Comprehensive Molecular Profiling of Tumors

Alterations in KRAS exon 2, BRAF exon 15 and EGFR exons 18–21 observed by HRMΑ, were identified by Pyrosequencing using the Pyromark Gold Q24 Reagent kit with the Q24 Pyrosequencer (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol as previously described [31 (link),32 (link)]. Sanger Sequencing was used to identify mutations in BRAF (except Val600Glu) and PIK3CA gene exons 9 and 20. Briefly PCR products positive by HRMA were sequenced using the BigDye terminator cycle sequencing kit (Applied Biosystems, CA, USA) in order to confirm the presence of mutations. The sequencing products were analysed on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). PCR primers were also used for sequencing analysis. Results were verified by sequencing analysis of at least two independent PCR products. PIK3CA gene exon 9 sequence analysis did not show amplification of the pseudogene.