Tumor Samples and BRAF Mutation Analysis

To be included in the study, all individuals had to have an adequate tumor sample for BRAF mutation testing. This sample could have been previously collected at the time of the diagnosis and stored in the department of anatomical pathology of the respective hospital or could be obtained after recruitment in the present study. In this second scenario, tumor samples were collected according to routine clinical practice to ensure the observational nature of the study. Tumor samples were collected from metastases, primary tumor, or relapses. Tumor samples were considered adequate for the study when they fulfilled the following methodology: 1) The sample was received immediately after it was collected, without being fixed, and handled under sterile conditions. 2) At least 100 g of tissue was provided for mutation analyses. If there was enough tissue, it was divided into two 1-cm side cubes from different areas and was subsequently divided into 4 pieces. 3) 10 unfixed sections were created, and the remaining tumor tissue was fixed in formalin as control for the analysis. 4) Samples were placed in a sterile culture for DNA and cytogenetics. 5) Tissue samples were frozen in liquid nitrogen and stored at −80°C until analysis.

Data collection and management of BRAF mutation analysis were conducted using the Biomarker point online platform available at www.biomarkerpoint.es. Hospitals lacking appropriate molecular techniques and resources for the analysis of BRAF mutation used this online platform, which provided these hospitals with the possibility of performing mutation analysis by means of: 1) collection of samples; 2) shipment to referral hospitals where the analysis is performed (Vall d'Hebron University Hospital, Barcelona, 12 de Octubre University Hospital, Madrid, and Virgen Macarena University Hospital, Seville; and 3) online submission of reports.

The type of sample could be paraffin-embedded blocks, paraffin blocks slides, or cytological slides. DNA extraction and BRAF mutation detection in tumor samples were performed using the Cobas BRAF Mutation Test® (Roche Molecular Systems, Inc., Branchburg, NJ). Other alternative methods could be used for BRAF mutation testing when the Cobas BRAF Mutation Test was not available (i.e., Sanger sequencing and pyrosequencing).