Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comparison of ddPCR and sdPCR systems for the analysis of liquid biopsy samples of patients affected by lung and colorectal cancer.

Author(s): Crucitta S, Ruglioni M, Novi C, Manganiello M, Arici R, Petrini I, Pardini E, Cucchiara F, Marmorino F, Cremolini C, Fogli S, Danesi R, Del Re M

Publication: Clin Chim Acta, 2023, Vol. , Page 117239

PubMed ID: 36736684 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare mutation detection using two different digital PCR technologies (dPCR and ddPCR) in cfDNA isolated from plasma collected from patients with colorectal and lung cancer. Blood was collected from 22 patients with colorectal cancer (10 patients had a RAS mutation detected in a biopsy) and 20 patients with non-small cell lung cancer (17 patients were diagnosed with an EGFR activating mutation and 3 patients were diagnosed with a KRAS mutation in a biopsy) and plasma was separated by centrifugation at 1900 × g for 10 min at 4 °C within 2 h. Plasma was stored at -80°C for an unspecified time. After thaw, plasma was centrifugated at 1900 × g for 15 min and cfDNA was isolated with the QIAamp Circulating Nucleic Acid Kit. EGFR and KRAS mutations were detected using the ddPCR KRAS G12X/G13X, ddPCR NRAS G12X/G13X, ddPCR NRAS Q61X and ddPCR EGFR Exon 19 deletions screening kits and individual ddPCR assays for BRAF p.V600E, EGFR p.L858R, EGFR p.T790M and EGFR p.C797S on a Bio-Rad QX-200 (ddPCR) and a QIAcuity dPCR.

   Summary of Findings:

   While ddPCR detected 10 of the 17 EGFR mutations evaluated, all 17 were detected by dPCR with the seven discordant cases detected at ≤200 copies/mL by dPCR.  Copies per/mL of the EGFR mutation were strongly correlated between the two detection platforms (R=0.990).  Overall, there was moderate agreement in EGFR mutation status between the two detection platforms (κ=0.54), but while perfect agreement was observed for EGFR exon 19 deletions (κ=1), agreement was only moderate for the EGFR p.T790M and p.C797S mutations (κ = 0.39). RAS mutations were detected in plasma from all 3 NSCLC patients regardless of the detection method.  In plasma from patients with colorectal cancer, RAS mutations were detected in 16 of 22 specimens by ddPCR and 19 of 22 specimens by dPCR, with two of the three cases missed by ddPCR having ≤200 copies/mL by dPCR and the third having >200 copies/mL by ddPCR. The number of copies of RAS mutations per mL was modestly correlated between detection platforms (R=0.694).  The overall agreement for RAS mutations status between detection platforms was low (κ=0.34), with low agreement (κ=0.32) observed for RAS p.G12X/G13X and p.Q61X mutations. In plasma of colorectal cancer patients, the BRAF p.V600E mutation was detected in one specimen by ddPCR and three specimens by dPCR, resulting in moderate agreement between detection platforms (κ=0.5).

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Digital PCR Specific	Targeted nucleic acid	ddPCR KRAS G12X/G13X screening kit ddPCR NRAS G12X/G13X screening kit ddPCR NRAS Q61X screening kit ddPCR EGFR Exon 19 deletions screening kit BRAF p.V600E EGFR p.L858R EGFR p.T790M EGFR p.C797S
   Digital PCR Specific	Technology platform	ddPCR Beam, Emulsion, Amplification, Magnetics (dPCR)

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