Profiling HRAS Mutations in Adnexal Tumors

Areas with overgrowth of epithelial or myoepithelial cells of three AMEs were selected based on p63 expression and morphology. The epithelium and myoepithelium of only the selected areas was separately microdissected from eight micron-thick FFPE histological sections under a stereomicroscope (Olympus SZ61), following careful histologic review and use of the corresponding p63 IHC stains to highlight the myoepithelium, as reference. DNA was extracted using the DNAeasy Blood and Tissue Kit (Qiagen), following manufacturers’ instructions. The presence of mutations affecting the HRAS Q61 hotspot locus was assessed by Sanger sequencing. In brief, PCR amplification was conducted using AmpliTaq Gold 360 Master Mix Kit (Life Technologies), as previously described^{16 (link)}. Following purification with exoSAP-IT, PCR products were subjected to Sanger sequencing using previously validated primers^{17 (link)} encompassing the HRAS Q61 hotspot locus (Supplementary Table 1). Sequence electropherograms corresponding to the forward and reverse strands were manually analyzed.