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1.  Sorafenib Inhibits Many Kinase Mutations Associated with Drug-Resistant Gastrointestinal Stromal Tumors 
Molecular cancer therapeutics  2012;11(8):1770-1780.
Sorafenib has substantial clinical activity as third- or fourth-line treatment of imatinib- and sunitinib-resistant gastrointestinal stromal tumors (GIST). Because sorafenib targets both angiogenesis-related kinases (VEGFR) and the pathogenetic kinases found in GIST (KIT or PDGFRA), the molecular basis for sorafenib efficacy in this setting remains unknown. We sought to determine the spectrum of activity of sorafenib against different mutant kinases associated with drug-sensitive and drug-resistant GIST. We compared the activity of imatinib and sorafenib against transiently expressed mutant forms of KIT and PDGFRA, including various secondary mutations that have been identified in imatinib-resistant or sunitinib-resistant GISTs. We also examined these drugs against four GIST cell lines, three of which are imatinib resistant. In our in vitro studies, we determined that sorafenib inhibited imatinib-resistant mutations in exons encoding the ATP/drug-binding pocket and in exons encoding the activation loop, with the exception of substitutions at KIT codon D816 and PDGFRA codon 842. Notably our data indicate that sorafenib is more effective than imatinib or sunitinib for inhibiting the kinase activity of drug-resistant KIT mutants (as assessed by biochemical IC50). We hypothesize that a major determinant of the efficacy of sorafenib for treatment of advanced GIST is the activity of this agent against KIT or PDGFRA-mutant kinases. These results have implications for the further development of treatments for drug-resistant GIST.
PMCID: PMC3992122  PMID: 22665524
2.  Quadruple wild-type (WT) GIST: defining the subset of GIST that lacks abnormalities of KIT, PDGFRA, SDH, or RAS signaling pathways 
Cancer Medicine  2014;4(1):101-103.
A subset of GISTs lack mutations in the KIT/PDGFRA or RAS pathways and yet retain an intact succinate dehydrogensase (SDH) complex. We propose that these KIT/PDGFRA/SDH/RAS-P WT GIST tumors be designated as quadruple wild-type (WT) GIST. Further molecular and clinicophatological characterization of quadruple WT GIST will help to determine their prognosis as well as assist in the optimization of medical management, including clinical test of novel therapies.
PMCID: PMC4312123  PMID: 25165019
BRAF; gastrointestinal stromal tumors; GIST; NF-1; quadruple negative; quadruple WT; RAS; SDH deficiency; SDHA; SDHB; wild type
3.  Efficacy and Safety of Regorafenib in Patients With Metastatic and/or Unresectable GI Stromal Tumor After Failure of Imatinib and Sunitinib: A Multicenter Phase II Trial 
Journal of Clinical Oncology  2012;30(19):2401-2407.
Metastatic GI stromal tumor (GIST) is a life-threatening disease with no therapy of proven efficacy after failure of imatinib and sunitinib. Regorafenib is a structurally unique inhibitor of multiple cancer-associated kinases, including KIT and platelet-derived growth factor receptor (PDGFR), with broad-spectrum anticancer activity in preclinical and early-phase trials. Because KIT and PDGFR-α remain drivers of GIST after resistance to imatinib and sunitinib, we performed a multicenter single-stage phase II trial of regorafenib in patients with advanced GIST after failure of at least imatinib and sunitinib.
Patients and Methods
Patients received regorafenib orally, 160 mg daily, on days 1 to 21 of a 28-day cycle. Disease assessment was performed every two cycles per RECIST 1.1. Primary end point was clinical benefit rate (CBR), defined as objective responses (ie, complete or partial response [PR] as well as stable disease [SD] ≥ 16 weeks). Serial tumor biopsies were obtained from consenting patients whenever possible.
From February to December 2010, 34 patients were enrolled at four US centers. As of July 28, 2011, 33 patients had received at least two cycles of regorafenib (range, two to 17 cycles). CBR was 79% (95% CI, 61% to 91%). Four patients achieved PR, and 22 exhibited SD ≥ 16 weeks. Median progression-free survival was 10.0 months. The most common grade 3 toxicities were hypertension and hand-foot-skin reaction.
Regorafenib has significant activity in patients with advanced GIST after failure of both imatinib and sunitinib. A phase III trial of regorafenib versus placebo is ongoing to define more fully the safety and efficacy of regorafenib in this setting.
PMCID: PMC3675695  PMID: 22614970
4.  Gene expression of the IGF pathway family distinguishes subsets of gastrointestinal stromal tumors wild type for KIT and PDGFRA 
Cancer Medicine  2013;2(1):21-31.
Gastrointestinal stromal tumors (GISTs) arise from the interstitial cells of Cajal (ICCs) and are the most common mesenchymal neoplasm of the gastrointestinal tract. While the majority of GISTs harbor activating mutations in either the v-kit Hardy-Zuckerman feline sarcoma viral oncogene homolog (KIT) or platelet-derived growth factor receptor alpha (PDGFRA) tyrosine kinases, approximately 10–15% of adult GISTs and 85% of pediatric GISTs lack such mutations. These “wild-type” GISTs have been reported to express high levels of the insulin-like growth factor 1 receptor (IGF1R), and IGF1R-targeted therapy of wild-type GISTs is being evaluated in clinical trials. However, it is not clear that all wild-type GISTs express IGF1R, because studies to date have predominantly focused on a particular subtype of gastric wild-type GIST that is deficient in the mitochondrial succinate dehydrogenase (SDH) complex. This study of a series of 136 GISTs, including 72 wild-type specimens, was therefore undertaken to further characterize wild-type GIST subtypes based on the relative expression of transcripts encoding IGF1R. Additional transcripts relevant to GIST biology were also evaluated, including members of the IGF-signaling pathway (IGF1, IGF2, and insulin receptor [INSR]), neural markers (CDH2[CDH: Cadherin], neurofilament, light polypeptide, LHX2 [LHX: LIM homeobox], and KIRREL3 [KIRREL: kin of IRRE like]), KIT, PDGFRA, CD34, and HIF1A. Succinate dehydrogenase complex, subunit B protein expression was also assessed as a measure of SDH complex integrity. In addition to the previously described SDH-deficient, IGF1Rhigh wild-type GISTs, other SDH-intact wild-type subpopulations were defined by high relative expression of IGF1R, neural markers, IGF1 and INSR, or low IGF1R coupled with high IGF2. These results underscore the complexity and heterogeneity of wild-type GISTs that will need to be factored into molecularly-targeted therapeutic strategies.
PMCID: PMC3797556  PMID: 24133624
Gastrointestinal stromal tumor; IGF1R; wild type
5.  Integrated genomic study of quadruple-WT GIST (KIT/PDGFRA/SDH/RAS pathway wild-type GIST) 
BMC Cancer  2014;14(1):685.
About 10-15% of adult gastrointestinal stromal tumors (GIST) and the vast majority of pediatric GIST do not harbour KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations (J Clin Oncol 22:3813–3825, 2004; Hematol Oncol Clin North Am 23:15–34, 2009). The molecular biology of these GIST, originally defined as KIT/PDGFRA wild-type (WT), is complex due to the existence of different subgroups with distinct molecular hallmarks, including defects in the succinate dehydrogenase (SDH) complex and mutations of neurofibromatosis type 1 (NF1), BRAF, or KRAS genes (RAS-pathway or RAS-P).
In this extremely heterogeneous landscape, the clinical profile and molecular abnormalities of the small subgroup of WT GIST suitably referred to as quadruple wild-type GIST (quadrupleWT or KITWT/PDGFRAWT/SDHWT/RAS-PWT) remains undefined. The aim of this study is to investigate the genomic profile of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, by using a massively parallel sequencing and microarray approach, and compare it with the genomic profile of other GIST subtypes.
We performed a whole genome analysis using a massively parallel sequencing approach on a total of 16 GIST cases (2 KITWT/PDGFRAWT/SDHWT and SDHBIHC+/SDHAIHC+, 2 KITWT/PDGFRAWT/SDHAmut and SDHBIHC-/SDHAIHC- and 12 cases of KITmut or PDGFRAmut GIST). To confirm and extend the results, whole-genome gene expression analysis by microarray was performed on 9 out 16 patients analyzed by RNAseq and an additional 20 GIST patients (1 KITWT/PDGFRAWTSDHAmut GIST and 19 KITmut or PDGFRAmut GIST). The most impressive data were validated by quantitave PCR and Western Blot analysis.
We found that both cases of quadrupleWT GIST had a genomic profile profoundly different from both either KIT/PDGFRA mutated or SDHA-mutated GIST. In particular, the quadrupleWT GIST tumors are characterized by the overexpression of molecular markers (CALCRL and COL22A1) and of specific oncogenes including tyrosine and cyclin- dependent kinases (NTRK2 and CDK6) and one member of the ETS-transcription factor family (ERG).
We report for the first time an integrated genomic picture of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, using massively parallel sequencing and gene expression analyses, and found that quadrupleWT GIST have an expression signature that is distinct from SDH-mutant GIST as well as GIST harbouring mutations in KIT or PDGFRA. Our findings suggest that quadrupleWT GIST represent another unique group within the family of gastrointestintal stromal tumors.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-685) contains supplementary material, which is available to authorized users.
PMCID: PMC4181714  PMID: 25239601
Gastrointestinal stromal tumors (GIST); Wild-type; KIT; PDGFRA; Succinate dehydrogenase; SDHA; RAS; QuadrupleWT
7.  Phase II Trial of Neoadjuvant/adjuvant Imatinib Mesylate for Advanced Primary and Metastatic/recurrent Operable Gastrointestinal Stromal Tumors: Long-term Follow-up Results of Radiation Therapy Oncology Group 0132 
Annals of surgical oncology  2011;19(4):1074-1080.
Imatinib inhibits the KIT and PDGFR tyrosine kinases, resulting in its notable antitumor activity in gastrointestinal stromal tumor (GIST). We previously reported the early results of a multi-institutional prospective trial (RTOG 0132) using neoadjuvant/adjuvant imatinib either in primary resectable GIST or as a planned preoperative cytoreduction agent for metastatic/recurrent GIST.
Patients with primary GIST (≥5 cm, group A) or resectable metastatic/recurrent GIST (≥2 cm, group B) received neoadjuvant imatinib (600 mg/day) for approximately 2 months and maintenance postoperative imatinib for 2 years. We have now updated the clinical outcomes including progression-free survival, disease-specific survival, and overall survival at a median follow-up of 5.1 years, and we correlate these end points with duration of imatinib therapy.
Sixty-three patients were originally entered (53 analyzable: 31 in group A and 22 in group B). Estimated 5-year progression-free survival and overall survival were 57% in group A, 30% in group B; and 77% in group A, 68% in group B, respectively. Median time to progression has not been reached for group A and was 4.4 years for group B. In group A, in 7 of 11 patients, disease progressed >2 years from registration; 6 of 7 patients with progression had stopped imatinib before progression. In group B, disease progressed in 10 of 13 patients >2 years from registration; 6 of 10 patients with progressing disease had stopped imatinib before progression. There was no significant increase in toxicity compared with our previous shortterm analysis.
This long-term analysis suggests a high percentage of patients experienced disease progression after discontinuation of 2-year maintenance imatinib therapy after surgery. Consideration should be given to studying longer treatment durations in intermediate- to high-risk GIST patients.
PMCID: PMC3800166  PMID: 22203182
8.  Regorafenib Induces Rapid and Reversible Changes in Plasma Nitric Oxide and Endothelin-1 
American Journal of Hypertension  2012;25(10):1118-1123.
Hypertension is a toxicity of antiangiogenic therapies and a possible biomarker that identifies patients with superior cancer outcomes. Understanding its mechanism will aid in treatment and could lead to the development of other biomarkers for predicting toxicity and anticancer efficacy. Recent evidence implicates nitric oxide (NO) suppression and endothelin-1 (ET-1) stimulation as potential mechanisms leading to antiangiogenic therapy-induced hypertension. The aim of this study was to evaluate the effects of regorafenib, a novel broad-spectrum kinase inhibitor with activity against multiple targets, including vascular endothelial growth factor receptor 2 inhibition, on NO and ET-1 levels.
Regorafenib was administered to 32 subjects with gastrointestinal stromal tumor on a 3-week-on, 1-week-off basis. Plasma levels of NO and ET-1 were measured at baseline, 2, 4, and 6 weeks of therapy. Data analysis was by Wilcoxon rank-sum and paired t-tests.
Twenty subjects (63%) developed regorafenib-induced hypertension. Two weeks after starting regorafenib therapy, plasma ET-1 levels increased (25% increase, P < 0.05) and NO was suppressed (20% decrease, P < 0.05). These normalized after 1-week washout but ET-1 rose again by 30% (P < 0.05) and NO fell by 50% (P < 0.05) after restarting regorafenib.
These findings indicate that regorafenib induces a coordinated and reversible suppression of NO and stimulation of ET-1. Whether NO and ET-1 might predict therapeutic efficacy in these patients requires further study.
American Journal of Hypertension, advance online publication 12 July 2012. doi:10.1038/ajh.2012.97
PMCID: PMC3578478  PMID: 22785409
antiangiogenic therapy; blood pressure; endothelin-1; hypertension; nitric oxide
9.  Mitotic recombination as evidence of alternative pathogenesis of gastrointestinal stromal tumours in neurofibromatosis type 1 
Journal of Medical Genetics  2007;44(1):e61.
Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder resulting in the growth of a variety of tumours, and is inherited in an autosomal dominant pattern. Gastrointestinal stromal tumours (GISTs) are mesenchymal tumours that commonly harbour oncogenic mutations in KIT or PDGFRA and are thought to arise from the interstitial cells of Cajal (ICC; the pacemaker cells of the gut).
To characterise two patients with NF1 and GISTs.
Two patients were genotyped for germline mutations in NF1. GISTs from both patients were genotyped for somatic mutations in KIT and PDGFRA. Loss of heterozygosity (LOH) of NF1 in one GIST was assessed by genotyping seven microsatellite markers spanning 2.39 Mb of the NF1 locus in the tumour and in genomic DNA. The known germline mutation in NF1 was confirmed in GIST DNA by sequencing. The copy number of the mutated NF1 allele was determined by multiplex ligand‐dependent probe amplification.
GISTs from both patients were of wild type for mutations in KIT and PDGFRA. In the GIST with adequate DNA, all seven markers were informative and showed LOH at the NF1 locus; sequencing of NF1 from that GIST showed no wild‐type sequence, suggesting that it was lost in the tumour. Multiplex ligand‐dependent probe amplification analysis showed that two copies of all NF1 exons were present.
This is the first evidence of mitotic recombination resulting in a reduction to homozygosity of a germline NF1 mutation in an NF1‐associated GIST. We hypothesise that the LOH of NF1 and lack of KIT and PDGFRA mutations are evidence of an alternative pathogenesis in NF1‐associated GISTs.
PMCID: PMC2597901  PMID: 17209131
Interstitial cell of Cajal; loss of heterozygosity; mitotic recombination
10.  Correlation of Kinase Genotype and Clinical Outcome in the North American Intergroup Phase III Trial of Imatinib Mesylate for Treatment of Advanced Gastrointestinal Stromal Tumor: CALGB 150105 Study by Cancer and Leukemia Group B and Southwest Oncology Group 
Journal of Clinical Oncology  2008;26(33):5360-5367.
Imatinib mesylate is standard treatment for patients who have advanced gastrointestinal stromal tumor (GIST), but not all patients benefit equally. In previous studies, GIST genotype correlated with treatment outcome and optimal imatinib dosing.
Patients and Methods
We examined the relationship between kinase genotype and treatment outcome for 428 patients enrolled on the North American phase III study SWOG S0033/CALGB 150105 and treated with either 400 mg or 800 mg daily doses of imatinib.
The presence of KIT exon 11–mutant genotype (n = 283) correlated with improved treatment outcome when compared with KIT exon 9–mutant (n = 32) and wild-type (WT; n = 67) genotypes for objective response (complete response [CR]/partial response [PR], 71.7% v 44.4% [P = .007]; and 44.6% [P = .0002], respectively); time to tumor progression (TTP; median 24.7 months v 16.7 and 12.8 months, respectively); and overall survival (OS; median 60.0 months v 38.4 and 49.0 months, respectively). The survival outcomes for patients with exon 9–mutant, exon 11–mutant or WT GIST were not affected by imatinib dose. However, there was evidence of improved response rates for patients with exon 9–mutant tumors treated with imatinib 800 mg versus 400 mg (CR/PR, 67% v 17%; P = .02). Patients who had CD117-negative GIST had similar TTP but inferior OS compared with patients who had CD117-positive disease, which suggests that patients who have CD117-negative GIST may benefit from imatinib treatment. In addition, we identified novel but rare mutations of the KIT extracellular domain (exons 8 and 9).
We confirmed the favorable impact of KIT exon 11 genotype when compared with KIT exon 9 and wild-type genotype for patients with advanced GIST who are treated with imatinib.
PMCID: PMC2651078  PMID: 18955451
11.  Primary and Secondary Kinase Genotypes Correlate With the Biological and Clinical Activity of Sunitinib in Imatinib-Resistant Gastrointestinal Stromal Tumor 
Journal of Clinical Oncology  2008;26(33):5352-5359.
Most gastrointestinal stromal tumors (GISTs) harbor mutant KIT or platelet-derived growth factor receptor α (PDGFRA) kinases, which are imatinib targets. Sunitinib, which targets KIT, PDGFRs, and several other kinases, has demonstrated efficacy in patients with GIST after they experience imatinib failure. We evaluated the impact of primary and secondary kinase genotype on sunitinib activity.
Patients and Methods
Tumor responses were assessed radiologically in a phase I/II trial of sunitinib in 97 patients with metastatic, imatinib-resistant/intolerant GIST. KIT/PDGFRA mutational status was determined for 78 patients by using tumor specimens obtained before and after prior imatinib therapy. Kinase mutants were biochemically profiled for sunitinib and imatinib sensitivity.
Clinical benefit (partial response or stable disease for ≥ 6 months) with sunitinib was observed for the three most common primary GIST genotypes: KIT exon 9 (58%), KIT exon 11 (34%), and wild-type KIT/PDGFRA (56%). Progression-free survival (PFS) was significantly longer for patients with primary KIT exon 9 mutations (P = .0005) or with a wild-type genotype (P = .0356) than for those with KIT exon 11 mutations. The same pattern was observed for overall survival (OS). PFS and OS were longer for patients with secondary KIT exon 13 or 14 mutations (which involve the KIT-adenosine triphosphate binding pocket) than for those with exon 17 or 18 mutations (which involve the KIT activation loop). Biochemical profiling studies confirmed the clinical results.
The clinical activity of sunitinib after imatinib failure is significantly influenced by both primary and secondary mutations in the predominant pathogenic kinases, which has implications for optimization of the treatment of patients with GIST.
PMCID: PMC2651076  PMID: 18955458
12.  Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains 
Biological membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-associated proteins. The membrane-binding N-terminal amphipathic helix-containing BIN/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quantitative experimental characterization.
We have measured tube pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR) bound to tubular membranes pulled from micropipette-aspirated giant vesicles. We observed that fluorescently-labeled DA-N-BAR showed significantly higher protein density on tubules compared to the connected low-curvature vesicle membrane. Furthermore, we found the equilibrium tube pulling force to be systematically dependent on the aqueous solution concentration of DA-N-BAR, thereby providing the first quantitative assessment of spontaneous curvature generation. At sufficiently high protein concentrations, pulled tubes required no external force to maintain mechanical equilibrium, in agreement with the qualitative spontaneous tubulation previously reported for amphiphysin.
PMCID: PMC3679405  PMID: 23772271
curvature sorting; curvature sensing; pulling force; GUV; membrane mechanics
13.  Molecular Target Modulation, Imaging, and Clinical Evaluation of Gastrointestinal Stromal Tumor Patients Treated with Sunitinib Malate after Imatinib Failure 
To evaluate sunitinib activity and potential cellular and molecular correlates in gastrointestinal stromal tumor (GIST) patients after imatinib failure, in addition to assessing the safety and pharmacokinetics (PK) of different dose schedules.
Experimental Design
In this open-label, dose-ranging, phase I/II study, 97 patients with metastatic imatinib-resistant/intolerant GIST received sunitinib at doses of 25, 50, or 75 mg/d on one of three schedules. Serial tumor imaging was done using computed tomography and [18F]fluoro-2-deoxy-d-glucose positron emission tomography scanning. PK and cell proliferation and KIT phosphorylation status in tumor biopsies were also analyzed.
Clinical benefit was observed in 52 patients (54%: 7 objective partial responses, 45 stable disease ≥6 months). Decreased tumor glycolytic activity was shown in most patients within 7 days of starting sunitinib using [18F]fluoro-2-deoxy-d-glucose positron emission tomography. Sunitinib treatment was associated with reduced tumor cell proliferation by >25% in 52% of cases analyzed and reduced levels of phospho-KIT in tumor biopsies (indicating target modulation). The recommended dose schedule was 50 mg/d for 4 weeks followed by 2 weeks off treatment. On the 50-mg dose across all schedules, 79% of PK-evaluable patients achieved total drug trough concentrations above the target concentration (50 ng/mL) within 14 days of dosing. In addition, adverse events were generally mild to moderate in severity.
Cellular and molecular analyses showed that sunitinib clinical activity is associated with inhibition of KIT in GIST following imatinib failure, illustrating the rational approach used to develop a therapy aimed at the underlying oncogenic signaling pathway aberrancy.
PMCID: PMC3417101  PMID: 19737946
14.  The multi-targeted receptor tyrosine kinase inhibitor, Linifanib (ABT-869), induces apoptosis through an AKT and Glycogen Synthase Kinase 3β-dependent pathway 
Molecular cancer therapeutics  2011;10(6):949-959.
The FMS-like receptor tyrosine kinase 3 (FLT3) plays an important role in controlling differentiation and proliferation of hematopoietic cells. Activating mutations in FLT3 occur in patients with acute myeloid leukemia (15-35%) resulting in abnormal cell proliferation. Furthermore, both adult and pediatric patients with acute myeloid leukemia (AML) harboring the FLT3 internal tandem duplication (ITD) mutation have a poor prognosis. Several inhibitors have been developed to target mutant FLT3 for the treatment of AML, yet the molecular pathways affected by drug inhibition of the mutated FLT3 receptor alone have not yet been characterized. Linifanib (ABT-869) is a multi-targeted tyrosine kinase receptor inhibitor that suppresses FLT3 signaling. In this paper, we demonstrate that treatment with Linifanib inhibits proliferation and induces apoptosis in ITD mutant cells in vitro and in vivo. We show that treatment with Linifanib reduces phosphorylation of AKT and glycogen synthase kinase 3β (GSK3β). In addition, we show that inhibition of GSK3β decreases Linifanib-induced apoptosis. This study demonstrates the importance of GSK3 as a potential target for AML therapy, particularly in patients with FLT3 ITD mutations.
PMCID: PMC3112478  PMID: 21471285
AML; FLT3 Inhibitor
15.  A Phase II Trial of Imatinib Mesylate in Merkel Cell Carcinoma (Neuroendocrine Carcinoma of the Skin): A Southwest Oncology Group Study (S0331) 
Imatinib mesylate (Gleevec®) was evaluated as a treatment for Merkel cell carcinoma (MCC, neuroendocrine carcinoma of the skin) based on the identification of strong c-KIT staining of these neoplasms.
Eligibility included patients with measurable metastatic or unresectable MCC, c-KIT (CD117) expression and a Zubrod performance status of 0–2. Imatinib 400 mg daily was administered orally in 28-day cycles to 23 patients.
Overall, imatinib was well tolerated with Grade 1 or 2 nausea, diarrhea, and hematologic toxicity as the most frequent side effects. A partial response was seen in 1 patient (4%; 95% CI: 0% – 22%). Median progression-free survival was 1 month (95% CI: 1–2 months). Median overall survival was 5 months (95% CI 2–8 months). One patient achieved a partial response and another had prolonged disease stabilization while receiving treatment.
The majority of patients progressed rapidly within 1–2 cycles of treatment. The observed progression-free survival and overall survival were not adequate to conclude that this agent was active in advanced MCC, and thus the planned second stage of patient accrual was not opened.
PMCID: PMC2978644  PMID: 20019577
16.  Survey of Activated FLT3 Signaling in Leukemia 
PLoS ONE  2011;6(4):e19169.
Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.
PMCID: PMC3084268  PMID: 21552520
17.  Met receptor sequence variants R970C and T992I lack transforming capacity 
Cancer research  2010;70(15):6233-6237.
High-throughput sequencing promises to accelerate the discovery of sequence variants, but distinguishing oncogenic mutations from irrelevant "passenger" mutations remains a major challenge. Here we present an analysis of two sequence variants of the MET receptor (hepatocyte growth factor receptor) R970C and T992I (also designated R988C and T1010I). Previous reports indicated these sequence variants are transforming and contribute to oncogenesis. We screened patients with chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), colorectal cancer, endometrial cancer, thyroid cancer, or melanoma as well as individuals without cancer and found these variants at low frequencies in most cohorts, including normal individuals. No evidence of increased phosphorylation or transformative capacity by either sequence variant was found. Since small-molecule inhibitors for MET are currently in development, it will be important to distinguish between oncogenic sequence variants and rare single-nucleotide polymorphisms to avoid the use of unnecessary and potentially toxic cancer therapy agents.
PMCID: PMC2913476  PMID: 20670955
cancer genetics; cancer genomics; targeted therapy
18.  Gene Expression Signatures and Response to Imatinib Mesylate in Gastrointestinal Stromal Tumor 
Despite initial efficacy of imatinib mesylate (IM) in most gastrointestinal stromal tumor (GIST) patients, many experience primary/secondary drug resistance. Therefore, clinical management of GIST may benefit from further molecular characterization of tumors before and after IM treatment.
Experimental Design
As part of a recent Phase II Trial of neoadjuvant/adjuvant IM treatment for advanced primary and recurrent operable GISTs (RTOG-S0132), gene expression profiling using oligonucleotide microarrays was performed on tumor samples obtained before and after IM therapy. Patients were classified according to changes in tumor size after treatment based on CT scan measurements. Gene profiling data were evaluated with Statistical Analysis of Microarrays (SAM) to identify differentially expressed genes (in pre-treatment GIST samples).
Based on SAM (FDR=10%), thirty-eight genes were expressed at significantly lower levels in the pre-treatment biopsy samples from tumors that significantly responded to 8 to 12 weeks of IM, i.e., ≥25% tumor reduction. Eighteen of these genes encoded KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. Importantly, ten KRAB-ZNF genes mapped to a single locus on chromosome 19p, and a subset predicted likely response to IM-based therapy in a naïve panel of GISTs. Furthermore, we found that modifying expression of genes within this predictive signature can enhance the sensitivity of GIST cells to IM.
Using clinical pre-treatment 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 predictive of and functionally associated with likely response to short term IM treatment.
PMCID: PMC2822341  PMID: 19671739
GISTs; imatinib mesylate; microarray; KRAB-ZNF genes
19.  Phase II Trial of Neoadjuvant/Adjuvant Imatinib Mesylate (IM) for Advanced Primary and Metastatic/Recurrent Operable Gastrointestinal Stromal Tumor (GIST) – early results of RTOG 0132 
Journal of surgical oncology  2009;99(1):42-47.
Therapy for gastrointestinal stromal tumors (GIST) has changed significantly with the use of Imatinib Mesylate (IM). Despite the success of this drug in metastatic GIST, disease progression remains a perplexing clinical issue suggesting the need for multimodality management. There have been no prospective studies either evaluating the neoadjuvant use of IM in primary GIST or as a preoperative cytoreduction agent for metastatic GIST.
RTOG 0132 was a prospective phase II study evaluating safety and efficacy of neoadjuvant IM (600 mg/day) for patients with primary GIST or the preop use of IM in patients with operable metastatic GIST. The trial continued postop IM for 2 years.
63 patients were entered (52 analyzable), 30 patients with primary GIST (Group A) and 22 with metastatic GIST (Group B). Response (RECIST) in Group A was (7% partial, 83% stable, 10% unknown), in Group B (4.5% partial, 91% stable, 4.5% progression). Two year progression free survival (Group A 83%, Group B 77%). Estimated overall survival (Group A 93%, Group B 91%). Complications of surgery and IM toxicity were minimal.
This trial represents the first prospective report of preop IM in GIST. This approach is feasible, requires multidisciplinary consultations, is not associated with notable postop complications.
PMCID: PMC2606912  PMID: 18942073
GIST; neoadjuvant imatinib; locally advanced GIST; metastatic GIST
20.  Phase II Study of Imatinib in Patients With Recurrent Gliomas of Various Histologies: A European Organisation for Research and Treatment of Cancer Brain Tumor Group Study 
Journal of Clinical Oncology  2008;26(28):4659-4665.
To evaluate the safety and the efficacy of imatinib in recurrent malignant gliomas.
Patients and Methods
This was a single-arm, phase II study. Eligible patients had recurrent glioma after prior radiotherapy with an enhancing lesion on magnetic resonance imaging. Three different histologic groups were studied: glioblastomas (GBM), pure/mixed (anaplastic) oligodendrogliomas (OD), and low-grade or anaplastic astrocytomas (A). Imatinib was started at a dose of 600 mg/d with dose escalation to 800 mg in case of no toxicity; during the trial this dose was increased to 800 mg/d with escalation to 1,000 mg/d. Trial design was one-stage Fleming; both an objective response and 6 months of progression-free survival (PFS) were considered a successful outcome to treatment.
A total of 112 patients (51 patients with GBM, 25 patients with A, and 36 patients with OD) were enrolled. Imatinib was in general well tolerated. The median number of cycles was 2.0 (range, 1 to 43 cycles). Five patients had an objective partial response, including three patients with GBM; all had 6 months of PFS. The 6-month PFS rate was 16% (95% CI, 8.0% to 34.0%) in GBM, 4.0% (95% CI, 0.3% to 15.0%) in OD, and 9% (95% CI, 2.0% to 25.0%) in A. The exposure to imatinib was significantly lower in patients using enzyme-inducing antiepileptic drugs. The presence of ABCG2 point mutations were not correlated with pharmacokinetic findings. No somatic activating mutations of KIT or platelet-derived growth factor receptor–A or –B were found.
In the dose range of 600 to 1,000 mg/d, single-agent imatinib is well tolerated but has limited antitumor activity in patients with recurrent gliomas.
PMCID: PMC2653126  PMID: 18824712
21.  Ménétrier disease and gastrointestinal stromal tumors: hyperproliferative disorders of the stomach 
Ménétrier disease and gastrointestinal stromal tumors (GISTs) are hyperproliferative disorders of the stomach caused by dysregulated receptor tyrosine kinases (RTKs). In Ménétrier disease, overexpression of TGF-α, a ligand for the RTK EGFR, results in selective expansion of surface mucous cells in the body and fundus of the stomach. In GISTs, somatic mutations of the genes encoding the RTK KIT (or PDGFRA in a minority of cases) result in constitutive kinase activity and neoplastic transformation of gut pacemaker cells (interstitial cells of Cajal). On the basis of the involvement of these RTKs in the pathogenesis of these disorders, Ménétrier disease patients have been effectively treated with a blocking monoclonal antibody specific for EGFR and GIST patients with KIT and PDGFRA tyrosine kinase inhibitors.
PMCID: PMC1716220  PMID: 17200708
22.  Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell–dependent antitumor effects 
Journal of Clinical Investigation  2004;114(3):379-388.
Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell–dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-γ production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.
PMCID: PMC489961  PMID: 15286804
23.  Kaposi's Sarcoma-Associated Herpesvirus-Induced Upregulation of the c-kit Proto-Oncogene, as Identified by Gene Expression Profiling, Is Essential for the Transformation of Endothelial Cells 
Journal of Virology  2002;76(16):8383-8399.
Kaposi's sarcoma (KS), the most frequent malignancy afflicting AIDS patients, is characterized by spindle cell formation and vascularization. Infection with KS-associated herpesvirus (KSHV) is consistently observed in all forms of KS. Spindle cell formation can be replicated in vitro by infection of dermal microvascular endothelial cells (DMVEC) with KSHV. To study the molecular mechanism of this transformation, we compared RNA expression profiles of KSHV-infected and mock-infected DMVEC. Induction of several proto-oncogenes was observed, particularly the receptor tyrosine kinase c-kit. Consistent with increased c-Kit expression, KHSV-infected DMVEC displayed enhanced proliferation in response to the c-Kit ligand, stem cell factor (SCF). Inhibition of c-Kit activity with either a pharmacological inhibitor of c-Kit (STI 571) or a dominant-negative c-Kit protein reversed SCF-dependent proliferation. Importantly, inhibition of c-Kit signal transduction reversed the KSHV-induced morphological transformation of DMVEC. Furthermore, overexpression studies showed that c-Kit was sufficient to induce spindle cell formation. Together, these data demonstrate an essential role for c-Kit in KS tumorigenesis and reveal a target for pharmacological intervention.
PMCID: PMC155158  PMID: 12134042

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