Liquid biopsy in colon cancer: comparison of different circulating DNA extraction systems following absolute quantification of <i>KRAS</i> mutations using Intplex allele-specific PCR.
Author(s): Kloten V, Rüchel N, Brüchle NO, Gasthaus J, Freudenmacher N, Steib F, Mijnes J, Eschenbruch J, Binnebösel M, Knüchel R, Dahl E
Publication: Oncotarget, 2017, Vol. 8, Page 86253-86263
PubMed ID: 29156792 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of using serum rather than plasma, DNA extraction method, and inclusion of carrier RNA on circulating cell-free DNA (cfDNA) yield, fragment size, and allele frequencies. Additionally, the KRAS mutational status of matched serum and formalin-fixed and paraffin-embedded (FFPE) specimens was compared and it was investigated if cfDNA or circulating tumor DNA (ctDNA) from serum could distinguish between healthy patients and those with colorectal carcinoma (CRC). The effects of analytical method on quantification of cfDNA and determination of copy number were also investigated.
Conclusion of Paper
Although serum yielded more cfDNA than plasma, it also resulted in higher quantification of KRAS than expected and lower calculated allele frequencies. Use of magnetic bead-based kits rather than silica membrane-based kits for cfDNA extraction from serum resulted in a significantly higher yield of small cfDNA fragments compared to large fragments and a higher percentage of fragments <600 bp. cfDNA yields using either kit type were unaffected by the use of carrier RNA.
The concordance of KRAS status between tumor and serum specimens was 66%. The specificity of serum for KRAS status in tumors was 70% but the sensitivity was only 50%. cfDNA concentrations were higher in serum from patients with CRC than healthy controls and appeared to rise further at occurrence of a second metastasis. ctDNA yield and allele frequency were not capable of discriminating among CRC stages.
cfDNA amounts and KRAS wildtype copy numbers were very strongly correlated between Intplex PCR and Qubit and digital PCR, respectively, but only 50% of KRAS G12D mutations identified with Intplex were found at similar copy numbers by digital PCR.
Studies
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Study Purpose
The purpose of this study was to investigate the effect of extraction kit type, inclusion of carrier RNA, and using plasma versus serum on the isolation of cell-free DNA and circulating tumor DNA from healthy patients and those with CRC. Blood was obtained by venipuncture from eight healthy individuals and 38 CRC patients in S-Monovette tubes containing clot activator (serum) or K3EDTA (plasma) and centrifuged within 3 h of collection. Serum and plasma were obtained by centrifugation at 2000 x g for 10 min and transferred to 1.5 mL tubes. Specimens were centrifuged again at 16000 x g for 10 min, transferred to new tubes, and frozen at -80˚C. DNA was extracted from plasma and serum using the magnetic bead based Maxwell RSC ccfDNA Plasma kit and the silica-membrane based QIAamp Free Circulating Nucleic Acid kit with or without 1 µg carrier RNA. DNA carrying KRAS G12S or G12D mutation was spiked in at various concentrations to investigate the effects on allelic frequency. DNA was stored at -80˚C until analysis. DNA was analyzed by Qubit, Intplex allele-specific PCR, and droplet digital PCR amplification of KRAS.
Summary of Findings:
No differences in total cfDNA yields from serum were found between silica membrane-based and magnetic bead-based kits by Qubit. While a slightly higher yield of fragments <600 bp was found using the silica-membrane-based rather than magnetic bead-based kits, there was also a higher yield of fragments >600 bp, indicating additional lymphocyte contamination. Consequently, a significantly higher yield of small cfDNA fragments compared to large (>600 bp) was only observed when magnetic bead-based kits were used and use of magnetic beads resulted in a higher percentage of fragments <600 bp. cfDNA yields using either kit type were unaffected by the use of carrier RNA.
As expected, use of serum resulted in 6.7-fold higher cfDNA yields than plasma when extracted using the magnetic bead-based kits and 3.7-fold higher using silica membrane-based kits. While quantified copies of G12D and G12S in serum were slightly higher than expected for both kit types, they were closer to expected values in plasma specimens but only when the magnetic bead-based systems were used for extraction. Quantification of spiked-in ctDNA by Intplex PCR showed lower than expected allele frequencies in serum, likely due to increased cfDNA.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform DNA Digital PCR DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Real-time qPCR Specific Technology platform Digital PCR
Intplex PCR
Qubit
Analyte Extraction and Purification Analyte isolation method Silica-membrane based extraction kit
Magnetic bead based extraction kit
With carrier RNA
Without carrier RNA
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Study Purpose
This study compared the KRAS mutational status of matched serum and FFPE specimens and investigated if cfDNA or ctDNA from serum could distinguish between healthy patients and those with CRC and if they could be used to discriminate among CRC stage. Additionally, the suitability of Intplex PCR was investigated by comparisons to fluorometry for determination of cfDNA yield and to digital PCR for copy numbers. Blood was obtained by venipuncture from eight healthy individuals and 50 CRC patients in S-Monovette tubes containing clot activator (serum) and centrifuged at 2000 x g for 10 min within 3 h of collection. Serum was transferred to a new 1.5 mL tube and centrifuged again at 16000 x g for 10 min, transferred to new tubes, and then frozen at -80˚C. DNA was extracted from serum using the magnetic bead-based Maxwell RSC ccfDNA Plasma kit and the silica membrane-based QIAamp Free Circulating Nucleic Acid kit. DNA was extracted from five 10 µm sections of matched FFPE tumor using the QIAamp DNA mini kit. DNA was stored at -80˚C until analysis. DNA was analyzed by Qubit, Intplex allele-specific PCR, and droplet digital PCR amplification of KRAS.
Summary of Findings:
Based on analysis of the serum specimens, 16 patients were identified to carry KRAS mutations: 12 with G12D, 3 with G12S and 1 with both. Of 40 patients whose tumor was found to be wildtype, 28 also did not have a mutation in the serum. Of the 10 patients with KRAS mutations identified in the tissue specimens, the mutation was not found in the serum of five. Consequently, the specificity of serum for KRAS tumors was 70% but the sensitivity was 50%. Overall concordance between KRAS status in serum and tumor specimens was 66%.
Compared to serum from healthy individuals, cfDNA concentrations were 4.7-fold higher in serum from patients with lymph node negative CRC (P<0.05), 6.7-fold higher in serum from patients with lymph node positive CRC (P<0.01), and 9.1-fold higher in serum from patients with distant metastasis (P<0.001). In three patients with metastasis, cfDNA concentrations were higher at occurrence of a second metastasis. Although ctDNA yield was highest in patients with metastasis, yield and allele frequency were not capable of discriminating among CRC stages.
cfDNA amounts in serum were very strongly correlated between Qubit and the Intplex PCR system (r=0.976, P<0.001). KRAS wildtype copy numbers in tissue and serum specimens were very strongly correlated between Intplex and digital PCR (r=0.989, P<0.001), but only 50% of G12D mutations identified with Intplex were found at similar copy numbers by digital PCR.
Biospecimens
Preservative Types
- Frozen
- Formalin
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Digital PCR DNA Real-time qPCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Healthy
Lymph node negative CRC
Lymph node positive CRC
CRC with distant metastasis
CRC with second metastasis
Real-time qPCR Specific Technology platform Intplex
Digital PCR
Qubit
Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
Frozen
Biospecimen Acquisition Biospecimen location Tumor
Serum