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Evaluation of a liquid biopsy protocol using ultra-deep massive parallel sequencing for detecting and quantifying circulation tumor DNA in colorectal cancer patients.

Author(s): Nguyen HT, Tran DH, Ngo QD, Pham HT, Tran TT, Tran VU, Pham TN, Le TK, Le NT, Nguyen NM, Vo BT, Nguyen LT, Nguyen TV, Bui QTN, Nguyen HN, Le LGH, Do DM, Do TT, Truong Dinh K, Phan MD, Tran LS, Giang H, Nguyen HN

Publication: Cancer Invest, 2020, Vol. , Page 1-21

PubMed ID: 31939681 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the mutational profiles of matched plasma FFPE biopsy specimens and compare mutation detection and VAF results in plasma specimens analyzed by MPS and ddPCR. Blood was collected from 56 patients with colorectal carcinoma into K₂EDTA tubes and stored for up to 8 h (95% <4 h) before plasma isolation by centrifugation at 2000 x g for 10 min followed by 16,000 x g for 10 min. Plasma was stored at -80°C until DNA extraction using the MagMAX Cell-Free DNA Isolation Kit. Matched tissue biopsies from 50 of the same patients were formalin-fixed and paraffin-embedded. Areas with >50% tumor content identified by hematoxylin and eosin staining were microdissected for DNA extraction using the QIAamp DNA FFPE Tissue Kit. Extracted DNA was quantified by the QuantiFluor dsDNA System. Sequencing libraries were prepared from cfDNA using Accel-NGS 2S Plus DNA Library Kit and xGen Predesigned Gene Capture Pools for all the exons of KRAS, NRAS, and BRAF and from the FFPE DNA using the NEBNext Ultra II FS DNA Library Prep Kit and xGenLockdown probes for KRAS, NRAS, and BRAF. Libraries were sequenced using a NextSeq 500/550 High Output Kit v2 on an Illumina NextSeq 550 System at 10,000 X coverage for cfDNA and 1,000 X coverage for cfDNA. ddPCR KRAS G12/G13 Screening Kit and the ddPCR BRAF V600 Screening Kit were used to detect seven mutation subtypes in KRAS and three mutation subtypes in BRAF identified in 18 specimens by MPS.

   Summary of Findings:

   Matching mutation profiles in cfDNA and FFPE specimens were found for 13 patients (11 KRAS, 2BRAF) using MPS, but four patients had a KRAS mutation that were only identified in the FFPE specimens. No mutations were identified by MPS in cfDNA and FFPE specimens from 33 patients. No NRAS mutations were found by MPS in any of the patients. The overall concordance rate for mutation detection was 92% with a sensitivity and specificity of cfDNA for detecting biopsy mutations of 76.5% and 100%, respectively. Of the 18 plasma specimens with mutations detected in KRAS or BRAF by MPS (VAF 0.3-30%), only 15 had mutations detected by ddPCR. The VAF of all three specimens negative by ddPCR was ≤ 1% by MPS, indicating that the lack of detection could be sampling differences but other mutations with lower VAF were detected successfully by ddPCR. For those 15 with mutations detected in cfDNA using both MPS and ddPCR, the VAF was very strongly correlated between the two methods (R²=0.97, P<0.001), with Bland-Altman analysis showing a high level of agreement.

   Biospecimens

   • Tissue - Colorectal
   • Bodily Fluid - Plasma

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Plasma Tumor
   Digital PCR Specific	Targeted nucleic acid	BRAF (V600E, V600K, and V600R) KRAS (G12A, G12C, G12D, G12R, G12S, G12V and G13D)
   Next generation sequencing Specific	Targeted nucleic acid	KRAS NRAS BRAF
   Next generation sequencing Specific	Technology platform	ddPCR

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