Tumor samples and mutation analyses

Mutations were tested preferentially in distant or lymph node metastases as heterogeneity of the mutational status between metastatic and primary tumor sites have been reported and as BRAF mutations have predictive value for the use of BRAFi and MEKi in metastatic disease [26, 27]. If no such samples were available, analyses were performed in primaries. Areas of tumor tissue were identified from 10 μm-thick formalin-fixed, paraffin-embedded sections. To enrich for a content of > 75%, tumors were manually micro-dissected with an ultra-thin cannula. DNA was isolated with extraction buffer (Tris-HCl pH 7.4 0.1 M, EDTA 0.5 mM, Tween 20 0.5% in distilled water) after proteinase K hydrolysis for 16 h (Thermo Fisher Scientific, Darmstadt, Germany). Samples were amplified using polymerase chain reactions (PCR) covering BRAF exon 15 (Codon 600), NRAS exon 2 (codon 12, 13), and NRAS exon 3 (codon 61) using specific primers (Additional file 1: Tables S1 and S2). PCR products were subsequently subjected to pyrosequencing with the PyroMark Q24 System (Qiagen, Hilden, Germany) together with specific primers (Additional file 1: Table S2). If sequencing results were unclear or if mutations were detected in both genes, pyrosequencing was followed by additional Sanger sequencing to confirm the mutational status which was performed by MWG Operon (Ebersberg, Germany).