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Comparison of Fresh Frozen Tissue With Formalin-Fixed Paraffin-Embedded Tissue for Mutation Analysis Using a Multi-Gene Panel in Patients With Colorectal Cancer.

Author(s): Gao XH, Li J, Gong HF, Yu GY, Liu P, Hao LQ, Liu LJ, Bai CG, Zhang W

Publication: Front Oncol, 2020, Vol. 10, Page 310

PubMed ID: 32232001 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared gene mutation detection in snap-frozen and FFPE colorectal cancer tumor tissue using NGS. Specimens were collected from surgically resected primary colorectal tumors from 118 patients (median age=62, 72 males and 46 females). Resected tissue was divided into two parts. One part was frozen in liquid nitrogen <30 min after surgery and stored at −80°C until DNA was extracted using the QIAamp DNA Mini Kit. The other half was fixed in 4% formalin for 24–72 h, embedded in paraffin, and stored at room temperature. Ten consecutive 10 μm sections were obtained from FFPE blocks. A slide was prepared from one section and stained with hematoxylin and eosin for histopathological analysis by two pathologists and only sections with ≥40% neoplastic cells were included in the study. DNA was extracted from the remaining nine sections using the GeneRead DNA FFPE Kit. DNA was quantified with a Qubit 3.0 fluorometer and dsDNA HS Assay Kit. Gene-specific PCR was performed using a commercially available 22-gene panel. Sequencing libraries were generated using a MiSeq system and evaluated using a bioanalyzer. Variants at a locus with coverage of <200 or with a variant frequency <0.05 were excluded and the remaining mutations were assessed using the Catalog of Somatic Mutations in Cancer (COSMIC) database.

   Summary of Findings:

   Overall, one or more variants were identified in 117 patients (99.2%). At least one variant was identified in the FFPE specimen of 112 patients (94.9%) and in the snap-frozen specimen of 105 patients (89.0%). Mutation rates were highly concordant between preservation types with >94% (96/129) of variants identified in both the FFPE and snap-frozen specimen and a Kappa coefficient >0.500 for 64.3% (83/129) of variants. Of the 129 total variants identified, 27 were found in FFPE only and 6 were found only in the snap-frozen specimen. At the gene level, concordance ranged from 73.8 to 100.0%, with a Kappa coefficient >0.500 in 81.3% (13/16) of genes.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Next generation sequencing
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Next generation sequencing Specific	Targeted nucleic acid	AKT1 ALK BRAF CTNNB1 DDR2 EGFR ERBB2 ERBB4 FBXW7 FGFR1 FGFR2 FGFR3 KRAS MAP2K1 MET NOTCH1 NRAS PIK3CA PTEN SMAD4 STK11 TP53
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen Formalin (buffered)

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