Published OnlineFirst August 13, 2013; DOI: 10.1158/1078-0432.CCR-12-3863 Clinical Cancer Human Cancer Biology Research Novel Clinically Relevant Genes in Gastrointestinal Stromal Tumors Identified by Exome Sequencing Sebastian F. Schoppmann1, Ursula Vinatzer1, Niko Popitsch5, Martina Mittlbock€ 2, Sandra Liebmann-Reindl1, Gerd Jomrich1, Berthold Streubel3, and Peter Birner4 Abstract Purpose: Chromosomal gains and losses resulting in altered gene dosage are known to be recurrent in gastrointestinal stromal tumors (GIST). The aim of our study was the identification of clinical relevant genes in these candidate regions. Material and Methods: A cohort of 174 GIST was investigated using DNA array (n ¼ 29), FISH (n ¼ 125), exome sequencing (n ¼ 13), and immunohistochemistry (n ¼ 145). Results: Array analysis revealed recurrent copy number variations (CNVs) of chromosomal arms 1p, 1q, 3p, 4q, 5q, 7p, 11q, 12p, 13q, 14q, 15q, and 22q. FISH studies of these CNVs showed that relative loss of 1p was associated with shorter disease-free survival (DFS). Analysis of exome sequencing concentrating on target regions showing recurrent CNVs revealed a median number of 3,404 (range 1,641–13,602) variants (SNPs, insertions, deletions) in each tumor minus paired blood sample; variants in at least three samples were observed in 37 genes. After further analysis, target genes were reduced to 10 in addition to KIT and PDGFRA. Immunohistochemical investigation showed that expression of SYNE2 and DIAPH1 was associated with shorter DFS, expression of RAD54L2 with shorter and expression of KIT with longer overall survival. Conclusion: Using a novel approach combining DNA arrays, exome sequencing, and immunohis- tochemistry, we were able to identify 10 target genes in GIST, of which three showed hithero unknown clinical relevance. Because the identified target genes SYNE2, MAPK8IP2, and DIAPH1 have been shown to be involved in MAP kinase signaling, our data further indicate the important role of this pathway in GIST. Clin Cancer Res; 19(19); 5329–39. Ó2013 AACR. Introduction known to inhibit both KIT and PDGFRA receptors and Gastrointestinal stromal tumors (GIST) are the most used in the treatment of recurrent and metastatic GIST or common mesenchymal tumors of the gastrointestinal tract GIST with high risk of progression (5). Effectiveness of (1). They are thought to arise from Cajal cells or their imatinib mesylate depends on the mutational status of KIT PDGFRA precursors and characteristically harbor gain of function and (3). In contrast to metastatic GIST, mutations in KIT leading to constitutive activation of the radical surgery seems to be the best treatment option for KIT receptor (2). localized tumors (6). The recurrence rate after radical An alternative mutation in a related tyrosine kinase, surgery seems to depend mainly on tumor localization, PDGFRA, is found in 35% of KIT mutation negative GIST size, and mitotic activity and ranges between 5% and (3, 4). Imatinib mesylate is a molecularly targeted drug 75% with a poor clinical outcome in relapsed patients (7, 8). Current classifications take into account tumor size, mitotic rate, and tumor location but do not include Authors' Affiliations: 1Department of Surgery; 2Center for Medical Sta- mutational data nor protein expressions of tumor cells 3 tistics, Informatics, and Intelligent Systems; Department of Obstetrics and (9–11). Gynecology and Core Unit Next Generation Sequencing; 4Clinical Institute of Pathology, Medical University of Vienna; and 5Center for Integrative The discovery of KIT and PDGFRA activating mutations in Bioinformatics Vienna (CIBIV), Max F Perutz Laboratories, University of the majority of GIST represents a significant progress in Vienna & Medical University of Vienna, & Faculty of Computer Science, University of Vienna, Vienna, Austria understanding their biological behavior. Although further molecular mechanisms underlying the development and Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). progression of GIST are not fully understood, they are nec- essary due to the wide range of clinical behavior. Chromo- Corresponding Author: Berthold Streubel, Department of Obstetrics and Gynecology, Medical University of Vienna, Wahringer€ Gurtel€ 18-20, A-1090 somal gains and losses resulting in altered gene dosage are Vienna, Austria. Phone: 43-1404002821; Fax: 43-1404002862; E-mail: known to be recurrent in GIST and believed to have a role [email protected] in the molecular pathogenesis of these tumors (12–19). doi: 10.1158/1078-0432.CCR-12-3863 Nevertheless, the target genes remain to be identified within Ó2013 American Association for Cancer Research. these regions. www.aacrjournals.org 5329 Downloaded from clincancerres.aacrjournals.org on October 3, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst August 13, 2013; DOI: 10.1158/1078-0432.CCR-12-3863 Schoppmann et al. Microarray analysis Translational Relevance High-quality genomic DNA was obtained from 29 fresh- Gastrointestinal stromal tumors (GIST) are the most frozen tumor samples using the DNeasy Blood & Tissue Kit common mesenchymal tumors of the gastrointestinal (Qiagen) and subjected to microarray analysis using the tract, characterized by uncertain clinical behavior. In commercially available Affymetrix Genome-Wide Human addition to KIT, there is a strong need for further SNP Array 6.0 (Affymetrix Inc.) following the protocols therapeutic targets in GIST. Using a technical approach provided by the manufacturer. Data analysis was conducted combining DNA array analysis, next generation exome with Affymetrix Genotyping Console 3.0.1 using the Bird- sequencing, and immunohistochemistry, we were able seed Algorithm and Affymetrix Chromosome Analysis Suite to identify 10 novel, frequently mutated genes in GIST, 1.01 at a resolution of 500 kb. Genome annotations applied of which 3 showed hithero unknown clinical rele- in data analysis referred to the human reference assembly vance. Because a part of the identified target genes has GRCh37/hg19 as provided by the Affymetrix annotation file been shown to be involved in MAP kinase signaling, release na31. The reference model file used for data nor- our data further indicate the important role of this malization with GTC was generated from 39 healthy control pathway in GIST. So the inclusion of MAP kinase individuals. CNVs showing an overlap greater than 80% pathway parameters in future clinical studies might with benign CNVs of the Database of Genomic Variants be of potential benefit for patients as selective inhibi- (http://projects.tcag.ca/variation/) were excluded from our tors are available. analysis. Somatic CNV status of matching tumor-blood/normal tissue samples was used to confirm gains and losses using TaqMan real-time PCR or FISH. The aim of our study was the identification of putative prognostic markers within the regions with recurrent FISH analysis gains or losses. We measured copy number variations Tissue microarrays from 125 GIST paraffin-embedded (CNVs) and defined exact chromosomal breakpoints for samples were used for FISH. Screening for CNVs was con- the most common alterations in GIST. CNVs were screened ducted using commercially available probes for TP73 (chro- in a large single-center cohort of GIST and correlated with mosomal band 1p36), ABL2 (1q25), CCND1 (11q13), clinical outcome. Exome sequencing identified mutated DLEU (13q14), IGH (14q32), SNRPN (15q11), and CLTCL1 candidate genes in the regions of interest, and protein (22q11.2; Abbott and Kreatech). FISH procedures and anal- products of identified target genes were investigated immu- yses were conducted according to standard protocols. nohistochemically in our large single-center cohort of GIST. Immunohistochemical analysis Tissue microarrays containing 145 GIST samples were used for evaluation of protein expression. Supplementary Patients and Methods Table S2 summarizes the antibodies used. Patients Immunohistochemical analysis was conducted using a We studied a total of 174 cases of GIST treated at the Benchmark Ultra Immunostainer (Ventana), except for exp- Medical University of Vienna between August 1992 and ression of RB, where a DAKO autostainer (DAKO) was used. February 2011 in this retrospective observational study. A specimen was considered to be positive if the vast majority Clinical data and follow-up were available for 145 of 174 of cells (>80%) showed distinct staining. The number of cases. All cases were restaged according to UICC TNM cases differed between antibodies due to the use of tissue classification of malignant tumors 7th edition and risk microarrays, and for investigation of FLT4 and AP1B1 the was evaluated according to Fletcher and Miettinen (9– blocks had to be recut, resulting in the loss of several spots. 11). As this cohort of patients has been used in several previous studies, clinical data in correlation with known Exome studies risk factors have been reported previously, as well as the Thirteen of the 29 GIST of the microarray study were results of the sequence analysis about mutations of KIT subjected to high-throughput sequencing. In all these cases, (exons 9, 11, 13, and 17) and PDGFRA (exons 12 and 18; matched control DNA obtained from the peripheral blood refs. 20–22). Tissue microarrays were established for or normal gastric tissue of patients were sequenced in immunohistochemical screening in all 145 cases. FISH parallel. Exome enrichment was conducted