Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis

Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker a...

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Published inThe Journal of molecular diagnostics : JMD Vol. 23; no. 11; pp. 1545 - 1552
Main Authors Till, Jacob E., Black, Taylor A., Gentile, Caren, Abdalla, Aseel, Wang, Zhuoyang, Sangha, Hareena K., Roth, Jacquelyn J., Sussman, Robyn, Yee, Stephanie S., O'Hara, Mark H., Thompson, Jeffrey C., Aggarwal, Charu, Hwang, Wei-Ting, Elenitoba-Johnson, Kojo S.J., Carpenter, Erica L.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 01.11.2021
American Society for Investigative Pathology
Subjects
Alleles
Biomarkers, Tumor - blood
Biomarkers, Tumor - genetics
Blood Specimen Collection - methods
Carcinoma, Pancreatic Ductal - blood
Carcinoma, Pancreatic Ductal - genetics
Carcinoma, Pancreatic Ductal - pathology
Case-Control Studies
Circulating Tumor DNA - blood
Circulating Tumor DNA - genetics
Circulating Tumor DNA - isolation & purification
Cohort Studies
Humans
Molecular Diagnostic Techniques - methods
Mutation
Neoplasm Metastasis
Pancreatic Neoplasms - blood
Pancreatic Neoplasms - genetics
Pancreatic Neoplasms - pathology
Real-Time Polymerase Chain Reaction - methods
Regular
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Abstract Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at −80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.
AbstractList Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.
Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.
Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at −80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.
Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at −80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g ) or slower-speed (4100 × g ) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.
Author Gentile, Caren
Sussman, Robyn
Black, Taylor A.
Sangha, Hareena K.
Abdalla, Aseel
Yee, Stephanie S.
Thompson, Jeffrey C.
Aggarwal, Charu
Carpenter, Erica L.
Wang, Zhuoyang
Till, Jacob E.
Hwang, Wei-Ting
Roth, Jacquelyn J.
Elenitoba-Johnson, Kojo S.J.
O'Hara, Mark H.
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  surname: Thompson
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  surname: Elenitoba-Johnson
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  email: erical@upenn.edu
  organization: Division of Hematology-Oncology, Department of Medicine, Raymond and Ruth Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
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Snippet Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream...
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SubjectTerms Alleles
Biomarkers, Tumor - blood
Biomarkers, Tumor - genetics
Blood Specimen Collection - methods
Carcinoma, Pancreatic Ductal - blood
Carcinoma, Pancreatic Ductal - genetics
Carcinoma, Pancreatic Ductal - pathology
Case-Control Studies
Circulating Tumor DNA - blood
Circulating Tumor DNA - genetics
Circulating Tumor DNA - isolation & purification
Cohort Studies
Humans
Molecular Diagnostic Techniques - methods
Mutation
Neoplasm Metastasis
Pancreatic Neoplasms - blood
Pancreatic Neoplasms - genetics
Pancreatic Neoplasms - pathology
Real-Time Polymerase Chain Reaction - methods
Regular
Title Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis
URI https://www.clinicalkey.com/#!/content/1-s2.0-S1525157821002634
https://dx.doi.org/10.1016/j.jmoldx.2021.08.007
https://www.ncbi.nlm.nih.gov/pubmed/34454115
https://www.proquest.com/docview/2566027040
https://pubmed.ncbi.nlm.nih.gov/PMC8647427
Volume 23
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