Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Müller, A.
BRAF V600E Testing in Malignant Melanoma: Analytical Comparison of Immunohistochemistry versus Genotyping
Humanmedizin; [ Diplomarbeit ] ; 2015. pp.
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Aigelsreiter Ariane
- Wolf Peter
- Altmetrics:
- Abstract:
- Introduction: Malignant melanoma is the sixth most diagnosed cancer and has a yearly increase in incidence of 4 to 6% in fair skinned populations in the western world. Approximately 50% of all melanoma carry a V600E mutation on the BRAF gene. Melanoma patients in advanced tumour stages with this specific mutation are eligible for targeted therapy with BRAF-inhibitors, such as Vemurafenib and Dabrafenib, instead of conventional chemotherapy with Dacarbazin. The clinical relevance of this research is based on the need for specific treatment of patients in terms of personalised medicine. The goal was to quantify the amount of cells carrying a V600E mutation in malignant melanoma tissue. Methods: 62 melanoma samples were analysed by the means of immunohistochemistry with a mutation specific antibody (VE1) that detects BRAF V600E mutated cells with high specificity and sensitivity. After optimisation of immunohistochemical staining methods “on the bench” the chromogen Nova Red was used in all samples; 31 of the samples were additionally stained with the chromogen Vina Green. Subsequently, images were taken of the immunohistochemically stained samples with a camera system integrated in a microscope (Aperio XT ScanScope). Visual examination was performed and with the help of an image analysis software (Aperio ImageScope) an attempt was made to create an algorithm for the quantification of the BRAF expression in melanoma. To validate the results obtained by immunohistochemistry, 13 samples were tested with genotyping by restriction digest method. Further, the results of immunohistochemistry and restriction digest were compared to results produced by genotyping with MALDI-TOF as the gold standard. Results: MALDI-TOF found 18 of the 62 (29%) samples to be positive for the V600E mutation. By visual evaluation of immunohistochemistry 15 of 62 (24%) were positive with Nova Red but only 2 of 31 (6%) with Vina Green. Compared to MALDI-TOF, immunohistochemistry with Nova Red showed a sensitivity of 66,7% and a specificity of 93,1%. The calculation of Cohen’s Kappa resulted in kappa=0,63 (95% CI, 0,41 to 0,85) with a p-value of 0,000000589. Immunohistochemistry with Vina Green on the other hand had a sensitivity of 100%, a specificity of 33,3% and a Cohen’s Kappa of 0,45 (95% CI, 0,02 to 0,87) with a p-value of 0,00284. Computer-aided evaluation of immunohistochemical stains produced higher median „positivity values” (percentage of positive pixels per total area analysed) in BRAF V600E positive samples than in negative ones. Conclusion: In our hands, immunohistochemistry with visual or computer-aided evaluation and the restriction digest method were inferior to the genetic method of MALDI-TOF in detecting BRAF V600E mutations in melanoma samples. Thus MALDI-TOF remains the gold standard to detect this specific BRAF mutation as a prerequisite for BRAF-inhibitor treatment.