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A Study of Pre-Analytical Variables and Optimization of Extraction Method for Circulating Tumor DNA Measurements by Digital Droplet PCR.

Author(s): Cavallone L, Aldamry M, Lafleur J, Lan C, Gonzalez Ginestet P, Alirezaie N, Ferrario C, Aguilar-Mahecha A, Basik M

Publication: Cancer Epidemiol Biomarkers Prev, 2019, Vol. , Page

PubMed ID: 30824523 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the efficiency of cfDNA extraction using the QIAamp circulating nucleic acid (CNA) kit, the QIAamp DSP virus kit, and an in-house hybrid protocol. Plasma was obtained from a healthy patient by centrifugation of K₂EDTA blood at 1500 x g followed by immediate centrifugation at 16000 x g. Plasma was spiked with known amounts of reference DNA from cell lines or tumors from breast cancer patients participating in a clinical trial. cfDNA was extracted from 1 mL of plasma using the QIAamp CNA and DSP kits following the manufacturer’s instructions or using an in-house hybrid protocol. The in-house hybrid protocol included a 15 min Proteinase K digestion at 56˚C using the buffer from the DSP kit with the addition of carrier RNA from the CNA kit, addition of ACB buffer, vortexing, incubation on ice, followed by purification using the DSP kit MiniElute columns, and elution with AVE buffer. DNA was stored at -20˚C until DNA quantification by droplet digital (dd)PCR amplification of CDH5, MAP1LC3B, PARK2, TP53, and ROBO2.

   Summary of Findings:

   The average recovery of the spiked-in DNA was significantly higher using the hybrid method than the QIAamp CNA (89% versus 46.9%, P<0.0001) or DSP (89% versus 75.4%, P<0.01) kits. When each of the five variants were examined individually, the efficiency varied between the genes but was always highest with the hybrid protocol and lowest with the CNA protocol.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Digital PCR Specific	Targeted nucleic acid	CDH5 MAP1LC3B PARK2 TP53 ROBO2
   Analyte Extraction and Purification	Analyte isolation method	QIAamp CNA kit QIAamp DSP virus kit Hybrid protocol

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2. Study Purpose

   This study investigated the effects of tube type, storing plasma at -80˚C for two weeks before the second centrifugation step, and the speed of the second centrifugation on the yield of cfDNA from plasma. To investigate the effects of blood processing protocol, blood was collected from nine healthy volunteers into Vacutainer K₂EDTA tubes which were inverted ten times and then plasma was obtained by centrifugation immediately at 1500 x g for 10 min. The supernatant was aliquoted and immediately centrifuged at 3000 x g or 16000 x g for 10 min or after frozen storage at -80˚C for 2 weeks. To investigate the effects of tube type, blood was collected from 7 healthy volunteers into two EDTA and two BD vacutainer CTAD tubes, inverted ten times, centrifuged at 1500 x g for 10 min followed by 16000 x g for 10 min. DNA was extracted from 1 mL of plasma using an in-house developed QIAamp CNA/DSP hybrid method and quantified by ddPCR amplification of wildtype TP53.

   Summary of Findings:

   Slightly more copies of TP53 were obtained from EDTA plasma than CTAD plasma but the difference was not significant (mean of 905.62 versus 805.83, P=0.43). There was no effect of freezing plasma for 2 weeks between centrifugation steps or of the speed of the second centrifugation on the amount of cfDNA obtained.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Citrate-theophylline-adenosine-dipyridamole EDTA
   Storage	Storage duration	0 h 2 weeks
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Multiple speeds compared
   Biospecimen Acquisition	Type of collection container/solution	CTAD vacutainer EDTA vacutainer

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3. Study Purpose

   This study investigated the effects of preamplification and WGA of plasma and tumor DNA on the fractional abundance of variants. Blood was collected into CTAD tubes from four patients with breast cancer participating in a clinical trial and plasma was obtained by centrifugation at 1500 x g for 10 min within 30 min of collection. Plasma was frozen at -80˚C and aliquots centrifuged at 16000 x g before DNA extraction using an in-house QIAamp CNA/DSAP hybrid protocol. Matched frozen needle core biopsy tumor specimens were collected from the same four patients and DNA was obtained by an unspecified method. DNA was subjected to targeted preamplification of TP53 using the PreAmp Supermix. DNA from tissue and plasma were WGA using the Illustra GenomiPhi V2 DNA Amplification Kit. TP53 variant allele frequency was determined by ddPCR.

   Summary of Findings:

   The VAFs were very strongly correlated between cfDNA specimens that were preamplified and those that were not preamplified (R2=0.9937, P=0.0032). Similarly, the VAFs were strongly correlated between tumor DNA specimens that were preamplified and those that were not preamplified (R2=0.9972, P≤0.0001). While the VAF was only modestly correlated between WGA and matched non-amplified cfDNA (R2=0.4337, P=0.3415), the VAF was very strongly correlated between WGA tumor DNA and matched non-amplified tumor DNA.

   Biospecimens

   • Tissue - Breast
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Whole genome amplification
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Digital PCR Specific	Template modification	Preamplified Not preamplified WGA Not amplified
   Biospecimen Acquisition	Biospecimen location	Plasma Tumor

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