BRAF+ melanoma
What makes it different, makes it vulnerable

BRAF mutation testing: published evaluations

The different methods for assessing BRAF mutations can be compared based on:

  • Performance characteristics
    • Specificity and sensitivity (analytical and clinical)
      • Specificity (true negative rate) – ability of the test to correctly identify patients without the condition1
      • Sensitivity (true positive rate) – ability of the test to correctly identify patients with the condition1
    • Reproducibility – between-laboratory precision
    • Repeatability – within-laboratory precision
  • Target: what exactly they detect (number of mutations, which mutations)
  • Regulatory status (i.e. US [FDA], European [CE marking], or country-specific [Laboratory Developed Tests (LDT)])
  • Instrumentation required to run the test
  • Throughput (how many samples can be tested at once and within a given time)
  • Percentage of invalid tests (where no result is generated, which can be due to limitation of the method and/or quality of the sample)
  • Cost of testing and time to result

Below are some of the published results of different expert teams, comparing different methods.

Publication

Methods compared

Comparison of targeted next generation sequencing (NGS) versus isolated BRAF V600E analysis in patients with metastatic melanoma2

Allele-specific PCR, NGS

Droplet digital PCR for detection of BRAF V600E mutation in formalin-fixed, paraffin-embedded melanoma tissues: a comparison with Cobas® 4800, Sanger sequencing, and allele-specific PCR3

Cobas 4800 BRAF Mutation Test; ddPCR, Sanger sequencing, allele-specific PCR

Rapid BRAF mutation tests in patients with advanced melanoma: comparison of immunohistochemistry, Droplet Digital PCR, and the Idylla Mutation Platform4

Idylla BRAF Mutation Test; IHC, droplet digital PCR (ddPCR), HRM/sequencing

BRAF V600 mutation detection in melanoma: a comparison of two laboratory testing methods5

Ventana Optiview 3’3-DAB IHC detection kit, cobas 4800 BRAF V600 Mutation Test; PCR, IHC

Comparison of five different assays for the detection of BRAF mutations in formalin-fixed paraffin embedded tissues of patients with metastatic melanoma6

Cobas 4800 V600 Mutation Test; HRM, multiplexed RT-ASA, IHC, NGS

Towards personalized medicine in melanoma: implementation of a clinical next-generation sequencing panel7

NGS

Heterogeneity and frequency of BRAF mutations in primary melanoma: comparison between molecular methods and immunohistochemistry8

Real-time PCR, IHC, classical capillary sequencing

A comparison of immunohistochemical and molecular methods used for analyzing the BRAF V600E gene mutation in malignant melanoma in Taiwan9

PCR, IHC

Detection of BRAF mutations using a fully automated platform and comparison with high resolution melting, real-time allele specific amplification, immunohistochemistry and next generation sequencing assays, for patients with metastatic melanoma10

IdyllaTM assay; HRM, real-time allele-specific amplification (RT-ASA), NGS, IHC

Comparison between two widely used laboratory methods in BRAF V600 mutation detection in a large cohort of clinical samples of cutaneous melanoma metastases to the lymph nodes11

Cobas 4800 BRAF V600 Mutation Test, direct Sanger sequencing; PCR/Sanger sequencing, real-time PCR

Performance comparison of three BRAF V600E detection methods in malignant melanoma and colorectal cancer specimens12

LightMix® Kit BRAF V600E/K; IHC, Sanger sequencing, single-probe high-resolution melting assay

Comparison of molecular testing methods for detecting BRAF V600 mutations in melanoma specimens with challenging attributes13

Cobas 4800 BRAF V600 Mutation Test, Qiagen BRAF RGQ PCR kit version 2; real-time PCR

Comparison of Testing Methods for the Detection of BRAF V600E Mutations in Malignant Melanoma: Pre-Approval Validation Study of the Companion Diagnostic Test for Vemurafenib14

Cobas BRAF Mutation Test; ABI BRAF test; and bidirectional direct sequencing

Comparison of Targeted Next-Generation Sequencing (NGS) and Real-Time PCR in the Detection of EGFR, KRAS, and BRAF Mutations on Formalin-Fixed, Paraffin-Embedded Tumor Material of Non-Small Cell Lung Carcinoma—Superiority of NGS15

NGS, real-time PCR

Detection of BRAF V600 mutations in metastatic melanoma: comparison of the Cobas 4800 and Sanger sequencing assays16

Cobas BRAF Mutation Test; Sanger sequencing

Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing17

NGS

Validation of Next Generation Sequencing Technologies in Comparison to Current Diagnostic Gold Standards for BRAF, EGFR and KRAS Mutational Analysis18

NGS, Sanger sequencing, real-time PCR

Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations19

HRM, NGS, pyrosequencing, IHC, Sanger sequencing

Comparative evaluation of the new FDA approved THxID™-BRAF test with high resolution melting and sanger sequencing20

THxID BRAF test; Real-time PCR, HRM, Sanger sequencing

KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA21

HRM, real-time PCR

Evaluation of BRAF Mutation Testing Methodologies in Formalin-Fixed, Paraffin-Embedded Cutaneous Melanomas22

Cobas 4800 BRAF V600 Mutation Test; Sanger sequencing, pyrosequencing, real-time PCR

Detection of BRAF p.V600E Mutations in Melanomas Comparison of Four Methods Argues for Sequential Use of Immunohistochemistry and Pyrosequencing23

IHC, Pyrosequencing, Sanger Sequencing, real-time PCR

Implementation of a companion diagnostic in the clinical laboratory: The BRAF example in melanoma24

Cobas 4800 BRAF V600 Mutation Test, ThxID BRAF test; HRM, real-time PCR, sequencing, NGS

  1. Lalkhen AG, McCluskey A. Clinical tests: sensitivity and specificity. Contin Educ Anaesth Crit Care Pain. 2008;8(6):221-2.
  2. Zhu M-L, et al. Comparison of targeted next generation sequencing (NGS) versus isolated BRAF V600E analysis in patients with metastatic melanoma. Virchows Arch. 2018;473(3):371–377.
  3. Malicherova B, et al. Droplet digital PCR for detection of BRAF V600E mutation in formalin-fixed, paraffin-embedded melanoma tissues: a comparison with Cobas® 4800, Sanger sequencing, and allele-specific PCR. Am J Transl Res. 2018;10(11):3773-3781.
  4. Bisschop C, et al. Rapid BRAF mutation tests in patients with advanced melanoma: comparison of immunohistochemistry, Droplet Digital PCR, and the Idylla Mutation Platform. Melanoma Res. 2018;28(2):96-104.
  5. O’Brien O, et al. BRAF V600 mutation detection in melanoma: a comparison of two laboratory testing methods. J Clin Pathol. 2017;70(11):935-40.
  6. Franczak C, et al. Comparison of five different assays for the detection of BRAF mutations in formalin-fixed paraffin embedded tissues of patients with metastatic melanoma. Mol Diagn Ther. 2017;21(2):209-16.
  7. de Unamuno Bustos B, et al. Towards personalized medicine in melanoma: implementation of a clinical next-generation sequencing panel. Sci Rep. 2017;7(1):495.
  8. Bruno W, et al. Heterogeneity and frequency of BRAF mutations in primary melanoma: comparison between molecular methods and immunohistochemistry. Oncotarget. 2017;8(5):8069-82.
  9. Huang W-K, et al. A comparison of immunohistochemical and molecular methods used for analyzing the BRAF V600E gene mutation in malignant melanoma in Taiwan. Asia Pac J Clin Oncol. 2016;12(4):403-8.
  10. Harlé A, et al. Detection of BRAF mutations using a fully automated platform and comparison with high resolution melting, real-time allele specific amplification, immunohistochemistry and next generation sequencing assays, for patients with metastatic melanoma. PLoS One. 2016;11(4):e0153576. doi: 10.1371/journal.pone.0153576. eCollection 2016.
  11. Jurkowska M, et al. Comparison between two widely used laboratory methods in BRAF V600 mutation detection in a large cohort of clinical samples of cutaneous melanoma metastases to the lymph nodes. Int J Clin Exp Pathol. 2015;8(7):8487-93.
  12. Løes IM, et al. Performance comparison of three BRAF V600E detection methods in malignant melanoma and colorectal cancer specimens. Tumor Biol. 2015;36(2):1003-13.
  13. Longshore J, et al. Comparison of molecular testing methods for detecting BRAF V600 mutations in melanoma specimens with challenging attributes. J Mol Biomark Diagn. 2015;6(1):625.
  14. Lopez-Rios F, et al. Comparison of Testing Methods for the Detection of BRAF V600E Mutations in Malignant Melanoma: Pre-Approval Validation Study of the Companion Diagnostic Test for Vemurafenib. PLoS ONE. 2013;8(1): e53733.
  15. Tuononen K, et al. Comparison of Targeted Next-Generation Sequencing (NGS) and Real-Time PCR in the Detection of EGFR, KRAS, and BRAF Mutations on Formalin-Fixed, Paraffin-Embedded Tumor Material of Non-Small Cell Lung Carcinoma—Superiority of NGS.Genes, Chromosomes & Cancer. 2013;52:503–11.
  16. Qu K, Pan Q, Zhang X, et al. Detection of BRAF V600 mutations in metastatic melanoma: comparison of the Cobas 4800 and Sanger sequencing assays. J Mol Diagn. 2013;15:790–5.
  17. Lin MT, et al. Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing. Am J Clin Pathol. 2014;141(6):856–66.
  18. McCourt CM, et al. Validation of Next Generation Sequencing Technologies in Comparison to Current Diagnostic Gold Standards for BRAF, EGFR and KRAS Mutational Analysis. PLoS ONE. 2013;8(7):e69604.
  19. Ihle MA, et al. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations. BMC Cancer. 2014;14:13.
  20. Marchant J, et al. Comparative evaluation of the new FDA approved THxID™-BRAF test with high resolution melting and sanger sequencing. BMC Cancer 2014.14:519.
  21. Heideman DA, et al. KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA. The Journal of molecular diagnostics: JMD. 2012;14(3):247–55.
  22. Lade-Keller J, et al. Evaluation of BRAF Mutation Testing Methodologies in Formalin-Fixed, Paraffin-Embedded Cutaneous Melanomas. J Mol Diagn. 2013;15:70–80.
  23. Colomba E, et al. Detection of BRAF p.V600E Mutations in Melanomas Comparison of Four Methods Argues for Sequential Use of immunohistochemistry and Pyrosequencing. J Mol Diagn. 2013;15:94–100.
  24. Mancini I, et al. Implementation of a companion diagnostic in the clinical laboratory: The BRAF example in melanoma. Clinica Chimica Acta. 2014;439:128–36.