BRAF V600 Mutation Detection by Immunohistochemistry Shows Tumor Homogeneity between Primary and Metastatic Sites
Lucile Boursault, Veronique Haddad, Thomas Jouary, Beatrice Vergier, Severine Verdon, Antoine de Mascarel, Jean Philippe Merlio. CHU et Université, Bordeaux, France, Metropolitan
Background: Metastatic melanoma is associated with a poor prognosis. First line therapy using the BRAFV600E kinase inhibitor increase overal survival of patients with metastatic melanoma and BRAFV600 mutation. Thus, rapid and accurate detection of BRAFV600 mutation is critical in such patients. However, intra or inter-tumor heterogeneity may account for different rates of BRAF mutation detection among differents sites. We have evaluated the potential use of immunohistochemistry(IHC) for detecting BRAFV600 mutations by comparison with molecular detection in a parallel blinded study. Moreover, we also evaluated whether intra and inter tumor heterogeneity may account for discrepancies between primary and metastatic material.
Design: We studied 96 metastatic melanoma patients with AJCC stage IIIc or IV, with a total of 221 samples, including primary melanoma(n=86) and different metastatic localization(n=135).BRAF mutation testing was performed on sections from FFPE material using two techniques: HRM analysis followed by Sanger sequencing of variant profiles and anti-BRAFV600E immunostaining.
Results: As reported by others, IHC was positive in all samples harboring V600E and V600E2 mutations and negative for all other mutations, including V600K(n=3, L597S(n=2), K601E(n=3) and pD594N(n=2). The cytoplasmic staining was either strongly positive in most tumour cells of V600E and V600E2 mutated cases or completely negative. It appeared strong brown with small dots. IHC staining and/or HRM analysis showed a perfect concordance between matched primary melanoma and metastasis.The prevalence of each BRAF mutation was V600E(40.6%), V600E2(3.1%), V600K(1.04%) and others(1.04%) respectively. Concordance between the two techniques was 99,5%. Sensitivity for IHC taking into account all V600BRAF mutations was 97% while specificity was 100%.
Conclusions: This study confirms the performance of IHC for BRAFV600E and V600E2 mutation detection using the VE1 antibody positivity. It provide evidence that VE1 IHC may be a cost-effective method of BRAFV600 status assessment, although some cases harbouring the V600K mutation were missed in patients who may benefit from Vemurafenib. Unlike previous reports, IHC was not found more sensitive for BRAFV600E mutation detection than its molecular detection as the two techniques matched perfectly. Moreover, using IHC we report for the first time the concordance between BRAF status at primary and metastatic sites that together with the homogeneous staining observed by IHC would suggest tumour homogeneity rather than heterogeneity at least for this primary genetic alteration.
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 35, Tuesday Morning