Published OnlineFirst August 11, 2009; DOI: 10.1158/1535-7163.MCT-09-0193 2172 Gene expression signatures and response to imatinib mesylate in gastrointestinal stromal tumor Lori Rink,1 Yuliya Skorobogatko,1 on Statistical Analysis of Microarrays [False Discovery Rate Andrew V. Kossenkov,1 Martin G. Belinsky,1 (FDR), 10%], 38 genes were expressed at significantly low- Thomas Pajak,2 Michael C. Heinrich,3 er levels in the pretreatment biopsy samples from tumors Charles D. Blanke,4 Margaret von Mehren,1 that significantly responded to 8 to 12 weeks of imatinib Michael F. Ochs,5 Burton Eisenberg,6 mesylate, that is, >25% tumor reduction. Eighteen of these genes encoded Krüppel-associated box (KRAB) domain con- and Andrew K. Godwin1 taining zinc finger (ZNF) transcriptional repressors. Impor- 1Department of Medical Oncology, Fox Chase Cancer Center, tantly, 10 KRAB-ZNF genes mapped to a single locus on and 2Radiation Therapy Oncology Group, Philadelphia, chromosome 19p, and a subset predicted likely response Pennsylvania; 3Oregon Health and Science University, Portland, – 4 to imatinib mesylate based therapy in a naïve panel of GIST. Oregon; University of British Columbia and the British Columbia Furthermore, we found that modifying expression of genes Cancer Agency, Vancouver, British Columbia, Canada; 5The Sidney Kimmel Cancer Center, Johns Hopkins University, within this predictive signature can enhance the sensitivity Baltimore, Maryland; and 6Norris Cotton Cancer Center, of GIST cells to imatinib mesylate. Using clinical pretreat- Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire ment biopsy samples from a prospective neoadjuvant phase II trial, we have identified a gene signature that includes KRAB-ZNF 91 subfamily members that may be both predic- Abstract tive of and functionally associated with likely response to Despite initial efficacy of imatinib mesylate in most gastro- short-term imatinib mesylate treatment. [Mol Cancer Ther intestinal stromal tumor (GIST) patients, many experience 2009;8(8):2172–82] primary/secondary drug resistance. Therefore, clinical man- agement of GIST may benefit from further molecular char- acterization of tumors before and after imatinib mesylate Introduction treatment. As part of a recent phase II trial of neoadju- Gastrointestinal stromal tumors (GIST) are the most com- vant/adjuvant imatinib mesylate treatment for advanced mon mesenchymal tumors of the digestive tract, with be- primary and recurrent operable GISTs (Radiation Therapy tween 3,300 to 6,000 new cases diagnosed each year in the Oncology Group S0132), gene expression profiling using ol- United States (1). The most common primary sites for these – igonucleotide microarrays was done on tumor samples ob- neoplasms are the stomach (60 70%; refs. 2, 3), followed by – tained before and after imatinib mesylate therapy. Patients the small intestine (25 35%; refs. 4, 5), and, to a much lesser were classified according to changes in tumor size degree, the colon and rectum (10%; ref. 6). GISTs have also after treatment based on computed tomography scan mea- been observed in the mesentery, omentum, esophagus, and surements. Gene profiling data were evaluated with Sta- the peritoneum (2, 7). GISTs occur most frequently in pa- tistical Analysis of Microarrays to identify differentially tients older than 50 years, with a median age of presentation expressed genes (in pretreatment GIST samples). Based of 58 years; however, GISTs have also been observed in the pediatric population (8). These tumors contain smooth mus- cle and neural elements, as described originally by Mazur and Clark in 1983, and are thought to arise from the inter- Received 12/3/08; revised 5/4/09; accepted 6/3/09; published OnlineFirst 8/11/09. stitial cells of Cajal (9, 10). GISTs express and are clinically Grant support: NIH grants (CA106588 and a supplement from the diagnosed by immunohistochemical (IHC) staining of the Radiation Therapy Oncology Group to U10 CA21661; A.K. Godwin), 145-kDa transmembrane glycoprotein, KIT, by the CD117 LM009382 (M.F. Ochs), an award by the Fox Chase Cancer Center antibody. Most (∼70%) GISTs possess gain-of-function mu- Translational Research Committee as part of the Fox Chase Cancer Center core grant (P30 CA006927; M. vonMehren and A.K. Godwin), and the tations in c-KIT in either exons 9, 11, 13, or 17, causing con- NIH Training Grant (Institutional National Research Service Award) stitutive activation of the kinase receptor, whereas smaller Appointment (CA009035-31; L. Rink). subsets of GISTs possess either gain-of-function mutations The costs of publication of this article were defrayed in part by the PDGFRA ∼ payment of page charges. This article must therefore be hereby marked in (exons 12, 14, or 18; 10%) or no mutations advertisement in accordance with 18 U.S.C. Section 1734 solely to in either KIT or PDGFRA and are therefore referred to as indicate this fact. wild-type GISTs (∼15–20%; refs. 11–14). The primary treat- Note: L. Rink and Y. Skorobogatko contributed equally to this work. ment for GIST is surgical resection, which is often not cura- Current address for A.V. Kossenkov: Wistar Institute, Philadelphia, PA. tive in high risk GIST because of a high incidence of Requests for reprints: Andrew K. Godwin, Department of Medical reoccurrence (15, 16). Since 2002, imatinib mesylate, an oral Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. Phone: 215-728-2756; Fax: 215-728-2741. 2-phenylaminopyrimidine derivative that works as a selec- E-mail: [email protected] tive inhibitor against mutant forms of type III tyrosine Copyright © 2009 American Association for Cancer Research. kinases, such as KIT, PDGFRA, and BCR/ABL, has become doi:10.1158/1535-7163.MCT-09-0193 a standard treatment for patients with metastatic and/or Mol Cancer Ther 2009;8(8). August 2009 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst August 11, 2009; DOI: 10.1158/1535-7163.MCT-09-0193 Molecular Cancer Therapeutics 2173 unresectable GIST, with objective responses or stable Therapy Oncology Group S0132 trial from 18 institutions. disease obtained in >80% of patients (17, 18). Response to Patients' GIST samples were screened for CD117 (KIT) pos- imatinib mesylate has been correlated to the genotype of a itivity by standard IHC before participation in the clinical given tumor (14). GIST patients with exon 11 KIT mutations trial. Patients were required to have adequate hematologic, have the best response and disease-free survival, whereas renal, and hepatic function, as well as measurable disease other KIT mutation types and wild-type GIST have worse for response evaluation. All patients signed informed con- prognoses. Despite the efficacy of imatinib mesylate, some sent following Institutional Review Board approval for this patients experience primary and/or secondary resistance to study and were consented to provide baseline biopsies and the drug. 18F-fluorodeoxyglucose-positron emission tomog- operative tissue. raphy can be used to rapidly assess tumor response to im- Collection of Samples atinib mesylate (19); however, there are cases in which GISTs Tumor samples were obtained from pre–imatinib mesylate do not take up significant amounts of the glucose precursor, core needle biopsies (pretreatment samples) and from the and therefore, this scanning method is of questionable value surgical specimen obtained at the time of resection following in evaluating response in this group of patients. Strategies for neoadjuvant/preoperative imatinib mesylate (posttreatment treatment of progressive disease can include imatinib mesy- samples). A total of 48 pre- and 34 post-imatinib-treated sam- late dose escalation (20), imatinib mesylate in combination ples were collected and banked. All patients received imati- with surgery, and alternative KIT/PDGFRA inhibitors, in- nib mesylate at 600 mg daily by mouth, which was continued cluding sunitinib (21). There are also options to participate daily until the day of surgery, with dose modifications for in clinical trials evaluating nilotinib (22), dasatinib (23), and protocol-defined toxicities. Fresh-frozen pre- and posttreat- HSP90 inhibitors (24). What may eventually prove to be the ment GIST samples were collected from all participating in- most effective paradigm in the clinical management of GIST stitutions and shipped to the Radiation Therapy Oncology is the development of individualized treatment approaches Group tissue bank before evaluation. based on KIT and PDGFRA mutational status and/or predic- RNA Isolation tive gene signatures of drug response. Ideally, in the future, Total RNA was isolated from all available pre- and post- patients may be preselected for treatment with imatinib me- frozen tissue samples using TRIzol reagent, according to the sylate or additional first- and second-line therapies based on protocols provided by the manufacturer (Invitrogen Corp.). these tumor-specific response markers. RNA quantification and quality assessment were done on The question of whether imatinib mesylate can be safe 2100 Bioanalyser (Agilent Technologies). Because of the and effective as a rapid cytoreductive agent if adminis- high variability in tissue collection and handling, storage tered before surgical resection has been evaluated in a re- and shipping procedures among the 18 institutions in- cent novel phase II trial (Radiation Therapy Oncology volved in the study, and the tumor cellularity