Leukemia (2004), 1 11 & 2004 Nature Publishing Group All rights reserved /04 $

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1 (2004), 1 11 & 2004 Nature Publishing Group All rights reserved /04 $ REVIEW Imatinib therapy in chronic myelogenous leukemia: strategies to avoid and overcome resistance A Hochhaus 1 and P La Rosée 1 1 III Medizinische Klinik, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany Imatinib is a molecularly targeted therapy that inhibits the oncogenic fusion protein BCR-ABL, the tyrosine kinase involved in the pathogenesis of chronic myelogenous leukemia (CML). Selective inhibition of BCR-ABL activity by imatinib has demonstrated efficacy in the treatment of CML, particularly in chronic phase. Some patients, however, primarily those with advanced disease, are either refractory to imatinib or eventually relapse. Relapse with imatinib frequently depends not only on re-emergence of BCR-ABL kinase activity but may also indicate BCR-ABL-independent disease progression not amenable to imatinib inhibition. Results from phase 2/3 trials suggest that rates of resistance and relapse correlate with the stage of disease and with the monitoring parameters hematologic, cytogenetic and molecular response. These observations and more recent trials with imatinib, combined with insights provided by an increased understanding of the molecular mechanisms of resistance, have established the rationale for strategies to avoid and overcome imatinib resistance in the management of CML patients. To prevent resistance, early diagnosis and prompt treatment with appropriate initial dosing is essential. Management of resistance may include therapeutic strategies such as dose escalation to achieve individual optimal levels, combination therapy, as well as treatment interruption. advance online publication, 24 June 2004; doi: /sj.leu Keywords: drug resistance; imatinib; tyrosine kinase inhibitors; chronic myelogenous leukemia; clinical strategies Introduction Chronic myelogenous leukemia (CML) is attributed to the chromosomal translocation t(9;22)(q34;q11), yielding the Philadelphia (Ph) chromosome that is also present in 20 40% of cases of adult acute lymphoblastic leukemia (ALL). 1,2 This chromosomal translocation generates a fusion gene that encodes BCR-ABL, a constitutively active protein tyrosine kinase. Signal transduction pathways stimulated by BCR-ABL kinase activity promote cell survival and proliferation while inhibiting apoptosis. The discovery of the BCR-ABL-mediated pathogenesis of CML provided the rationale for the design of an inhibitory agent that targets BCR-ABL kinase activity. 3 Imatinib mesylate (formerly STI571; Glivec s, Gleevec s, Novartis Pharmaceuticals, Basel Switzerland) is a selective inhibitor of ABL and its derivative BCR-ABL, as well as certain other tyrosine kinases. 4,5 It is effective as a single agent for the treatment of patients in all stages of CML, with the most encouraging results seen in patients in chronic phase (CP) Correspondence: Professor Dr A Hochhaus, III Medizinische Klinik Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Wiesbadener Strasse 7-11, Mannheim 68305, Germany; Fax: þ ; hochhaus@uni-hd.de Received 5 April 2004; accepted 25 May 2004 disease. 6 9 Hematologic and cytogenetic responses to imatinib for the treatment of CP CML have permitted imatinib to be registered as first-line treatment for newly diagnosed CML. Compared with previous therapies excluding allogeneic stem cell transplantation (SCT), responses to imatinib have raised the standard of response from suppression of cells bearing the Ph chromosome to reductions in the levels of BCR-ABL mrna transcripts A minority of CML patients in CP and a substantial proportion in advanced disease phases are either initially refractory to imatinib treatment or lose imatinib sensitivity over time and experience relapse. 11,12 Resistance to imatinib in patients has been associated with a heterogeneous array of mechanisms that range from nonspecific multidrug resistance to BCR-ABL inherent genetic alterations. The most frequently identified mechanism of acquired imatinib resistance is BCR-ABL kinase domain point mutations that impair imatinib binding either by interfering with an imatinib-binding site or by stabilizing a BCR- ABL conformation with reduced affinity to imatinib. 13,14 Clinical use of imatinib started in June and since then, it is clear that resistance to imatinib does not dominate clinical experience. It does not necessarily occur and strategies can be employed to prevent or overcome it This review aims to discuss clinical strategies to avoid resistance and to improve responses with imatinib treatment in CML. While these recommendations are based on the authors laboratory and clinical experience, they do not represent an approved consensus of practicing hematologists. Resistance to imatinib in CML defined Resistance to imatinib can be categorized according to the time of onset: primary (intrinsic) resistance is a lack of efficacy from the onset of treatment with imatinib and secondary (acquired) resistance (relapse) is defined as an initial response followed by a loss of efficacy with the time of exposure to imatinib. According to the clinical and laboratory criteria used for detection, resistance should be further subdivided into hematologic, cytogenetic and molecular resistance. In most studies, the definition of hematologic resistance depends on whether the disease phase is chronic or advanced. In CP, hematological resistance is defined as the lack or loss of normalization of peripheral blood counts, the differential leukocyte count and spleen size. In advanced phases of CML, hematologic resistance means a lack of return to CP or hematologic relapse after initial response. Cytogenetic resistance can be defined according to the level of cytogenetic response, which is the aim of the therapy at certain time points; owing to a lack or loss of major cytogenetic response (MCR, p34% Ph-positive metaphases) or complete cytogenetic response (CCR, 0% Ph-positive metaphases).

2 2 Molecular resistance could be defined as the lack or loss of complete molecular response. In qualitative terms complete molecular response means undetectable BCR-ABL transcripts by either real-time or nested reverse transcriptase-polymerase chain reaction (RT-PCR). However, based on variability in the quality of blood samples and RNA or cdna extraction, results may fluctuate between positive and negative. Positive results must not be judged as relapse or lack or loss of major molecular response (MMR) unless detected in at least two consecutive samples after more than one negative result obtained using nested PCR. 19 MMR is defined quantitatively as a X3 log reduction of BCR-ABL transcripts or a BCR-ABL/ABL ratio of o0.1%. 11,12 Molecular relapse, in complete cytogenetic responders, is considered to be an increase in BCR-ABL transcript levels by at least one log. Resistance to imatinib monotherapy CP CML Early phase 2 studies involved CP CML patients in whom interferon-alpha (IFN) therapy had failed. 9 Of 454 patients with a confirmed diagnosis of CP CML who were treated with imatinib following IFN failure or intolerance, 5% did not achieve a CHR and 40% had less than an MCR after a median follow-up of 18 months, suggesting the presence of primary resistance to imatinib. The 24-month follow-up data demonstrate that the overall rate of failure to achieve CHR was 4% and failure to achieve MCR was 36% among patients in CP previously treated with IFN (Kantarjian et al. Blood 2003; 102(Suppl. 1): 905a, abstract). The frequency of hematologic resistance to imatinib according to disease phase after 2 years is summarized in Figure 1. Estimated progression-free survival at 24 months among CP patients previously treated with IFN was 87%, indicating that the secondary resistance or relapse rate was approximately 13% (Figure 2). The International Randomized Study of Interferon and STI571 (IRIS) investigated imatinib efficacy in 553 patients newly diagnosed with CP CML and treated with imatinib without Figure 1 Frequency of hematologic primary resistance and relapse/progression at 2 years of imatinib therapy. CP, AP and My BC denote chronic, accelerated and myeloid BC phases of CML, respectively. (Cervantes et al. Blood 2003; 102(Suppl. 1): 181a 182a; Kantarjian et al. Blood 2003; 102(Suppl. 1): 905a; Talpaz et al. Blood 2003; 102(Suppl. 1): 905a; Sawyers et al. Blood 2003; 102(Suppl. 1): 905a 906a, abstracts). Figure 2 Estimated progression-free survival of CML patients treated with imatinib at 2 years of imatinib therapy. CP, AP and BC denote chronic, accelerated and myeloid BC phases of CML, respectively. (Cervantes et al. Blood 2003; 102(Suppl. 1): 181a 182a; Kantarjian et al. Blood 2003; 102(Suppl. 1): 905a; Talpaz et al. Blood 2003; 102(Suppl. 1): 905a; Sawyers et al. Blood 2003; 102(Suppl. 1): 905a 906a, abstracts). prior therapy. 8 The rate of primary resistance to achieving a CHR was approximately 5% after 18 months. The estimated rate of failure to achieve an MCR was 12% after 24 months of follow-up. The estimated rate of relapse or progression was 10% after 24 months in those treated with imatinib as first-line therapy (Cervantes et al. Blood 2003; 102(Suppl. 1): 181a 182a, abstract). Taken together with the phase 2 study results, the probability of refractoriness or relapse during imatinib therapy is lower among newly diagnosed CML patients treated with imatinib without prior therapy than among patients previously treated with IFN. These clinical studies were accompanied by laboratory studies investigating the role of cytogenetics and PCR for the prediction of the outcome. Among 261 CP CML patients previously treated with IFN estimated 3-year survival rates from the start of imatinib therapy after 3 months of treatment were 98% among patients with MCR compared with 84% among patients with cytogenetic resistance. 20 The relationship between BCR-ABL transcript levels and eventual relapse was explored in a phase 2 study of CP CML patients treated with imatinib after IFN treatment failed. 21 BCR- ABL transcript levels, detected by quantitative RT-PCR, were examined in 120 patients after 2 months of imatinib therapy. Those with a BCR-ABL/ABL ratio X20% had a significantly (P ¼ 0.007) lower probability of MCR after 6 months compared to patients with a BCR-ABL/ABL ratio o20%. In the IRIS study, 39% of newly diagnosed CP CML patients achieved a X3-log reduction in the level of BCR-ABL transcripts after 12 months of imatinib therapy as detected by quantitative RT-PCR. 11 Among patients with CCR and a X3-log reduction in BCR-ABL, the probability of progression-free survival at 24 months was 100% compared to 95% in patients with CCR but o3-log reduction of BCR-ABL levels. The dynamics of BCR-ABL mrna expression during imatinib therapy was investigated in an analysis of a subset of patients enrolled at 17 German centers participating in the IRIS study. 12 Among patients treated with imatinib as first-line therapy, 13% did not achieve a CCR after a median treatment interval of 24 months. Among patients with

3 CCR, 27% experienced recurrence of Ph-positive metaphases after a median period of 6 months. The median BCR-ABL/ABL ratios were 0.029% among patients with continued CCR compared with 0.24% among those who achieved a CCR but relapsed thereafter. Together, these results demonstrate that among patients who achieve cytogenetic responses, with or without prior IFN therapy, higher BCR-ABL/ABL ratios are associated with a greater risk of relapse during imatinib therapy. Advanced phases of CML The rates of both primary and secondary resistance to imatinib increase with CML disease progression. 7 In a phase 2 study of 181 accelerated phase (AP) CML patients, imatinib treatment failed to achieve CHR in 66% of patients and failed to attain MCR in 76% of patients at 12 months. A phase 2 trial involving 229 myeloid blast crisis (BC) CML patients taking either 400 or 600 mg/day of imatinib resulted in an approximately 93% failure rate to achieve a CCR. A total of 84% failed to reach MCR. 6 Together, these studies demonstrate that rates of resistance and relapse directly correlate with disease progression. These data were confirmed by another trial of imatinib as treatment for BC CML, which reported that approximately 40% of patients treated with 600 mg/day imatinib failed to achieve a sustained hematologic remission. 22 A long interval between diagnosis of BC and imatinib therapy was associated with significantly shorter rates of event-free survival. Summary of clinical data These trials provide evidence that relapse and resistance rates in CP CML are lower with first-line imatinib therapy compared with treatment after failure with IFN after 2 years of follow-up. This may reflect the second observation emerging from these studies that rates of resistance and relapse increase with CML disease progression. Among patients who achieve cytogenetic response, molecular responses are linked with lower rates of resistance and relapse. Early molecular responses correlate with prolonged cytogenetic responses and cytogenetic responses are associated with long-term survival. Assessing resistance Cytogenetics Periodic (every 6 months) cytogenetic monitoring for karyotypic abnormalities is critical throughout imatinib therapy to detect clonal evolution even in cases of early CCR. 23 In several studies, the propensity for relapse in the subset of patients with both a cytogenetic and molecular response was lower than for those with only a cytogenetic response. 11,12,21 Minimal residual disease Once cytogenetic response is achieved, minimal residual disease can be assessed by molecular monitoring. Detecting BCR-ABL transcripts with a degree of sensitivity that defines a MMR requires methodology capable of detecting a single BCR- ABL-positive cell among normal cells, which can be achieved with various PCR techniques. 19 A quantitative PCR reaction including ABL amplification as an internal control can be used to express BCR-ABL as a function of total ABL. Standardized cytogenetic and molecular assays should be performed prospectively and peripheral blood should be assayed for BCR-ABL expression using quantitative and nested PCR every 3 months during therapy. 11,12,24 BCR-ABL reactivation In cases where BCR-ABL-dependent resistance to imatinib is suspected, reactivation of BCR-ABL kinase activity should be demonstrable. BCR-ABL kinase activity in patients can be assayed by measuring phosphorylation of BCR-ABL substrates such as CRKL or Stat These assays facilitate the distinction between BCR-ABL-dependent and -independent mechanisms of imatinib resistance. Genomic BCR-ABL amplification Amplification of the BCR-ABL gene can be determined by interphase fluorescence in situ hybridization (FISH) using fluorescently labeled probes for BCR and ABL genes. 25,28 BCR-ABL mutations The detection of BCR-ABL mutants prior to and during the course of imatinib therapy may aid in risk stratification as well as in determining therapeutic strategies. A screen for mutations is indicated in patients lacking or losing hematologic response. Mutations are more frequently isolated from relapsed compared to primary resistant patients. 13,25 The observation that 89% of patients with mutations eventually relapsed suggests that harboring any BCR-ABL mutation has prognostic value with respect to disease progression. 29 Thus, a search for mutations could be performed even when a 3-log reduction in BCR-ABL transcripts is not achieved or there is a two-fold increase in BCR- ABL transcript levels. Conversely, the likelihood of detecting BCR-ABL mutations increases with CML disease progression. In early CP, mutations are rare. 25,29 The proportion of patients with mutations in late CP, AP and BC certainly depends on the inclusion criteria for such molecular studies and ranges between 22 and 53%. 25,29,30 Mutations can be reliably and sensitively detected by selection and expansion of specific clones followed by DNA sequencing. However, this process is too time consuming and labor intensive to be routinely feasible. 28,31 Alternatively, sequencing of nested PCR-amplified BCR-ABL products can reveal mutations with a high degree of sensitivity and fidelity. Mutation-specific restriction digest analysis of RT-PCR-amplified products can also be used as a tool for surveillance of BCR-ABL with a sensitivity of about 2% 25 (Kreil et al. Blood 2003; 102(Suppl. 1): 71a, abstract). Highly sensitive detection methods can increase the detection rate of point mutations. For example, allele-specific oligonucleotide PCR methods or the analysis of a significant number of clones have permitted the detection of BCR-ABL kinase domain mutations prior to imatinib therapy in patients with CML and ALL. 34 Mutations can also be detected in an automated manner by denaturing high-performance liquid chromatography (D-HPLC). D-HPLC compares favorably to DNA sequencing as samples can be rapidly analyzed for single-nucleotide polymorphisms. 35,36 Screening for mutations from samples obtained from imatinibresistant patients has thus far been confined to analysis of products amplified using primers spanning various regions of the 3

4 4 Table 1 BCR-ABL point mutations associated with imatinib resistance detected in CML or ALL patients Nucleotide change a Amino-acid change b No. of cases (detected/tested) References A1094G M244V 3/125 25, 29, 31 C1106G L248V 2/29 29, y G1113A G250E 6/87 29, 31, 37 G1112A G250R 1/117 # Q252R 1/32 31 G1120C/T Q252H 12/125 25, 29, 31 T1121C Y253H 9/154 25, 31, 37, 42 A1122T Y253F 6/125 25, 29, 31 G1127A E255K 28/182 25, 29, 30, 31, 37, 42 A1128T E255V 3/101 25, 29,37,40 A1191G D276G 1/33 41, y A1194G T277A 1/117 # V289A 39 T1494C F311L 1/24 32 C1308T T315I 27/194 25,29,37,41,42 C1308A T315N 1/33 41 C1315G F317L 4/60 31, 37 T1392C M343T 1/32 31 T1416C M351T 24/204 25,29,31,32,37 T1428G E355G 4/25 25,29,31 T1439G F359V 4/59 29,31 T1440G F359A 2/150 41, # G1499A V379I 1/32 31 T1508C F382L 1/32 31 T1523A L387M 2/ G1523C L387F 3/117 # A1551C H396P 39,42 A1551G H396R 5/12 25,29,31 A1558C A397P 1/117 # C1614A S417Y 1/27 29 G1739A E459K 1/27 29 T1821C F486S 1/27 29 a Nucleotide positions according to GenBank accession number M b Amino-acid positions, denoted with the single letter code, are those for GenBank sequence (accession number AAB60394) and correspond to ABL type 1a. Table adapted (Melo et al). 44 Additional mutations from ASH 2003: Kreil et al. Blood 2003; 102(Suppl. 1): 71a # ; Chu et al. Blood 2003; 102(Suppl. 1): 70a y, abstracts and a review article by Nardi et al. 39 BCR-ABL kinase domain (amino acids ). 25,31 To date, approximately 30 different point mutations that code for distinct single amino-acid substitutions in the BCR-ABL kinase domain have been isolated from relapsed CML patients resistant to imatinib treatment 25,14,28 32,37 41 (Bories et al. Blood 2003; 102(Supp;. 1): 414a; Chu et al. Blood 2003; 102(Supp. 1): 70a; Gruber et al. Blood 2003; 102(Supp. 1): 414a; Kreil et al. Blood 2003; 102(Supp. 1): 71a, abstracts), as well as from patients with ALL 42,43 (Table 1). This number will probably increase with time as technology for detection improves and the scope of the search widens. Usually, a single point mutation is detected but occasional patients carry more than one BCR-ABL point mutation. 13,25,16,29,31,37 Preclinical studies have demonstrated that mutations outside the kinase domain can also result in molecular conformations of BCR-ABL that impair imatinib binding. 45 Therefore, screening for mutations outside the kinase domain may be necessary in the future to fully account for imatinib resistance in patients. It is also important to note that in certain instances BCR-ABL point mutations may accompany, but cannot explain resistance to imatinib. 46,47 Genetic polymorphism in genes other than BCR-ABL may also be associated with responses to imatinib. A subset of patients in the IRIS study was analyzed for polymorphic loci in 26 different genes. A correlation was observed between cytogenetic responses and a particular polymorph of a yet-to-be characterized gene, suggesting that this genotype may have predictive value with respect to imatinib responsiveness in CML 48 Clinical management The goals of CML therapy and the methods used to monitor response can influence clinical management decisions. The following recommendations to avoid or combat imatinib resistance are based on evidence obtained with imatinib in clinical trials combined with laboratory observations on samples from imatinib-treated patients and preclinical studies. Several mechanisms of imatinib resistance may operate within an individual and these may interact with each other. Therefore, some of these recommendations may necessitate concomitant application. Strategies to prevent resistance Optimal dosing at diagnosis: Clinical evidence: Administration of full therapeutic doses of imatinib as early as possible is likely to promote maximal depletion of leukemic cells. Data from trials show that early use of imatinib for CML results in rapidly achieved, robust response rates with an encouraging duration. Cytogenetic responses and survival rates are higher

5 with first-line imatinib in early, rather than late, CP CML after IFN therapy had failed. 8,9 Anecdotal examples of resistance developing in patients who received suboptimal doses of imatinib at the outset of treatment or who did not comply with treatment have been reported. 25,49,50 Starting with at least the approved imatinib dosage 400 mg/day for CML in CP and 600 mg/day for advanced disease can aid in diminishing relapse risk. In the phase 1 dose-escalating trial of imatinib for CP CML, up to 1000 mg/day was administered without the identification of a maximum-tolerated dose; the efficacy results showed a dose response relationship. 49 While the current standard dose of imatinib is 400 mg once daily, the safety and toxicity profile indicates that considerably higher doses are tolerable. In a trial (n ¼ 36) investigating the efficacy of 800 mg/day imatinib in CP CML patients after failure with IFN therapy, 89% of patients achieved a CCR and 56% of patients reached MMR. 51 This response level was improved compared with previous studies in which CP CML patients, previously treated with IFN, were given 400 mg/day imatinib. 9 The use of 800 mg/day imatinib as first-line therapy in newly diagnosed early CP CML has also been investigated. 52 In all, 114 patients were treated with 400 mg imatinib twice daily. A total of 90% achieved a CCR, defined as 0% Ph-positive cells. After a median follow-up of 15 months, no patient progressed to advanced disease. Significantly improved rate of CCR (P ¼ ) and MMR (P ¼ ) were achieved with 800 mg/day imatinib compared with historical data from patients treated with 400 mg/day. Together, these studies suggest that either in newly diagnosed CML without prior therapy or subsequent to IFN failure, imatinib dose escalation may be the optimal approach to avoid imatinib resistance. Rationale: Early diagnosis and treatment minimizes the opportunity for genetic instability inherent in CML to contribute to imatinib resistance. Phase 1 trials investigated optimal dosing and showed that 95% of patients achieved CHR with 400 mg/ day imatinib, a dose that generated mean trough plasma levels of imatinib of 1.46 mm. 15,49 This concentration is consistent with preclinical studies demonstrating that the IC 50 of BCR-ABL kinase activity inhibition is in the micromolar range. 53 Therefore, doses of imatinib less than 300 mg may lead to plasma imatinib concentrations that suboptimally inhibit BCR-ABL kinase activity, favoring the selection of spontaneously arising resistant clones. Strategies to treat resistant patients Dose escalation: Clinical evidence: To investigate whether imatinib-resistant patients benefit from escalated dosing, 54 CML patients in CP, resistant or refractory to imatinib, were given 300 or 400 mg/day imatinib doses escalated from once to twice daily. 54 CHRs were obtained in 65% of patients treated for hematologic resistance. Of patients treated for cytogenetic resistance, 56% achieved CCR, demonstrating that CP CML patients resistant to imatinib can be brought into response by increasing the imatinib dosage. Previously mentioned phase 2 trials of imatinib demonstrated efficacy and feasibility of increased dosing in advanced phases of CML. 6,7 The 2-year follow-up data of AP patients taking 400 mg/day indicate that 87% failed to reach CCR and 82% failed to reach MCR, whereas among the group taking 600 mg/ day, 76% failed to achieve CCR and 67% failed to reach MCR (Talpaz et al. Blood 2003; 102(Supp. 1): 905a, abstract). After 24 months, the estimated progression-free survival is 32 and 49% for the 400 and 600 mg/day doses of imatinib, respectively (Figure 2). The 2-year follow-up results among patients in BC taking 400 mg/day imatinib indicate that 97% failed to reach CCR and 94% failed to reach MCR compared with the group taking 600 mg/day in which 92% failed to achieve CCR and 82% failed to reach MCR (Talpaz et al. Blood 2003; 102(Supp. 1): 905a, abstract), supporting dose escalation to minimize resistance in advanced phases of CML (Figure 2). Rationale: Mechanisms of imatinib resistance that have the theoretical potential to respond to increasing concentrations of imatinib have been observed in cell lines 55 and in patients. BCR-ABL kinase domain mutations are the most frequent mechanism associated with relapse during treatment with imatinib. Approximately 60% of relapsed patients have point mutations detected in one of the three main regions of the BCR- ABL kinase domain: (i) the amino-terminal P-loop, which includes amino acids that form the nucleotide binding loop for adenosine triphosphate (ATP), (ii) the catalytic domain and intervening sequences containing amino acids that contact imatinib, as well as (iii) the carboxy-terminal activation loop, which is critical for the control of catalytic activity. 1,25,14,16,28,30,31,39 Resistance is acquired by the selective expansion of clones bearing BCR-ABL point mutations less sensitive to imatinib compared with wild type. With few exceptions, the rank order of the frequency with which point mutations appear in patient samples generally correlates with the rank order of BCR-ABL imatinib insensitivity as indicated by an increase in IC 50 of imatinib inhibition of BCR- ABL kinase activity. 47 Mutations have been isolated from patients who have been shown to have diminished sensitivity to imatinib but could still be inhibited by concentrations of imatinib achievable in patients. When these mutants are the dominant clone detected during resistance, imatinib dose escalation may be justified. The relationship between imatinib resistance and the appearance of point mutations in BCR-ABL is not clearly understood because some kinase domain mutations isolated from patients have near wild-type sensitivity to imatinib. 47 This suggests that additional mechanisms of resistance may operate in these cases. For example, BCR-ABL genomic amplification and overexpression of BCR-ABL transcripts have also been reported in resistant patients. 13,25,28,56,57 To the extent that imatinib resistance is based on increased BCR-ABL levels in these patients, they are likely to respond to escalating concentrations of imatinib. Multidrug-resistance mechanisms are potentially involved in resistance to imatinib in patients because they limit intracellular drug concentrations. Low levels of the multidrug-resistance protein MRP1 have been shown to predict imatinib responses in patients with myeloid BC CML. 58 Overexpression of P- glycoprotein (PGP), a MDR1 gene product that functions as a drug efflux pump, has also been suggested to play a role in imatinib resistance. 59 Another mechanism of pharmacological interaction that may influence imatinib responses is the plasma levels of a1 acid glycoprotein (AGP), which binds imatinib and sequesters it from cells. The addition of erythromycin, a competitor for AGP binding, restored the ability of imatinib to inhibit BCR-ABL phosphorylation in samples derived from relapsed patients. 60 Therefore, the use of imatinib plasma levels to estimate intracellular imatinib concentrations is under debate. 61 Other studies have shown that AGP levels can be used to predict disease progression While AGP levels might have some prognostic value, reducing the protein-bound fraction of imatinib in patients has yet to be shown to recover responses 5

6 6 to imatinib therapy. It is conceivable, however, that increasing imatinib concentrations can assist in overcoming resistance attributable to elevated serum AGP. Differential compartmentation of imatinib in the body can also result, for example, in subtherapeutic concentrations of imatinib in the central nervous system (CNS) owing to the blood brain barrier. 65 This can lead to CNS BC in CML patients otherwise experiencing cytogenetic remission. Treatment options are limited in these cases, but dose escalation of imatinib should be considered. Interruption or cessation of imatinib therapy: Clinical evidence: Complete cessation or temporary interruption of imatinib therapy can be considered in certain instances of resistance. Discontinuation of imatinib therapy was found to significantly reduce a clone of cells bearing a BCR-ABL Y253H (P-loop) mutation in a resistant patient. 66 Further, a patient taking imatinib in CP progressed to BC and subsequent withdrawal of imatinib resulted in a spontaneous reversion to CP. 67 Rationale: Cessation or interruption of imatinib is most likely to be beneficial where relapse can be attributed to the expansion of an imatinib-resistant clone owing to a BCR-ABL point mutation that severely impairs imatinib binding. Owing to the high frequency of binding impairing mutations such as E255K or Y253F 1 and the poor prognosis portended by P-loop mutations 29,68 (Kreil et al. Blood 2003; 102(Suppl. 1): 71a; Branford et al. Blood 2003; 101(Suppl. 1): 71a, abstracts), it is conceivable that cessation or interruption of imatinib will become an important tool for clinical management of resistant patients. This approach relies on the reappearance of nonmutant BCR-ABL leukemic clones to suppress the mutant clone by removing its competitive advantage. Y253 was predicted to be an important point of interaction between BCR-ABL and imatinib based on crystal structure analyses of imatinib bound Table 2 Clinical studies of imatinib in combination with other agents CML phase References Drugs in combination Cytarabine CP 72 CP a CP b Interferon-a CP c CP d Pegylated interferon-a2a CP e Pegylated interferon-a2b CP 73 Cytarabine vs etoposide AP or BC f Arsenic trioxide AP or BC g Gemtuzumab ozogamicin my-bc h Mitoxantrone, etoposide and cytarabine my-bc I Idarubicin and low-dose cytarabine BC k Cyclophosphamide, vincristine, doxorubicin, dexamethasone ly-bc 74 Homoharringtonine CP l CP, AP, BC m Tipifarnib (farnesyltransferase inhibitor) CP n, o Second-line therapy Bortezomib CP or AP p Hydroxyurea, homoharringtonine, low-dose cytarabine CP, AP q Troxacitabine BC 75 Lonafarnib (farnesyltransferase inhibitor) CP, AP r Homoharringtonine AP s Decitabine CP, AP, BC t New signal transduction inhibitors (targets: ABL, SRC and mtor) a Monroy et al. Blood 2003; 102(Suppl. 1): 320b. b Druker et al. Blood 2001; 98(Suppl. 1): 845a. c Kyo et al. Blood 2003; 102(Suppl. 1): 318b. d O Dwyer et al. Blood 2001; 98(Suppl. 1): 846a. e Hochhaus et al. Meeting Proc ASCO 2003; 22: 569. f Ceballos et al. Blood 2003;102(Suppl. 1): 321b. g Ravandi-Kashani. Blood 2003; 102(Suppl. 1): 314b. h Sallah et al. Blood 2002; 100(Suppl. 1): 319b. i Fruehauf et al. Meeting Proc ASCO 2003; 22: 589. k Alvarez et al. Meeting Proc ASCO 2003; 22: 617. l Marin et al. Blood 2003; 102(Suppl. 1): 908a. m Maloisel et al. Blood 2003; 102(Suppl. 1): 909a. n Cortes et al. Blood 2003; 102(Suppl. 1): 909a. o Gotlib et al. Blood 2003; 102(Suppl. 1): 909a 910a. p Cortes et al. Blood 2003; 102(Suppl. 1): 312b. q Kreil et al. Blood 2003; 102(Suppl. 1): 71a. r Cortes et al. Blood 2002; 100(Suppl. 1): 164a. s Burton et al. Blood 2003; 102(Suppl. 1): 908a. t Issa et al. Blood 2003; 102(Suppl. 1): 910a, abstracts. Phase 1/2 studies in progress

7 to ABL. 69,70 Mutation T315I, which is frequently observed in resistant patients, is located in a predicted imatinib-binding site and not in the ATP-binding domain. This mutation has also been shown to confer complete insensitivity to imatinib as well as diminished intrinsic kinase activity owing to a decreased affinity to ATP. 28,46 Upfront combination therapy: Clinical evidence: Combination therapy is a third option to consider in combating imatinib resistance. Upfront combination therapy with cytotoxic agents, IFN or other signal transduction inhibitors has been investigated in several clinical trials. 71 (Table 2) Studies (eg the CML Study IV of the German CML Study Group or the STI571 Prospective International Randomized Trial SPIRIT) are currently underway to compare directly imatinib monotherapy vs imatinib plus cytarabine vs imatinib plus IFN as first-line therapy in thousands of randomized newly diagnosed BCR-ABL-positive CML patients. 76,77 Second-line combination therapy: Clinical evidence: While clinical data for second-line combination therapy are limited, this approach must be considered after imatinib monotherapy fails, despite imatinib dose escalation, to achieve MCR after 6 months, CCR after 12 months, a 3-log reduction after 18 months or relapse ensues (Table 2). In a significant proportion of patients lack of CR is associated with the development of cytopenias, which occurs frequently (15 40% of CP patients) during imatinib therapy. 78,79 Combination therapy with imatinib plus granulocyte colony-stimulating factor resulted in MCR in seven of 11 CP or AP patients, who had failed to achieve CR after 6 months of imatinib monotherapy. A recent novel approach to combination therapy involves stimulating the host immune response. 80 In a phase 2 trial, 14 patients with CP CML were vaccinated with a BCR-ABL fusion peptide to stimulate T-cell-mediated immune responses. Four patients, also treated with IFN or imatinib, in hematologic remission had a further CR, suggesting that a peptide-derived vaccine that elicits specific immune responses can be used as combination therapy. Rationale: Combination therapy with imatinib and other agents best targets BCR-ABL-independent resistance including disease progression arising from clonal evolution or activating mutations in genes encoding molecules downstream of BCR- ABL. In addition, combination therapy may address BCR-ABLdependent refractoriness or relapse arising from BCR-ABL point mutations or atypical BCR-ABL fusion genes 81 that abrogate sensitivity to imatinib. Disease progression in CML is associated with nonrandom, consistently observed, karyotypic abnormalities referred to as clonal evolution. In all, 60 80% of CML patients who progress exhibit secondary chromosomal abnormalities in addition to the Ph chromosome. 82 Clonal evolution has been demonstrated to occur during imatinib therapy and to be associated with advanced disease Recent reports indicate that chromosomal abnormalities are also emerging in Ph-negative cells in CML patients on imatinib therapy. 24,85 94 The underlying mechanism for the appearance of these clones during imatinib treatment is not completely understood. Imatinib may reveal the presence of pre-existing chromosomally abnormal cells. The emergence of cytogenetically unrelated Ph-negative clones with additional aberrations in CML may support the multistep model of leukemic transformation and the concept of genetic instability inherent in CML. Alternatively, these cells could arise as a consequence of the hematopoietic proliferative pressure applied to normal cells under conditions where Ph-positive cells are eradicated. That the same chromosomal abnormalities in Ph-negative cells have emerged with treatments for CML other than imatinib in patients with cytogenetic responses suggest that it is unlikely that imatinib is responsible for the initial presence of these chromosomal abnormalities. Most patients developing chromosomal abnormalities in Ph-negative cells were not found to progress until now. 88,89,91 Whether proliferation of these abnormal Ph-negative clones contributes to relapse or the development of other clonal hematologic disorders, like myelodysplastic syndromes, during imatinib therapy will require further investigation. Preclinical evidence supporting combination therapy Conventional chemotherapeutic agents: Prior to imatinib, CML treatment was frustrated in part by the antiapoptotic effects of BCR-ABL, which renders leukemic cells resistant to chemotherapy. 71 Imatinib chemosensitizes cells in vitro to cytotoxic agents by virtue of its ability to promote apoptosis. Several studies in Ph-positive cell lines have indicated that the cell killing effect of chemotherapeutic agents is additive or synergistic with that of imatinib. 71,95 98 Signal transduction inhibitors: Preclinical studies have demonstrated that inhibition of downstream signaling targets of BCR-ABL such as the Ras pathway using farnesyltransferase inhibitors may be a promising combination therapy with imatinib. 99,100 Other molecules that might serve as targets for inhibition of BCR-ABL downstream signaling include Jak2, MEK, and PI-3 kinases. 101 Chaperone inhibition: BCR-ABL depends on the molecular chaperone, heat-shock protein 90 (Hsp90), to attain its functional conformation. Treatment of leukemic cells, isolated from CML patients and resistant to imatinib by virtue of kinase domain point mutations, with geldanamycin, an Hsp60 inhibitor, has been shown to degrade wild-type and mutant BCR- ABL and to inhibit cell growth. 102 Stem cell transplantation: Allogeneic SCT remains the only proven cure for CML, but is limited by donor availability and treatment-related morbidity and mortality. 103 In biologically young patients with histocompatible donors, SCT is a viable option to consider as first-line therapy, after suboptimal response to imatinib, or in cases of intractable imatinib resistance. Treatment of minimal residual disease The criterion for complete molecular remission, undetectable BCR-ABL-positive leukemic cells, is associated with continued remission. 11,104 In the IRIS study, less than 5% of patients taking imatinib had undetectable BCR-ABL, indicating that this standard is rarely attained with imatinib therapy. Current limits of PCR sensitivity imply that even with a complete molecular response, a body load of 10 6 leukemic cells potentially remains. 19,105,106 The persistence of leukemic cells after imatinib therapy raises questions as to whether and how to treat minimal residual disease. Analysis of peripheral blood and bone marrow samples from CP CML patients has demonstrated the existence of primitive quiescent Ph-positive stem cells (CD34 þ ) 107 that have been postulated to contribute to residual disease because their 7

8 8 Table 3 Management strategies to consider for avoiding or overcoming resistance to imatinib in patients with CML Begin imatinib therapy in early chronic phase, given the correlation seen in imatinib-treated patients between increased rates of elimination of Ph-positive cells and earlier disease stage. Initiate imatinib therapy with, at minimum, the approved daily dose: 400 mg/day for chronic phase, 600 mg/day for accelerated phase or blast crisis. Increase the dosage, for example, from 400 to 600 or from 600 to 800 mg/day (400 mg twice daily) if relapse occurs at the initial dose or response is inadequate (eg lack of hematological response after X1 month of therapy, lack of any cytogenetic response after X6, lack of CCR or MCR after X12 months of therapy). Avoid subtherapeutic dosing (o300 mg/day in adult patients). Manage side effects promptly and aggressively to maximize adherence to optimal therapy; if treatment must be interrupted, consider rechallenge with imatinib at gradually increasing doses after side-effect resolution. Consideration of imatinib in combination with a second agent (eg cytarabine, homoharringtonine, IFN) may be warranted on a case-by-case basis (best in clinical trials), particularly if the patient s disease continues to be refractory to increased imatinib dosing. The improvement of long-term progression-free survival of preemptive combinations of imatinib with (pegylated) IFN and other agents is a matter of ongoing phase 2 and 3 trials. Consider interruption of therapy if resistant BCR-ABL mutants are detected. Therapeutic decisions should be based on cytogenetic and molecular data with regard to clonal evolution, point mutations, cytogenetic and molecular response, and dynamics of the resistant clone. proliferation can be induced by exposure to growth factors. BCR-ABL-positive hematopoietic progenitor cells persist in CML patients with CCR to imatinib. 108 The sensitivity of Ph- and CD34-positive stem cells to imatinib has been explored by treating stem cells, isolated from CP CML patients, with growth factors and imatinib. Imatinib appeared to have only an antiproliferative effect on the quiescent cell subpopulation, suggesting that cell-cycle-arrested stem cells are resistant to the cytocidal activity of imatinib. Another study, however, found no correlation between the proliferative status of BCR-ABL-positive cell lines and imatinib-induced apoptosis. A plausible explanation for these results is that a molecular mechanism rather than quiescence is responsible for imatinib insensitivity in stem cells. 109 BCR-ABL mutants have been detected in CML and ALL patients prior to exposure to imatinib. 32,34 The significance of these mutations, despite their low prevalence, was underscored by the observation that these patients eventually relapsed and that these mutations were the same as those previously isolated from relapsing patients. Treatment of minimal residual disease should consist mainly of continued inhibition of BCR-ABL kinase activity with imatinib combined with molecular surveillance. Future trials will determine the duration of treatment with imatinib necessary to sustain molecular response. Combination therapy may prove to be important in addressing the transition from minimal residual disease to resistance and eventual relapse. Trials are underway to evaluate combination therapies with imatinib and chemotherapeutic agents as first-line therapy. Whether resistance and relapse rates are reduced with combination therapy as a consequence of reducing minimal residual disease compared with imatinib monotherapy is currently an open issue. Conclusions and new directions Molecularly targeted therapy with imatinib has improved treatment of CML, particularly CP patients. Emergence of resistance and relapse indicates that adaptation nevertheless plays a role in the complex interaction between imatinib and the CML disease process. Accumulating experience has provided insight into the underlying mechanisms of, and risk factors for, imatinib resistance. These have guided recommendations for the management of patients in all phases of CML treated with imatinib (Table 3). The outlook for long-term treatments is likely to involve combination therapies with cytotoxic agents or agents that target multiple sites along the BCR-ABL signal transduction pathway. The oncogenicity of BCR-ABL will likely mandate that inhibition of its kinase activity remains as the cornerstone of treatment for CML. Improvements in the technologies used to characterize disease and monitor response, when integrated with results from clinical trials, will facilitate the design of future strategies aimed at optimizing the prognosis for patients with CML. Acknowledgements The study was supported by Novartis Pharma, East Hanover, NJ, USA, the Competence Network Acute and chronic leukemias, sponsored by the German Bundesministerium für Bildung und Forschung (Projektträger Gesundheitsforschung; DLR e.v.- 01 GI9980/6) and the European Net within the Sixth European Community Framework Programme for Research and Technological Development. References 1 Goldman JM, Melo JV. Chronic myeloid leukemia advances in biology and new approaches to treatment. N Engl J Med 2003; 349: Ottmann OG, Druker BJ, Sawyers CL, Goldman JM, Reiffers J, Silver RT et al. A phase 2 study of imatinib in patients with relapsed or refractory Philadelphia chromosome-positive acute lymphoid leukemias. Blood 2002; 100: Lydon NB, Druker BJ. Lessons learned from the development of imatinib. Res 2004; 28S1: S29 S38. 4 Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ et al. Abl protein kinase inhibitor STI571 inhibits in vitro signal

9 transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000; 295: Okuda K, Weisberg E, Gilliland DG, Griffen JD. ARG tyrosine kinase activity is inhibited by STI571. Blood 2001; 97: Sawyers CL, Hochhaus A, Feldman E, Goldman JM, Miller CB, Ottmann OG et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 2002; 99: Talpaz M, Silver RT, Druker BJ, Goldman JM, Gambacorti- Passerini C, Guilhot F et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 2002; 99: O Brien SG, Guilhot F, Larson RA, Gathmann I, Baccarani M, Cervantes F et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003; 348: Kantarjian H, Sawyers C, Hochhaus A, Guilhot F, Schiffer C, Gambacorti-Passerini C et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002; 346: Branford S, Rudzki Z, Harper A, Grigg A, Taylor K, Durrant S et al. Imatinib produces significantly superior molecular responses compared to interferon alfa plus cytarabine in patients with newly diagnosed chronic myeloid leukemia in chronic phase. 2003; 17: Hughes TP, Kaeda J, Branford S, Rudzki Z, Hochhaus A, Hensley ML et al. Frequency of major molecular responses to imatinib or interferon alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 2003; 349: Müller MC, Gattermann N, Lahaye T, Deininger MW, Berndt A, Fruehauf S et al. Dynamics of BCR-ABL mrna expression in first-line therapy of chronic myelogenous leukemia patients with imatinib or interferon alpha/ara-c. 2003; 17: Hochhaus A. Cytogenetic and molecular mechanisms of resistance to imatinib. Semin Hematol 2003; 40: Gambacorti-Passerini CB, Gunby RH, Piazza R, Galietta A, Rostagno R, Scapozza L. Molecular mechanisms of resistance to imatinib in Philadelphia-chromosome-positive leukaemias. Lancet Oncol 2003; 4: Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM et al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001; 344: von Bubnoff N, Peschel C, Duyster J. Resistance of Philadelphiachromosome positive leukemia towards the kinase inhibitor imatinib (STI571, Glivec): a targeted oncoprotein strikes back. 2003; 17: Weisberg E, Griffin JD. Resistance to imatinib (Glivec): update on clinical mechanisms. Drug Resist Updat 2003; 6: Goldman JM. Chronic myeloid leukemia still a few questions. Exp Hematol 2004; 32: van der Velden VH, Hochhaus A, Cazzaniga G, Szczepanski T, Gabert J, van Dongen JJ. Detection of minimal residual disease in hematologic malignancies by real-time quantitative PCR: principles, approaches, and laboratory aspects. 2003; 17: Kantarjian H, O Brien S, Cortes J, Giles F, Shan J, Rios MB et al. Survival advantage with imatinib mesylate therapy in chronicphase chronic myelogenous leukemia (CML-CP) after IFN-alpha failure and in late CML-CP, comparison with historical controls. Clin Cancer Res 2004; 10: Merx K, Müller MC, Kreil S, Lahaye T, Paschka P, Schoch C et al. Early reduction of BCR-ABL mrna transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon alpha. 2002; 16: Sureda A, Carrasco M, de Miguel M, Martinez JA, Conde E, Sanz MA et al. Imatinib mesylate as treatment for blastic transformation of Philadelphia chromosome positive chronic myelogenous leukemia. Haematologica 2003; 88: Lange T, Bumm T, Otto S, Al-Ali HK, Kovacs I, Krug D et al. Quantitative reverse transcription polymerase chain reaction should not replace conventional cytogenetics for monitoring patients with chronic myeloid leukemia during early phase of imatinib therapy. Haematologica 2004; 89: Schoch C, Haferlach T, Kern W, Schnittger S, Berger U, Hehlmann R et al. Occurrence of additional chromosome aberrations in chronic myeloid leukemia patients treated with imatinib mesylate. 2003; 17: Hochhaus A, Kreil S, Corbin AS, La Rosée P,Müller MC, Lahaye T et al. Molecular and chromosomal mechanisms of resistance to imatinib (STI571) therapy. 2002; 16: ten Hoeve J, Arlinghaus RB, Guo JQ, Heisterkamp N, Groffen J. Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia. Blood 1994; 84: Jacobberger JW, Sramkoski RM, Frisa PS, Ye PP, Gottlieb MA, Hedley DW et al. Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity. Cytometry 2003; 54A: Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN et al. Clinical resistance to STI-571 cancer therapy caused by BCR- ABL gene mutation or amplification. Science 2001; 293: Branford S, Rudzki Z, Walsh S, Parkinson I, Grigg A, Szer J et al. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood 2003; 102: Barthe C, Cony-Makhoul P, Melo JV, Reiffers J, Mahon FX. Roots of clinical resistance to STI-571 cancer therapy. Science 2001; 293: 2163a. 31 Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2002; 2: Roche-Lestienne C, Soenen-Cornu V, Grardel-Duflos N, Lai JL, Philippe N, Facon T et al. Several types of mutations of the Abl gene can be found in chronic myeloid leukemia patients resistant to STI571, and they can pre-exist to the onset of treatment. Blood 2002; 100: Kreuzer KA, Le Coutre P, Landt O, Na IK, Schwarz M, Schultheis K et al. Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique. Ann Hematol 2003; 82: Hofmann WK, Komor M, Wassmann B, Jones LC, Gschaidmeier H, Hoelzer D et al. Presence of the BCR-ABL mutation Glu255Lys prior to STI571 (imatinib) treatment in patients with Ph+ acute lymphoblastic leukemia. Blood 2003; 102: Deininger MW, McGreevey L, Willis S, Bainbridge TM, Druker BJ, Heinrich MC. Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography. 2004; 18: Soverini S, Martinelli G, Amabile M, Poerio A, Bianchini M, Rosti G et al. Denaturing HPLC-based assay for detection of ABL mutations in chronic myeloid leukemia patients resistant to imatinib. Clin Chem 2004, Apr 23 [Epub ahead of print]. 37 Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 2002; 99: Roumiantsev S, Shah NP, Gorre ME, Nicoll J, Brasher BB, Sawyers CL et al. Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. Proc Natl Acad Sci USA 2002; 99: Nardi V, Azam M, Daley GQ. Mechanisms and implications of imatinib resistance mutations in BCR-ABL. Curr Opin Hematol 2004; 11: Hochhaus A, Kreil S, Corbin AS, La Rosée P, Lahaye T, Berger U et al. Roots of clinical resistance to STI-571 therapy. Science 2001; 293: 2163a. 41 Al-Ali HK, Heinrich MC, Lange T, Krahl R, Müller M, Muller C et al. High incidence of BCR-ABL kinase domain mutations and 9