Dataset Information

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.

SUBMITTER: Risberg B

PROVIDER: S-EPMC6197164 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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

Risberg Bente B Tsui Dana W Y DWY Biggs Heather H Ruiz-Valdepenas Martin de Almagro Andrea A Dawson Sarah-Jane SJ Hodgkin Charlotte C Jones Linda L Parkinson Christine C Piskorz Anna A Marass Francesco F Chandrananda Dineika D Moore Elizabeth E Morris James J Plagnol Vincent V Rosenfeld Nitzan N Caldas Carlos C Brenton James D JD Gale Davina D

The Journal of molecular diagnostics : JMD 20180828 6

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 sam ...[more]

PMID: 30165204

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Project description:BackgroundTo evaluate the clinical research related to the level and integrity of circulating free DNA (cfDNA) in the plasma of patients with multiple myeloma (MM).MethodsThe plasma samples of 56 patients with newly diagnosed MM and 60 healthy volunteers were collected. ALU247 fragment and ALU115 fragment were used as target genes, and quantitative polymerase chain reaction (qPCR) was used to assess the plasma of the patient and healthy control groups. The cfDNA level in MM was analyzed, and the ALU247/ALU115 ratio was used to calculate the integrity of cfDNA. The correlation between the cfDNA level and integrity and the clinical characteristics of patients with primary MM was analyzed, and their value in efficacy monitoring and prognostic evaluation was evaluated.ResultsThe plasma concentrations of ALU247 and ALU115 and the integrity of cfDNA in patients with primary MM were significantly higher than those in the healthy controls (P<0.05). The ALU247 fragment concentration was markedly correlated with the Durie-Salmon (D-S), International Staging System (ISS), and Revised-International Staging System (R-ISS) stages (P<0.05). After three courses of induction chemotherapy, the levels of ALU247, ALU115, and cfDNA integrity in both groups were lower than those before chemotherapy (P<0.05). Patients with curative effects of CR, sCR, and VGPR were classified into the ≥ very good partial response (VGPR) group (n=38), while those with curative effects of PR and SD were allocated into the <VGPR group (n=18). In addition, after chemotherapy, the levels of ALU247, ALU115, and cfDNA integrity of patients in the ≥ VGPR group were significantly lower than those in the < VGPR group (P<0.05). The follow-up results showed that the progression-free survival (PFS) of MM patients with low ALU247 expression was considerably longer than that of MM patients with high ALU247 expression (33.59±1.15 vs. 27.31±2.16, P<0.05).ConclusionsCfDNA levels were significantly elevated in MM patients, and the ALU247 fragment concentration was remarkably correlated with multiple clinical features and had important clinical value for efficacy monitoring and prognostic assessment.

Dataset Information

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

Publications

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

Similar Datasets

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