Oncogene Mutation Profiling by Sequenom

Mutational analysis was done using the SequenomMassARRAY technology platform (OncoMap version 4.0), as previously described [13 (link), 14 (link)]. A pathologist (JK) reviewed formalin-fixed paraffin-embedded tissue and marked tumor portions, where DNA was extracted from using the QIAamp DNA Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Multiplex polymerase chain reaction amplification using iPLEXchemistry (#10134–2; Sequenom, San Diego, CA), and homogenous mass extension validation of mutation were implemented. Single-base extension was done and a MALDI-TOF mass spectrometer was used to determine the unique mass value according to the mutation site. Target genes were KRAS (exon 2, 3, and 4), NRAS (exon 2, 3, and 4), PIK3CA (exon 9 and 20), and BRAF (exon 15).