Imatinib as a Paradigm of Targeted Therapies

Imatinib as a Paradigm of Targeted Therapies Brian J. Druker Howard Hughes Medical Institute, Cancer Institute, Oregon Health and Science University, Portland, Oregon 97239

I. Introduction II. Identification of the BCR-ABL Tyrosine Kinase as a Therapeutic Target A. Chronic Myeloid Leukemia: Clinical Features B. Genetic Changes in Cancer Cells and the Philadelphia Chromosome C. Oncogenes and Mapping of the ABL Gene D. BCR-ABL, Tyrosine Kinase Activity, and CML E. BCR-ABL and Leukemia F. Animal Models of CML G. BCR-ABL Signaling and CML Pathogenesis H. BCR-ABL as a Therapeutic Target III. Development of an ABL-Specific Tyrosine Kinase Inhibitor A. Chemistry B. Preclinical Studies IV. Clinical Trials in CML A. Phase I Clinical Trials B. Phase II Studies C. Phase III Randomized Comparison of Imatinib with IFN-
Plus Ara-C V. Mechanisms of Relapse VI. Structural Basis of ABL Inhibition by Imatinib VII. Activity of Imatinib in Other Indications A. Gastrointestinal Stromal Tumors B. Other Malignancies VIII. Lessons Learned from the Clinical Trials of Imatinib A. Patient Selection B. Dose Selection IX. Translating the Success of Imatinib to Other Malignancies References

Imatinib (Gleevec) exemplifies the successful development of a rationally designed, molecularly targeted therapy for the treatment of a specific cancer. This article reviews the identification of the BCR-ABL tyrosine kinase as a therapeutic target in chronic myeloid leukemia and the steps in the development of an agent to specifically inactivate this abnormality. The clinical trials results are reviewed along with a description of resistance mechanisms. As imatinib also inhibits the tyrosine kinase activity of KIT and the platelet-derived growth factor receptors, the extension of imatinib to malignancies Advances in CANCER RESEARCH 0065-230X/04 $35.00

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Copyright 2004, Elsevier Inc. All rights reserved

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Brian J. Druker driven by these kinases will be described. Issues related to clinical trials of molecularly targeted agents are discussed, including patient and dose selection. Last, the translation of this paradigm to other malignancies is explored. ß 2004 Elsevier Inc.

I. INTRODUCTION A wealth of knowledge has emerged regarding the molecular events involved in human cancer. A major priority is the translation of this growing body of knowledge into therapeutics targeted specifically to these pathways. Perhaps the best example in which an understanding of the molecular pathogenesis of a human malignancy has been translated into clinical reality is imatinib for chronic myeloid leukemia (CML). The success of imatinib has also exemplified how the confluence of several fields in cancer research can result in significant advances. These include the investigations of chromosomal or genetic changes in cancer, the study of transforming retroviruses leading to the discovery of oncogenes, the availability of molecular techniques to map genes, and the biochemical evaluation of protein phosphorylation leading to the discovery of tyrosine kinases. All of these have contributed to the identification of the BCR-ABL oncogene as the causative molecular abnormality of CML and the development of a drug designed to inactivate this enzyme. This review traces these discoveries and discusses some of the lessons learned in the clinical development of a molecularly targeted agent and the implications for translating this success to other malignancies.

II. IDENTIFICATION OF THE BCR-ABL TYROSINE KINASE AS A THERAPEUTIC TARGET A. Chronic Myeloid Leukemia: Clinical Features CML is a clonal hematopoietic stem cell disorder with an annual incidence of 1 or 2 cases per 100,000 per year. The first description of CML was by two pathologists, Rudolf Virchow and John Hughes Bennett, in 1845 (Bennett, 1845; Virchow, 1845). Although a debate ensued as to whose description was first, Virchow acknowledged that Bennett’s case report had predated his (Geary, 2000). Of note, these first accounts of CML occurred before staining methods for blood, which were not developed until the late 1800s. The chronic, or stable, phase of CML is characterized by excess numbers of myeloid cells that differentiate and function normally. Approximately 90% of patients will be diagnosed in this phase of the disease; however, within an average of 4 to 6 years, the disease transforms through an

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‘‘accelerated phase’’ to an invariably fatal acute leukemia, also known as blast crisis. Disease progression is likely due to the accumulation of molecular abnormalities that lead to a progressive loss of the capacity for terminal differentiation of the leukemic clone (Faderl et al., 1999; Sawyers, 1999). Treatment choices for patients with CML, before the introduction of imatinib, included stem cell transplantation, hydroxyurea or interferon
(IFN-
)-based regimens, with allogeneic stem cell transplantation being the only proven curative therapy. As the average age of onset of CML is greater than 50 years, this factor, plus the inability to identify suitably matched donors, limits allogeneic stem cell transplantation to a minority of patients. Thus, less than 20% of CML patients are cured with these treatment options (Faderl et al., 1999; Sawyers, 1999). Patients in the blast phase of the disease are highly refractory to therapy. Response rates to standard chemotherapy are 20% or less and median survival is 2 to 3 months (Kantarjian et al., 1987; Sacchi et al., 1999).

B. Genetic Changes in Cancer Cells and the Philadelphia Chromosome In 1960, Peter Nowell and David Hungerford described a consistent chromosomal abnormality in CML patients, an acrocentric chromosome that was thought to be a chromosomal deletion (Nowell and Hungerford, 1960). This was the first example of a chromosomal abnormality linked to a specific malignancy. In this seminal article, the authors stated, ‘‘the findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemia’’ (Nowell and Hungerford, 1960). This prescient statement was met with skepticism as it was felt that the chromosome abnormality was an associated rather than causative phenomenon. As chromosomal banding techniques improved, it became apparent that the chromosome abnormality was a shortened chromosome 22. Then, in 1973, Janet Rowley determined that the shortened chromosome 22, the so-called Philadelphia (Ph) chromosome, was the product of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11) (Rowley, 1973) (Fig. 1A).

C. Oncogenes and Mapping of the ABL Gene The study of transforming retroviruses led to the recognition that mutations in normal cellular genes could be oncogenic. One such retrovirus, the Abelson murine leukemia virus, was initially described in 1970 (Abelson and Rabstein, 1970). Studies of this retrovirus led to the identification of its

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Fig. 1 (A) Schematic diagram of the translocation that creates the Philadelphia chromosome. The ABL and BCR genes reside on the long arms of chromosomes 9 and 22, respectively. As a result of the translocation, a BCR-ABL gene is formed on the derivative chromosome 22 (Philadelphia chromosome). (B) Structures of the BCR and ABL genes, showing locations of the breakpoints and various mRNAs created. The arrows indicate possible breakpoints within ABL. Regardless of the specific breakpoint in ABL, fusion mRNAs are produced that fuse BCR sequences to the second ABL exon. Fusions between BCR exon e13 (previously b2) or exon e14 (previously b3) and ABL exon a2 produce p210BCR-ABL that is characteristic of CML, whereas

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transforming gene, v-ABL, and to the cloning of its normal cellular homolog, c-ABL (Rosenberg and Witte, 1988). By mapping oncogenes to specific chromosomal locations, it was recognized that c-ABL, which normally resides on the long arm of chromosome 9, had been translocated to chromosome 22 in CML patients (de Klein et al., 1982). As the breakpoints on chromosome 22 clustered in a relatively small region that spanned 5.3 kilobases (kb), this region was named the breakpoint cluster region or BCR (Deininger et al., 2000; Groffen et al., 1984).

D. BCR-ABL, Tyrosine Kinase Activity, and CML The discovery of the translocation of c-ABL to chromosome 22 in CML patients prompted investigators to perform Northern blots with ABL probes. These studies demonstrated a larger than normal ABL mRNA in CML patients (Collins et al., 1984; Gale and Canaani, 1984). This was subsequently shown to be a chimeric mRNA that was a fusion of BCR and ABL sequences (Shtivelman et al., 1985). Similarly, a larger than normal ABL protein with tyrosine kinase activity was detected in CML cells and was identified as the product of the BCR-ABL mRNA (Ben-Neriah et al., 1986; Davis et al., 1985; Mes-Masson et al., 1986). These discoveries provided an important link between the study of oncogenes and the biochemistry of protein kinases, as it had previously been recognized that v-ABL possessed a novel kinase activity, the ability to phosphorylate tyrosine residues (Hunter and Cooper, 1985). BCR-ABL is now known to have elevated tyrosine kinase activity as compared with c-ABL, and the kinase activity of BCR-ABL is essential for its ability to transform cells (Konopka et al., 1984; Lugo et al., 1990).

E. BCR-ABL and Leukemia BCR-ABL can be detected in more than 95% of patients with CML. Most of the breakpoints in the ABL gene occur in a 200-kB region between two alternate first exons. Despite this, the first exon of ABL is not included in the BCR-ABL chimeric mRNA, presumably due to alternative splicing. Two slightly different chimeric BCR-ABL genes are present in patients with CML, depending on the precise location of the breakpoint in the BCR gene. Breaks can occur between exons b2 (also known as e13) and b3 (e14),

fusions between BCR exon e1 and ABL exon a2 give rise to p190BCR-ABL (found in two-thirds of patients with Ph-positive acute lymphoblastic leukemia). Rare CML patients have a breakpoint in the so-called micro-breakpoint cluster region (-BCR) and produce a 230-kDa BCR-ABL fusion protein (not shown).

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yielding a b2a2 fusion mRNA, whereas a break occurring between exons b3 and b4 produces a b3a2 fusion mRNA (Deininger et al., 2000) (Fig. 1B). Although the b3a2 mRNA encodes a BCR-ABL protein that is 25 amino acids larger than that encoded by the b2a2 transcript, both are referred to as p210BCR-ABL and have similar prognoses. The Ph chromosome and the BCR-ABL fusion gene are also found in 25–50% of adult patients with acute lymphoblastic leukemia (ALL) and rare cases of acute myeloid leukemia. In adults with Ph chromosome-positive ALL, one-third have BCR-ABL transcripts indistinguishable from those found in CML. In two-thirds, the genomic breakpoint on chromosome 22 occurs in the first intron of the BCR gene (between e1 and e2), resulting in a protein of 190 kDa (p190), also referred to as p185 (Fig. 1B) (Chan et al., 1987; Clark et al., 1987, 1988; Hermans et al., 1987). Approximately 5% of children with ALL are Ph chromosome positive and 95% of these patients have the p190 form of BCR-ABL. Other types of fusions have been observed in rare cases (Deininger et al., 2000).

F. Animal Models of CML In 1990, two experimental approaches demonstrated the ability of BCRABL, as the sole oncogenic abnormality, to cause leukemia. In one set of experiments, transgenic mice that express BCR-ABL were shown to develop a rapidly fatal acute leukemia (Heisterkamp et al., 1990). Using a different approach, a BCR-ABL-expressing retrovirus was used to infect murine bone marrow. These BCR-ABL-expressing marrow cells were used to repopulate irradiated mice. The transplanted mice developed a variety of myeloproliferative disorders, including a CML syndrome (Daley et al., 1990; Kelliher et al., 1990). Both of these approaches clearly demonstrated the leukemogenic potential of BCR-ABL; however, even in these models, it was still possible that secondary changes were required for leukemia to develop. More recently, Huettner et al. placed BCR-ABL under the control of a tetracycline-repressible promoter (Huettner et al., 2000). In these experiments, when tetracycline was withdrawn, the mice developed pre-B cell leukemia that completely remitted on administration of tetracycline. These experiments even more clearly demonstrate the leukemogenic potential of BCR-ABL as a sole oncogenic abnormality.

G. BCR-ABL Signaling and CML Pathogenesis Significant advances have been made in determining the signaling pathways that are impacted by BCR-ABL tyrosine kinase activity (Fig. 2). Numerous substrates and binding partners have been identified and current

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Fig. 2 Signaling pathways impacted by BCR-ABL expression. BAD, BCL-2 antagonist of cell death; BCL-XL, long form of BCL-X protein (BCL, B cell lymphoma); CBL, Casitas B-lineage lymphoma protein; CRKL, CRK-like; DOK, docking protein; ERK, extracellular signal-related protein kinase; GAP, GTPase-activating protein; GRB-2, growth factor receptor-bound protein 2; MEK, MAPK (mitogen-activated protein kinase)/ERK kinase; MYK, PI3K, phosphatidylinositol 3-kinase; SAPK, stress-activated protein kinase; SHC, SRC homology 2 domain-containing protein; SOS, ‘‘son of sevenless’’ (guanine nucleotide exchange factor); STAT, signal transducer and activator of transcription; AKT, CRK, MYC, RAS, and RAF are products of viral oncogene homologs, and their abbreviations are derived from the viral oncogene names.

efforts are directed at linking these pathways to the specific pathologic defects that characterize CML (Deininger et al., 2000). These defects include the following: increased proliferation or decreased apoptosis of a hematopoietic stem cell or progenitor cell, leading to a massive increase in myeloid cell numbers; premature release of immature myeloid cells into the circulation, postulated to be due to a defect in adherence of myeloid progenitors to marrow stroma; and genetic instability resulting in disease progression. An example of a cellular pathway that links to an increased proliferative rate is activation of the RAS pathway. Protection from programmed cell death may be mediated in part through STAT-5 upregulation of the antiapoptotic molecule BCL-XL and phosphorylation of and inactivation of the proapoptotic molecule BAD by AKT (Deininger et al., 2000). CML cells also exhibit reduced adhesion to fibronectin, possibly as a downstream effect of CRKL phosphorylation (Deininger et al., 2000).

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Despite the seemingly endless expansion of the list of pathways activated by BCR-ABL and the increasing complexity that is being revealed in these pathways, all of the transforming functions of BCR-ABL are dependent on its tyrosine kinase activity (Lugo et al., 1990).

H. BCR-ABL as a Therapeutic Target BCR-ABL possesses many characteristics of an ideal therapeutic target. It is expressed in the majority of patients with CML and it has been shown to be the cause of CML. BCR-ABL functions as a constitutively activated tyrosine kinase and mutagenic analysis has shown that this activity is essential for the transforming function of the protein. Thus, an inhibitor of the BCR-ABL kinase would be predicted to be an effective and selective therapeutic agent for CML. This recognition has relied on numerous fields of study and their convergence (Fig. 3). There is the field of tumor virology, from which oncogenes such as v-ABL were identified. The field of chromosome banding and gene mapping led to the recognition of BCR-ABL arising from the (9;22) chromosomal translocation. In addition, there is the entire field of protein phosphorylation, from serine/threonine kinases to tyrosine kinases, linking to oncogenes, leading to an understanding that BCR-ABL functions as a tyrosine kinase. This understanding generated interest in developing a specific inhibitor of this protein tyrosine kinase.

Fig. 3 Summary of the significant events leading to an understanding of the molecular pathogenesis of CML and to specific therapy for this disease. TK, tyrosine kinase.

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III. DEVELOPMENT OF AN ABL-SPECIFIC TYROSINE KINASE INHIBITOR A. Chemistry Given the success of imatinib and the enormous interest in protein kinase inhibitors, it is easy to forget the degree of skepticism that kinase inhibitors faced from the scientific community and the pharmaceutical industry in the 1980s and 1990s. Much of this skepticism was due to the prevailing thought that inhibitors of ATP binding would lack sufficient target specificity to be clinically useful. However, in 1988, Yaish et al. published a series of compounds, known as tyrphostins that demonstrated that specific tyrosine kinase inhibitors could be developed (Yaish et al., 1988). Working independently, scientists at Ciba-Geigy (now Novartis) under the direction of N. Lydon and A. Matter, performed high-throughput screens of chemical libraries, searching for compounds with kinase-inhibitory activity. From this time-consuming approach, a lead compound of the 2-phenylaminopyrimidine class was identified. The activity of the 2-phenylaminopyrimidine series was optimized for various kinases by synthesizing chemically related compounds and analyzing the relationship between their structure and activity (Fig. 4) (Buchdunger et al., 1996; Zimmermann et al., 1996, 1997). A key finding was that substitutions at the 6-position of the anilino phenyl ring led to loss of serine/threonine kinase inhibition, whereas the introduction of a methyl group at this position retained or enhanced activity against tyrosine kinases (Fig. 4C). The activity against the platelet-derived growth factor receptor (PDGFR) tyrosine kinase was further enhanced by the introduction of a benzamide group at the phenyl ring (Fig. 4B). These compounds were also found to possess inhibitory activity toward ABL, with CGP57148 (STI571, now imatinib mesylate, Gleevec, Glivec) emerging as the lead compound for clinical development. Introduction of N-methylpiperazine as a polar side chain greatly improved water solubility and oral bioavailability (Fig. 4D). Imatinib emerged from these efforts as the lead compound for preclinical development on the basis of its selectivity against CML cells in vitro and its drug-like attributes, including pharmacokinetic and formulation properties.

B. Preclinical Studies Imatinib showed inhibitory activity against ABL and its activated derivatives v-ABL, BCR-ABL, and TEL-ABL (Buchdunger et al., 1996; Carroll et al., 1996; Druker et al., 1996). Fifty percent inhibitory concentration

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Fig. 4 Optimization of the phenylaminopyrimidine lead structure and synthesis of imatinib. The initial 2-phenylaminopyrimidine lead inhibitor is shown (A), with steps (B) and (C) leading to imatinib (D). See text for details.

(IC50) values are in the range of 0.025 M, using in vitro kinase assays with immunoprecipitated or purified proteins. Activity against the PDGFR and KIT are in a similar range. In contrast, the IC50 values for a large number of other tyrosine and serine/threonine kinases were generally at least 100-fold higher, demonstrating that imatinib exhibits a high level of selectivity (Buchdunger et al., 2001). Cellular studies showed that imatinib specifically inhibited the proliferation of myeloid cell lines that express BCR-ABL or CML blast crisis cell lines (Deininger et al., 1997; Druker et al., 1996; Gambacorti-Passerini et al., 1997). The IC50 for BCR-ABL phosphorylation in intact cells is between 0.25 and 0.5 M (Buchdunger et al., 2001). Complete inhibition of proliferation with cell death through apoptotic mechanisms occurs between 0.5 and 1 M concentrations of imatinib. Further experiments showed that p185- and p210-expressing cells were equally sensitive to imatinib (Beran et al., 1998; Carroll et al., 1997). Concentrations of up to 10 M imatinib did not affect the growth of BCR-ABL-negative cell lines (Druker et al., 1996). To assess the effects of imatinib on committed hematopoietic progenitors, mononuclear cells from patients with CML and normal individuals were

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studied in assays of colony formation. These studies showed a marked decrease (92–98%) in the number of BCR-ABL-positive colonies formed with no inhibition of normal colony formation, using 1 M imatinib (Deininger et al., 1997; Druker et al., 1996). The differential sensitivity of CML versus normal cells to imatinib was also confirmed with long-term culture-initiating cells, which represent early hematopoietic progenitor cells (Kasper et al., 1999; Marley et al., 2000). Dose-dependent inhibition of tumor growth was seen in animals inoculated with BCR-ABL-expressing cells and treated daily with imatinib, with no effects against v-SRC-expressing tumors. Using a once-per-day injection schedule of up to 50 mg/kg, tumor growth was inhibited, but not eradicated (Druker et al., 1996). The reason for this modest in vivo activity became apparent from the murine pharmacokinetic profile of imatinib. This profiling revealed a short drug half-life in mice, which was not seen in other species (rat, dog, human). Thus, in nude mice a single dose of imatinib inhibited BCR-ABL kinase activity for only 2 to 5 h. A three times-per-day dosing schedule led to a continual block of BCR-ABL kinase activity, resulting in eradication of tumors in 87% of imatinib-treated mice (le Coutre et al., 1999). On the basis of these data, it was considered likely that continuous exposure to imatinib would be required for optimal antileukemic effects.

IV. CLINICAL TRIALS IN CML A. Phase I Clinical Trials A standard dose-escalation, phase I study of imatinib began in June 1998. The study population consisted of CML patients in chronic phase, refractory or resistant to IFN-
-based therapy or intolerant of this drug (Druker et al., 2001b). At later stages of the study, patients with CML in blast crisis and patients with Ph chromosome-positive ALL were also enrolled (Druker et al., 2001a). Imatinib was well tolerated, with the most common side effects including occasional nausea, periorbital edema, and muscle cramps. Despite dose escalation from 25 to 1000 mg in 14 cohorts of patients, a maximally tolerated dose could not be defined. Imatinib was administered once daily and pharmacokinetics showed a half-life of 13–16 h (Druker et al., 2001b). At doses of 300 mg and above, significant therapeutic benefits were observed. In chronic phase patients who had failed therapy with IFN-
, 53 of 54 (98%) patients treated at 300 mg and above achieved a complete hematologic response, and with 1 year of follow-up, only 1 of these patients relapsed (Druker et al., 2001b). In myeloid blast crisis

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patients, 21 of 38 (55%) patients treated at doses at or exceeding 300 mg/ day responded, with 18% having responses lasting beyond 1 year (Druker et al., 2001a).

B. Phase II Studies These remarkable phase I data led to rapidly accruing phase II clinical trials. These clinical trials confirmed the results seen in the phase I studies and led to Food and Drug Administration approval of imatinib in May 2001 (Fig. 5). In chronic phase patients who had failed IFN-
therapy, 95% of patients achieved a complete hematologic response and 60% a major cytogenetic response, defined as a reduction in the percentage of Ph chromosome-positive metaphases to less than 35%. With a median follow-up of 29 months, only 13% of these patients have relapsed (Kantarjian et al., 2002). In accelerated phase and blast crisis patients, the response rates were also quite high, but relapses have been much more common, with the majority of blast crisis patients relapsing during the first year of therapy (Sawyers et al., 2002; Talpaz et al., 2002).

Fig. 5 Phase II clinical trials of imatinib for CML. The results of the phase II studies are shown for chronic phase patients who failed interferon therapy, and for accelerated phase and blast crisis patients. Results shown are with a median follow-up of up to 30 months, and the percent of patients with disease progression is at 24 months (Kantarjian et al., 2002; Sawyers et al., 2002; Talpaz et al., 2002). CR (complete response) and PR (partial response) for cytogenetic responses include patients with Ph chromosome-positive metaphases of less than or equal to 35%.

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C. Phase III Randomized Comparison of Imatinib with IFN-a Plus Ara-C The next clinical trial performed was a phase III study of newly diagnosed chronic phase patients, comparing imatinib with the standard therapy of IFN-
plus cytarabine (Ara-C). This study accrued more than 1000 patients in a 7-month period. Five hundred and fifty-three patients were randomized to each of the two treatments, imatinib at 400 mg/day or IFN-
plus Ara-C. There were no significant differences in prognostic features between the two arms. With a median follow-up of 18 months, patients randomized to imatinib had significantly better results than did patients treated with IFN-
plus Ara-C in all parameters measured, including rates of complete hematologic response (97 versus 69%, p < 0.001), major and complete cytogenetic responses (87 and 76% versus 35 and 14%, p < 0.001), discontinuation of assigned therapy due to intolerance (3 versus 31%), and progression to accelerated phase or blast crisis (3 versus 8%, p < 0.001) (Table I) (O’Brien et al., 2003). Responses to imatinib are rapid, with most patients taking imatinib achieving complete hematologic responses within the first 4 to 6 weeks of therapy. In addition, more than 50% of patients obtain a complete cytogenetic response in 3 months. Despite the fact that 76% of patients randomized to imatinib achieve a complete cytogenetic response, the majority of these patients have detectable leukemia as analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for BCR-ABL (Hughes et al., 2003). When BCR-ABL transcript levels are analyzed by log reduction, 39% of patients achieved

Table I

Phase III Results of Imatinib versus IFN-
Plus Cytarabine for Newly Diagnosed Chronic Phase Patients with CMLa Imatinib percentage responding at 400 mg/day (n ¼ 553)

CHR MCR CCR Intolerance Progressive disease

97 87 76 3 3

IFN-
plus Ara-C (n ¼ 553) 69 35 14 31 8.5

aFrom O’Brien et al. (2003). Results are with a median follow-up of 18 months. Abbreviations: CHR, complete hematologic response; MCR, major cytogenetic response; CCR, complete cytogenetic response; intolerance, leading to discontinuation of first-line therapy; progressive disease, progressing to accelerated phase or blast crisis. All of these differences are highly significant, with p < 0.001.

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at least a 3-log reduction in levels, but only 13 and 3% achieved a 4- and 5-log reduction, respectively. Thus, most patients treated with imatinib have persistent disease at the molecular level. Remaining questions for chronic phase patients include the durability of responses and how to integrate imatinib therapy with allogeneic stem cell transplantation. The mechanisms of disease persistence and whether it will be possible to completely eradicate the disease are currently being investigated. However, for advanced phase patients, the more pressing question is, why do they relapse?

V. MECHANISMS OF RELAPSE Response rates to imatinib in chronic phase patients are quite high and thus far, responses have been durable. Response rates are also quite high in patients with advanced phase disease, but relapses, despite continued therapy with imatinib, have been common. This high response rate in advanced phase patients is encouraging as imatinib targets an early molecular change in a malignancy that presumably contains multiple molecular abnormalities. In all patients who have relapsed, the BCR-ABL kinase remains present. One of the most useful categorizations of relapse mechanisms has been to separate patients into those with persistent inhibition of the BCRABL kinase and those with reactivation of the BCR-ABL kinase (Fig. 6). Patients with persistent inhibition of the BCR-ABL kinase would be predicted to have additional molecular abnormalities besides BCR-ABL driving the growth and survival of the malignant clone. In contrast, patients with persistent BCR-ABL kinase activity or reactivation of the kinase would be postulated to have resistance mechanisms that either prevent imatinib from reaching the target or render the target insensitive to imatinib. In the former category are mechanisms such as drug efflux or protein binding of imatinib. In the latter category would be mutations of the BCR-ABL kinase that render BCR-ABL insensitive to imatinib or amplification of the BCR-ABL protein. To examine BCR-ABL kinase activity, an assay has been developed that looks at the major tyrosine-phosphorylated protein in CML patient samples, CRKL (Druker et al., 2001b; Oda et al., 1994). Using this assay, it has been determined that the majority of patients who respond to imatinib and then relapse have reactivation of the BCR-ABL tyrosine kinase (Gorre et al., 2001). In these studies, greater than 50% and perhaps as many as 90% of patients have BCR-ABL point mutations encoding at least 18 different amino acids scattered throughout the ABL kinase domain (Fig. 7) (Nardi et al., 2004; Shah and Sawyers, 2003). Other patients have amplification of

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Fig. 6 Distinguishing between potential mechanisms of relapse among advanced phase CML patients.

Fig. 7 Schematic of point mutations in the ABL kinase domain. The ABL kinase domain, from amino acid 240 to 500, is shown with the ATP-binding domain (P), the catalytic domain (C), and the activation loop (A). The numbers below the kinase domain indicate amino acids that are mutated in patients who relapsed on therapy with imatinib. The columns above the kinase domain indicate the number of times each amino acid has been found to be mutated as compiled from the literature (Al-Ali et al., 2004; Barthe et al., 2002; Branford et al., 2002, 2003; Gorre et al., 2001; Hochhaus et al., 2002; Hofmann et al., 2001; Roche-Lestienne et al., 2002; Shah et al., 2002; von Bubnoff et al., 2002). Adapted from Shah et al. (2002).

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Fig. 8 (A) Structure of the activation loop (in black) in the kinase domains of ABL, HCK, and IRK in their inactive states. Note that, in ABL, the activation loop is folded back over the catalytic center of the molecule and occludes the mouth of the kinase. In the inactive state, the activation loops have distinct configurations, which forms the basis for specific inhibition.

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BCR-ABL at the genomic or transcript level (Hochhaus et al., 2002). In contrast, in patients with primary resistance, that is, patients who do not respond to imatinib therapy, BCR-ABL-independent mechanisms are most common (Hochhaus et al., 2002). Whether these mechanisms of relapse will apply to the situation of disease persistence in chronic phase patients treated with imatinib is being investigated. Analysis of the ability of imatinib to inhibit the kinase activity of the BCR-ABL kinase domain mutations found in relapsed patients has shown that some might be sensitive to dose escalation, but that most are highly resistant to imatinib (Corbin et al., 2002, 2003). ABL kinase inhibitors with specificity that differs from that of imatinib have already been synthesized and one of these compounds, PD180970, is capable of inhibiting some, but not all, of the common BCR-ABL kinase mutations (La Rosee et al., 2002). These data suggest that it may be possible to treat patients with several different ABL kinase inhibitors to circumvent resistance.

VI. STRUCTURAL BASIS OF ABL INHIBITION BY IMATINIB The catalytic domains of kinases include the ATP-binding lobe (P-loop), the catalytic site, and the activation loop (A-loop). In active kinases, the A-loop is in an ‘‘open’’ conformation, as it swings away from the catalytic center of the kinase (Fig. 8A). Whereas the conformation of the A-loop is structurally similar in kinases when they are in the active, open conformation, there are considerable differences between their inactive (closed) conformations. The crystal structure of the catalytic domain of the ABL kinase in complex with an imatinib analog and with imatinib has been solved (Nagar et al., 2002; Schindler et al., 2000). The most important finding of these studies is that the compound binds to the inactive conformation of ABL, In contrast, tyrosine kinase domains are similar in their active states, when the activation loop is folded away from the catalytic center as in active LCK. (B) Structure of imatinib bound to the kinase domain of ABL. The arrow indicates the P-loop, and the arrowhead points to helix
C. The activation loop is shown in blue, with the conserved DFG motif in gold. Imatinib penetrates the center of the kinase, stabilizing the inactive conformation of the activation loop. Binding to the activation loop occurs without major steric clashes, whereas binding to the P-loop involves an induced fit mechanism. Imatinib would be unable to bind to the active configuration of the kinase (A). (C) Amino acids contacting imatinib. Imatinib: carbon is shown in green, nitrogen in blue, and oxygen in red. Carbons of the protein backbone are shown in orange. Hydrogen bonds are indicated as dashed lines. Residues that form hydrophobic interactions are circled. Imatinib contacts 21 amino acids within the ABL kinase domain (adapted from Nagar et al., 2002).

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contacting 21 amino acid residues. By exploiting the distinct inactive conformation of the A-loop of ABL, imatinib is able to achieve its high specificity. No major structural rearrangements are required for imatinib to bind to the A-loop. In contrast, there are significant structural alterations induced by imatinib binding in the P-loop, which is glycine rich and highly flexible (Fig. 8B). In examining imatinib-resistant mutations, they fall into several categories. One obvious set of mutations includes those at contact points between the ABL kinase domain and imatinib. Examples of this include T315I and E255K. Another set includes those that alter the conformation of the binding pocket such that imatinib can no longer bind or those that prevent the alterations in the P-loop required for imatinib binding. A related class includes mutations that stabilize the open, active conformation of ABL, such that imatinib can no longer bind. Interestingly, ABL inhibitors with activity against these mutants have been shown to bind to the open, active conformation of ABL (Nagar et al., 2002). These inhibitors are also inactive against mutations that impair their binding (La Rosee et al., 2002); thus to date, no inhibitor of T315I has been identified.

VII. ACTIVITY OF IMATINIB IN OTHER INDICATIONS A. Gastrointestinal Stromal Tumors In addition to inhibiting the ABL tyrosine kinase, imatinib inhibits the PDGFR and KIT tyrosine kinases. There are now several other cancers in which imatinib has shown clinical benefits that are based on the profile of kinases inhibited by imatinib and an understanding of the genetic defects causing various malignancies (Table II). One such disease is gastrointestinal stromal tumor (GIST). GISTs are mesenchymal neoplasms that can arise from any organ in the gastrointestinal tract or from the mesentery or omentum. There are approximately 5000 new cases per year in the United States. Although GISTs morphologically resemble leiomyosarcomas and nerve sheath tumors, they are a distinct entity (Fletcher et al., 2002). Published data suggest that the response rate of GISTs to single- or multiagent chemotherapy is less than 5%. More than 90% of GISTs express KIT and biochemical evidence of KIT activation can be found in almost all GISTs (Hirota et al., 1998; Rubin et al., 2001). In approximately 90% of cases, this activation is linked to somatic mutations of KIT, usually involving exon 9 or 11 (Rubin et al., 2001). Several lines of evidence indicate that KIT mutations are an early pathogenetic event in GISTs: (1) a similar frequency and spectrum of mutations is seen in histologically benign versus malignant tumors and early/localized

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Table II Target ABL KIT PDGFRA PDGFRB

Diseases Against Which Imatinib Has Shown Activity Disease

Mechanism of activation

CML ALL GIST GIST HES CMML DFSP

Chromosomal translocation t(9;22): BCR-ABL Chromosomal translocation t(9;22): BCR-ABL Point mutation Point mutation Intrachromosomal deletion: FIP1L1-PDGFRA Chromosomal translocation t(5;12): EVT6-PDGFRB Chromosomal translocation t(17;22): Col1A1-PDGFB

Abbreviations: ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; DFSP, dermatofibrosarcoma protuberans; GIST, gastrointestinal stromal tumor; HES, hypereosinophilic syndrome.

versus late/metastatic tumors; (2) familial syndromes of GIST are associated with germ line mutations of KIT; and (3) molecular studies suggest that KIT mutations are acquired before the development of cytogenetic abnormalities (Corless et al., 2002; Heinrich et al., 2002; Rubin et al., 2001). The concept that patients with GISTs might benefit from treatment with imatinib was based in part on two experimental observations: (1) treatment of GIST cell lines with imatinib inhibited proliferation and induced apoptosis, and (2) several GIST-associated mutant KIT isoforms were potently inhibited by imatinib in vitro at concentrations similar to wild-type KIT (Heinrich et al., 2000a; Tuveson et al., 2001). On the basis of the identification of mutated KIT as a therapeutic target in GIST and the lack of an effective conventional medical therapy, a patient with chemotherapy-resistant gastric GIST metastatic to omentum and liver was started on imatinib at 400 mg/day in March 2000 (Joensuu et al., 2001). The tumor in this patient expressed an exon 11 mutant KIT isoform and the patient responded dramatically to imatinib therapy. Spurred by this clinical success, larger clinical trials for this disease indication were performed. In these clinical trials, the objective response rate to imatinib as a single agent in patients with advanced GIST was 53 to 65%, with another 19 to 36% of patients having disease stabilization (Demetri et al., 2002; van Oosterom et al., 2001). These clinical trials have served as the basis for further exploration of the utility of imatinib in GISTs, including the adjuvant and neoadjuvant, because the recurrence rate of GISTs after surgery is quite high.

B. Other Malignancies Imatinib has also shown significant activity in patients with ALL who are BCR-ABL positive (Ottmann et al., 2002), but responses are generally transient. Translocations involving the PDGFRB gene have been identified

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in several myeloproliferative and myelodysplastic syndromes. The most common of these translocations, t(5;12)(q33;p13), is seen in a subset of patients with chronic myelomonocytic leukemia (CMML) and results in fusion of the EVT6 (TEL) and PDGFRB genes (Golub et al., 1994). Several patients with CMML containing the (5;12) translocation have been treated with imatinib and all achieved complete hematologic remissions (Apperley et al., 2002; Magnusson et al., 2002). The PDGFRB pathway is also a target in dermatofibrosarcoma protuberans (DFSP), a low-grade sarcoma of the dermis that often recurs after surgical excision. These tumors are characterized by a (17;22) translocation involving the COL1A1 and PDGFB genes, which results in overproduction of PDGF-BB and consequent hyperactivation of PDGFRB (Simon et al., 1997). It has been shown that imatinib inhibits the growth of DFSP cells both in culture and in immunodeficient mice (Sjoblom et al., 2001) and preliminary results in patients look promising (Maki et al., 2002; Rubin et al., 2002). Another disease caused by KIT mutations is systemic mastocytosis. The majority of tumors have a mutation of Asp-816 to valine (D816V) in the kinase domain of KIT, resulting in activation of KIT. Unfortunately, the kinase activity of the D816V mutant isoform is resistant to imatinib (Heinrich et al., 2000b; Ma et al., 2002), probably because this mutation induces conformational changes in the activation loop of KIT that prevent the binding of imatinib. Thus, imatinib is unlikely to be useful in this disorder, but an inhibitor of this KIT mutation would be predicted to be an effective therapy for this disorder. As KIT and the PDGFRs are expressed in many common tumors and are reported to be activated by both autocrine and paracrine mechanisms, there has been much interest in using imatinib broadly. As no other tumor has genetic evidence of involvement of these receptor systems, clinical trials with imatinib in these indications would be considered empiric clinical trials. Thus far, single-agent activity of imatinib in tumors such as smallcell lung cancer and breast, prostate, melanoma, and non-GIST sarcomas has been minimal (Johnson et al., 2003; Modi et al., 2003; Rao et al., 2003; Verweij et al., 2003; Wyman et al., 2003). One example in which empiric clinical trials of imatinib have been successful is hypereosinophilic syndrome (HES) (Gleich et al., 2002). The dramatic empiric results of imatinib in HES prompted investigations of the molecular basis for imatinib activity in this disease. Two groups independently arrived at the conclusion that an intrachromosomal deletion on chromosome 4 resulted in a fusion between a gene of unknown function, FIP1L1, and a truncated PDGFRA in a large percentage of patients with this disorder (Cools et al., 2003; Griffin et al., 2003). The resulting FIP1L1-PDGFRA fusion protein is a constitutively activated tyrosine kinase that is imatinib sensitive, thus accounting for the responsiveness of this disease to imatinib.

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VIII. LESSONS LEARNED FROM THE CLINICAL TRIALS OF IMATINIB A. Patient Selection One of the goals of cancer research is to develop the ability to match the right patient with the right drug, based on specific knowledge of the genetic abnormalities of a tumor. The impact of this was clear in the clinical trials of imatinib. As an ABL inhibitor was being tested, enrollment was limited to patients with activated ABL driving their cancer, and these patients could be identified easily as they had the Ph chromosome or BCR-ABL. In GIST, the situation was more complicated. In these clinical trials, virtually all patient tumors expressed KIT. The majority of tumors had activating KIT mutations in exon 11 and these patients had a partial response rate of close to 80%. In contrast, patients whose tumors expressed wild-type KIT, with no mutation, had a response rate of only 18% (Heinrich et al., 2003a). Thus, KIT mutational status correlated with response and in GIST, expression of KIT is not sufficient to predict responses; rather, a mutation in KIT is necessary for responses to be observed. It has also been instructive to determine why 18% of patients with wildtype KIT expression respond to imatinib. Examination of tumors from patients with wild-type KIT expression showed that one-third of these tumors had activating mutations of the PDGFRA gene (Heinrich et al., 2003b). These mutations occurred in two different exons. One set of mutations was imatinib sensitive and this accounted for responses observed in patients whose tumors expressed wild-type KIT (Heinrich et al., 2003b). Thus, careful evaluations of subsets of responding patients can yield important insights into disease pathogenesis and the mechanism of response to an agent.

B. Dose Selection In the phase I clinical trials of imatinib, a maximally tolerated dose was never reached. Even among those closely associated with the phase I studies there was a lack of consensus about when to discontinue dose escalation. There were some investigators who felt that no cap on the dose should be considered except the maximally tolerated dose, particularly because studies of solid tumors were planned and because penetration of imatinib into solid tumors might require higher doses. Others believed that alternative end points, such as optimal therapeutic response and/or pharmacokinetic end points, could be used. In evaluating pharmacokinetic end points,

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it was known from preclinical studies that continuous exposure of cells to imatinib doses of 1 M or higher resulted in maximal cell killing (Druker and Lydon, 2000). In the phase I clinical trials, a trough level of 1 M was reached at a dose level of 300 mg, which corresponds to a threshold for significant therapeutic benefits (Peng et al., 2004). Thus, pharmacokinetic parameters could have been used to predict therapeutic responses. With solid tumors, pharmacokinetic analyses of tumor samples might be necessary to address the issue of tumor penetrance. When using molecularly targeted agents, it would seem more reasonable to consider maximal modulation of the target as the therapeutic end point. In the case of CML and a BCR-ABL kinase inhibitor, the obvious choice would be to assess for maximal inhibition of the BCR-ABL kinase activity. Direct assays of BCR-ABL tyrosine phosphorylation in patient samples have been difficult; therefore, assays of CRKL phosphorylation have been used. CRKL, a 39-kDa SH2, SH3-containing adaptor protein, is the major novel tyrosine phosphorylated protein in CML neutrophils and is a direct substrate of BCR-ABL (Oda et al., 1994). Assays of CRKL phosphorylation, based on its migration in sodium dodecyl sulfate–polyacrylamide gels, has shown that the BCR-ABL kinase is inhibited by imatinib in vivo (Druker et al., 2001b). This assay has also been useful in evaluating relapse mechanisms (Gorre et al., 2001; Hochhaus et al., 2002). However, as the amount of inhibition is not easily quantitated, this assay has been less useful at defining an optimal dose of therapy.

IX. TRANSLATING THE SUCCESS OF IMATINIB TO OTHER MALIGNANCIES The clinical trials with imatinib are a dramatic demonstration of the potential of targeting molecular pathogenetic events in a malignancy. As this paradigm is applied to other malignancies, it is worth remembering that BCR-ABL and CML have several features that were critical to the success of this agent. One of these is that BCR-ABL tyrosine kinase activity has clearly been demonstrated to be critical to the pathogenesis of CML. Thus, not only was the target of imatinib known, but also the target is a critical factor required for the development of CML. Another important feature is that as with most malignancies, treatment earlier in the course of the disease yields better results. Specifically, the response rate and durability of responses have been greater in chronic phase patients as opposed to blast phase patients. Thus, for maximal utility as a single agent, the identification of crucial, early events in malignant progression is the first step in reproducing the success with imatinib in other malignancies. An equally important issue is the

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selection of patients for clinical trials on the basis of the presence of an appropriate target. Again, in the CML experience, patients with activation of BCR-ABL were easily identifiable by the presence of the Ph chromosome. In this regard, as reagents to analyze molecular end points are developed, these same reagents should be useful in identifying appropriate candidates for treatment with a specific agent. When all of these elements are put together, a critical pathogenetic target that is easily identifiable early in the course of the disease, remarkable results with an agent that targets this abnormality can be achieved. The obvious goal is to identify these early pathogenetic events in each malignancy and to develop agents that specifically target these abnormalities.

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Heinrich, M. C., Griffith, D. J., Druker, B. J., Wait, C. L., Ott, K. A., and Zigler, A. J. (2000a). Inhibition of c-Kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood 96, 925–932. Heinrich, M. C., Wait, C. L., Yee, K. W. H., and Griffith, D. J. (2000b). STI571 inhibits the kinase activity of wild type and juxtamembrane c-kit mutants but not the exon 17 D816V mutation associated with mastocytosis. Blood 96, 173b. Heinrich, M. C., Rubin, B. P., Longley, B. J., and Fletcher, J. A. (2002). Biology and genetic aspects of gastrointestinal stromal tumors: KIT activation and cytogenetic alterations. Hum. Pathol. 33, 484–495. Heinrich, M. C., Corless, C. L., Demetri, G. D., Blanke, C. D., von Mehren, M., Joensuu, H., McGreevey, L. S., Chen, C. J., Van den Abbeele, A. D., Druker, B. J., Kiese, B., Eisenberg, B., Roberts, P. J., Singer, S., Fletcher, C. D., Silberman, S., Dimitrijevic, S., and Fletcher, J. A. (2003a). Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J. Clin. Oncol. 21, 4342–4349. Heinrich, M. C., Corless, C. L., Duensing, A., McGreevey, L., Chen, C. J., Joseph, N., Singer, S., Griffith, D. J., Haley, A., Town, A., Demetri, G. D., Fletcher, C. D., and Fletcher, J. A. (2003b). PDGFRA activating mutations in gastrointestinal stromal tumors. Science 299, 708–710. Heisterkamp, N., Jenster, G., ten Hoeve, J., Zovich, D., Pattengale, P. K., and Groffen, J. (1990). Acute leukaemia in bcr/abl transgenic mice. Nature 344, 251–253. Hermans, A., Heisterkamp, N., von Lindern, M., van Baal, S., Meijer, D., van der Plas, D., Wiedemann, L. M., Groffen, J., Bootsma, D., and Grosveld, G. (1987). Unique fusion of bcr and c-abl genes in Philadelphia chromosome positive acute lymphoblastic leukemia. Cell 51, 33–40. Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S., Kawano, K., Hanada, M., Kurata, A., Takeda, M., Muhammad Tunio, G., Matsuzawa, Y., Kanakura, Y., Shinomura, Y., and Kitamura, Y. (1998). Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 279, 577–580. Hochhaus, A., Kreil, S., Corbin, A. S., La Rose´ e, P., Mu¨ ller, M. C., Lahaye, T., Hanfstein, B., Schoch, C., Cross, N. C. P., Berger, U., Gschaidmeier, H., Druker, B. J., and Hehlmann, R. (2002). Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. Leukemia 16, 2190–2196. Hofmann, W. K., Jones, L. C., Lemp, N. A., de Vos, S., Gschaidmeier, H., Hoelzer, D., Ottmann, O. G., and Koeffler, H. P. (2001). Phþ acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation. Blood 99, 1860–1862. Huettner, C. S., Zhang, P., Van Etten, R. A., and Tenen, D. G. (2000). Reversibility of acute B-cell leukaemia induced by BCR-ABL1. Nat. Genet. 24, 57–60. Hughes, T. P., Kaeda, J., Branford, S., Rudzki, Z., Hochhaus, A., Hensley, M. L., Gathmann, I., Bolton, A. E., van Hoomissen, I. C., Goldman, J. M., and Radich, J. P. (2003). Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N. Engl. J. Med. 349, 1423–1432. Hunter, T., and Cooper, J. A. (1985). Protein-tyrosine kinases. Annu. Rev. Biochem. 54, 897–930. Joensuu, H., Roberts, P. J., Sarlomo-Rikala, M., Andersson, L. C., Tervahartiala, P., Tuveson, D., Silberman, S., Capdeville, R., Dimitrijevic, S., Druker, B., and D. D. G. (2001). Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N. Engl. J. Med. 344, 1052–1056. Johnson, B. E., Fischer, T., Fischer, B., Dunlop, D., Rischin, D., Silberman, S., Kowalski, M. O., Sayles, D., Dimitrijevic, S., Fletcher, C., Hornick, J., Salgia, R., and Le Chevalier, T. (2003). Phase II study of imatinib in patients with small cell lung cancer. Clin. Cancer Res. 9, 5880–5887.

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