Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection.

Author(s): Barták BK, Kalmár A, Galamb O, Wichmann B, Nagy ZB, Tulassay Z, Dank M, Igaz P, Molnár B

Publication: Pathol Oncol Res, 2018, Vol. , Page

PubMed ID: 29374860 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared methylation rates of cfDNA extracted from plasma using the High Pure Viral Nucleic Acid Large Volume Kit and the Epi proColon 2.0 kits. The effects of diagnosis on methylation was also investigated.  Blood was collected from 10 healthy, 10 adenoma and 10 CRC patients in K3EDTA tubes. Within 4 h, plasma was obtained by double centrifugation at 1350 x g for 12 min) and then stored at -20˚C until DNA extraction. cfDNA was extracted from the EDTA plasma of using the High Pure Viral Nucleic Acid Large Volume Kit and the Epi proColon 2.0 kits. Bisulfite conversion was performed using the EZ DNA Methylation Direct Kit and converted DNA was used immediately or stored at -80˚C. Methylation of SFRP1, SFRP2, SDC2 and PRIMA1 promoters was quantified using the MethylLight PCR and methylated was defined as greater than 0.1% methylation.

   Summary of Findings:

   As expected, more than 0.1% methylation occurred at a lower rate in healthy controls (0-30%) than in patients with adenoma (10-100%) or CRC (30-90%) and while consistently higher in adenoma or CRC than in healthy controls values were dependent on extraction method and promoter. Extraction and bisulfite conversion using the Epi proColon kit resulted in lower average levels of methylation than the High Pure Viral Nucleic Acid Large Volume kit for SFRP1 (2.4% versus 5.5%), SFRP2 (0.4% versus 1.4%, SDC2 (0.8% versus 4.5%) and PRIMA1 (14.6% versus 22.8%).  Methylation of SFRP1 was comparable between kits in healthy (30%, both) and CRC patients (90%, both), but was higher when extraction was with HighPure in adenoma patients (100% versus 70%). Methylation of SFRP2 and SDC2 were higher when extraction was with HighPure rather than the Epi proColon kit in healthy (10% versus 0% and 20% versus 10%, respectively), adenoma (50% versus 10% and 90% versus 30%, respectively), and CRDC patients (90% versus 30% and 80% versus 50%, respectively).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   DNA	Bisulfite conversion assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	High Pure Viral Nucleic Acid Large Volume Kit Epi proColon 2.0 Kit
   Bisulfite conversion assay Specific	Targeted nucleic acid	SFRP1 SFRP2 SDC2 PRIMA1
   Preaquisition	Diagnosis/ patient condition	Normal Adenoma CRC

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2. Study Purpose

   This study compared methylation rates of four promoters after extraction of cfDNA from plasma using the High Pure Viral Nucleic Acid Large Volume Kit and InviGenius. The effects of diagnosis on methylation was also investigated.  Blood was collected from 27 healthy, 25 adenoma and 17 CRC patients in K3EDTA tubes. Within 4 h, plasma was obtained by double centrifugation at 1350 x g for 12 min) and then stored at -20˚C until DNA extraction. cfDNA was extracted from the EDTA plasma using the of High Pure Viral Nucleic Acid Large Volume Kit and InviGenius. cfDNA was quantified using the Qubit dsDNA high Sensitivity Assay kit. Bisulfite conversion was performed using the EZ DNA Methylation Direct Kit and converted DNA was used immediately or stored at -80˚C. Methylation of SFRP1, SFRP2, SDC2 and PRIMA1 promoters was quantified using the MethylLight PCR and methylated was defined as greater than 0.1% methylation.

   Summary of Findings:

   As expected, more than 0.1% methylation generally occurred at a lower rate in healthy controls (4-78%) than in patients with adenoma (0-52%) or CRC (6-940%), but values were dependent on extraction method and promoter.  cfDNA yield was higher using the InviGenius kit than the HighPure Manual extraction and increased with progression from adenoma to CRC Dukes A/B and then to CRC Dukes C/D. Methylation of SFRP1 was higher when DNA was extracted with InviGenius than High Pure (78% versus 48% for healthy, 88% versus 72% for adenoma patients, 82% versus 76% for CRC patients), but methylation of SFRP2 and SDC2 were higher when extraction was with HighPure (30-94% versus 0-6% and 11-75% versus 4-35%, respectively).  PRIMA1 methylation was higher in healthy patients and those with adenoma when extraction was with InviGenius rather than HighPure (15% versus 4% and 24% versus 16%, respectively), but in CRC patients more methylation was observed when extraction was with HighPure (53% versus 47%).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   DNA	Bisulfite conversion assay
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Normal Adenoma CRC
   Bisulfite conversion assay Specific	Targeted nucleic acid	SFRP1 SFRP2 SDC2 PRIMA1
   Analyte Extraction and Purification	Analyte isolation method	High Pure Viral Nucleic Acid Large Volume Kit InviGenius kit

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3. Study Purpose

   This study compared methylation rates of four promoters after extraction of cfDNA from plasma using the Zymo Quick cfDNA S&P Kit and the InviGenius Plus kit. The effects of diagnosis on methylation was also investigated.  Blood was collected from 10 healthy, 10 adenoma and 10 CRC patients in K3EDTA tubes. Within 4 h, plasma was obtained by double centrifugation at 1350 x g for 12 min) and then stored at -20˚C until DNA extraction. cfDNA was extracted using the Zymo Quick cfDNA S&P Kit and the InviGenius Plus kit. cfDNA was quantified using the Qubit dsDNA high Sensitivity Assay kit. Bisulfite conversion was performed using the EZ DNA Methylation Direct Kit and converted DNA was used immediately or stored at -80˚C. Methylation of SFRP1, SFRP2, SDC2 and PRIMA1 promoters was quantified using the MethylLight PCR and methylated was defined as greater than 0.1% methylation.

   Summary of Findings:

   cfDNA yields were higher using the InviGenius Plus kit rather than the Quick cfDNA Serum and Plasma kit, and in adenoma than healthy controls, but were surprisingly low using either kit in plasma from CRC patients. In healthy patients, methylation of all four promoters was higher using the InviGenius Plus kit rather than the Quick cfDNA kit, but for patients with adenoma methylation rates for all four promoters were highest when extraction was with the Quick cfDNA kit. In patients with CRC, the average methylation of SFRP1 and SFRP2 were higher when extraction was with the InviGenius Plus kit, but methylation of SDC2 and PRIMA were higher when extracted using the Quick cfDNA kit.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   DNA	Bisulfite conversion assay
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Adenoma CRC Healthy
   Bisulfite conversion assay Specific	Targeted nucleic acid	SFRP1 SFRP2 SDC2 PRIMA1
   Analyte Extraction and Purification	Analyte isolation method	InviGenius Plus kit Zymo Quick cfDNA S&P kit

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4. Study Purpose

   This study compared cfDNA yields and methylation in specimens collected in K3EDTA tubes and processed within 4 h with those collected in Streck BCT tubes and processed 48 h later. The effect of diagnosis on methylation was also investigated. To investigate the effect of tube type blood from 5 patients with colorectal cancer and 5 patients with adenoma were collected in Streck BCT tubes and EDTA tubes. Plasma was obtained by double centrifugation at 1350 x g for 12 min within 4h (EDTA specimens) or 48 h (Streck BCT specimens) and then stored at -20˚C until DNA extraction using the HighPure Viral Nucleic Acid LV kit. cfDNA was quantified using the Qubit dsDNA high Sensitivity Assay kit. Bisulfite conversion was performed using the EZ DNA Methylation Direct Kit and converted DNA was used immediately or stored at -80˚C. Methylation of SFRP1, SFRP2, SDC2 and PRIMA1 promoters was quantified using the MethylLight PCR and methylated was defined as greater than 0.1% methylation.

   Summary of Findings:

   cfDNA yields were comparable from plasma collected in K3EDTA tubes and processed within 4 h and that collected in Streck BCT and processed 48 h later. In adenoma patients, methylation levels were comparable between tube types. In CRC patients, plasma collected in K3EDTA tubes had higher methylation levels than that collected in Streck BCT tubes but the difference was only significant for the SFRP1 and SDC2 promoters (P<0.05). 

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Streck/BCT
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Bisulfite conversion assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Adenoma CRC
   Bisulfite conversion assay Specific	Targeted nucleic acid	SFRP1 SFRP2 SDC2 PRIMA1
   Biospecimen Acquisition	Type of collection container/solution	K3EDTA Vacuette Streck cell-free DNA BCT
   Storage	Storage duration	4 h 48 h

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