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NGS Analysis of Liquid Biopsy (LB) and Formalin-Fixed Paraffin-Embedded (FFPE) Melanoma Samples Using Oncomine™ Pan-Cancer Cell-Free Assay.

Author(s): Olbryt M, Rajczykowski M, Bal W, Fiszer-Kierzkowska A, Cortez AJ, Mazur M, Suwiński R, Widłak W

Publication: Genes (Basel), 2021, Vol. 12, Page

PubMed ID: 34356096 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare BRAF genotype in FFPE melanoma specimens as determined by real-time PCR and NGS and investigate the concordance of the tissue and plasma genotypes. The correlations among LDH levels, cfDNA levels, and progression-free survival were also investigated. Blood from 22 patients with Stage IV melanoma starting targeted therapy or immunotherapy was collected into K₂EDTA plasma preparation tubes (PPT). Plasma was obtained by centrifugation at 1100 x g for 10 min followed by 16,000 x g for 10 min within 1 h of collection and stored in cryogenic vials at -80°C. cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit without carrier RNA. cfDNA was quantified using the Qubit dsDNA HS Assay Kit and, when necessary, concentrated by precipitation with ethanol and glycogen. DNA was previously isolated from FFPE tissue (details of tissue procurement not provided) using the Maxwell RSC FFPE Plus DNA Kit. BRAF status was evaluated in the cfDNA and FFPE DNA from 14 patients using the NGS-based Oncomine Pan-Cancer Cell-Free Assay. Additionally, BRAF status was evaluated in the FFPE DNA using the real-time PCR-based AmoyDx BRAF V600 Mutation Detection Kit. Details of LDH analysis were not provided.

   Summary of Findings:

   cfDNA yield was modestly positively correlated with LDH (ρ=0.646, P=0.001) and this correlation was stronger when only patients with >220 units/L LDH were considered (ρ=0.755, P=0.001). LDH was modestly negatively correlated with progression-free duration of first-line therapy (ρ=-0.451, P=0.035). cfDNA level was not correlated with progression-free survival (P=0.99). Concordant genotypes were found using NGS and real-time PCR in 10 of 11 (91%) FFPE tumor specimens. The discordant case was V600 by real-time PCR and wildtype by NGS in the tumor DNA but plasma confirmed the presence of V600E with a VAF of 0.3%. Comparison of the plasma cfDNA NGS results with the genotype determined by real-time PCR in FFPE tumor was concordant in 13 of 14 specimens (93%). The VAF increased in 3 of 5 patients between the start of the first and second therapy, with one patient wildtype in both specimens. Further analysis found 11 of 14 patients harboring additional non-BRAF mutations detected in the FFPE tumor or cfDNA specimen. The tumor-plasma concordance in mutational status was 28% with only 5 of 18 mutations detected in both specimens, 11 detected only in tumor, and two detected only in plasma. The authors found a shorter average time to progression for the two patients with CTNNB1 mutations with a VAF >1% than with a VAF<1%.

   Biospecimens

   • Tissue - Skin
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Melanoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   Protein	Clinical chemistry/auto analyzer
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qPCR Specific	Targeted nucleic acid	BRAF genotyping
   Biospecimen Acquisition	Biospecimen location	Plasma FFPE tissue
   Next generation sequencing Specific	Technology platform	Real-time PCR NGS

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