1. Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis
- Author
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Zhuoyang Wang, Charu Aggarwal, Stephanie S. Yee, Aseel Abdalla, Jacob Till, Taylor A. Black, Robyn T. Sussman, Jacquelyn J. Roth, Kojo S.J. Elenitoba-Johnson, Erica L. Carpenter, Wei-Ting Hwang, Hareena Sangha, Caren Gentile, Jeffrey C. Thompson, and Mark H. O'Hara
- Subjects
Real-Time Polymerase Chain Reaction ,medicine.disease_cause ,Circulating Tumor DNA ,Pathology and Forensic Medicine ,Cohort Studies ,Biomarkers, Tumor ,medicine ,Humans ,Digital polymerase chain reaction ,Centrifugation ,Neoplasm Metastasis ,Alleles ,Whole blood ,Blood Specimen Collection ,Chemistry ,Regular Article ,Molecular biology ,Circulating Cell-Free DNA ,Pancreatic Neoplasms ,Real-time polymerase chain reaction ,Molecular Diagnostic Techniques ,Case-Control Studies ,Mutation ,Molecular Medicine ,Biomarker (medicine) ,Fresh frozen plasma ,KRAS ,Carcinoma, Pancreatic Ductal - 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.
- Published
- 2021