Heparinase enables reliable quantification of circulating tumor DNA from heparinized plasma samples by droplet digital PCR.
Author(s): Sefrioui D, Beaussire L, Clatot F, Delacour J, Perdrix A, Frebourg T, Michel P, Di Fiore F, Sarafan-Vasseur N
Publication: Clin Chim Acta, 2017, Vol. 472, Page 75-79
PubMed ID: 28729136 PubMed Review Paper? No
Purpose of Paper
This paper investigated if heparinase treatment would allow for amplification of cell-free DNA from heparinized blood without affecting the mutant allele frequency (MAF). The effects were further investigated by comparing MAF in matched heparin and EDTA blood.
Conclusion of Paper
Inclusion of heparinase in the pre-amplification step resulted in fewer cases with less than 200 copies/µL of ESR1 or KRAS. Importantly, the MAFs were strongly correlated in heparinase-treated and untreated specimens and MAFs were comparable in specimens with or without PCR inhibition. Finally, while all eight cases with matched EDTA specimens showed inhibition in the heparinized specimen, the KRAS MAFs were similar for all eight and none changed between positive and negative.
Studies
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Study Purpose
This study investigated if heparinase treatment would allow for amplification of cell-free DNA from heparinized blood without affecting the MAF and compared MAF in matched heparin and EDTA blood in a subset of specimens. Whole blood from 144 patients with hormone receptor-positive metastatic breast cancer (HR+MBC) and 50 patients with pancreatic adenocarcinoma (PA) was collected in heparinized tubes. A matched blood specimen from eight of the PA patients was collected into an EDTA tube. Within 2 h of collection, plasma was obtained from heparinized blood by single centrifugation at 2000 x g for 10 min at 4˚C and from EDTA blood by centrifugation at 1000 x g for 10 min followed by 16,000 x g for 10 min. Plasma was stored at -80˚C. DNA was extracted from 0.4-2.0 mL plasma using the QIAamp® Circulating Nucleic Acid Kit. Cell-free DNA was quantified by Quant-iT dsDNA assay. Estrogen receptor alpha (ESR1) and KRAS were preamplified with or without Heparinase I. Total copies of DNA and genotyping were then determined using droplet digital PCR.
Summary of Findings:
When heparinase was not included in the pre-amplification step, quantification showed fewer than 200 copies/µL in 63% of HR+MBC cases and 52% of PA cases. However, inclusion of heparinase in the pre-amplification resulted in a significant increase in the number of ESR1 copies/µL in HR+MBC (P<0.0001) and KRAS copies in PA cases (P<0.0001) such that more than 200 copies per µL were found in 22 additional HR+MBC cases and 13 additional PA cases. The MAFs were strongly correlated in heparinase-treated and untreated HR+MBC (R2= 0.886, P<0.0001) and PA (R2= 0.859, P<0.0001) cases when there was no PCR inhibition. Further, heparinase treatment did not create any false positives nor was there any difference in MAF between specimens showing inhibition or no inhibition in HR+MBC (7.6% versus 8.42%, P=0.670) or PA (5.97% versus 4.99%, P=0.801) cases. Finally, while all eight cases with matched EDTA specimens showed inhibition in the heparinized specimen, the KRAS MAFs were similar for all eight and none changed between positive and negative.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Hormone receptor-positive metastatic breast cancer
Pancreatic adenocarcinoma
Biospecimen Acquisition Anticoagulant EDTA
Heparin
Digital PCR Specific Template modification Heparinase treated
Heparinase untreated