Dataset Information

NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.

ABSTRACT: The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRAS(Q61R) and BRAF(V600E) proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRAS(Q61R) immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRAS(Q61R) and VE1 anti-BRAF(V600E) antibodies in the theranostic mutation screening of melanomas.142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry.23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively. The 23 NRAS (Q61R) and 23 BRAF (V600E) -mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative. Proportions and intensities of staining were varied. Other NRAS (Q61L) , NRAS (Q61K) , BRAF (V600K) and BRAF (V600R) mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data. Technical limitations are discussed to explain those discrepancies. Anyway we could not rule out real tumor heterogeneity.In our study, we showed that combining immunohistochemistry analysis targeting NRAS(Q61R) and BRAF(V600E) proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.

SUBMITTER: Uguen A

PROVIDER: S-EPMC4513673 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.

Uguen Arnaud A Talagas Matthieu M Costa Sebastian S Samaison Laura L Paule Laure L Alavi Zarrin Z De Braekeleer Marc M Le Marechal Cédric C Marcorelles Pascale P

Diagnostic pathology 20150725

<h4>Background</h4>The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRAS(Q61R) and BRAF(V600E) proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRAS(Q61R) immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRAS(Q61R) and VE1 anti-BRAF(V600E) antibodies ...[more]

PMID: 26204954

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Project description:Screening for theranostic biomarkers is mandatory for the therapeutic management of cutaneous melanoma. BRAF and NRAS genes must be tested in routine clinical practice. The methods used to identify these alterations must be sensitive to detect mutant alleles in a background of wild type alleles, and specific to identify the correct mutation. They should not require too much material, since in some cases the available samples are small biopsies. Finally, they should also be quick enough to allow a rapid therapeutic management of patients. Sixty five consecutive formalin-fixed paraffin-embedded (FFPE) melanoma samples were prospectively tested for BRAF mutations with the VE1 (anti-BRAF V600E) antibody and for both BRAF and NRAS mutations with the Idylla NRAS-BRAF-EGFR S492R Mutation Assay cartridges. Results were compared to our routine laboratory practice, allele specific amplification and/or Sanger sequencing and discordant cases confirmed by digital PCR. Excluding discordant by-design-mutations, system failures and DNA quantity or quality failures, BRAF IHC demonstrated an overall concordance of 89% for BRAF V600E mutation detection, the Idylla system gave a concordance of 100% for BRAF mutation detection and of 92.1% for NRAS mutation detection when compared to our reference. When discrepancies were observed, all routine results were confirmed by digital PCR. Finally, BRAF IHC positive predictive value (PPV) was of 82% and negative predictive value (NPV) of 92%. The Idylla cartridges showed a PPV and NPV of both 100% for BRAF mutation detection and a PPV and NPV of 100% and 87% respectively, for NRAS mutation detection. In conclusion, BRAF V600E immunohistochemistry is efficient for detecting the V600E mutation, but negative cases should be further evaluated by molecular approaches for other BRAF mutations. Since 3 NRAS mutations have not been detected by the Idylla NRAS-BRAF-EGFR S492R Mutation Assay, these cartridges should not be used as a substitute for traditional molecular methods in the conventional patient therapeutic care process without the expertise needed to have a critical view of the produced results.

Project description:BackgroundHigh frequencies of the BRAF V600E mutation have been reported in pleomorphic xanthoastrocytoma (PXA). Recently, a BRAF V600E mutation-specific antibody has been developed and validated. We evaluated the immunohistochemical (IHC) detection of BRAF V600E mutation in PXA by comparing to gold standard molecular analysis and investigating the interobserver variability of the IHC scoring. We performed BRAF V600E IHC in 46 cases, of which 37 (80%) cases had sufficient tumor tissue for molecular analysis. IHC detection was performed using monoclonal mouse antibody VE1 (Spring Bioscience). IHC slides were scored independently by four reviewers blind to molecular data, including a primary (gold standard) and three additional reviewers. BRAF V600E mutation status was assessed by allele-specific polymerase chain reaction (PCR) with fragment analysis.ResultsAll 46 cases showed interpretable BRAF V600E IHC results: 27 (59%) were positive (strong cytoplasmic staining), 19 (41%) were negative (6 of these cases with focal/diffuse weak cytoplasmic staining, interpreted as nonspecific by the primary reviewer). By molecular analysis, all 37 cases that could be tested had evaluable results: 22 (59%) cases were positive for BRAF V600E mutation and were scored as "IHC-positive", and 15 (41%) were negative (including 11 cases scored as "IHC-negative" and 4 cases scored as negative with minimal nonspecific staining). IHC detection of BRAF V600E mutant protein was congruent in all 37 cases that were successfully evaluated by molecular testing (sensitivity and specificity of 100%). Agreement for IHC scoring among the 4 reviewers was almost perfect (kappa 0.92) when cases were scored as "positive/negative" and substantial (kappa 0.78) when minimal nonspecific staining was taken into account.ConclusionsWe conclude that detection of BRAF V600E mutation by immunohistochemistry is highly sensitive and specific. BRAF V600E IHC interpretation is usually straightforward, but awareness of possible nonspecific staining is necessary and training is recommended. It is a practical rapid method that may avoid the need of labor-intensive molecular testing and may be most valuable in small biopsies unsuitable for molecular analysis.

Project description:BackgroundSentinel node biopsy (SNB) is an important step in melanoma staging and prognostication. It is commonly performed for patients with intermediate thickness melanomas, based on clinicopathological features. However, only 20-25% of patients eventually demonstrate nodal involvement. The aim of this study was to evaluate whether tissue biomarkers with links to melanoma biology, together with clinicopathological parameters, could aid in the prediction of sentinel node involvement and improve selection of patients for SNB. In addition, we examined the role of these clinical or biological markers in disease outcome.MethodsWe collected a case-control cohort of 140 intermediate thickness (Breslow 0,9-4,0mm) melanoma patients with or without SNB involvement matched for age, gender, Breslow thickness and location. From this cohort, we tested the predictive value of common clinicopathological parameters (ulceration, mitotic count and tumor regression) and FMNL-2, ezrin and BRAF V600E immunoreactivity, for sentinel node involvement and survival. We further analyzed the correlations in the superficial spreading melanoma subtype.ResultsBased on our case control analysis, of the markers, BRAF V600E status (p = 0.010) and mitotic count (p = 0.036) correlated with SNB involvement. SNB status was a strong independent prognosticator for recurrence free survival (RFS p<0.001), melanoma specific survival (MSS p = 0.000) and overall survival (OS p = 0.029). In the superficially spreading melanoma subgroup, BRAF V600E positivity indicated poorer RFS (p = 0.039) and OS (p = 0.012). By combining the Breslow thickness, mitotic count and BRAF immunohistochemistry, we identified a group of superficially spreading melanomas with an excellent survival probability independent of SNB status.ConclusionsThese results demonstrate that BRAF immunohistochemistry could serve as a useful addition to a marker panel for selecting intermediate thickness melanoma patients for SNB.

Dataset Information

NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.

Publications

NRAS (Q61R), BRAF (V600E) immunohistochemistry: a concomitant tool for mutation screening in melanomas.

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