GSTT1 Copy Number Gain and ZNF Overexpression Are Predictors of Poor Response to Imatinib in Gastrointestinal Stromal Tumors Eui Jin Lee1¤☯, Guhyun Kang1,4☯, Shin Woo Kang1,5, Kee-Taek Jang1, Jeeyun Lee2, Joon Oh Park2, Cheol Keun Park1, Tae Sung Sohn3, Sung Kim3, Kyoung-Mee Kim1* 1 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 2 Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 3 Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 4 Department of Pathology, Sanggye Paik Hospital, Inje University College of Medicine, Seoul, Korea, 5 Department of Mathematics, Korea University, Seoul, Korea Abstract Oncogenic mutations in gastrointestinal stromal tumors (GISTs) predict prognosis and therapeutic responses to imatinib. In wild-type GISTs, the tumor-initiating events are still unknown, and wild-type GISTs are resistant to imatinib therapy. We performed an association study between copy number alterations (CNAs) identified from array CGH and gene expression analyses results for four wild-type GISTs and an imatinib-resistant PDGFRA D842V mutant GIST, and compared the results to those obtained from 27 GISTs with KIT mutations. All wild-type GISTs had multiple CNAs, and CNAs in 1p and 22q that harbor the SDHB and GSTT1 genes, respectively, correlated well with expression levels of these genes. mRNA expression levels of all SDH gene subunits were significantly lower (P≤0.041), whereas mRNA expression levels of VEGF (P=0.025), IGF1R (P=0.026), and ZNFs (P<0.05) were significantly higher in GISTs with wild-type/PDGFRA D842V mutations than GISTs with KIT mutations. qRT-PCR validation of the GSTT1 results in this cohort and 11 additional malignant GISTs showed a significant increase in the frequency of GSTT1 CN gain and increased mRNA expression of GSTT1 in wild-type/PDGFRA D842V GISTs than KIT-mutant GISTs (P=0.033). Surprisingly, all four malignant GISTs with KIT exon 11 deletion mutations with primary resistance to imatinib had an increased GSTT1 CN and mRNA expression level of GSTT1. Increased mRNA expression of GSTT1 and ZNF could be predictors of a poor response to imatinib. Our integrative approach reveals that for patients with wild-type (or imatinib-resistant) GISTs, attempts to target VEGFRs and IGF1R may be reasonable options. Citation: Lee EJ, Kang G, Kang SW, Jang K-T, Lee J, et al. (2013) GSTT1 Copy Number Gain and ZNF Overexpression Are Predictors of Poor Response to Imatinib in Gastrointestinal Stromal Tumors. PLoS ONE 8(10): e77219. doi:10.1371/journal.pone.0077219 Editor: Rui Manuel Reis, University de Minho, Portugal Received March 21, 2013; Accepted September 1, 2013; Published October 4, 2013 Copyright: © 2013 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the National Research Foundation of Korea (http://www.nrf.re.kr)(S-2010-0503-000-1) and Samsung Biomedical Research Institute (http://www.sbri.or.kr)(C-A9-203-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] ☯ These authors contributed equally to this work. ¤ Current address: Department of Pathology, Incheon St. Mary Hospital, The Catholic University of Korea, Incheon, Korea Introduction considerable attention due to their sensitivity to tyrosine kinase inhibitors. Gastrointestinal stromal tumors (GISTs) are the most Oncogenic KIT and PDGFRA mutations in GISTs correlate common mesenchymal tumor of the gastrointestinal tract with with tumor phenotype, prognosis, and therapeutic responses to an annual incidence ranging from 11 to 19.6 per million tyrosine kinase inhibitors [4,5]. However, kinase mutation population, which corresponds to between 3,300 and 6,000 status does not fully explain the complex biology of GISTs. new cases per year in the United States [1]. The gold standard Moreover, approximately 85% of pediatric GISTs and 10-15% for treating a localized primary GIST is surgical resection [2]. of adult GISTs do not harbor mutations of KIT or PDGFRA However, tumor recurrence is common and usually occurs in genes (so called ‘wild-type’ GISTs) [6–8]. Although mutations in the liver and/or the peritoneum [3]. GISTs have received BRAF, RAS, and the succinate dehydrogenase (SDH) subunits PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e77219 Integrated aCGH and Expression Profiling of GISTs have recently been identified in a subset of these tumors, the version 4.0. A pool of normal genomic DNA (Promega, tumor-initiating events in wild-type GISTs are still not fully Madison, WI, USA) was used as a reference according to the understood [1]. Moreover, wild-type GISTs are less sensitive to patient’s gender. Data were obtained using Agilent feature imatinib than GISTs harboring mutations in exon 11 of KIT extraction software and analyzed with Agilent Genomic gene. This may in part be due to differences in the ability of Workbench version 6.0 software using the ADM-2 algorithm imatinib to inhibit wild-type versus mutant forms of KIT, but with a sensitivity threshold of 6.0 and a moving average there may be other underlying mechanisms that could be window of 2 Mb or 20 Kb. Minimal overlapping regions of gain uncovered by high-throughput approaches [9–11]. In the future, and loss were determined by assessing the smallest alteration patients with progressive disease may be preselected for regions identified in three or more of the samples [18]. The treatment with imatinib or alternative and/or additional copy number (CN) data are available in Gene Expression therapies based on their KIT/PDGFRA mutational status and Omnibus (GEO) with the accession number GSE47912. predictive gene signatures of drug response [12]. Previous comparative genomic hybridization (CGH) studies Gene expression profiling have shown frequent loss of 14q, 22q, 1p, and 9p (including the genes PARP2, APEX1, NDRG2, SIVA, NF2, ENO1 and Gene expression analysis was carried out using the Agilent CDKN2A/2B), and gain of 8q (including MYC) [13]. Array- 44K Human Gene Expression Array that contains over 41,000 based studies have also demonstrated site-dependent human genes and transcripts. Total mRNAs were extracted chromosomal imbalances in GISTs, indicating that frequent from 15 fresh tissues of three wild-type, one PDGFRA-mutant, losses at 14q are associated with gastric GISTs and losses of and 11 KIT-mutant GISTs using the RNeasy Mini Kit (Qiagen, 1p are related to intestinal GISTs and an aggressive clinical Valencia, CA, USA). RNAs (~200 ng) were reverse-transcribed course [14–16]. However, all previous studies focused on KIT- into cDNAs and quantified using a NanoDrop ND-1000 mutant GISTs, and no studies on wild-type GISTs have been (Thermo, Fisher Scientific, Waltham, MA, USA). A reference reported. To explore potential target genes or mechanisms pool was made by combining equal amounts of RNAs from underlying imatinib resistance in wild-type GISTs, we three schwannomas and four leiomyomas from the stomach. integrated CGH and expression profiling in 32 gastric GISTs, The microarray was hybridized using an Agilent SureHyb including four wild-type GISTs and one imatinib-resistant chamber and incubated in a Rotisserie hybridization oven. PDGFRA D842V mutant GIST. Slides were washed in Gene Expression Wash Buffers and then scanned on an Agilent microarray scanner. Data were Materials and Methods extracted with Agilent feature extraction software and normalized by quantile and VSN (variance stabilizing Case selection normalization) method using Agilent Genespring GX software Written informed consent was obtained from all patients, and 11.5 and Bioconductor packages, respectively. Differences in the present study was conducted after the approval from the expression level between wild-type/PDGFRA-mutant and KIT- Institutional Review Board of Samsung Medical Center. Thirty- mutant GISTs were calculated using the Mann-Whitney U test. two cases of primary gastric GIST were selected based on the The P values were adjusted using the Benjamini and Hochberg availability of fresh-frozen tissue among patients who had false discovery rate method. Differentially expressed genes undergone complete surgical resection (R0) at the institution were selected based on ≥10 fold change with P value <0.001 between 2006 and 2010. Eighteen patients were male and 14 and ≥2 fold change with P value <0.05 for quantile and VSN were female with ages ranging from 34 to 81 years (mean, 62 normalized data, respectively. The expression data are also years). Four risk groups stratified by tumor size and mitotic available in GEO under the accession number GSE47911. counts comprised eight cases each. The presence of mutations in exons 9, 11, 13, and 17 of KIT Quantitative real-time PCR and exons 12, 14, and 18 of PDGRFA were investigated by sequencing these exons using an ABI 3700 automated Genomic DNA and total mRNA were extracted from 43 sequencer (Applied Biosystems, Foster City, CA, USA) as formalin-fixed, paraffin-embedded GISTs using the QIAamp described previously [17]. KIT exon 11 mutations were DNA Mini Kit and RNeasy FFPE Kit (Qiagen). The CN of observed in 26 cases (81%), and consisted of 10 deletion, 5 GSTT1 (Assay ID Hs00659429_cn) was determined using the duplication/insertion, and 11 missense mutations. One case Taqman Copy Number Assay (Applied Biosystems). Total RNA each harbored a missense mutation in KIT exon 17 (N822K) was reverse transcribed using the High Capacity cDNA and PDGFRA exon 18 (D842V). The remaining four (13%) Reverse Transcription Kit (Applied Biosystems) and then GISTs were wild-type for both KIT and PDGFRA genes.