Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients

Abstract

Circulating tumor DNA (ctDNA) offers new opportunities for noninvasive cancer management. Detecting ctDNA in plasma is challenging because it constitutes only a minor fraction of the total cell-free DNA (cfDNA). Pre-analytical factors affect cfDNA levels contributed from leukocyte lysis, hence the ability to detect low-frequency mutant alleles. This study investigates the effects of the delay in processing, storage temperatures, different blood collection tubes, centrifugation protocols, and sample shipment on cfDNA levels. Peripheral blood (n = 231) from cancer patients (n = 62) were collected into K3EDTA or Cell-free DNA BCT tubes and analyzed by digital PCR, targeted amplicon, or shallow whole-genome sequencing. To assess pre-analytic effects, plasma was processed under different conditions after 0, 6, 24, 48, 96 hours, and 1 week at room temperature or 4°C, or using different centrifugation protocols. Digital PCR showed that cfDNA levels increased gradually with time in K3EDTA tubes, but were stable in BCT tubes. K3EDTA samples stored at 4°C showed less variation than room temperature storage, but levels were elevated compared with BCT. A second centrifugation at 3000 × g gave similar cfDNA yields compared with higher-speed centrifugation. Next-generation sequencing showed negligible differences in background error or copy number changes between K3EDTA and BCT, or following shipment in BCT. This study provides insights into the effects of sample processing on ctDNA analysis. Circulating tumor DNA (ctDNA) offers new opportunities for noninvasive cancer management. Detecting ctDNA in plasma is challenging because it constitutes only a minor fraction of the total cell-free DNA (cfDNA). Pre-analytical factors affect cfDNA levels contributed from leukocyte lysis, hence the ability to detect low-frequency mutant alleles. This study investigates the effects of the delay in processing, storage temperatures, different blood collection tubes, centrifugation protocols, and sample shipment on cfDNA levels. Peripheral blood (n = 231) from cancer patients (n = 62) were collected into K3EDTA or Cell-free DNA BCT tubes and analyzed by digital PCR, targeted amplicon, or shallow whole-genome sequencing. To assess pre-analytic effects, plasma was processed under different conditions after 0, 6, 24, 48, 96 hours, and 1 week at room temperature or 4°C, or using different centrifugation protocols. Digital PCR showed that cfDNA levels increased gradually with time in K3EDTA tubes, but were stable in BCT tubes. K3EDTA samples stored at 4°C showed less variation than room temperature storage, but levels were elevated compared with BCT. A second centrifugation at 3000 × g gave similar cfDNA yields compared with higher-speed centrifugation. Next-generation sequencing showed negligible differences in background error or copy number changes between K3EDTA and BCT, or following shipment in BCT. This study provides insights into the effects of sample processing on ctDNA analysis. Circulating tumor DNA (ctDNA) in plasma offers new opportunities for noninvasive cancer management. Recent studies have demonstrated its potential for molecular stratification, monitoring tumor response, identifying resistance mutations, and patients at risk of relapse.1Wan J.C.M. Massie C. Garcia-Corbacho J. Mouliere F. Brenton J.D. Caldas C. Pacey S. Baird R. Rosenfeld N. Liquid biopsies come of age: towards implementation of circulating tumour DNA.Nat Rev Cancer. 2017; 17: 223-238Crossref PubMed Scopus (1336) Google Scholar, 2Siravegna G. Marsoni S. Siena S. Bardelli A. Integrating liquid biopsies into the management of cancer.Nat Rev Clin Oncol. 2017; 14: 531-548Crossref PubMed Scopus (1114) Google Scholar Detecting ctDNA in plasma is challenging because it constitutes only a minor fraction of the total cell-free DNA (cfDNA), particularly in early-stage cancers and in the minimal residual disease setting.3Garcia-Murillas I. Schiavon G. Weigelt B. Ng C. Hrebien S. Cutts R.J.