Application of circulating tumor DNA in prospective clinical oncology trials - standardization of preanalytical conditions.
Author(s): van Dessel LF, Beije N, Helmijr JC, Vitale SR, Kraan J, Look MP, de Wit R, Sleijfer S, Jansen MP, Martens JW, Lolkema MP
Publication: Mol Oncol, 2017, Vol. 11, Page 295-304
PubMed ID: 28164427 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of blood collection tube type and delayed centrifugation on cell-free DNA (cfDNA) concentration, DNA fragment size distribution, and variant detection in plasma from patients with metastatic cancer.
Conclusion of Paper
cfDNA concentration and DNA fragment size distribution increased significantly when blood was stored in K2EDTA tubes for 96 h before plasma isolation but there were no effects due to storage in BCT or CellSave tubes. cfDNA concentrations were strongly correlated with an increase in wild-type copy numbers and modestly correlated with an increase in variant copy numbers. Although there was no effect of collection tube type or delayed centrifugation on the detection of variants or variant copy numbers, the variant allele frequency (VAF) decreased significantly when blood was stored in K2EDTA tubes for 96 h before plasma isolation.
Studies
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Study Purpose
This study investigated the effects of blood collection tube type and delayed centrifugation on cfDNA concentration, DNA fragment size distribution, and variant detection in plasma from patients with metastatic cancer. Sixteen patients with metastatic cancer (one with cholangiocarcinoma, one with pancreatic cancer, one with breast cancer, six with melanoma, five with colorectal cancer, and two with non-small cell lung cancer) were selected for study based on the availability of TaqMan digital PCR assays for the genetic variant identified in their primary or metastatic lesion. Blood specimens were collected from each patient into three each of K2EDTA Vacutainer tubes, Streck Cell-Free BCT, and CellSave Preservative tubes. Plasma was isolated by sequential centrifugation (1711 x g for 10 min and 12000 x g for 10 min) from one specimen of each type 1 h, 24h, and 96 after blood collection and stored at -80˚C. Plasma was thawed at 4˚C and cfDNA was isolated using the QIAamp Circulating Nucleic Acid kit, quantified using the Quant-iT dsDNA high-sensitivity assay, and stored at -20˚C. DNA was thawed at room temperature and SNPs were detected using TaqMan digital PCR assays. Fragmentation was investigated using the TaqMan β-actin fragmentation assay which compares amplification of 136 bp and 402 bp regions of β-actin.
Summary of Findings:
Storage of blood in K2EDTA tubes for 72 h resulted in a significant increase in the plasma cfDNA concentration (P<0.001) compared to blood stored for 1 h, but there were no effects due to storage in BCT or CellSave tubes. Similarly, there was an increase in the amount of >420 and >2000 bp DNA present with storage of blood in K2EDTA tubes at room temperature (P<0.0001), indicating the increase in DNA was from lysed leukocytes. An increase in amplification of all DNA >136 bp (P=0.002) was also noted, but as this is the same size as cfDNA the increase in amplification was less pronounced. There was no increase in DNA fragment size distribution when blood was stored in BCT or CellSave tubes. cfDNA concentrations were strongly correlated with an increase in wild-type copy numbers (ρ=0.85, P<0.001) and modestly correlated with an increase in variant copy numbers (ρ=0.42, P<0.001).
The expected somatic variants were detected in the plasma of 11 of the 16 patients (69%) when the blood was collected in EDTA Vacutainer tubes and processed to plasma within 1 h but VAFs were not correlated with those observed in tissue specimens. For one of the two patients with multiple somatic variants, both variants were found in K2EDTA plasma obtained within 1 h, but only two of three variants were found for the other patient. Importantly, all variants detected in K2EDTA plasma processed after 1 h were also observed when blood was stored in BCT or CellSave tubes for 1 h. The VAFs declined significantly when blood was stored in K2EDTA tubes for 96 h instead of 1 h (P=0.003) but most VAFs were not significantly affected by storage of blood in BCT or CellSave tubes. There was no effect of blood storage in the various tube types on the variant copy numbers.
Biospecimens
Preservative Types
- None (Fresh)
- Streck/BCT
Diagnoses:
- Neoplastic - Melanoma
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Digital PCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck BCT
CellSave preservative tube
K2EDTA Vacutainer tube
Biospecimen Preservation Type of fixation/preservation Blood collection tube additive
EDTA
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Time at room temperature 1 h
24 h
96 h
Digital PCR Specific Length of gene fragment 136 bp
420 bp
2000 bp