Measuring KRAS Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing.
Author(s): Demuth C, Spindler KG, Johansen JS, Pallisgaard N, Nielsen D, Hogdall E, Vittrup B, Sorensen BS
Publication: Transl Oncol, 2018, Vol. 11, Page 1220-1224
PubMed ID: 30086420 PubMed Review Paper? No
Purpose of Paper
This paper compared using next generation sequencing (NGS) versus droplet digital PCR (ddPCR) for the detection of KRAS mutations in circulating tumor DNA (ctDNA). The effects of DNA dilution and using a smaller plasma volume were also investigated.
Conclusion of Paper
Twenty-four of the 28 plasma specimens allowed for sequencing and 19 of these had concordant genotype by both methods to that previously found in the matched tissue specimen. One specimen was found by NGS to contain a different mutation than that which was identified in the tissue specimen (pGlu12Ala instead of pGly12Ser and pGly12Arg) and this was confirmed by ddPCR. NGS also found a c.38_39delGCinsAT instead of c38G>A, but ddPCR was not performed. The remaining discrepancies were due to specimens with low allele frequencies. The allele frequencies determined by ddPCR and NGS were very strongly correlated (R2=0.91). Dilution did not affect genotyping for 24 of 26 specimens, with the two discordant cases losing detection of a low allele frequency mutation. When a lower volume of plasma was used for extraction, there was a slightly lower amount of DNA than expected.
Studies
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Study Purpose
This study compared KRAS cfDNA genotyping data obtained by NGS and ddPCR with previously determined KRAS status in metastatic colorectal tumor specimens and investigated the effects of DNA dilution on genotype determination. Plasma from 28 patients known to have a KRAS mutation in codon 12 or 13 based on the real-time PCR based Therascreen analysis were obtained from a Danish clinical trial. DNA was extracted from all specimens using the QIAamp Circulating Nucleic acid kit and stored at -80˚C, along with a 10-fold dilution. Total DNA was quantified by ddPCR quantification of beta-2 microglobulin (B2M). Lymphocyte contamination was quantified by a ddPCR based immunoglobulin gene assay. KRAS mutations were quantified using the Bio-Rad KRAS PrimePCR ddPCR mutation assays. NGS libraries were prepared using the Oncomine Solid Tumor DNA kit and sequenced using an Ion Personal Genome Machine. DNA concentration was based on copy numbers of beta-2 microglobulin (B2M).
Summary of Findings:
Twenty-four of the 28 specimens allowed for sequencing and 19 of these had concordant genotype by both methods to that previously found in the matched tissue specimen. When the sequence was manually visualized, three of the specimens did have the correct genotype, but the allele frequency was low. One of the specimens was found by NGS to contain a different mutation than that which was identified in the tissue specimen (pGlu12Ala instead of pGly12Ser and pGly12Arg) and this was confirmed by ddPCR. The remaining discrepancy was a c.38_39delGCinsAT by NGS instead of c38G>A. ddPCR genotype was concordant with the tissue specimen for 24 of the 27 specimens with one discrepancy due to detection of the same mutation found by NGS rather than that found in the tissue (pGlu12Ala instead of pGly12Ser and pGly12Arg). Two additional specimens failed to identify the mutation and each of these initially were not identified by NGS due to low allele frequency. The allele frequencies determined by ddPCR and NGS were very strongly correlated (R2=0.91). For 24 of the 26 specimens that were diluted, the genotype was concordant between the DNA and the diluted DNA and for the remaining two specimens, the mutation had a low allele frequency.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Next generation sequencing DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Digital PCR Specific Template modification Diluted DNA
Undiluted
Next generation sequencing Specific Technology platform ddPCR
Therascreen PCR kit
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Study Purpose
This study compared cfDNA measurements in matched 200 µL and 2 mL plasma specimens by ddPCR. Plasma from 16 patients with metastatic colorectal cancer and known to have a KRAS mutation in codon 12 or 13 based on the real-time PCR based Therascreen analysis were obtained from a Danish clinical trial. Specimens were divided to produce 2 mL and 0.2 mL aliquots, each of which were used for DNA extraction. DNA was extracted using the QIAamp Circulating Nucleic acid kit and stored at -80˚C. Total DNA was quantified by ddPCR quantification of beta-2 microglobulin (B2M). Lymphocyte contamination was quantified by a ddPCR-based immunoglobulin gene assay. KRAS mutations were quantified using the Bio-Rad KRAS PrimePCR ddPCR mutation assays.
Summary of Findings:
An average of 1.15 copies of B2M were found in the 2 mL specimens relative to the 200 µL specimen. Importantly, six of the sixteen 2 mL specimens tested showed some immunoglobulin positive droplets
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR DNA Digital PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Aliquot size/volume 200µL
2 mL