Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited. Multiplex mutation screening by mass spectrometry in gastrointestinal stromal tumours GUHYUN KANG*jj,JEEYUN LEE{jj,KI TAEK JANG*, CAROL BEADLING{, CHISTOPHER L. CORLESS{,MICHEAL C. HEINRICH§,JOON OH PARK{,WON KI KANG{, CHEOL KEUN PARK* AND KYOUNG-MEE KIM* *Department of Pathology, and {Division of Hematology and Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; zDepartment of Pathology and §Division of Hematology and Oncology, Oregon Health and Science University, Portland, OR, USA; jjthese authors contributed equally to this paper Summary Aims: Clinical decision making and optimal clinical trial design based on cancer genetic information will be increas- ingly informed by the mutational status of multiple genes. Methods: We performed mutation screening on 22 fresh frozen gastrointestinal stromal tumours (GISTs) using a multiplexed oncogene screening panel with a mass spectroscopy readout (MassARRAY). The panel can detect 390 known mutations across 30 genes, including several known to contribute to intracellular signalling in cancers (BRAF, PIK3CA, KRAS HRAS, NRAS, AKT1, CTNNB1, GNAQ, CDK4, MAP2K1 and MAP2K2). Results: Direct Sanger sequencing confirmed that 16 cases (73%) harboured KIT mutations, affecting exon 11, 13 and 17, and the remaining six were wild-type for both KIT and PDGFRA. The sensitivity of the multiplexed oncogene screening panel was 100% for identifying missense mutations in KIT. Only 17% of the deletion mutations were detected, because the panel was not designed for detecting these. A substitution in FBX4 exon 1 (S8R), representing a germline single-nucleotide polymorphism, was observed in a case with KIT exon 11 missense mutation. No other mutations were identified, including in the six wild-type GISTs. Conclusions: Our results indicate that mutations other than KIT or PDGFRA are rare in GISTs. Although multiplex mutation screening by mass spectrometry detected missense mutations accurately, it is not sufficient to screen mutations because deletion mutations are common in GISTs. Key words: Gastrointestinal stromal tumour, high-throughput screening, mass spectrometry, mutation, sequencing. Received 29 December 2011, revised 31 January, accepted 2 February 2012 INTRODUCTION As many compelling targets are shared across several cancer types, a new clinical trial is likely to be more beneficial when patients are recruited on the basis of genotype, not solely on the basis of histological subtype. 1 In most molecular diagnostic laboratories, mutations are currently tested one at a time, and not all types of mutations are detected by the same method. The application of targeted therapy requires identifi- cation of relevant molecular subtypes, and thus new approaches are necessary to simultaneously profile any tumour for pivotal mutations in multiple cancer genes. 2,3 For multiplexed analyses of missense mutations, one of the major platforms in use is the MassARRAY system (Sequenom, USA). The system supports the analysis of multiplexed panels for common oncogene mutations reported in the Catalogue of Somatic Mutations in Cancer (COSMIC; http://www.sanger.ac.uk/genetics/CGP/ cosmic) database. 2,3 Compared with direct sequencing for all of the mutations represented in a panel, this approach is robust, rapid, relatively inexpensive, and can readily identify mutations with limited amounts of DNA. 1,3 Gastrointestinal stromal tumours (GISTs) are the most com- mon mesenchymal tumours in the gastrointestinal tract, and are generally KIT positive and driven by KIT or platelet-derived growth factor receptor alpha (PDGFRA) activating mutations. 4 Most KIT mutations involve exon 11 (66.9%), followed by exon 9 (18.1%), exon 13 (1.6%) and exon 17 (1.6%). Nearly 7% of GISTs have mutations in PDGFRA, and the most common of these is exon 18 substitution D842 V. 5,6 About 85% and 10– 15% of GISTs occurring in children and adults, respectively, lack KIT or PDGFRA mutation, in which tumour-initiating events are not fully understood. 7,8 Recently, BRAF exon 15 V600E mutation was reported in a subgroup of adult wild-type GISTs and it seems to show a predilection for the small intestine. 9–11 GISTs in Carney–Stratakis syndrome and some wild-type tumours without a personal or familial history of paraganglioma are found to be associated with germline mutation of succinate dehydrogenase (SDH) subunit B, C or D. 12–14 From a therapeutic point of view, imatinib is less effective against wild-type GISTs and sunitinib only rarely results in objective responses in these tumours. 15,16 Moreover, imatinib resistance is an increasing clinical problem with more prolonged follow-up. 17 These observations underscore the need for a broad-spectrum genotyping approach to identify the mechanism of oncogenesis in wild-type GISTs and resistance to targeted therapies. Investigating molecular genetic abnormalities beyond KIT and PDGFRA might lead to the discovery of novel molecular events in GIST oncogenesis and help to identify new therapeutic targets. Therefore, we performed mutation screening on 22 fresh frozen GIST samples using a mass spectrometry-based panel of multiplex assays interrogating 390 mutations across 30 cancer genes. This approach was compared to the Sanger sequencing method for the detection of KIT mutations. Pathology (August 2012) 44(5), pp. 460–464 ANATOMICAL PATHOLOGY Print ISSN 0031-3025/Online ISSN 1465-3931 # 2012 Royal College of Pathologists of Australasia DOI: 10.1097/PAT.0b013e3283559c45