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BMC Cancer
Published in: BMC Cancer 1/2014

Open Access 01-12-2014 | Research article

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 BRAFmutations

Authors: Michaela Angelika Ihle, Jana Fassunke, Katharina König, Inga Grünewald, Max Schlaak, Nicole Kreuzberg, Lothar Tietze, Hans-Ulrich Schildhaus, Reinhard Büttner, Sabine Merkelbach-Bruse

Published in: BMC Cancer | Issue 1/2014

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Abstract

Background

The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.

Methods

Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).

Results

There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.

Conclusion

Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.
Appendix
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Metadata
Title
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 BRAFmutations
Authors
Michaela Angelika Ihle
Jana Fassunke
Katharina König
Inga Grünewald
Max Schlaak
Nicole Kreuzberg
Lothar Tietze
Hans-Ulrich Schildhaus
Reinhard Büttner
Sabine Merkelbach-Bruse
Publication date
01-12-2014
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2014
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/1471-2407-14-13

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