Molecular Diagnosis & Therapy

, Volume 21, Issue 2, pp 209–216 | Cite as

Comparison of Five Different Assays for the Detection of BRAF Mutations in Formalin-Fixed Paraffin Embedded Tissues of Patients with Metastatic Melanoma

  • Claire Franczak
  • Julia Salleron
  • Cindy Dubois
  • Pierre Filhine-Trésarrieu
  • Agnès Leroux
  • Jean-Louis Merlin
  • Alexandre Harlé
Original Research Article

Abstract

Background

Metastatic or unresectable melanoma is a serious and deadly disease. Anti-BRAF and immunotherapy improved overall survival in patients with metastatic disease. Thus, BRAF genotyping is important to choose the right therapy.

Methods

In our study, we assessed and compared BRAF mutations in 59 formalin-fixed and paraffin-embedded tumor samples of patients with metastatic melanoma with next-generation sequencing (NGS), Cobas® 4800 BRAF V600 mutation test CE-IVD commercial kit, high-resolution melting PCR (HRM), multiplex real-time allele specific amplification (multiplexed RT-ASA) and immunohistochemistry (IHC).

Results

Thirty-one samples were found bearing a BRAF mutation with NGS (52.5%), 28 with Cobas® test (47.5%), 28 with HRM (47.5%), 29 with multiplexed RT-ASA (49.2%) and 27 with IHC (45.8%). Based on NGS data, 26 (81.2%) were c.1799 T>A (p.Val600Glu), 3 (9.4%) were c. 1798-1799 GT>AA (p.Val600Lys), 1 was c.1789_1790 CT>TC (p.Leu597Ser) and 2 were complex mutations. Sensitivity was 90.3% for Cobas® test, 93.1% for multiplexed RT-ASA and 87.1% for IHC and HRM. Specificity was 100% for Cobas® test, IHC and multiplexed RT-ASA and 96.4% for HRM. The reference assay was NGS. Rare mutations were detected with NGS and HRM: c.1789_1790 CT>TC (p.Leu597Ser) mutation and the complex mutation c.1796 A>T; c.1797_1798 insACT (p.Thr599Thr; p.Thr599_Val600insThr). Our data suggest that multiplexed RT-ASA is the most sensitive assay but specific primers for each mutation are needed. HRM can detect all exon 15 mutations but has a lower sensitivity. Because of its specificity for Val600Glu mutation, IHC may be considered only as a screening tool and testing should be completed by a method able to detect other V600 mutations. BRAF Cobas® assay is Val600Glu-specific and has poor sensitivity for the other V600 mutations; thus, it looks important to use multiplex assays able to detect all V600 mutations because a false-negative result will deprive the patient of an important treatment option.

Notes

Compliance with Ethical Standards

Conflict of interest

The authors (CF, JS, CD, P F-T, AL, J-L M & AH) declare no potential conflict of interest.

Funding

No funding has been received for this study.

Ethical approval and informed consent

The authors assure that accepted principles of ethical and professional conduct have been followed for this study. All patients gave their consent for BRAF genotyping and study has been approved by Institut de Cancérologie de Lorraine scientific board.

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Copyright information

© Springer International Publishing Switzerland 2017

Authors and Affiliations

  • Claire Franczak
    • 3
  • Julia Salleron
    • 1
    • 4
  • Cindy Dubois
    • 3
  • Pierre Filhine-Trésarrieu
    • 3
  • Agnès Leroux
    • 1
    • 2
    • 3
  • Jean-Louis Merlin
    • 1
    • 2
    • 3
  • Alexandre Harlé
    • 1
    • 2
    • 3
  1. 1.Université de LorraineNancyFrance
  2. 2.CNRS UMR 7039 CRANNancyFrance
  3. 3.Institut de Cancérologie de Lorraine, Service de BiopathologieVandoeuvre-lès-Nancy CedexFrance
  4. 4.Institut de Cancérologie de Lorraine, Cellule data management et biostatistiqueVandoeuvre-lès-NancyFrance

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