Universität zu Köln

Ihle, Michaela Angelika, Fassunke, Jana, Koenig, Katharina, Gruenewald, Inga, Schlaak, Max, Kreuzberg, Nicole, Tietze, Lothar, Schildhaus, Hans-Ulrich, Buettner, Reinhard and Merkelbach-Bruse, Sabine (2014). 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, 14. LONDON: BMC. ISSN 1471-2407

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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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Ihle, Michaela Angelika UNSPECIFIED UNSPECIFIED UNSPECIFIED Fassunke, Jana UNSPECIFIED UNSPECIFIED UNSPECIFIED Koenig, Katharina UNSPECIFIED UNSPECIFIED UNSPECIFIED Gruenewald, Inga UNSPECIFIED UNSPECIFIED UNSPECIFIED Schlaak, Max UNSPECIFIED UNSPECIFIED UNSPECIFIED Kreuzberg, Nicole UNSPECIFIED UNSPECIFIED UNSPECIFIED Tietze, Lothar UNSPECIFIED UNSPECIFIED UNSPECIFIED Schildhaus, Hans-Ulrich UNSPECIFIED UNSPECIFIED UNSPECIFIED Buettner, Reinhard UNSPECIFIED UNSPECIFIED UNSPECIFIED Merkelbach-Bruse, Sabine UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-448610
DOI:	10.1186/1471-2407-14-13
Journal or Publication Title:	BMC Cancer
Volume:	14
Date:	2014
Publisher:	BMC
Place of Publication:	LONDON
ISSN:	1471-2407
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language KRAS MUTATIONS; MALIGNANT-MELANOMA; V600E MUTATIONS; RAS MUTATIONS; INHIBITOR; PATHWAY; ASSAY; VEMURAFENIB; SENSITIVITY; PERFORMANCE Multiple languages Oncology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/44861