LS and AM drafted the manuscript

LS and AM drafted the manuscript. a fully automatized immunohistochemical method. We correlated the data to the clinicopathologic characteristics of patients and their clinical outcome. Results The sensitivity and the specificity of the Ventana? VE1 antibody were 89.2 and 96.2% respectively, while the positive predictive value and negative predictive value were 97.1 and 86.2%, respectively. For six mutated patients the histological sample before treatment and when disease progressed was available. The immunohistochemical BRAF V600E expression in the specimens when disease progressed was less intense and more heterogeneous compared to the basal expression. Multivariate analysis revealed that a less intense grade of positive expression is an impartial predictor of a less aggressive stage at diagnosis (mutations and 90% of these involve a specific missense substitution of valine by glutamic acid at codon 600 (V600E). This mutation constitutively activates the protein and the downstream MAPK signaling pathway in a RAS-independent manner, promoting proliferation, survival and distributing of tumor cells [2]. Metastatic melanoma patients harboring this hot spot mutation can be effectively treated ZINC13466751 with BRAF inhibitors ZINC13466751 alone or in combination with MEK inhibitors [3, 4] because this genetic alteration is usually predictive to therapeutic response. Therefore, quick screening for status in patients with unresectable or metastatic melanoma has recently become integral to treatment decisions and essential for optimal patient care. In clinical practice the platinum standard and the most ZINC13466751 commonly used method to assess status is based on DNA molecular assays. The most common ones are the classical Sanger sequencing, pyrosequencing and the FDA-approved cobas? 4800 mutation test. Each method has its own sensitivity, specificity, cost and response delay [5, 6]. However, the molecular methods are often more time consuming and not usually routinely available in all anatomic pathology laboratories. Moreover, some diagnostic samples are still unsuitable for molecular screening because of their inadequate tumor content and the variable quality of DNA extracted due to fragmentation that occurs with tissue processing. The effect of melanin pigment on molecular assays is also important. Thus, you will find circumstances ZINC13466751 when the alternative diagnostic mutation detection method may have power [7]. Recently, a mutation-specific mouse monoclonal antibody (clone VE1), which does not detect other mutant BRAF V600 epitopes or the wild type form but only the BRAF V600E mutated protein, has been developed [8] and it is now commercially available from Spring Bioscience and Ventana?. It has been previously shown ZINC13466751 that immunohistochemistry (IHC) with this antibody is usually sensitive and specific for the detection of the genomic BRAF V600E mutation [9C11]. This obtaining has permitted the use of IHC, which is a potentially faster, less expensive, and more available methodology to assess BRAF status in the formalin-fixed and paraffin-embedded tissue of melanoma patients [2]. The aim of our study was to confirm the clinical value of the VE1 Ventana? antibody as we plan to use an immunohistochemical method to preliminary detect BRAF status in our routine diagnostic procedures. We intend to use a fully automatized immunohistochemical method, marked CE-IVD, to analyze a retrospective series of metastatic melanoma samples previously investigated for genetic status by molecular techniques and to compare the results obtained by the two methods, as today a univocal validated and accredited immunohistochemical procedure and criteria for analysis does not exist. Moreover, we propose to explore the biological meaning of BRAF immunohistochemical labeling both as a marker predictive of response to target therapy and as a player of acquired tumor drug resistance. Therefore, we intend to correlate the data to the clinicopathologic characteristics of patients and their clinical outcome, and to compare, in a small subset of patients, BRAF labeling before treatment and when disease progressed. Methods Patients and samples Our retrospective study included 64 patients enrolled from June 2008 to April 2015 with histologically confirmed metastatic melanoma treated at the of Bari, with a median age at diagnosis of 61?years (range 22C82 years); 35 patients were male (54.7%) while 29 were female (45.3%). Clinical and follow-up data were collected and evaluated in the entire set of patients, according to approval by the local Ethics Committee L1CAM antibody of the of Bari (prot. no. 515/EC of May 12, 2015). All patients signed informed consent form authorizing the Institute to utilize biological materials for research purpose according to ethical requirements. The study was conducted in accordance with the international requirements of good clinical practice. The immunohistochemical analysis of BRAF status was conducted after the end of treatment and, thus, did not influence.