Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Characterization of Plasma Cell-Free DNA Integrity Using Droplet-Based Digital PCR: Toward the Development of Circulating Tumor DNA-Dedicated Assays.

Author(s): Poulet G, Garlan F, Garrigou S, Zonta E, Benhaim L, Carrillon MJ, Didelot A, Le Corre D, Mulot C, Nizard P, Ginot F, Boutonnet-Rodat A, Blons H, Bachet JB, Taïeb J, Zaanan A, Geromel V, Pellegrina L, Laurent-Puig P, Wang-Renault SF, Taly V

Publication: Front Oncol, 2021, Vol. 11, Page 639675

PubMed ID: 34094923 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the fragment size of wildtype and KRAS mutant cfDNA in plasma from healthy patients and those with colorectal carcinoma. Fragment size was also compared in plasma from healthy individuals collected in Streck BCT and K₂EDTA tubes and between plasma collected from both healthy individuals and those with CRC in K₂EDTA tubes and extracted with the QIAamp Circulating Nucleic Acids Kit and plasma collected in Streck BCT tubes and extracted with the Maxwell RSC ccfDNA Plasma Kit. Blood was collected from 24 healthy controls and 34 patients with colorectal carcinoma with KRAS G12D, G12V, or G13D mutations in BCT Streck tubes (AGEO RASANC study) and from 25 healthy controls and 12 patients with colorectal carcinoma with KRAS G12V or G13D mutations in K₂EDTA tubes (PLASCOL study). Plasma was obtained by centrifugation at 1600 x g for 20 min followed by 15,000 x g for 15 min and stored at -80°C. DNA was extracted from AGEO RASANC plasma with Maxwell RSC ccfDNA Plasma Kit and from PLACOL plasma with the QIAamp Circulating Nucleic Acids Kit. DNA integrity was evaluated using DNA from both cohorts by ddPCR amplification of 60, 86, 164, and 302 bp fragments of KRAS with wildtype and mutant specific probes. cfDNA fragment size was also evaluated in plasma from 10 healthy patients and 33 metastatic colorectal carcinoma patients in the AGEO RASANC study by BIAbooster capillary electrophoresis. To determine the effects of collection tube type, blood was collected from eight healthy patients into both Streck BCT and K₂EDTA tubes and DNA was extracted from plasma using the QIAamp Circulating Nucleic Acids Kit.

   Summary of Findings:

   The number of copies of cfDNA detected was dependent on amplicon size with a decrease in copy number observed with increasing amplicon size. However, mutant and wildtype copies were not equally affected with the ratios of 302 bp to 60 bp product of 0.09 and 0.05 for the  wildtype and mutant DNA, respectively (P=0.0075), and the ratios of 302 bp to 86 bp products of 0.11 and 0.04 for  wildtype and mutant DNA, respectively (P=0.03). Importantly, the ratio of 164/60 bp and 164/86 bp products were not significantly different for mutant and wildtype DNA (P=0.14 and P=0.9, respectively). There was no difference in the number of copies of cfDNA when plasma was collected in Streck BCT versus K₂EDTA tubes and extracted with the QIAamp Circulating Nucleic Acids Kit. While more copies of 302 bp cfDNA were found when blood was collected in K₂EDTA tubes and extracted with the QIAamp Circulating Nucleic Acids Kit than when collected in Streck BCT tubes and extracted with Maxwell RSC ccfDNA Plasma Kit (P=0.0038), there was no difference in copies of the 60 bp fragment (P=0.045). Further, the ratio of the 302 bp to 60 bp amplicon was higher when blood was collected in K₂EDTA tubes and extracted with the QIAamp Circulating Nucleic Acids Kit than when collected in Streck BCT tubes and extracted with Maxwell RSC ccfDNA Plasma Kit (P=0.0017). The ratio of the 302 bp to 60 bp amplicon was lower in cfDNA isolated from patients with metastatic CRC than healthy patients in the AGEO RASANC study (P=2.5e−08) and the PLACOL study (P=1.2e−05) but differences in the ratio of the 302 bp to 60 bp amplicon between copies of wildtype and mutant KRAS were only found in the PLCAOL study (P=0.0058). BIAbooster System analysis of the specimens from the AGEO RASANC study found more 75-110 bp fragments in cfDNA from CRC patients than healthy controls but no difference in 110-239 bp fragments.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	Streck BCT K2EDTA
   Digital PCR Specific	Length of gene fragment	62 bp 86 bp 164 bp 302 bp
   Digital PCR Specific	Targeted nucleic acid	KRAS p.G12V KRAS pG12D KRAS pG13D
   Analyte Extraction and Purification	Analyte isolation method	RSC Maxwell Kit QIAamp Circulating Nucleic Acid Kit

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