Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC).
Author(s): Sherwood JL, Corcoran C, Brown H, Sharpe AD, Musilova M, Kohlmann A
Publication: PLoS One, 2016, Vol. 11, Page e0150197
PubMed ID: 26918901 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of blood collection tube type, delayed centrifugation, double centrifugation, plasma volume, and DNA extraction method on the DNA yield and detection of KRAS mutations in plasma from patients with non-small cell lung cancer (NSCLC).
Conclusion of Paper
A 72 h centrifugation delay resulted in higher DNA yields from plasma and lower KRAS mutation detection rates, regardless of the collection tube type, but the magnitude of the changes was larger when the specimens were stored in EDTA tubes rather than in Streck tubes. There was no significant difference in DNA yield when specimens in EDTA tubes were centrifuged once versus twice, but a trend toward higher yields after a single centrifugation step was found in specimens stored for 72 h. DNA yields were highest when extraction was with the QIAamp Circulating Nucleic Acid Kit and increased with increasing plasma input volume. Importantly, KRAS mutation detection rates were low regardless of plasma processing method.
Studies
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Study Purpose
This study investigated the effects of blood collection tube type, delayed centrifugation, and single versus double centrifugation on plasma DNA yield and detection of KRAS mutations. Blood from 20 patients with NSCLC was collected into K2EDTA tubes and Streck tubes. Specimens were immediately split and stored at room temperature for 2 or 72 h before plasma separation by two centrifugations at 2000 x g for 10 min (all specimens) or by a single centrifugation (5 EDTA specimens). Plasma was stored in 1 mL aliquots at -80˚C before DNA isolation using the QIAamp Circulating Nucleic Acid Kit and quantification with the RNAse P real-time PCR assay. KRAS mutation status was confirmed using the Therascreen real-time PCR kit in FFPE specimens of 10 patients known to harbor KRAS mutations.
Summary of Findings:
DNA yields from plasma were comparable when blood was stored in either K2EDTA or Streck tubes for 2 h before centrifugation, but when blood was stored for 72 h in EDTA tubes the DNA yield was higher than when stored for 72 h in Streck tubes (P<0.01). The plasma DNA yield was significantly higher when blood was stored for 72 h rather than 2 h in EDTA tubes or Streck tubes (P<0.01, both), although the magnitude of the increase was much smaller when the pre-centrifugation storage was in Streck tubes rather than EDTA tubes (~0.4 versus 5-fold). KRAS DNA yield followed the same trends as for the DNA yield assay, indicating increased contaminating DNA after centrifugation delays. Less than 50% of the KRAS mutations were detected in plasma DNA. Although detection numbers were too low to establish significance, mutations were detected in a higher percentage of specimens collected in Streck tubes than EDTA tubes, both when centrifugation was delayed by 2 h (50% versus 40%) or by 72 h (40% versus 20%). There was no significant difference in DNA yield between specimens centrifuged once or twice when blood in EDTA tubes was centrifuged after 2 h or 72 h, but non-significantly higher DNA levels were found after double centrifugation in the specimens stored for 72 h compared to those after single centrifugation (35.47 ng/µL versus 19.35 ng/µL, P=0.058).
Biospecimens
Preservative Types
- Streck/BCT
- Other Preservative
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution K2EDTA tube
Streck BCT tube
Storage Time at room temperature 2 h
72 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Different number of centrifugation steps compared
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Study Purpose
This study explored the effects of DNA extraction kit and plasma volume on DNA yield and KRAS detection in plasma from 15 patients with NSCLC. Blood was collected in EDTA tubes and plasma was isolated by a single centrifugation at 1300 x g for 10 min. Plasma was stored in 1 mL aliquots at -80˚C. DNA was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit,the JENA PME free-circulating DNA Extraction Kit or the Qiagen DSP Virus/Pathogen Midi Kit on a QIAsymphony. DNA was extracted from two matched 5µm FFPE sections using the QIAamp DNA FFPE Tissue Kit. DNA was quantified using the RNAse P real-time PCR assay and KRAS mutation status was determined in the using the Therascreen real-time PCR kit
Summary of Findings:
DNA yields were higher when DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit rather than the JENA PME free-circulating DNA Extraction Kit (3.03 ng/µL versus 0.83 ng/µL, P<0.001) or the Qiagen DSP Virus/Pathogen Midi Kit on QIAsymphony (3.03 ng/µL versus 0.53 ng/µL, P<0.01). As expected, the DNA yield and KRAS detection increased when DNA was extracted from 2 mL rather than 1 mL plasma (P<0.01 and P<0.001, respectively) or from 3 mL rather than 2 mL plasma (P<0.01 and P<0.001, respectively). KRAS mutations were identified in 7 of the 15 patients using the FFPE specimens but only in one patient using the plasma specimen and the mutation was detected in DNA extracted from as little as 1 mL of plasma.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Aliquot size/volume 1 mL
2 mL
3 mL
Biospecimen Acquisition Biospecimen location Plasma
FFPE NSCLC tumor
Analyte Extraction and Purification Analyte isolation method QIAamp Circulating Nucleic Acid Kit
JENA PME free-circulating DNA Extraction Kit
QIAamp Circulating Nucleic Acid Kit on the QIAsymphony