© 2002 by Oxford University Press
Journal of the National Cancer Institute, Vol. 94, No. 22, 1673-1679,
November 20, 2002
© 2002 Oxford University Press
ARTICLE |
Potential Use of Imatinib in Ewing’s Sarcoma: Evidence for In Vitro and In Vivo Activity
Affiliation of authors: M. S. Merchant, C.-W. Woo, C. L. Mackall, C. J. Thiele, Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD.
Correspondence to: Melinda S. Merchant, M.D., Ph.D., 10 Center Dr., MSC 1298, Bldg. 10/13N240, Bethesda, MD 20892 (e-mail: merchanm{at}mail.nih.gov).
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ABSTRACT |
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 TopNotes Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Background: Ewing’s sarcoma cells express c-kit, a receptor tyrosine kinase, and its ligand, stem cell factor (SCF), creating a potential autocrine loop that may promote tumor survival. We thus examined whether the specific tyrosine kinase inhibitor imatinib mesylate (hereafter imatinib; formerly STI571) could inhibit the proliferation of Ewing’s sarcoma cells in vitro and in vivo. Methods: The effect of imatinib on c-kit expression and phosphorylation in Ewing’s sarcoma cells was examined by immunoblotting. The effect of imatinib on cell growth and apoptosis was examined with an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and with a morphologic test and Annexin V staining, respectively. The effect of imatinib oral therapy (every 12 hours for 5–7 days) on primary tumor growth was assessed in Ewing’s sarcoma xenografts in SCID/bg mice (5 or 10 mice per group). Results: All Ewing’s sarcoma cell lines tested were sensitive to imatinib-mediated apoptosis with a concentration inhibiting growth by 50% (IC50) of 10–12 µM. Imatinib inhibited SCF-mediated c-kit phosphorylation (IC50 = 0.1–0.5 µM). In the xenograft model, imatinib treatment resulted in the regression or control of primary Ewing’s sarcomas. After 6 days of treatment, the mean lower extremity volume including xenograft tumor was 3744 mm3 (95% confidence interval [CI] = 3050 to 4437 mm3), 1442 mm3 (95% CI = 931 to 1758 mm3), and 346 mm3 (95% CI = 131 to 622 mm3) in mice treated with carrier alone or with imatinib at 50 mg/kg or at 100 mg/kg, respectively. Conclusions: Imatinib interferes with growth of all Ewing’s sarcoma cell lines tested in vitro and in vivo. Targeted inhibition of tyrosine kinase-dependent autocrine loops, therefore, may be a viable therapeutic strategy for Ewing’s sarcoma.
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INTRODUCTION |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tyrosine kinases are important intracellular mediators of proliferation, survival, and differentiation signals and have been shown to play a role in the growth of several malignancies (1,2). In some cases, constitutive activation of a tyrosine kinase is a sentinel event in neoplastic transformation. The bcr-abl gene fusion (i.e., BCR-ABL) on the Philadelphia chromosome in patients with chronic myelogenous leukemia results in the constitutive expression of an abnormal protein tyrosine kinase (3), and a mutation in the c-kit gene (i.e., KIT; encoding the stem cell factor [SCF] receptor, a receptor tyrosine kinase) expressed in gastrointestinal stromal tumors leads to constitutive activity of this receptor tyrosine kinase (4). Even in the absence of proximal transforming events involving tyrosine kinases, tyrosine kinase signaling may contribute to the survival advantage of transformed cells. For example, overexpression of epithelial growth factor receptors have been implicated in the growth of breast and ovarian cancers (1,5), and growth loops involving the insulin-like growth I receptor have been shown to play a role in several malignancies (2). An autocrine growth loop resulting from the coexpression of the c-kit receptor tyrosine kinase and its ligand, SCF, is found in Ewing’s sarcoma and in small-cell lung cancer cells and likely contributes to the growth of these transformed cells (6,7).
Ewing’s sarcoma is a small, round, blue-cell tumor of bone and soft tissue, with peak incidence during childhood and adolescence. Despite frequent responses to initial multimodality therapy, the majority of patients with metastatic Ewing’s sarcoma succumb to recurrent disease, which highlights the need for effective alternative therapies directed at minimal residual Ewing’s sarcoma. Interaction of c-kit with its ligand, SCF, has been implicated in the growth, survival, and metastatic potential of Ewing’s sarcoma (6,8). Cell lines and fresh isolates from the Ewing’s sarcoma family of tumors express cell surface c-kit (8) and SCF in both soluble and membrane-bound forms (6). Expression of both receptor and ligand suggests an autocrine or juxtacrine loop that may contribute to the unregulated growth of Ewing’s sarcoma cells. Blockade of c-kit signaling by monoclonal antibodies or antisense oligonucleotides inhibits cell growth and leads to apoptosis in cultures of Ewing’s cell lines (8).
The c-abl, platelet-derived growth factor receptor (PDGFR), and c-kit tyrosine kinases are specifically inhibited by imatinib mesylate (hereafter imatinib, formerly STI571, Gleevec; Novartis Pharmaceuticals, East Hanover, NJ), approved by the U.S. Food and Drug Administration (9,10). Both preclinical and clinical studies have shown that imatinib induces apoptosis and is associated with remission in patients with bcr-abl-positive chronic myelogenous leukemia as well as tumor shrinkage in patients with c-kit-positive gastrointestinal stromal tumors (11–13). Imatinib can inhibit the ligand-dependent growth of small-cell lung cancer cells in vitro by blocking SCF-mediated c-kit phosphorylation (7). In this study, we examined the effects of imatinib on the in vitro proliferation of Ewing’s sarcoma cell lines, the induction of apoptosis in these lines, and the in vivo growth of Ewing’s sarcoma xenografts.
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MATERIALS AND METHODS |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Lines and Cell Growth
The following Ewing’s sarcoma cell lines were used: TC71, TC32, RD-ES, 5838, A4573, EWS-925, NCI-EWS-94, and NCI-EWS-95 (14). NCI-EWS-011 and NCI-EWS-021 cell lines were generated at the National Cancer Institute from tumor tissue obtained from recurrent Ewing’s sarcomas. Both resected tumors and the recently generated cell lines were positive for the t(11;22) EWS/FLI-1 translocation. The rhabdomyosarcoma line RD4A (15) and the neuroblastoma cell lines CHP-212 and KCNR (16) were used where indicated as negative controls. Cell lines were grown in RPMI-1640 medium supplemented with 2 mM L-glutamine and 0.1% or 10% fetal calf serum (Life Technologies, Gaithersburg, MD). Cells were plated 24 hours before treatment and cultured in medium containing SCF at 100 ng/mL (Intergen Co., Purchase, NY) or medium alone in the presence or absence of imatinib. Cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay (Sigma Chemical Co., St. Louis, MO) 24–72 hours after treatment.
Detection of Surface Receptor Expression and Apoptosis
Adherent cells were removed from culture dishes by treatment with 0.05% trypsin/0.53 mM EDTA•4Na (Life Technologies) and washed in flow cytometry buffer (phosphate-buffered saline [PBS] containing 2% bovine serum albumin and 0.1% NaN3). Approximately 1–5 x 105 cells were stained with phycoerythrin-conjugated anti-CD117 (c-kit), anti-CD140a (PDGFR
), or anti-CD140b (PDGFR
) (BD Biosciences Pharmingen, San Diego, CA) or with Annexin V-conjugated fluorescein isothiocyanate and propidium iodide (PI) in calcium-containing buffer. One- or two-color immunofluorescence was detected with a FACSCalibur (BD Biosciences, Burlington, MA). Data from a minimum of 10 000 cells were acquired and analyzed with CellQuest software (BD Biosciences Immunocytometry Systems, Franklin Lakes, NJ). For the morphologic assessment of apoptosis by fluorescence microscopy, 10 µL of Hoechst 33342 (50 µg/mL; Sigma Chemical Co.) was added to each culture well and incubated for 10 minutes at 37 °C. PI was then added (50 µg/mL), and samples were analyzed for nuclear condensation and fragmentation by fluorescence microscopy.
Immunoprecipitation and Immunoblotting
Two million cells were cultured on 150-mm tissue culture plates for 3 days at 37 °C in an atmosphere of 5% CO2/95% air. Cells were preincubated for 30 minutes with imatinib as indicated before stimulation with SCF at 100 ng/mL (Pierce, Rockford, IL) for 7 minutes at 37 °C. After two washes with ice-cold PBS, total cell lysates were prepared in 1 mL of 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris–HCl [pH 8.0], 500 µM sodium orthovanadate [pH 9.0], 140 mM NaCl, 10% glycerol, aprotinin at 10 µg/mL, leupeptin at 1 µg/mL, and 1 mM phenylmethylsulfonyl fluoride; Sigma Chemical Co.) at 4 °C for 30 minutes. Insoluble material was removed by centrifugation at 4 °C for 15 minutes at 10 000g. Protein concentrations were determined with the Bradford protein assay. The protein lysates (1 mg) were immunoprecipitated with 1 µg of monoclonal anti-Kit antibody (K45; NeoMarkers, Fremont, CA) for 2 hours at 4 °C. The immune complexes were then collected by rocking in 20 µL of 10% (vol/vol) protein A-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C for 2 hours and washed twice with lysis buffer followed by a single wash with H2O. Precipitates were resuspended in 20 µL of 2x Tris glycine/sodium dodecyl sulfate (SDS) buffer (Invitrogen, Carlsbad, CA), and proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred to 0.45-µm (pore size) Protran membranes (Schleicher & Schuell, Keene, NH). Protein blots were incubated with anti-phosphotyrosine antibody (pY99; Santa Cruz Biotechnology) at a 1 : 1000 dilution for 1 hour at room temperature, washed with Tris-buffered saline containing Tween-20 (20 mM Tris–HCl [pH 7.4], 150 mM NaCl, and 0.5% Tween-20), and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (Santa Cruz Biotechnology) at a 1 : 2000 dilution for 1 hour. Blots were analyzed with an enhanced chemiluminescence (ECL) detection system (Amersham Corp., Piscataway, NJ). The blots were stripped of antibody in 62.5 mM Tris (pH 6), 2% SDS, and 100 mM 2-mercaptoethanol at 50 °C for 30 minutes and then reprobed with polyclonal anti-c-kit antibody (DAKO, Carpinteria, CA).
In Vivo Tumor Growth
Tumor cells were cultured to a confluence of 75%, harvested with trypsin/EDTA, and then washed twice with PBS. Two million Ewing’s sarcoma cells were injected in 100 µL of PBS into the gastrocnemius of 4- to 8-week-old female SCID/bg mice (Taconic, Germantown, NY). Each mouse had a single palpable tumor evident at 21–28 days after inoculation. At a tumor volume of 100–500 mm3, mice were randomly assigned to receive oral gavage with imatinib (100 mg/kg per dose or 50 mg/kg per dose) or vehicle alone (5 or 10 mice per group). Doses of imatinib were administered by gavage in 100 µL of distilled H2O at 12-hour intervals for 5–7 days, and dosages were obtained from previously reported studies (17) in which imatinib was administered to SCID mice. Tumor dimensions were measured every 1 or 2 days with digital calipers to obtain two diameters of the tumor sphere. The lower extremity volume at the site of the tumor was determined by the formula (D x d2/6) x 
, where D is the longer diameter and d is the shorter diameter. Lower extremity volumes without tumor were approximately 50 mm3. Xenograft studies were approved by the National Cancer Institute’s Animal Care and Use Committee, and all animal care was in accordance with institutional guidelines.
Statistical Analysis
One-way analysis of variance was performed with the use of Prism 3.0 software (GraphPad Software, Inc., San Diego, CA). Tumor growth curves were compared with a post-test Bonferroni comparison of groups to reduce the overall chance of a type I error (18). Data were considered statistically significant at P<.05. All statistical tests were two-sided.
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RESULTS |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surface Expression of c-kit and PDGFRs by Ewing’s Sarcoma Cell Lines
Flow cytometry was used to confirm the presence of receptor tyrosine kinases on our panel of Ewing’s sarcoma cell lines. Surface c-kit expression was detected on all cell lines in this panel (Fig. 1
and Table 1). Although c-kit expression was generally homogeneous within a given cell line, considerable variability in c-kit expression was evident among cell lines. Surface expression of PDGFR also varied among these Ewing’s sarcoma cell lines, with seven of 10 lines expressing PDGFR. Like the majority of established Ewing’s sarcoma lines tested, the early-passage cell line EWS-011 consistently expressed c-kit and PDGFR. Conversely, we were not able to detect cell surface expression of PDGFR in this panel of Ewing’s sarcoma cell lines (Fig. 1).
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Growth Inhibition of Ewing’s Sarcoma Cell Lines
Because of previous data showing that the c-kit-mediated SCF autocrine loop was important for cell survival and growth in Ewing’s sarcoma (8), we tested the effects of imatinib on the growth of Ewing’s sarcoma cell lines. When Ewing’s sarcoma cell lines were treated with imatinib from 0 to 20 µM during the linear growth phase, the proliferation of each Ewing’s sarcoma cell line was inhibited in a concentration-dependent manner (Fig. 2, A). The inhibition of cell growth by imatinib was similar for all 10 Ewing’s sarcoma cell lines tested, including early passages of EWS-021. However, not all tumor cell lines were sensitive to imatinib at these drug concentrations, as shown by imatinib-resistant CHP-212 neuroblastoma cells (Fig. 2, A). The concentration of imatinib inhibiting cell growth by 50% (IC50) was 10–12 µM for the 10 Ewing’s sarcoma cell lines tested (five experiments).
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To determine whether growth inhibition reflected cytostasis or cell death, cultures were microscopically inspected after treatment with imatinib. We observed morphologic changes consistent with the induction of cell death, such as loss of adherence properties, rounded cells, and nuclear condensation (Fig. 2, B
). Hoechst dye staining further revealed nuclear condensation (data not shown). To verify apoptosis, cells were harvested at regular intervals and assessed for apoptosis by Annexin V staining. Treatment with imatinib at concentrations greater than 7.5 µM produced many Annexin V-positive, PI-negative apoptotic cells (Fig. 3). Apoptosis was observed as early as 12 hours after treatment with imatinib at concentrations greater than its IC50 value, and the peak level of apoptosis occurred between 36 and 72 hours after imatinib treatment (Fig. 3, B). The degree of Annexin V staining at peak apoptosis was dependent on the concentration of imatinib (Fig. 3, C and D). By 4 days after treatment, all cells cultured with greater than 10 µM imatinib were nonviable (data not shown). Thus, imatinib induced apoptosis in Ewing’s sarcoma with an IC50 value of 10–12 µM.
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Inhibition of c-kit Phosphorylation by Imatinib in Ewing’s Sarcoma Cell Lines
Because all Ewing’s sarcoma cell lines examined were sensitive to imatinib and expressed cell surface c-kit, the ability of imatinib to inhibit c-kit phosphorylation was examined. Baseline c-kit phosphorylation was detected minimally or was absent in Ewing’s sarcoma cell lines cultured in 0.1% fetal calf serum. Treatment with SCF (100 µg/mL) substantially increased c-kit phosphorylation in Ewing’s sarcoma cell lines (Fig. 4), but pretreatment with imatinib blocked SCF-mediated c-kit phosphorylation in all cell lines tested (Fig. 4, A). The imatinib inhibition of c-kit phosphorylation was concentration-dependent, with an IC50 value of 0.1–0.5 µM (Fig. 4, B). These results confirmed c-kit expression and signaling activity in Ewing’s sarcoma.
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Inhibition of Tumor Growth In Vivo
The in vivo activity of imatinib was tested by administering imatinib orally to mice with Ewing’s sarcoma xenografts. SCID/bg mice with a palpable Ewing’s sarcoma xenograft tumor were given imatinib at 50 mg/kg or 100 mg/kg or water, as a control, by oral gavage every 12 hours for 7 days. Tumor dimensions were measured every 1 or 2 days and are presented as lower extremity volumes. Although tumors in sham-treated mice grew exponentially, tumors in the group receiving imatinib doses of 50 mg/kg were stable or grew only minimally throughout the course of treatment (Fig. 5), and tumors of mice receiving imatinib doses of 100 mg/kg regressed. In fact, four of 10 mice receiving imatinib doses of 100 mg/kg had no palpable tumor at the termination of treatment (lower extremity volume <50 mm3). A comparison of groups at day 6 of therapy showed a lower extremity volume, including xenograft tumor, of 3744 mm3 (95% confidence interval [CI] = 3050 to 4437 mm3), 1442 mm3 (95% CI = 931 to 1758 mm3), or 346 mm3 (95% CI = 131 to 622 mm3) in mice treated with carrier alone or with imatinib at 50 mg/kg or at 100 mg/kg, respectively. The high-dose arm of this study was prematurely stopped because of high mortality. Six of 10 mice died on therapy between days 3 and 6, and the remaining mice appeared wasted and sluggish. No gross abnormalities or metastatic tumors were found on autopsy of these animals. Similar results were obtained with imatinib doses of 100 mg/kg in the tumor-free mice and in mice xenografted with EWS-95 cells (data not shown). In contrast, no wasting and only one death were observed in the TC71 xenograft mice treated with imatinib doses of 50 mg/kg, and no death was observed in control mice treated with carrier only. Of note, in human clinical trials of imatinib, minimal toxicities were detected (11), but studies in imatinib-treated mice have shown substantially different pharmacokinetics. In spite of these difficulties, these studies provide proof of principle that imatinib has antitumor activity against Ewing’s sarcoma in vivo.
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DISCUSSION |
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Imatinib has had remarkable success in early clinical trials targeting tumors in which sentinel, transforming events involve constitutive activation of susceptible tyrosine kinases. It remains unknown whether targeting pathways that contribute to growth and/or survival advantages of neoplastic cells with imatinib will result in a clinical benefit. Our studies provide evidence that imatinib can impact tumor survival even in tumors lacking sentinel, transforming c-kit mutations. These results suggest that targeted inhibition of tyrosine kinase-dependent autocrine growth loops may be a viable therapeutic strategy for Ewing’s sarcoma.
All Ewing’s sarcoma cell lines tested were sensitive to the cytotoxic effects of imatinib, which causes apoptosis in a concentration-dependent manner, even though some of these Ewing’s sarcoma tumor lines have been shown to be chemoresistant and/or Fas resistant [(19) and data not shown]. This observation is especially important because chemoresistance is a major contributor to the rate of treatment failure in recurrent Ewing’s sarcoma. Fas resistance in Ewing’s cell lines has been attributed to decreased ligand expression or initiator caspase expression (14). The fact that Fas resistance did not alter the ability of cells to respond to imatinib implies that the apoptosis initiated by the inhibition of this growth factor is not dependent on an intact Fas pathway but effectively activates downstream caspases despite proximal deficits in the extrinsic death pathway.
The results presented in this article are from cell lines and, thus, may not be representative of parent tumors; however, several lines of evidence suggest that nascent Ewing’s sarcoma tumors may be targeted by imatinib. First, we have studied an early-passage cell line, EWS-021 (studied at passages 3 and 10), derived from fresh tumor and found that these cells were as sensitive as more established cell lines to imatinib-induced apoptosis. In addition, imatinib inhibition of growth in our studies was not limited to in vitro assays, as shown by the in vivo sensitivity of imatinib-treated xenografts.
Although the inhibition of c-kit phosphorylation in our studies occurred at concentrations similar to the IC50 values previously reported for tyrosine kinase inhibition (9,20), the inhibition of Ewing’s sarcoma cell growth required a higher concentration of imatinib (IC50 = 10 µM) than that required in studies of chronic myelogenous leukemia or gastrointestinal stromal tumors (IC50 = 1 µM). At least two possibilities may explain these results. One possibility is that a pharmacologic limitation may restrict the accessibility of imatinib to c-kit in Ewing’s sarcoma cells by limiting uptake or inhibiting its action once inside the cell. However, as shown in Fig. 4, inhibition of SCF-mediated c-kit phosphorylation occurred at the relatively low IC50 of 0.5 µM, providing evidence that phosphorylation of c-kit could be inhibited in Ewing’s sarcoma cells at these low doses. Another possibility is that the action of imatinib in Ewing’s sarcoma may involve inhibition of other tyrosine kinases. Immunohistochemical studies found that only approximately 30% of Ewing’s sarcoma clinical specimens were strongly positive for c-kit, although up to 71% of Ewing’s sarcomas showed at least some c-kit staining (21,22). In fact, the heterogeneity of c-kit expression, the low or absent level of baseline c-kit phosphorylation, and the uniformity of imatinib sensitivity in Ewing’s sarcoma cell lines would support an alternative target.
Because imatinib is also a specific inhibitor of PDGFR, a particularly attractive possibility is the inhibition of signaling by PDGF-C, a growth factor with inherent transforming properties that is induced by EWS/FLI1 transfection (23,24). Currently, it is not clear whether autocrine or paracrine growth is responsible for PDGF-C-mediated transformation (24). Our results are consistent with recent reports that PDGFR is expressed on the surface of some but not all Ewing’s sarcoma cell lines (25). However, in these studies, we observed no evidence for cell surface PDGFR expression, a component that is apparently necessary for PDGF-C signaling (26,27). Of note, cell lines that did not express surface PDGFR or PDGFR in these studies were nonetheless sensitive to imatinib-induced apoptosis. In addition, imatinib-mediated cytotoxicity occurred at higher concentrations of imatinib than concentrations reported for PDGFR inhibition (20), making it unlikely to fully account for the induction of cell death in this setting. PDGF-C may bind to another, perhaps uncharacterized, receptor with tyrosine kinase activity that is inhibited by imatinib, or a separate growth pathway that is critical for proliferation may be inhibited by imatinib. Indeed, although imatinib is commonly cited as specifically inhibiting v-abl, bcr-abl, PDGFR, and c-kit because of their exquisite sensitivity at doses less than 1 µM, other targets have been reported (28). Epidermal growth factor-dependent growth of BALB/MK cells is inhibited by imatinib at concentrations similar to those required in our Ewing’s sarcoma experiments (IC50 = 12.7 µM) (20), and the SCF-mediated or insulin-like growth factor I-mediated growth of small-cell lung cancer cells is inhibited by similar concentrations of imatinib (7). Whether these or other, as yet undescribed, tyrosine kinases contribute to the effect of imatinib in Ewing’s sarcoma is currently under study.
The fact that higher concentrations of imatinib were required to kill Ewing’s sarcoma cells than other tumor cells does not preclude future clinical evaluation of imatinib in these tumors. The toxicity of imatinib at this level was reported to be minimal to nonexistent for normal cells (9,29), providing evidence that the effects observed are likely to be relatively tumor-specific. Although the IC50 values determined in these studies are twofold higher than the concentrations achieved in early clinical trials (i.e., 4.6 µM) (11), a clear, maximally tolerated dose of this drug has not been determined, and trials are underway to assess efficacy and toxicity of increased doses to treat gastrointestinal stromal tumors. Finally, even if the toxicity of higher doses of imatinib proves intolerable for clinical translation of these results, the identification of the target or targets of imatinib that lead to cytotoxicity in Ewing’s sarcoma may allow the design of related compounds with increased specificity to induce death of Ewing’s sarcoma cells.
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NOTES |
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We thank Andrew Reff for his technical assistance and Dr. Chand Khanna from the Pediatric Oncology Branch, National Cancer Institute, for contributing the patient-derived cell line EWS-011. We also thank Drs. Pamela Cohen and Elisabeth Buchdunger from Novartis Pharmaceuticals for their helpful discussions.
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TopNotesAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Manuscript received April 3, 2002; revised September 3, 2002; accepted September 11, 2002.
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S. Basciani, M. Brama, S. Mariani, G. De Luca, M. Arizzi, L. Vesci, C. Pisano, S. Dolci, G. Spera, and L. Gnessi Imatinib Mesylate Inhibits Leydig Cell Tumor Growth: Evidence for In vitro and In vivo Activity Cancer Res., March 1, 2005; 65(5): 1897 - 1903. [Abstract] [Full Text] [PDF]
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S. Uccini, O. Mannarino, H. P. McDowell, U. Pauser, R. Vitali, P. G. Natali, P. Altavista, T. Andreano, S. Coco, R. Boldrini, et al. Clinical and Molecular Evidence for c-kit Receptor as a Therapeutic Target in Neuroblastic Tumors Clin. Cancer Res., January 1, 2005; 11(1): 380 - 389. [Abstract] [Full Text] [PDF]
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E. Tamborini, L. Bonadiman, T. Negri, A. Greco, S. Staurengo, P. Bidoli, U. Pastorino, M. A. Pierotti, and S. Pilotti Detection of Overexpressed and Phosphorylated Wild-Type Kit Receptor in Surgical Specimens of Small Cell Lung Cancer Clin. Cancer Res., December 15, 2004; 10(24): 8214 - 8219. [Abstract] [Full Text] [PDF]
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M. S. Merchant, X. Yang, F. Melchionda, M. Romero, R. Klein, C. J. Thiele, M. Tsokos, H. U. Kontny, and C. L. Mackall Interferon {gamma} Enhances the Effectiveness of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor Agonists in a Xenograft Model of Ewing's Sarcoma Cancer Res., November 15, 2004; 64(22): 8349 - 8356. [Abstract] [Full Text] [PDF]
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I. Gonzalez, E. J. Andreu, A. Panizo, S. Inoges, A. Fontalba, J. L. Fernandez-Luna, M. Gaboli, L. Sierrasesumaga, S. Martin-Algarra, J. Pardo, et al. Imatinib Inhibits Proliferation of Ewing Tumor Cells Mediated by the Stem Cell Factor/KIT Receptor Pathway, and Sensitizes Cells to Vincristine and Doxorubicin-Induced Apoptosis Clin. Cancer Res., January 15, 2004; 10(2): 751 - 761. [Abstract] [Full Text] [PDF]
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M. S. Merchant and C. J. Thiele RESPONSE: Re: Potential Use of Imatinib in Ewing's Sarcoma: Evidence for In Vitro and In Vivo Activity J Natl Cancer Inst, July 16, 2003; 95(14): 1088 - 1089. [Full Text] [PDF]
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E. Tamborini, L. Bonadiman, V. Albertini, M. A. Pierotti, and S. Pilotti Re: Potential Use of Imatinib in Ewing's Sarcoma: Evidence for In Vitro and In Vivo Activity J Natl Cancer Inst, July 16, 2003; 95(14): 1087 - 1088. [Full Text] [PDF]
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G. Singer, P. Schraml, C. Belgard, A. Raggi, S. Dirnhofer, P. Went, M. J. Mihatsch, and H. Moch KIT in Ovarian Carcinoma: Disillusion About a Potential Therapeutic Target J Natl Cancer Inst, July 2, 2003; 95(13): 1009 - 1010. [Full Text] [PDF]
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K. Scotlandi, M. C. Manara, R. Strammiello, L. Landuzzi, S. Benini, S. Perdichizzi, M. Serra, A. Astolfi, G. Nicoletti, P.-L. Lollini, et al. c-kit Receptor Expression in Ewing's Sarcoma: Lack of Prognostic Value but Therapeutic Targeting Opportunities in Appropriate Conditions J. Clin. Oncol., May 15, 2003; 21(10): 1952 - 1960. [Abstract] [Full Text] [PDF]
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B. J. Druker Taking Aim at Ewing's Sarcoma: Is KIT a Target and Will Imatinib Work? J Natl Cancer Inst, November 20, 2002; 94(22): 1660 - 1661. [Full Text] [PDF]
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