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Originally published as JCO Early Release 10.1200/JCO.2006.06.2265 on September 5 2006 © 2006 American Society of Clinical Oncology. Molecular Correlates of Imatinib Resistance in Gastrointestinal Stromal Tumors
From the Division of Hematology/Oncology, Department of Pathology, Oregon Health & Science University Cancer Institute, Oregon Health & Science University; Portland Veterans Affairs Medical Center, Portland, OR; Dana-Farber Cancer Institute; Harvard Cancer Center; Department of Pathology, Brigham & Women's Hospital, Boston, MA; Division of Oncologic Surgery, Hitchcock-Dartmouth Medical Center, Lebanon, NH; Division of Medical Oncology, Fox-Chase Cancer Center, Philadelphia, PA; Novartis Pharmaceuticals Corporation, Hanover, NJ; University of Helsinki, Helsinki; and the Department of Surgery, University of Turku, Turku, Finland Address reprint requests to Michael C. Heinrich, MD, R&D-19 3710 U.S. Veterans Hospital Rd, Portland, OR 97239; e-mail: Heinrich{at}ohsu.edu
PURPOSE: Gastrointestinal stromal tumors (GISTs) commonly harbor oncogenic mutations of the KIT or platelet-derived growth factor alpha (PDGFRA) kinases, which are targets for imatinib. In clinical studies, 75% to 90% of patients with advanced GISTs experience clinical benefit from imatinib. However, imatinib resistance is an increasing clinical problem. PATIENTS AND METHODS: One hundred forty-seven patients with advanced, unresectable GISTs were enrolled onto a randomized, phase II clinical study of imatinib. Specimens from pretreatment and/or imatinib-resistant tumors were analyzed to identify molecular correlates of imatinib resistance. Secondary kinase mutations of KIT or PDGFRA that were identified in imatinib-resistant GISTs were biochemically profiled for imatinib sensitivity. RESULTS: Molecular studies were performed using specimens from 10 patients with primary and 33 patients with secondary resistance. Imatinib-resistant tumors had levels of activated KIT that were similar to or greater than those typically found in untreated GISTs. Secondary kinase mutations were rare in GISTs with primary resistance but frequently found in GISTs with secondary resistance (10% v 67%; P = .002). Evidence for clonal evolution and/or polyclonal secondary kinase mutations was seen in three (18.8%) of 16 patients. Secondary kinase mutations were nonrandomly distributed and were associated with decreased imatinib sensitivity compared with typical KIT exon 11 mutations. Using RNAi technology, we demonstrated that imatinib-resistant GIST cells remain dependent on KIT kinase activity for activation of critical downstream signaling pathways. CONCLUSION: Different molecular mechanisms are responsible for primary and secondary imatinib resistance in GISTs. These findings have implications for future approaches to the growing problem of imatinib resistance in patients with advanced GISTs.
Gastrointestinal stromal tumors (GISTs) are the most common type of sarcoma arising in the digestive tract and are generally distinguished from other abdominal sarcomas by the expression of KIT receptor tyrosine kinase. This kinase is important not only as a diagnostic marker for GISTs, but serves as a primary oncogene in approximately 80% of these tumors, as evidenced by activating mutations of the KIT gene. Imatinib (Gleevec; Novartis Pharmaceuticals, Basel, Switzerland) inhibits the enzymatic activity of several oncogenic tyrosine kinases, including KIT and platelet-derived growth factor alpha (PDGFRA). In clinical studies, 75% to 90% of patients with advanced GISTs treated with imatinib experienced a clinical benefit (stable disease + complete/partial objective response).1-3 These imatinib-induced responses correlated with tumor kinase mutational status.4,5 Patients with KIT exon 11-mutant GIST have a higher response rate and a significantly longer median survival compared with patients with exon 9-mutant GISTs, and those whose GISTs lack KIT or PDGFRA mutations.4 Although imatinib has revolutionized the treatment of advanced GISTs, clinical resistance to this drug has proved to be a significant problem with more prolonged follow-up. Secondary kinase mutations acquired during imatinib treatment have been reported in several small series.6-15 In this study we evaluated molecular markers of imatinib resistance in a series of well-documented GIST samples from a phase II study of imatinib.
Patient Materials Tumor samples were obtained from patients enrolled in the CSTI571 B2222 phase II trial of imatinib (sponsored by Novartis, Basel, Switzerland) for the treatment of advanced GIST.2,4 Of 147 original patients, 92 had documented disease-related treatment failure as of May, 2005. Samples were obtained from 43 progressive-disease patients who consented to analysis of their tumors. Per the clinical protocol, tumor progression was defined according to traditional Southwest Oncology Group response criteria.16 Specifically, progressive disease was defined as either a 50% or greater increase in the of sum of the products of all measurable lesions over the smallest sum observed (or baseline if no decrease was observed); clear worsening from previous examinations of any assessable disease; reappearance of any lesion that had disappeared; appearance of a new lesion; or the failure to return for evaluation due to death or deteriorating clinical condition. The appearance of a nodule within a mass on follow-up imaging studies was not considered to meet the protocol-specified definition of progression. The study was approved by the local institutional review board of each participating institution, and written informed consent was obtained from each patient. In addition, informed consent for the analysis of tumor-associated genetic alterations was obtained independently of patient consent for participation in the clinical study. Imatinib response correlates were obtained using paired GIST biopsies taken during the week before initiation of imatinib and biopsies taken during the first week of therapy. Imatinib resistance correlates were evaluated in biopsies taken at the time of GIST progression while patients were still receiving imatinib. Routine pathology review, including KIT immunostaining, was performed on all biopsies.
Reagents and Cell Lines
KIT Short Hairpin RNA Studies
Immunoblotting Studies
Mutation Detection Methods
PCR Evaluations of the Allelic Relationships of Primary and Secondary Mutations Allelic relationships of primary and secondary KIT mutations were evaluated by reverse transcriptase-PCR (exons 8-18) or genomic PCR (exons 11 to 13) using primer pairs listed in Table 1. The resultant PCR products were cloned and individual bacterial colonies were bidirectionally sequenced.23
Description of Patient Population The study population consisted of 147 patients treated in a randomized, phase II study of imatinib for patients with advanced GISTs.2,24 As of May 2005, the median time to treatment failure for the entire phase II study population was 19.3 months and the median survival was estimated to be 57 months. Late treatment failure was primarily due to disease progression rather than discontinuation of therapy related to medication intolerance. Treatment failures were divided into two groups. Patients who never achieved a partial remission and showed continued tumor growth during the first 180 days of treatment were defined as having primary imatinib resistance. Disease progression after a minimum of 6 months of partial remission or stable disease was defined as secondary imatinib resistance. This study is based on comparative analyses of tumor samples obtained from both these groups before and after treatment failure.
KIT Kinase Is Inhibited by Imatinib in Responding GISTs, but Not in Tumors With Primary Resistance
We analyzed the activation status of KIT-dependent signaling pathways in biopsies obtained from two patients whose GISTs had a KIT exon 9 mutation and showed primary imatinib resistance (Fig 2A and Table 3). Frozen tumor samples were not available from either patient pretreatment; nevertheless, on-treatment biopsies contained phosphorylated KIT protein in quantities equivalent to that typically present in untreated GIST specimens.21 The pattern of intracellular signaling was similar to that seen in untreated GISTs (Fig 1 and Duensing et al21), indicating that primary resistance is associated with persistent KIT phosphorylation and activation of downstream AKT and MAPK pathways.
Reactivation of KIT and KIT-Dependent Signaling in Secondary Resistance Two patients who had objective responses to imatinib underwent tumor biopsy after documented progression at 18 and 32 months. Matched pretreatment biopsies were available for both patients (Fig 2B and Table 2). Patient 1 had a primary KIT exon 11 deletion. Patient 14 had two activating KIT mutations in multiple pretreatment specimens (K642E and N822K). This is the only such example of double mutations in our series of more than 1,000 imatinib-naïve patients with GISTs.17,25 We speculate that one mutation was the initiating mutation and the other developed during malignant progression. The imatinib-resistant tumors contained abundant amounts of activated KIT. Notably, the ratio of phosphorylated-AKT (P-AKT) to AKT was markedly increased in the progression samples compared with matched pretreatment samples. In contrast, MAPK was only significantly activated in one of the progression samples (patient 14).
Secondary Kinase Mutations Are Common in Secondary but Not Primary Imatinib Resistance In 10 patients with primary resistance the median time to treatment failure was 3.6 months (range, 0.8 to 5.8; Table 3). No secondary mutations of KIT or PDGFRA kinase domain were found in specimens obtained from these patients at the time of progression. Patient 39 had primary progression on a dose of 400 mg of imatinib. No secondary kinase mutations were found at the time of progression on 400 mg, and cross-over to 600 mg led to disease stablilization. Twenty-three months later, the patient had tumor progression and ultimately underwent surgical debulking. Specimens from this procedure had multiple secondary kinase mutations (Table 3 and Fig 3).
In thirty-three patients with secondary resistance, the median time to treatment failure was 20.2 months (range, 7.2 to 52.7). Sixty-one progression specimens obtained from these 33 patients were judged to contain GISTs. The original mutation, which was known for 29 of 33 patients based on analysis of preimatinib specimens, was confirmed in all progression biopsies (54 of 54 samples). Overall, 22 (67%) of 33 patients with secondary resistance had one or more secondary kinase mutations (21 KIT and 1 PDGFRA; Table 2 and Fig 3). This frequency is distinctly higher than that in patients with primary resistance (10% v 67%; P = .002). Notably, all secondary KIT kinase mutations were found in GISTs with an underlying primary KIT mutation and these secondary mutations were not present in pretreatment specimens. No secondary mutations were identified in one GIST lacking a primary KIT or PDGFRA mutation. In addition, the only secondary PDGFRA mutation identified was in a GIST with a primary PDGFRA V561D mutation. The secondary KIT kinase mutations were nonrandom and involved either the adenosine triphosphate (ATP) binding pocket of the kinase domain (V654A, T670I) or the kinase activation loop (C809G, D816H, D820A/E/G, N822K/Y, Y823D). Figure 3 summarizes the spectrum and frequency of secondary KIT mutations in this series and other published reports. In contrast to primary resistance, two of three KIT exon 9 mutant GISTs were found to have additional KIT kinase mutations in biopsies obtained after the development of secondary imatinib resistance. However, the frequency of secondary KIT mutations in KIT exon 9 mutant GISTs was not obviously different between cases with primary resistance versus secondary resistance (1 of 7 v 2 of 3; P = .18).
Evidence for Clonal Evolution and/or Polyclonal Secondary Resistance Mutations in Individual Patients
Patient 5 had a baseline KIT V560D substitution on one allele and a single adenine base insertion in KIT exon 13 of the other allele, resulting in a frameshift and protein truncation. Thus, this patient's tumor was functionally homozygous due to loss of the wild-type allele. In specimens obtained at the time of imatinib resistance, a V654A mutation was present in two biopsy specimens and D816H in a third specimen. All three specimens had the original preimatinib KIT exon 11 and 13 mutations.
Allelic Distribution of Secondary Kinase Mutations
Secondary Kinase Mutations Lead to Imatinib Resistance In Vitro Several of the secondary KIT mutations identified in our series have been previously identified in human cancers: D816H, D820A, N822H/K, and Y823D.4,17,18,26-30 In contrast, there are no reports of V654A, T670I, or C809G mutations occurring as a primary mutation.
The isolated KIT V560D mutation was very sensitive to imatinib (IC50
A number of amino acids located in or near the KIT activation loop were mutated in imatinib-resistant tumors (Fig 3 and Table 2). In vitro profiling of isolated activation loop mutations revealed a spectrum of imatinib sensitivity ranging from relatively sensitive (IC50 of 100-200 nmol/L, N882K, Y823D) to highly resistant (IC50 > 5,000 nmol/L, D816H). When coexpressed with V560D, all of the double mutant kinases were extremely resistant to imatinib, with the exception of V560D + N822K which was only moderately imatinib resistant (Fig 5A). Similar results were obtained using KIT exon 9 AY-insertion or K642E as the primary mutation (Fig 5B). Notably, both primary mutations were intrinsically imatinib sensitive, but coexpression of D820G with KIT exon 9 resulted in moderate imatinib resistance. Coexpression of the imatinib-sensitive K642E mutation with D816H resulted in extreme imatinib resistance.
A doubly mutant (K642E + N822H) kinase modeled on patient 14 was moderately resistant to imatinib (IC50 of  2 µmol/L). Notably, patient 14 received a dose of 600 mg daily of imatinib and experienced an objective partial response that lasted 500 days. The C809G mutation found at the time of progression proved resistant in vitro (IC50 > 5 µmol/L), both in isolation and when coexpressed with KIT K642E + N822H. In patient 7, a primary PDGFRA V561D mutation (known to be sensitive to imatinib) was accompanied by a PDGFRA D842V mutation at the onset of progression. The doubly mutant V561D + D842V kinase proved imatinib-resistant with an IC50 similar to the isolated D842V isoform (Fig 6). 17
GIST Cell Lines With Secondary Kinase Mutations Have In Vitro Imatinib Resistance To further validate our biochemical studies, we generated cell lines from two patients with secondary imatinib resistance (neither patient was part of the phase II study). The GIST48 cell line has a combination of mutations (homozygous V560D + heterozygous D820A) similar to that documented in samples from patient 19. Concentrations of imatinib higher than 1 µmol/L were required for complete inhibition of KIT activation in this cell line (Fig 7A). This concentration is 10-fold greater than that necessary to block exon 11 mutant isoforms of KIT in GIST cells.8,21,31 Activation of AKT was partially but not completely inhibited by imatinib doses of 0.1 to 5 µmol/L.
The GIST430 cell line is heterozygous for a KIT exon 11 deletion mutation and the V654A substitution (both on the same allele). Eight samples from imatinib-resistant tumors in our study had a similar combination of KIT exon 11 and V654A mutations. The doubly mutant KIT isoform expressed by GIST430 cells was imatinib resistant (IC50 of 2.5 µmol/L; IC90 of > 5 µmol/L). AKT activation in this cell line was only partially inhibited by imatinib doses higher than 2.5 µmol/L (Fig 7A). We used RNAi to determine whether KIT expression was still required for activation of signaling pathways in imatinib-resistant GIST cells. shRNA knockdown of total KIT expression in the previously described imatinib-sensitive GIST882 cell line (homozygous KIT K642E mutation)31 resulted in parallel decreases in phospho-KIT, phospho-AKT, phospho-p70S6K, and the proliferation marker cyclin A expression (Fig 7B). In contrast, there was no change in the expression of p70S6K, AKT, or PI3K. Comparable findings were obtained after KIT shRNA knockdown in GIST430 cells, demonstrating that activation of proliferation/survival signaling pathways remains KIT dependent in this imatinib-resistant cell line. KIT knockdown in the cell lines also induced flow-cytometric evidence for G1 block, decreased S phase, and markedly increased apoptosis (data not shown).
We analyzed genomic mechanisms of imatinib resistance in a cohort of patients that were part of a randomized phase II study of imatinib for treatment of advanced, unresectable GIST. Forty-three (46.7%) of 92 patients with disease-related treatment failures consented to studies of their tumor samples. The resulting collection of 83 specimens from well-monitored patients receiving standardized imatinib treatment is the largest examined to date for molecular mechanisms related to drug resistance. Imatinib-resistant GISTs show activation of the same signaling pathways that are operative in untreated GISTs. Interestingly, primary imatinib resistance is infrequently associated with secondary kinase mutations (1 of 10; 10%). PDGFRA D842V and KIT exon 9 mutations were significantly over-represented in this group compared with secondary imatinib resistance. GISTs with these genotypes are less responsive to imatinib therapy than GISTs with an associated KIT exon 11 mutation.4,5 The PDGFRA D842V mutation has moderate to high level in vitro resistance to imatinib explaining why this mutation was responsible for primary imatinib resistance in the two patients with a primary PDGFRA D842V mutation.4,8,32 This conclusion is strengthened by our observation that the PDGFRA D842V mutation was associated with secondary imatinib resistance in a GIST harboring a primary PDGFRA V561D mutation. Debiec-Rychter et al8 also reported a case of imatinib-resistant GIST associated with acquisition of a PDGFRA D842V mutation in a GIST with a primary KIT exon 11 mutation. The molecular mechanisms underlying primary resistance in GISTs with KIT exon 9 mutations are as yet unidentified, but may be related to differences in the underlying biology of this subset of GISTs. We speculate that some KIT exon 9 mutant GISTs have an alternative mechanism of KIT activation that does not require the enzymatic activity of KIT. Notably, imatinib only binds to the inactive form of KIT.33,34 Therefore, KIT-independent mechanisms of KIT phosphorylation could stabilize the protein in an active, imatinib-resistant conformation. GISTs with KIT exon 11 mutations commonly become imatinib resistant due to acquisition of secondary kinase mutations located on the same allele as the original gain of function KIT mutation. These secondary kinase mutations appear to be the predominant mechanism for late imatinib resistance in KIT exon 11 mutant GISTs. Fluorescence in situ hybridization assays (data not shown) revealed low-level KIT amplification (3 to 4 copies per cell), accompanied by KIT protein overexpression, in only two patients, suggesting that genomic KIT amplification has only a minor role in GIST imatinib resistance. We believe that the difference in the frequency of secondary mutations between GISTs with underlying KIT exon 11 versus KIT exon 9 mutations is reflective of the underlying biology of these tumors—namely, that KIT exon 11 mutant GISTs are more addicted to KIT signaling than KIT exon 9 mutant GISTs. In this model, KIT exon 9 mutant GISTs are able to use alternative signaling mechanisms to drive cellular proliferation. In contrast, KIT exon 11 mutant GISTs are more restricted in their signaling requirements and the most common mechanism of resistance is expansion of a clone with an imatinib-resistant kinase mutation. Using RNAi KIT knockdown, we demonstrated that imatinib-resistant GIST cell lines with secondary kinase mutations remain dependent on KIT signaling for growth and survival. These findings have implications for future approaches to the growing problem of imatinib resistance in patients with advanced GISTs.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-$99,999 (C)
We thank the Fletcher laboratory: Nora Joseph and Bryna McConarty for assistance with cell culture and immunoblotting studies; the Heinrich-Corless laboratories: Diana Griffith, Ajia Town, Troy Bainbridge, Laura McGreevey, Tina Harrell, Arin Schroeder, Amy Harlow, Claudia Le; and Ashley Edwards for assistance in creating the figures for this article.
published online ahead of print at www.jco.org on September 5, 2006. Supported in part by Novartis Pharmaceuticals, VA Merit Review Grant (M.C.H.), GIST Cancer Research Fund (M.C.H.), B.P., Lester and Regina John Foundation (M.C.H.). Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Verweij J, Casali PG, Zalcberg J, et al: Progression-free survival in gastrointestinal stromal tumours with high-dose imatinib: Randomised trial. Lancet 364:1127-1134, 2004[CrossRef][Medline] 2. Demetri GD, von Mehren M, Blanke CD, et al: Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors. N Engl J Med 347:472-480, 2002 3. Van Oosterom AT, Judson I, Verweij J, et al: Safety and efficacy of imatinib (STI571) in metastatic gastrointestinal stromal tumours: A phase I study. Lancet 358:1421-1423, 2001[CrossRef][Medline] 4. Heinrich MC, Corless CL, Demetri GD, et al: Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 21:4342-4349, 2003 5. Debiec-Rychter M, Dumez H, Judson I, et al: Use of c-KIT/PDGFRA mutational analysis to predict the clinical response to imatinib in patients with advanced gastrointestinal stromal tumours entered on phase I and II studies of the EORTC Soft Tissue and Bone Sarcoma Group. Eur J Cancer 40:689-695, 2004[CrossRef][Medline] 6. Antonescu CR, Besmer P, Guo T, et al: Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation. Clin Cancer Res 11:4182-4190, 2005 7. Chen LL, Trent JC, Wu EF, et al: A missense mutation in KIT kinase domain 1 correlates with imatinib resistance in gastrointestinal stromal tumors. Cancer Res 64:5913-5919, 2004 8. Debiec-Rychter M, Cools J, Dumez H, et al: Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants. Gastroenterology 128:270-279, 2005[CrossRef][Medline] 9. Tamborini E, Bonadiman L, Greco A, et al: A new mutation in the KIT ATP pocket causes acquired resistance to imatinib in a gastrointestinal stromal tumor patient. Gastroenterology 127:294-299, 2004[CrossRef][Medline] 10. Tamborini E, Gabanti E, Lagonigro MS, et al: KIT/Val654 Ala receptor detected in one imatinib-resistant GIST patient. Cancer Res 65:1115, 2005 11. Wakai T, Kanda T, Hirota S, et al: Late resistance to imatinib therapy in a metastatic gastrointestinal stromal tumour is associated with a second KIT mutation. Br J Cancer 90:2059-2061, 2004[Medline] 12. Wardelmann E, Thomas N, Merkelbach-Bruse S, et al: Acquired resistance to imatinib in gastrointestinal stromal tumours caused by multiple KIT mutations. Lancet Oncol 6:249-251, 2005[Medline] 13. Grimpen F, Yip D, McArthur G, et al: Resistance to imatinib, low-grade FDG-avidity on PET, and acquired KIT exon 17 mutation in gastrointestinal stromal tumour. Lancet Oncol 6:724-727, 2005[Medline] 14. McLean SR, Gana-Weisz M, Hartzoulakis B, et al: Imatinib binding and cKIT inhibition is abrogated by the cKIT kinase domain I missense mutation Val654Ala. Mol Cancer Ther 4:2008-2015, 2005 15. Wardelmann E, Merkelbach-Bruse S, Pauls K, et al: Polyclonal evolution of multiple secondary KIT mutations in gastrointestinal stromal tumors under treatment with imatinib mesylate. Clin Cancer Res 12:1743-1749, 2006 16. Green S, Weiss GR: Southwest Oncology Group standard response criteria, endpoint definitions and toxicity criteria. Invest New Drugs 10:239-253, 1992[CrossRef][Medline] 17. Corless CL, Schroeder A, Griffith D, et al: PDGFRA mutations in gastrointestinal stromal tumors: Frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol 23:5357-5364, 2005 18. Heinrich MC, Corless CL, Duensing A, et al: PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299:708-710, 2003 19. National Institutes of Health: Recombinant DNA and gene transfer: Guidelines for research involving recombinant DNA molecules. http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html 20. Duensing A, Joseph NE, Medeiros F, et al: Protein kinase C theta (PKCtheta) expression and constitutive activation in gastrointestinal stromal tumors (GISTs). Cancer Res 64:5127-5131, 2004 21. Duensing A, Medeiros F, McConarty B, et al: Mechanisms of oncogenic KIT signal transduction in primary gastrointestinal stromal tumors (GISTs). Oncogene 23:3999-4006, 2004[CrossRef][Medline] 22. Corless CL, McGreevey L, Town A, et al: KIT gene deletions at the intron 10-exon 11 boundary in GI stromal tumors. J Mol Diagn 6:366-370, 2004 23. Thompson JR, Marcelino LA, Polz MF: Heteroduplexes in mixed-template amplifications: Formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Res 30:2083-2088, 2002 24. Joensuu H, Roberts PJ, Sarlomo-Rikala M, et al: Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 1052:1052-1056, 2001 25. Corless CL, Fletcher JA, Heinrich MC: Biology of gastrointestinal stromal tumors. J Clin Oncol 22:3813-3825, 2004 26. Tian Q, Frierson HFJ, Krystal GW, et al: Activating c-kit gene mutations in human germ cell tumors. Am J Pathol 154:1643-1647, 1999 27. Kemmer K, Corless CL, Fletcher JA, et al: KIT mutations are common in testicular seminomas. Am J Pathol 164:305-313, 2004 28. Rubin BP, Singer S, Tsao C, et al: KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res 61:8118-8121, 2001 29. Hirota S, Nishida T, Isozaki K, et al: Familial gastrointestinal stromal tumors associated with dysphagia and novel type germline mutation of KIT gene. Gastroenterology 122:1493-1499, 2002[CrossRef][Medline] 30. Goemans BF, Zwaan CM, Miller M, et al: Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia. Leukemia 19:1536-1542, 2005[CrossRef][Medline] 31. Tuveson DA, Willis NA, Jacks T, et al: STI571 inactivation of the gastrointestinal stromal tumor c-KIT oncoprotein: Biological and clinical implications. Oncogene 20:5054-5058, 2001[CrossRef][Medline] 32. Hirota S, Ohashi A, Nishida T, et al: Gain-of-function mutations of platelet-derived growth factor receptor alpha gene in gastrointestinal stromal tumors. Gastroenterology 125:660-667, 2003[CrossRef][Medline] 33. Mol CD, Dougan DR, Schneider TR, et al: Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase. J Biol Chem 279:31655-31663, 2004 34. Mol CD, Fabbro D, Hosfield DJ: Structural insights into the conformational selectivity of STI-571 and related kinase inhibitors. Curr Opin Drug Discov Devel 7:639-648, 2004[Medline] Submitted February 17, 2006; accepted June 23, 2006.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
R8.91, and pMD.G helper virus packaging plasmids (at a 10:10:1 ratio) into 293T cells. These transfections were performed using lipofectamine and PLUS reagent (Invitrogen, Carlsbad, CA), and lentivirus supernatants were harvested at 24, 36, 48, and 60 hours. Viral titers were determined in GIST882 cells, according to a protocol from Invitrogen. GIST882 or GIST430 cells were infected, in the presence of 8 µg/mL of polybrene, and were then lysed for western blot analysis at 72 hours postinfection. 
$100,000 (N/R) Not Required
