DNA in blood is a potential screening and diagnostic maker of breast cancer: a meta-analysis

Comparative Evaluation of Diagnostic Performance of Circulating free DNA by Conventional vs. Real Time PCR in Gallbladder Cancer

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