A Study of Pre-Analytical Variables and Optimization of Extraction Method for Circulating Tumor DNA Measurements by Digital Droplet PCR.
Journal
Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology
ISSN: 1538-7755
Titre abrégé: Cancer Epidemiol Biomarkers Prev
Pays: United States
ID NLM: 9200608
Informations de publication
Date de publication:
05 2019
05 2019
Historique:
received:
05
06
2018
revised:
13
08
2018
accepted:
25
02
2019
pubmed:
3
3
2019
medline:
8
7
2020
entrez:
3
3
2019
Statut:
ppublish
Résumé
Circulating free DNA (cfDNA) is an exciting novel method to diagnose, monitor, and predict resistance and response to cancer therapies, with the potential to radically alter the management of cancer patients. To fulfill its potential, greater knowledge about preanalytical variables is required to optimize and standardize the collection process, and maximize the yield and utility of the small quantities of cfDNA extracted. To this end, we have compared the cfDNA extraction efficiency of three different protocols, including a protocol developed in house (Jewish General Hospital). We evaluated the impact on cfDNA levels of preanalytical variables including speed and timing of the second centrifugation and the use of k-EDTA and CTAD blood collection tubes. Finally, we analyzed the impact on fractional abundance of targeted pre-amplification and whole genome amplification on tumor and circulating tumor DNA (ctDNA) from patients with breast cancer. Making use of a novel protocol for cfDNA extraction we increased cfDNA quantities, up to double that of commercial kits. We found that a second centrifugation at 3,000 g on frozen plasma is as efficient as a high-speed (16,000 g) centrifugation on fresh plasma and does not affect cfDNA levels. These results allow for the implementation of protocols more suitable to the clinical setting. Finally, we found that, unlike targeted gene amplification, whole genome amplification resulted in altered fractional abundance of selected ctDNA variants. Our study of the preanalytical variables affecting cfDNA recovery and testing will significantly enhance the quality and application of ctDNA testing in clinical oncology.
Sections du résumé
BACKGROUND
Circulating free DNA (cfDNA) is an exciting novel method to diagnose, monitor, and predict resistance and response to cancer therapies, with the potential to radically alter the management of cancer patients. To fulfill its potential, greater knowledge about preanalytical variables is required to optimize and standardize the collection process, and maximize the yield and utility of the small quantities of cfDNA extracted.
METHODS
To this end, we have compared the cfDNA extraction efficiency of three different protocols, including a protocol developed in house (Jewish General Hospital). We evaluated the impact on cfDNA levels of preanalytical variables including speed and timing of the second centrifugation and the use of k-EDTA and CTAD blood collection tubes. Finally, we analyzed the impact on fractional abundance of targeted pre-amplification and whole genome amplification on tumor and circulating tumor DNA (ctDNA) from patients with breast cancer.
RESULTS
Making use of a novel protocol for cfDNA extraction we increased cfDNA quantities, up to double that of commercial kits. We found that a second centrifugation at 3,000 g on frozen plasma is as efficient as a high-speed (16,000 g) centrifugation on fresh plasma and does not affect cfDNA levels.
CONCLUSIONS
These results allow for the implementation of protocols more suitable to the clinical setting. Finally, we found that, unlike targeted gene amplification, whole genome amplification resulted in altered fractional abundance of selected ctDNA variants.
IMPACT
Our study of the preanalytical variables affecting cfDNA recovery and testing will significantly enhance the quality and application of ctDNA testing in clinical oncology.
Identifiants
pubmed: 30824523
pii: 1055-9965.EPI-18-0586
doi: 10.1158/1055-9965.EPI-18-0586
doi:
Substances chimiques
Biomarkers, Tumor
0
Circulating Tumor DNA
0
Reagent Kits, Diagnostic
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
909-916Informations de copyright
©2019 American Association for Cancer Research.