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Oregon Health and Science University Cancer Institute, Portland, Oregon
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I. Introduction |
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Chronic myelogenous leukemia (CML1) was described in 1845 by Hughes Bennet, a physician from Edinburgh who thought that the disease was an infection (Bennett, 1845
). Rudolf Virchow, who published a similar case only a few weeks later, postulated that the disease was noninfectious and later coined the term leukemia (from 
u
o
µ = white blood) (Virchow, 1845). That the leukemic cells originated from the bone marrow was recognized by Neumann in 1870 (Neumann, 1870). In 1960, Nowell and Hungerford (1960), two Philadelphia researchers noted that an abnormally small chromosome was consistently present in the cells of CML patients, and this chromosome was subsequently called the Philadephia (Ph)1 chromosome. This was the first time that a chromosomal abnormality had been associated with a malignant disease. In 1973, Janet Rowley recognized that the Ph1 chromosome was indeed the product of a reciprocal translocation between the long arms of chromosomes 9 and 22, the t(9;22)(q34;q11). The early 1980s saw the identification of the two genes that flank the translocation breakpoint. The ABL gene from chromosome 9 had been known as the human homolog of a murine leukemia virus (Abelson and Rabstein, 1970; Bartram et al., 1983); the translocation partner from chromosome 22 was termed BCR for breakpoint cluster region, since DNA breaks occurred in a relatively small genomic region (Groffen et al., 1984). Of paramount importance was the discovery that the protein derived from the chimeric BCR-ABL gene had protein-tyrosine kinase (PTK) activity that was deregulated compared with normal Abl and correlated with its ability to transform cells to a malignant phenotype (Lugo et al., 1990). In 1990, several groups reported that a CML-like disease could be induced in mice transplanted with bone marrow infected with a BCR-ABL retrovirus (Daley et al., 1990; Heisterkamp et al., 1990). This proved the point that BCR-ABL is the causative agent and not just a marker of the disease. In 1996, Druker and colleagues described CGP57148, a highly specific pharmacologic inhibitor of the Abl-tyrosine kinase that selectively suppressed the growth of BCR-ABL-positive cells. This compound, first renamed STI571 and then imatinib, has revolutionized the treatment of CML and set a precedent for the development of targeted therapies for malignant diseases.
The incidence of CML is approximately 1 to 1.5/105 (Sawyers, 1999). Thus, between 3500 and 5000 new cases per year are expected in the United States. The incidence rises slowly with age until the middle forties when it starts to rise more rapidly, resulting in a median age at diagnosis of about 60 years. There is no geographical or ethnic background that predisposes to CML. The only well characterized risk factor is exposure to ionizing radiation. An increased incidence of CML was observed approximately 8 years after the atomic bombings of Hiroshima and Nagasaki (Heyssel et al., 1960). Patients exposed to Thorotrast, an -emitter that was used as a contrast medium in radiology in the 1930s, also have an increased risk of developing CML (Van Kaick et al., 1990), as do patients treated with radiation therapy (Corso et al., 1995). BCR-ABL fusion transcripts can be induced in vitro by high-dose ionizing radiation (Deininger et al., 1998). In contrast to acute myelogenous leukemia, there is no convincing evidence to support a link between exposure to organic solvents and CML.
The clinical hallmarks of CML are leukocytosis, a left shift in the differential count, and splenomegaly. Importantly, in contrast to acute myelogenous leukemia, the disease is not restricted to the myeloid compartment, since the Philadelphia chromosome is regularly demonstrable in megakaryocytes and erythroid precursor cells. Thus, high platelet counts are frequent, but for unknown reasons, erythrocytosis is rarely seen. CML runs a three-phased course. During the initial chronic phase, there is gross expansion of the myeloid cell compartment, but the cells still retain the capacity to differentiate and function normally. Symptoms in the chronic phase are generally mild and many patients are asymptomatic, being diagnosed by routine blood sampling (Cervantes et al., 1999). After an average of 4 to 5 years, the disease typically progresses to accelerated phase, characterized by the appearance of more immature cells in the blood, frequent constitutional symptoms, and a less favorable response to therapy. The diagnostic criteria for accelerated phase are not universal, reflecting that disease progression from chronic to accelerated phase is a continuous process rather than a single step. Although the duration of accelerated phase varies from weeks to years, the disease inexorably progresses to the final stage of blast crisis, where immature cells dominate and survival is measured in weeks to months.
Given the highly diverse treatment options available for CML and its variable clinical course, methods have been developed to predict the biological behavior of individual cases based on information available at diagnosis. The most commonly used prognostic scores are the Sokal (Sokal et al., 1984) and European (Hasford et al., 1998) risk scores. They are based on parameters with a known adverse effect on outcome such as spleen size, platelet count, blast-, basophil-, and eosinophil counts as well as age. Although these scores are capable of separating patients into low, intermediate, and high risk groups, they are relatively crude indicators that have limited value for outcome prediction in individual patients. Moreover, it is not clear if they retain their predictive value in patients treated with imatinib. An important focus of CML research is the development of risk scores based on molecular rather than clinical features of the disease.
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II. Pathogenesis of Chronic Myelogenous Leukemia |
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A. The Philadelphia Translocation and the BCR-ABL Fusion Gene
As a result of the exchange of genetic material, two fusion genes are produced: BCR-ABL on the derivative chromosome 22 and ABL-BCR on the derivative 9 (Fig. 1, A and B). Although ABL-BCR mRNA is expressed in approximately two-thirds of CML patients (Melo et al., 1993), expression of the protein has never been documented. Thus, the pathogenetically relevant principle is the BCR-ABL fusion gene and its cognate Bcr-Abl protein. All DNA breakpoints occur within introns, and regardless of their precise location, two types of fusion mRNA are generated that contain the first 13 or 14 exons of BCR fused to ABL exon 2 (e13a2 and e14a2 fusions, respectively) (Fig. 2) (Deininger et al., 2000a). Very rarely in CML, and much more frequently in acute lymphoblastic leukemia (ALL), the break in BCR occurs between the first and second exons, resulting in an e1a2 fusion mRNA (Melo et al., 1994). Yet other types of fusion have been described in isolated cases (Pane et al., 1996; Al Ali et al., 2002). The variation in the BCR part of the fusion mRNA contrasts with the constant ABL part. This is in itself an indication that ABL is likely to carry the relevant transforming principle.
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Classical cytogenetics is still the mainstay of diagnosing and monitoring CML. However, fluorescence-in situ hybridization (FISH), used to detect the BCR-ABL translocation, has become an important complementary method (Fig. 1C). FISH is able to detect the 5% or so of CML cases with a masked translocation that escapes conventional cytogenetics. Moreover, it is useful for samples in which metaphases cannot be obtained. Its main limitation is the fact that it does not detect abnormalities other than the BCR-ABL translocation.
Reverse transcription-polymerase chain reaction (RTPCR) is used for both diagnostic purposes and follow-up, particularly after allogeneic stem cell transplantation (Cross et al., 1993). It is the most sensitive method for monitoring residual disease. Improved methods of quantification enable reliable monitoring of leukemic burden and allow for therapeutic interventions before cytogenetic or hematological relapse occurs (Dazzi and Goldman, 1999).
The function of the various structural motifs in the Bcr and Abl proteins have recently been reviewed (Deininger et al., 2000a). Both Bcr and Abl are multidomain proteins. The physiological function of Bcr is not well understood. The N terminus of the protein has serine/threonine kinase activity (Maru and Witte, 1991) and a dimerization domain (McWhirter et al., 1993). The central portion of the protein contains dbl-like and pleckstrin homology domains that stimulate GDP-GTP exchange on rho guanidine exchange factors (Ron et al., 1991) (Fig. 3A). The C terminus has GTPase activity for Rac, a Ras-family protein that activates an NADPH oxidase in neutrophils (Diekmann et al., 1991). Altogether, this suggests a function for Bcr in signal transduction. However, apart from an increased neutrophilic burst, the phenotype of mice with homozygous deletion of BCR is normal (Voncken et al., 1995).
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The Abl protein contains several domains such as the Src homology domains 2 and 3 (SH2 and SH3), prolinerich regions in the center, and the actin-binding domain at the C terminus, which allow for interactions with other proteins (Fig. 3B). In addition, there is a DNA-binding domain as well as nuclear localization signals. The SH1 domain has the PTK activity that may be regulated by the NH2-terminal SH3 domain. The data regarding the physiological function of Abl are complex (recently reviewed in Van Etten, 1999). The nuclear fraction appears to play an inhibitory role in cell cycle regulation, which led to the notion that ABL is a tumor suppressor gene (Sawyers et al., 1994). The cytoplasmic pool may function in the transmission of integrin-mediated signals from the cellular environment (Lewis and Schwartz, 1998). Importantly, Abl interacts with several proteins involved in DNA repair such as ataxia telangiectasia-mutated (Atm) (Baskaran et al., 1997; Shafman et al., 1997), DNA-PK (Kharbanda et al., 1997), and Rad51 (Yuan et al., 1998; Chen et al., 1999). It appears that Abl kinase activity is important for the induction of apoptosis in response to genotoxic stress such as ionizing radiation (Yuan et al., 1997). ABL null mice have a high neonatal mortality, and the survivors exhibit a variety of defects such as disturbed immune function, bone defects, and a rather ill-defined wasting syndrome (Schwartzberg et al., 1991; Tybulewicz et al., 1991). There is, however, no increased incidence of tumors in these mice, which argues against the concept of ABL as a tumor suppressor. It is possible that ARG (ABL-related gene), a close homolog of ABL, is capable of compensating for the loss of some of the functions of ABL. Notably, homozygous deletion of both the ABL and the ARG locus results in embryonic lethality due to a block of neuronal development (Koleske et al., 1998).
C. Essential Features of Bcr-Abl
Several motifs within the chimeric protein are essential for malignant transformation. In Abl, they include the SH1 (kinase) domain (Lugo et al., 1990) and actin-binding domain (McWhirter and Wang, 1993). In Bcr, the dimerization domain is crucial (McWhirter et al., 1993); it can be replaced by other sequences that also allow for dimerization, such as Tel in the Tel-Abl fusion protein that is seen in rare patients with acute lymphoblastic leukemia (Papadopoulos et al., 1995). In addition, a tyrosine at position 177 of Bcr is essential for transformation of myeloid cells (Million and Van Etten, 2000). The requirement of specific features for transformation of cells by Bcr-Abl is dependent on the cellular background. For example, the SH2 domain is crucial for fibroblast transformation (Afar et al., 1995) but not for transformation of cytokine-dependent hematopoietic cells to factor independence (Ilaria and Van Etten, 1995). However, malignant transformation is absolutely dependent upon the kinase domain, although more subtle effects of the Bcr-Abl protein, such as the adhesion defect, may be independent of its tyrosine kinase activity (Wertheim et al., 2002). There is no consensus as to how Abl kinase is regulated under physiological circumstances. Both trans- and cis-acting mechanisms have been implicated. The fact that purified Abl protein is kinase active (Sawyers et al., 1992) is indirect evidence for a trans-acting inhibitory factor. Several proteins have been shown to bind to Abl in vivo (Dai and Pendergast, 1995; Shi et al., 1995; Wen and Van Etten, 1997), but their true relevance is presently not completely understood. Other data suggest an inhibitory role for the SH3 domain and more 5' regions of the molecule (Mayer and Baltimore, 1994; Barila and Superti Furga, 1998). The SH3 domain may either bind Abl, leading to a kinase-inactive conformation, or it may bind to an inhibitory factor. In v-abl, the SH3 domain is deleted and replaced with viral Gag sequences, which leads to kinase activation and again argues for an inhibitory role of the SH3 domain.
D. Consequences of Deregulated Tyrosine Kinase Activity
The phosphorylation of cellular proteins on tyrosine residues is an important mechanism of intracellular signal transduction, used by many growth factor receptors. Normally, less than 1% of cellular tyrosine residues are phosphorylated, and the activity of tyrosine kinases is counterbalanced by the activity of tyrosine phosphatases. In cells that express a constitutively active tyrosine kinase, this tight regulation is undermined, leading to a situation that resembles chronic growth factor stimulation. In the case of Bcr-Abl, a multitude of signal transduction pathways is activated. They appear to target three major cellular functions.
1. Increased Proliferation.
Many cell lines derived from CML patients in blast crisis proliferate in the absence of growth factors. The defect in the chronic phase appears to be more subtle. These cells are not completely factor-independent, but compared with normal cells, they proliferate at lower cytokine concentrations (Jonuleit et al., 1998) or they may have interleukin-3-driven autocrine loops (Jiang et al., 1999). This may enable them to outpace normal hematopoiesis over time.
2. Reduced Apoptosis.
Bcr-Abl stimulates pathways that transduce survival signals, leading to a decreased rate of apoptosis (Bedi et al., 1994). Again, this phenomenon is more easily demonstrable in cell lines (Cortez et al., 1996) than in primary chronic phase cells, which still require cytokines for survival. This may explain, why some studies found CML progenitor cells no more resistant to apoptosis induced by growth-factor withdrawal and ionizing radiation than normal cells (Amos et al., 1995). Bcr-Abl effects may also include a prolonged arrest in the G2 phase of the cell cycle that allows for extensive DNA repair after DNA damage, whereas a normal cell would undergo apoptosis under the same circumstances (Bedi et al., 1995).
3. Disturbed Interaction with the Extracellular Matrix.
Among the proteins that are tyrosine-phosphorylated in BCR-ABL-positve cells are several that are involved in the organization of the cytoskeleton, such as paxillin (Salgia et al., 1995) and focal adhesion kinase (Gotoh et al., 1995). Importantly, the COOH-terminal portion of Bcr-Abl binds actin (McWhirter and Wang, 1993). Thus, it is not surprising that BCR-ABL-positive cells show abnormalities in motility as well as adhesion to integrins and other components of the extracellular matrix (Wertheim et al., 2002). The extramedullary hematopoiesis that characterizes CML may be a consequence of a defective interaction with the bone marrow stroma (Gordon et al., 1987).
It is evident that some clinical features of CML are explicable by its molecular pathogenesis. However, a number of questions remain unsolved. For example, it is not clear why a lesion that occurs in a hematopoietic stem cell predominantly targets the myeloid compartment. Even more important, the genetic events that underlie progression to blast crisis are poorly understood. Cytogenetic abnormalities in addition to the Philadelphia chromosome are seen in at least 50% of patients (Johansson et al., 2002). The causative significance of such abnormalities for disease progression is frequently not clear. Deletion and inactivation of tumor suppresser genes such as p53 (Feinstein et al., 1991) and p16 (Sill et al., 1995) is seen in some patients but no universal genetic lesion has been associated with progression. Even less is known about the biological basis of the "genetic instability" that is thought to render chronic phase CML cells prone to the acquisition of further genetic damage. There is evidence that DNA repair may be less efficient in CML cells (Canitrot et al., 1999; Takedam et al., 1999), and the threshold for the induction of apoptosis in response to DNA damage may be unduly high. Finally, the high cell turnover in itself might predispose to a higher frequency of mutations.
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III. Conventional Treatment Options for Chronic Myelogenous Leukemia |
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As with any other disease, the ultimate measure of the efficacy of a therapy is survival. However, for a disease with a relatively long course such as CML, surrogate markers are often used to allow for an earlier assessment of efficacy. From historical data, it has been estimated that the median survival of CML patients without any treatment is between 2 and 3 years. Three levels of disease control can be defined in CML.
Complete hematological response (CHR) is defined as the normalization of the blood counts and the white cell differential as well as the disappearance of all symptoms and signs of disease.
Complete cytogenetic response (CCR) means that no Ph-positive metaphases are detectable using classical cytogenetics, with at least 20 metaphases available for analysis. Major cytogenetic response (MCR) is the presence of less than 35% Ph-positive metaphases.
Molecular remission implies that no BCR-ABL transcripts are detectable by RT-PCR. This assay is far less standardized than the other tests, and its sensitivity varies greatly between laboratories. However, there is a general consensus that PCR negativity requires a level of sensitivity that allows for detection of one BCR-ABL-positive cell in 105 to 106 normal cells (Bose et al., 1998).
There is good evidence that achievement of a major cytogenetic response on interferon- therapy predicts improved survival, unless the patient belongs to the high risk group of patients (Hehlmann et al., 1994; Italian Cooperative Study Group on CML, 1994). Thus, cytogenetic response instead of survival is frequently used as a surrogate marker to assess efficacy. It must be stressed that such endpoints have been validated only in interferon-treated patients.
B. Conventional Cytotoxic Drugs
The first effective treatment for CML was Fowler's solution, which was widely used in the 19th century and contains arsenic as the active component. Recent in vitro studies have confirmed activity of arsenicals against CML cells, and the agent may see a comeback in the future (La Rosee et al., 2002a). With the advent of radiotherapy, splenic irradiation became popular in the 1920s and 1930s. It offered symptomatic relief but probably did not prolong life. The first synthetic compound with activity in CML was busulfan, an alkylating agent. Busulfan is extremely toxic to stem cells, which may explain why it is particularly effective in the stem cell disease CML; it was the first therapeutic modality that offered a definitive survival benefit, although no randomized study was carried out. Interestingly, there are anecdotal cases of long-term remissions after high-dose busulfan (Djaldetti et al., 1966). Although superseded by more effective and less toxic alternatives, busulfan is still used in preparative regimens for allogeneic stem cell transplantation. The next effective drug to be introduced for CML was hydrea. Compared with busulfan, hydrea does not cause prolonged cytopenias, since it primarily targets the more mature myeloid cells. It also has a far more benign nonhematological toxicity profile than busulfan. A survival advantage for hydrea over busulfan was shown in a controlled randomized trial (Hehlmann et al., 1994). Another drug with significant single agent activity in CML is cytarabine, although it never became widely used. Neither busulfan, hydrea, nor cytarabine produced cytogenetic remissions in a significant number of cases. The 1970s saw a number of trials using acute leukemia-type multiagent chemotherapy. In contrast to conventional chemotherapy, a proportion of patients achieved some degree of Ph-negative hematopoiesis (Kantarjian et al., 1985). However, as a rule, these were transient responses. Given the very considerable toxicity of polychemotherapy in CML, this approach was abandoned for patients in chronic phase.
At the beginning of the 1980s, interferon- was introduced as a therapy for CML. In contrast to other drug treatments, interferon- produced sustained cytogenetic responses in up to one-third of patients (Talpaz et al., 1991). The initial single center results were subsequently confirmed in randomized trials that demonstrated a survival advantage for interferon- over hydrea and busulfan (Hehlmann et al., 1994; Italian Cooperative Study Group on CML, 1994). A large randomized trial suggested that the combination of interferon- and cytarabine is superior to interferon alone (Guilhot et al., 1997), a finding that was not confirmed in a subsequent study (Baccarani et al., 2002). The cytogenetic remissions induced by interferon are durable in a proportion of patients, sometimes even after discontinuation of the agent (Bonifazi et al., 2001). Although, with RT-PCR, BCR-ABL mRNA is still detectable, these long-lasting remissions amount to a biological although not molecular cure of the disease.
D. Allogeneic Stem Cell Transplantation
A comprehensive evaluation of allografting for CML is beyond the scope of this review. As of now, allografting is the only treatment capable of disease eradication, with the majority of patients achieving RT-PCR negativity (Savage and Goldman, 1997). Long-term disease-free survival is in the range of 50 to 80% in most studies (Savage and Goldman, 1997; Hansen et al., 1998). However, allografting is limited to patients who have a suitable donor and are medically fit to undergo the procedure, which involves high-dose chemotherapy and total body irradiation. Less toxic (nonmyeloablative) transplant regimens make allografting an option for patients who do not qualify for a conventional transplant (McSweeney et al., 2001; Or et al., 2003). It is not yet clear if the durability of remissions with nonmyeloablative regimens equals that of conventional transplants, and transplant-related complications remain a problem (Bornhauser et al., 2001). Although no randomized trials have been conducted, there is no doubt that decisively more patients are long-term survivors after an allograft than with nontransplant therapies.
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IV. Imatinib |
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Given that the Bcr-Abl protein has deregulated tyrosine kinase activity, it was logical to search for specific pharmacological inhibitors. In 1993, Anafi and colleagues (1993) reported a tyrphostin, related to erbstatin, that inhibited the PTK activity of Bcr-Abl and suggested that it might be possible to design specific compounds for the treatment of Abl-associated human leukemias (Anafi et al., 1993). In a more extensive analysis of number of tyrphostins, the compounds AG568, AG957, and AG1112 proved to be the most specific agents. Growth inhibition of the CML cell line K562 occurred at micromolar concentrations and was associated with inhibition of Bcr-Abl tyrosine kinase activity (Kaur et al., 1994). Tyrphostins are competitive toward ATP or substrate, or both (Kovalenko et al., 1997). Although active in vitro, tyrphostins have not been developed for clinical use.
Another compound with activity toward Bcr-Abl is herbimycin A, an antibiotic derived from Streptomyces hygroscopicus. Its efficacy in inhibiting transforming tyrosine kinases was recognized as early as 1988 (Uehara et al., 1988). Herbimycin was originally thought to inhibit Bcr-Abl PTK (Okabe et al., 1992), but it was subsequently shown that its mode of action is the acceleration of Bcr-Abl protein degradation (Shiotsu et al., 2000). Selective inhibition of primary CML cells was also shown for genistein, a flavonoid (Carlo Stella et al., 1996).
In 1995 and 1996, Buchdunger and colleagues reported the synthesis of a series of compounds that exhibited specific inhibitory activity against the platelet-derived growth factor receptor (PDGF-R) (Buchdunger et al., 1995) and Abl (Buchdunger et al., 1996). These compounds emerged from a high-throughput screen of chemical libraries with the goal of identifying kinase inhibitors. From this time-consuming approach, a lead compound of the 2-phenylaminopyrimidine class was identified. This lead compound had weak inhibitory activity against both serine/threonine and tyrosine kinases, but served as a starting point for the synthesis of other related compounds. A key finding was that substitutions at the 6-position of the anilino phenyl ring led to loss of serine/threonine kinase inhibition, while the introduction of a methyl group at this position retained or enhanced activity against tyrosine kinases. The activity against the platelet-derived growth factor receptor tyrosine kinase was further enhanced by the introduction of a benzamide group at the phenyl ring. 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 (Fig. 4). Introduction of N-methylpiperazine as a polar side chain greatly improved water solubility and oral bioavailability. Studies in our laboratory demonstrated that imatinib had extremely potent and specific in vitro and in vivo activity against BCR-ABL-transformed cells (Druker et al., 1996), results that were independently confirmed by other groups (Deininger et al., 1997; Gambacorti-Passerini et al., 1997).
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B. Preclinical Evaluation of Imatinib
1. In Vitro Studies
a. Kinase Assays.
The effects of imatinib on a number of serine/threonine as well as tyrosine kinases was tested using in vitro kinase assays with immunoprecipitated or purified proteins (Table 1), which are independent of the specific cellular environment. Imatinib showed activity toward Abl and its activated derivatives v-Abl, Bcr-Abl (Buchdunger et al., 1996; Druker et al., 1996) and Tel-Abl (Carroll et al., 1997), with IC50 values in the range of 0.025 µM for protein autophosphorylation. Activity against PDGF-R and c-Kit was found to be in a similar range. By 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.
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b. Studies in Cell Lines.
To determine whether imatinib was able to penetrate the cell membrane, studies were extended to intact cells. Most of these investigations used cell lines engineered to express Bcr-Abl or cell lines derived from CML patients in blast crisis. These experiments showed that the IC50 values for inhibition of Bcr-Abl tyrosine phosphorylation are in the range of 0.25 to 0.5 µM, approximately 10-fold higher than those measured in the in vitro kinase assays (Druker et al., 1996; Beran et al., 1998; Dan et al., 1998; Deininger et al., 2000b). The precise reason for this difference is not known, but it may be related to drug efflux or to intracellular regulatory mechanisms that affect the binding of imatinib to the Abl kinase.
Incubation of BCR-ABL-positive cell lines resulted in growth inhibition and induction of apoptosis in almost all lines studied (Druker et al., 1996; Deininger et al., 1997; Gambacorti-Passerini et al., 1997). The IC50 values for inhibition of cell proliferation mirrored those for inhibition of Bcr-Abl tyrosine phsophorylation in cellular assays. Importantly, the IC50 values seen in these assays are well below the concentrations that can be achieved in patients. Of the many cell lines studied, only the KCL22 line derived from a CML patient in myeloid blast crisis, and SD1, an Epstein-Barr virus-transformed lymphoblastoid line derived from a patient with acute lymphoblastic leukemia, were primarily resistant to imatinib (Deininger et al., 1997) (Fig. 5). Given the fact that cell lines derived from CML blast crisis patients harbor multiple genetic abnormalities in addition to the Ph chromosome, these findings were impressive. Equally relevant, concentrations of up to 10 µM imatinib did not affect the growth of BCR-ABL-negative cell lines.
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c. Studies in Primary Cells.
To assess the effects of imatinib on committed hematopoietic progenitors, mononuclear cells from CML patients and normal individuals were studied in assays of colony formation. The formation of myeloid and erythroid colonies was reduced in CML samples using imatinib concentrations of up to 10 µM, whereas there was relatively little effect on normal cells (Fig. 6) (Druker et al., 1996; Deininger et al., 1997). A dose of approximately 1 µM offered the maximal differential effect. In re-plating experiments, it was also demonstrated that imatinib selectively suppressed the growth of secondary colonies from CML patients, similar to interferon- (Marley et al., 2000). The differential sensitivity of CML versus normal cells was also confirmed using long-term culture-initiating cells (LTC-IC) that represent early hematopoietic progenitor cells (Kasper et al., 1999).
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2. Animal Studies.
The myeloid murine cell line 32D, engineered to express Bcr-Abl, forms tumors in syngeneic mice. Imatinib, at 10 to 50 mg/kg, given once daily intraperitoneally (i.p.), starting 1 week after the injection of 32DBCR-ABL, caused dose-dependent inhibition of tumor growth. In contrast, imatinib (50 mg/kg) was ineffective against tumor formation by v-Src-transformed 32D cells, again demonstrating the specificity of the compound (Druker et al., 1996). Another study tested imatinib against the human CML cell line KU812 injected into nude mice. These experiments demonstrated that continuous inhibition of the Bcr-Abl tyrosine kinase is required for maximal antitumor effect (le Coutre et al., 1999). In this model, a treatment schedule of three times daily administration of 50 mg/kg i.p. or daily administration of 160 mg/kg p.o. for 11 consecutive days, resulting in continuous inhibition of p210BCR-ABL tyrosine kinase activity and led to tumor-free survival after an injection of KU812 cells. Moreover, 160 mg/kg p.o. every 8 h for 11 days inhibited tumor growth even in the presence of advanced disease. Tumor nodules began to regress 48 h after the initiation of treatment; by day 8, no treated animal had measurable disease. However, 4 of 12 animals relapsed on days 48 through 60, whereas 8 animals remained tumor free after 200 days of followup. The specificity of the effect was demonstrated by the fact that imatinib did not inhibit tumor growth after injection of BCR-ABL-negative U937 cells.
Imatinib was also tested in a refined version of the transplantation model of CML (Pear et al., 1998). In these experiments, murine bone marrow is infected with a BCR-ABL retrovirus and subsequently transplanted into syngeneic recipients. Mice typically die within 3 weeks from CML. By contrast, animals treated with imatinib have prolonged survival. However, responses were variable, and 25% of mice showed primary resistance to imatinib (Wolff and Ilaria, 2001). In no case was the compound able to prevent the disease, even if treatment was started as early as 48 h after injection of the BCR-ABL-infected bone marrow. No universal cause of resistance could be established in this study. Analysis of clonality showed, however, that there was "clonal depletion" in animals that responded to imatinib, indicating that the compound was able to successfully target some but not all leukemic clones.
1. Phase I.
In June 1998, a phase I trial was initiated at three centers in the United States. The study population included CML patients in chronic phase who were resistant to or intolerant of interferon-. The initial dose was 25 mg of imatinib daily. Hematological responses were seen with daily doses above 85 mg. Almost all patients (53/54, 98%) treated with at least 300 mg per day achieved complete hematological response. Thirty-one percent of patients obtained major cytogenetic responses, and 13% complete cytogenetic response. Importantly, responses were durable, with only 2/53 patients relapsing with a median follow-up of 265 days (Druker et al., 2001a).
The encouraging results in patients with CML in chronic phase led to an expansion of the protocol to 58 patients in blast crisis or with Ph-positive acute lymphoblastic leukemia. The minimum dose in this cohort was 300 mg of imatinib daily. Hematological responses were seen in 21/38 patients (55%) with myeloid phenotype and 14/20 of patients (70%) with lymphoid phenotype, including four complete responses in each group. Twelve percent of patients achieved a major and 8% a complete cytogenetic response. In contrast to patients treated in the chronic phase, approximately 50% of responders with myeloid disease and all but one responder with lymphoid disease relapsed between 42 and 193 days after initiating imatinib therapy (Druker et al., 2001b).
2. Phase II.
Three international multicenter phase II protocols were initiated in the second half of 1999. They enrolled patients with CML in myeloid blast crisis, relapsed Ph-positive ALL, CML in accelerated phase, and patients with CML who had failed interferon-.
In myeloid blast crisis, the results largely confirmed the results seen in the phase I study (Table 2) (Sawyers et al., 2002). Since no controlled trials are available for this patient population, it is difficult to conclude how the results of imatinib compare with conventional chemotherapeutic agents. However, the 1-year survival rate of 30% is better than in any reported study (Kantarjian et al., 1987, 1992, 1997). In addition, since imatinib is well tolerated (see below) and usually administered as an outpatient treatment, it can be assumed that there is a large gain in quality of life compared with conventional chemotherapy, which is associated with considerable toxicity. In contrast to myeloid blast crisis, where a small proportion of patients achieve durable remissions, there are practically no durable responses in blast crisis with a lymphoid phenotype and in Ph-positive acute lymphoblastic leukemia (Ottmann et al., 2002). The only exception may be patients treated for relapse after an allograft (Wassmann et al., 2001).
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Patients in accelerated phase had not been studied in the phase I protocols. Accelerated phase was diagnosed when at least one of the following criteria was present: 1) bone marrow or peripheral blood blasts more than 15% but less than 30%; 2) blast combined with promyelocytes more than 30%; 3) basophils in the blood or marrow more than 20%; 4) platelets less than 100 x 109/liter (unrelated to therapy). Not surprisingly, the results for this group fall between those observed in myeloid blast crisis and chronic phase (Table 2) (Talpaz et al., 2002). As with myeloid blast crisis, there are very few controlled studies available for comparison. However, the 1-year survival rate of 74% with imatinib is twice as high as in the study with the best results previously published (Kantarjian et al., 1992). Moreover, cytogenetic responses in accelerated phase patients have rarely been observed, but 17% of patients treated with imatinib obtained a complete cytogenetic response. This might indicate that some of the responses may be maintained, a notion that is corroborated by the fact that achievement of a major cytogenetic response at 3 months was predictive of progression-free survival. The accelerated phase study had been initiated with an imatinib dose of 400 mg per day. When additional safety data became available, this dose was increased to 600 mg per day. Thus, a retrospective comparison between the two dose cohorts was possible and showed a significanty longer time to progression and overall survival for the 600 mg cohort.
The largest of the phase II trials enrolled patients in chronic phase who had previously failed interferon--based therapy. The patients were grouped as hematologically resistant or refractory, cytogenetically resistant or refractory and intolerant of interferon-. Refractoriness implies that a response was never achieved, whereas resistance refers to loss of a response. The rate of complete hematological responses in all the patients was 95%, with an 89% progression-free survival at 18 months. Moreover, the rate of complete cytogenetic response was 41% with 60% major cytogenetic responses. The best results were seen in patients who had previously achieved a cytogenetic response to interferon- (Table 3).
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3. Phase III.
A randomized study was initiated in June 2000 that compared imatinib to interferon- plus cytarabine in newly diagnosed patients with CML in chronic phase. At that time, the combination of interferon and cytarabine was considered the best nontransplant therapy for CML (Guilhot et al., 1997). Results from the phase III study show that imatinib is vastly superior with respect to the rates of complete hematological remission, major cytogenetic remission, and complete cytogenetic remission. Most importantly, there was also a highly significant difference in the rate of progression to accelerated phase or blast crisis at 18 months (Table 4) (O'Brien et al., 2003). Based on these results, the FDA has approved imatinib as first-line treatment for newly diagnosed CML in December 2002.
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Imatinib is generally very well tolerated. Although side effects are quite common, they are usually mild and only rarely lead to discontinuation of therapy. Side effects are more common in advanced phases of CML, reflecting the poorer performance status of many of these individuals. Toxicity can be broadly divided in hematological and nonhematological adverse events, and the following overview summarizes the observations from the controlled clinical trials as well as "expanded access" protocols. These less stringently controlled trials bridged the gap between the end of enrollment into the phase II trials and regulatory approval of imatinib.
1. Nonhematological Toxicity
a. Edema and Fluid Retention.
Superficial edema, most frequently in the form of periorbital edema, is seen in approximately 50% of patients treated with imatinib. In some cases, more severe forms of fluid retention occurred, such as pleural and pericardial effusions, pulmonary edema, ascites, anasarca, and cerebral edema. The more serious adverse reactions necessitate interruption of therapy, while in the milder forms, diuretics may be used. The pathogenesis of the edema is not precisely understood, but current thinking links it to kinases inhibited by imatinib. For example, the PDGF-R has a role in the maintenance of the integrity of vessel walls, since mice with homozygous deletions of PDGF-R have defective blood vessels and, as a consequence, suffer from edema (Lindahl et al., 1997). In addition, mice with homozygous deletions of ABL and the ABL-related gene ARG, are also prone to edema (Koleske et al., 1998).
b. Gastrointestinal Side Effects.
Mild nausea is common, particularly if imatinib is taken on an empty stomach. However, with studies indicating that there is no difference in absorption, when imatinib is taken together with food (Reckmann et al., 2002), the recommendation is now to take the drug with the largest meal of the day. It is thought that nausea and also the abdominal pain that some patients experience are due to the local irritative effects of imatinib. Mild diarrhea, also relatively common, may also be caused by local irritation. An alternative explanation, although unproven, would be that it results from the inhibition of c-Kit on the interstitial cells of Cajal, the pacemaker cells for gastrointestinal motility.
c. Skin Reactions.
Skin rashes are seen in about one-third of patients. They vary greatly in appearance, and may be quite severe, with one patient developing Stevens-Johnson syndrome (Hsiao et al., 2002). Most of the rashes are mild and self-limited or respond to anti-histamines or steroids. Rashes do not necessarily recur when therapy is resumed after being discontinued; however, skin reactions are the most frequent reason for permanent discontinuation of imatinib therapy. Apart from these drug-induced reactions, an urticarial rash has been seen at the start of treatment in patients with high basophil counts. It is likely that this reaction is the consequence of histamine release from the basophils, and it tends to subside when remission is induced. In a few cases, changes of skin pigmentation and darkening of the hair have been seen (Etienne et al., 2002). This may be due to imatinib effects on melanocytes that express c-kit.
d. Arthralgia, Myalgia, and Bone Pain.
Bone, joint, and muscle pain are frequent side effects, although they are rarely severe enough to require discontinuation of treatment. Muscle cramps are very common, can be quite unpleasant, and frequently respond well to calcium supplements or quinine. Their pathogenesis is not known.
e. Liver Toxicity.
Preclinical studies in rats and dogs suggested that liver toxicity would be a major problem in clinical trials of imatinib. Fortunately, liver toxicity has been relatively uncommon. One patient with advanced CML died of liver failure; besides imatinib, he was taking large quantities of acetaminophen, thus a causal relation to imatinib could not be established with certainty. Nonetheless, monitoring of liver function tests is mandatory and should be continued routinely for as long as patients are on imatinib, since toxicity may develop late. Histology has been consistent with a toxic drug reaction, without any peculiarities specific to imatinib.
2. Hematological Toxicity.
Myelosuppression may reflect a therapeutic effect but could also be due to toxicity to normal hematopoietic cells. Generally, severe neutropenia and thrombocytopenia are more common in advanced disease, particularly blast crisis. This may be due to the smaller numbers of residual Ph-negative stem cells that are available for the reestablishment of normal hematopoiesis (Petzer et al., 1996). Another observation that supports this view is that in some cases the peripheral blood counts recover, when the patient achieves a cytogenetic response while remaining on imatinib therapy. The management of imatinib-induced myelosuppression requires experience. The guiding principle should be to match the aggressiveness of therapy with the aggressiveness of the disease. This implies that in patients with advanced disease it may be justified to continue imatinib in the face of myelosuppression although in early chronic phase interrupting therapy is advisable. Myeloid growth factors have been used successfully to treat neutropenia and do not seem to adversely affect prognosis (Mauro et al., 2001).
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V. Pharmakokinetics |
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). The half-life of the drug is approximately 18 h. With once daily administration of 400 mg orally per day, median peak plasma concentrations at steady state are 5.4 µM, and median trough levels are 1.43 µM (Druker et al., 2001a). Thus, concentrations are achieved that are several times higher than the IC50 values in assays of intracellular tyrosine phosphorylation. Imatinib is metabolized by the CYP3A4/5 enzyme system. Thus inhibitors and inducers of this system are expected to alter plasma concentrations. One patient who was treated with phenytoin and imatinib failed to achieve a hematological response and was found to have drug levels that were 4-fold lower than expected. He obtained a complete hematological remission after discontinuation of phenytoin and a 1.5-fold dose increase (Druker et al., 2001a). Imatinib may also alter the plasma levels of other drugs that are metabolized by the CYP3A4/5 system, such as cyclosporine A or simvastatin. The interaction with cyclosporine A may be particularly relevant if imatinib is used in patients after allogeneic stem cell transplantation. |
VI. Monitoring Patients on Imatinib |
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), has been used for monitoring (Druker et al., 2001a). This is a cumbersome test that requires immunoblotting of protein from patient cells and is difficult to use in a routine setting. Other options that are being developed include fluorescence-activated cell sorting analysis and enzyme-linked immunosorbent assay tests that might be more suitable for routine detection of cellular phosphotyrosine. The second important area of development is monitoring for mutations of the BCRABL kinase domain. As discussed below, resistance is frequently caused or associated with kinase domain mutations that interfere with imatinib binding. Early detection of such mutations would be desirable, since it might influence therapeutic decisions. |
VII. Imatinib in Drug Combinations |
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) and CML patients (
) in the presence of imatinib.