Management of Chronic Myeloid Leukemia with BCR/ABL Inhibitors: Current Status and Future Perspectives

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1 Clinical Medicine Insights: Therapeutics R e v i e w Open Access Full open access to this and thousands of other papers at Management of Chronic Myeloid Leukemia with BCR/ABL Inhibitors: Current Status and Future Perspectives Peter Valent Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna and Ludwig Boltzmann Cluster Oncology, Vienna, Austria. peter.valent@meduniwien.ac.at Abstract: Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome (Ph) and the BCR/ABL oncoprotein. In chronic phase (CP) CML, leukemic cells display multilineage differentiation and maturation capacity. The BCR/ABL inhibitor imatinib exerts profound antileukemic effects in these patients and is considered standard frontline therapy. However, not all patients show a long-lasting response to this drug. Rather, resistance to imatinib has been recognized as an emerging clinical problem in CML. While CML stem cells exhibit intrinsic resistance against imatinib and thus survive therapy, one or more stem cell-derived subclones may acquire additional hits over time, so that an overt relapse occurs. A major triggering hit in such subclones are BCR/ABL mutations. For these patients, novel multikinase inhibitors targeting BCR/ABL such as nilotinib, dasatinib, bosutinib, bafetinib, as well as Aurora kinase inhibitors have been developed and shown to exert antileukemic effects in imatinib-resistant CML. In addition, hematopoietic stem cell transplantation (HSCT) remains an important treatment option for drug-resistant patients. The decision concerning second- or third line therapy, selection of drugs, and HSCT, is usually based on the presence and type of BCR/ABL mutation(s), phase of disease, other disease-related factors, and patient-related variables including age and co-morbidity. The current article provides an overview on current diagnostic and therapeutic strategies for patients with drug-naïve and drug-resistant CML. Keywords: CML, imatinib, BCR/ABL mutations, dasatinib, nilotinib Clinical Medicine Insights: Therapeutics 2010: This article is available from the author(s), publisher and licensee Libertas Academica Ltd. This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited. The authors grant exclusive rights to all commercial reproduction and distribution to Libertas Academica. Commercial reproduction and distribution rights are reserved by Libertas Academica. No unauthorised commercial use permitted without express consent of Libertas Academica. Contact tom.hill@la-press.com for further information. Clinical Medicine Insights: Therapeutics 2010:2 365

2 Valent Introduction Chronic myeloid leukemia (CML) is a stem cellderived myeloid neoplasm in which leukemic cells retain multilineage differentiation and maturation capacity over a certain time period. Cytogenetically, the disease is characterized by the Philadelphia chromosome (Ph) that results from the reciprocal translocation t(9;22). 1 3 The specific fusion-gene product, BCR/ABL, is a 210 kda-sized cytoplasmic oncoprotein that contributes essentially to the pathogenesis of CML. 3 5 The clinical course in CML is divided into a chronic phase (CP), accelerated phase (AP), and a blast phase (BP) that behaves as an acute leukemia. 6 8 During the past decades, several effective treatment approaches have been introduced for patients with CML An effective cytoreductive agent that was used as standard treatment in the past, is interferon-alpha. 14,15 An approach with curative potential is hematopoietic stem cell transplantation (HSCT) which can be offered to a subgroup of patients HSCT is associated with enhanced disease-free survival, but also with transplant-related mortality and transplant-related morbidity Today, imatinib (Gleevec ) is prescribed as firstline therapy in most patients with freshly diagnosed CML. This BCR/ABL tyrosine kinase inhibitor (TKI) produces major cytogenetic responses in a majority of these patients In addition, imatinib exerts anti-cml activity in patients refractory to interferon-alpha, and is even effective in some patients with AP or BP However, unfortunately, CML stem cells display intrinsic resistance to imatinib As a consequence, imatinib can suppress leukemia cell growth for prolonged time periods but is usually not capable of eradicating the malignant clone Thus, life-long imatinib appears to be required in responding patients, even when a major molecular response is obtained. 21,22 In addition, even as slowly cycling cells, leukemic stem cells (LSC) can acquire further hits in initially small-sized subclones, and later, when these hits lead to clonal expansion and enhanced proliferation, frank resistance and thus an overt relapse develops. 21 In many cases, such stem cell-derived subclones exhibit BCR/ABL mutations and/or other resistance mechanisms Because HSCT is a potentially curative treatment option, it is of importance to define the exact prognosis as early as possible in each patient, to predict drug responses, to monitor the levels of minimal residual disease (MRD), and to define whether the patient would be a potential candidate for HSCT. During the past few years, a number of novel BCR/ ABL TKI have been developed and used in preclinical and clinical trials Two of these second generation TKI, dasatinib and nilotinib are now approved for treatment of patients with imatinib-resistant or imatinib-intolerant CML. Other new agents such as bosutinib (SKI-606), bafetinib (INNO-406), and the Aurora kinase inhibitors, are currently being tested in clinical studies. In several instances, targeted drugs are combined with other treatment approaches or HSCT. All in all, substantial progress has been made in the diagnosis, management, and therapy of CML in recent years. In the current article, optimal management and therapy of CML is discussed, with special focus on established and novel TKI. In addition, the article provides an overview on forthcoming new agents and future strategies to improve antileukemic therapy in freshly diagnosed and drug-resistant CML. Investigations in Freshly Diagnosed Patients Blood examinations at diagnosis include a complete blood count with microscopic evaluation of leukocyte subsets, BCR/ABL, serum chemistry, and HLA-typing. The demonstration of BCR/ABL p210 confirms the diagnosis CML. Initial staging includes a bone marrow (BM) examination, an ultrasound of the spleen and a chest X-ray. Depending on the overall situation and symptoms, further staging investigations such as a computed tomography, or neurologic tests may be required. In patients with primary BP, immunophenotyping of blast cells by flow cytometry (lymphoid versus myeloid) is mandatory. 41 A thorough investigation of the basophil compartment should be performed in each case, especially in suspected AP. Morphologic assessment of basophils in blood- and BM smears is regarded standard, although immature basophils may escape counting, especially in AP Biochemical and immunohistochemical markers of basophils may be useful to overcome this problem Cellular blood histamine levels are elevated at diagnosis in CML and correlate with basophil numbers. 48 An interesting parameter is the serum tryptase level 366 Clinical Medicine Insights: Therapeutics 2010:2

3 Management and therapy of CML which is a marker of immature basophils in CML, and may correlate with (imminent) AP and prognosis. 49 Therefore, whole blood histamine- and serum tryptase levels, although not considered routine tests in CML, might be of interest and should be assessed at diagnosis when the assays are available. Screening for BCR/ABL mutations at diagnosis is presently not recommended. A BM biopsy and aspiration should be performed in all patients at diagnosis. The BM histology and immunohistochemistry are of importance and may reveal fibrosis or multifocal accumulations of CD34+ blasts. 49,50 If blast cells are CD34-negative, KIT is used as a progenitor-marker. 49 In addition, the histologic evaluation of the BM is useful to assess the morphology and numbers of basophils and megakaryocytes as well as the microvessel density. 46,47,49 Immunohistochemical evaluation of the BM in CML is of particular importance when a dry tap or non-representative smear was obtained (example: BM fibrosis). The aspirate smear is used to perform morphologic studies, including the percentage of blasts, promyelocytes, and basophils, and to screen for signs of dysplasia. The aspirate is also used to perform karyotyping and fluorescence in situ hybridization (FISH) The karyogram should be based on at least 20 metaphases and should report the percentage of Ph+ cells and, if present, additional chromosome abnormalities (ACA), which are prognostic and often seen in AP. 54,55 Chromosome abnormalities in other (Ph-) cells (OCA) may also be detected, 56 but such (sub)clones usually become detectable (develop) later during (not before) therapy. BCR/ABL transcripts are quantitated by real-time PCR (qpcr) using ABL as reference or an alternative reference gene A most important question in freshly diagnosed patients is whether the disease is early stage disease (CP), exhibits signs of an (imminent) AP, or is frank AP. Table 1 provides an overview of parameters indicative of imminent and frank AP. During the past few decades, several scores have been developed for prognostication in CML. 10,61 64 In most scores, the Table 1. Parameters indicative of an accelerated phase (AP) of CML. Parameters and findings indicating the presence of Accelerated phase of CML (AP) Blast cells 10% 19% in the PB or BM Blood basophils 20% in PB Persistent thrombocytopenia ( /µL) unrelated to therapy or persistent marked thrombocytosis unresponsive to therapy Marked BM fibrosis*** Features of imminent or early AP* Blast cells 5% 10% in the PB or BM Basophils 15% 19%; numerous immature basophils; elevated serum tryptase level** Marked megakaryocyte dysplasia or clusters and sheets of megakaryocytes in marrow sections (+/- JAK2 V617F) Mild BM fibrosis*** BM multilineage dysplasia ACA in a prominent subclone during therapy ACA at dignosis or in a small subclone during therapy**** (clonal evolution)**** Suboptimal Response or Treatment failure according to ELN guidelines***** BCR/ABL mutant(s) in subclone(s) Persistent marked leukocytosis or increasing WBC despite therapy Increasing splenomegaly unresponsive to therapy, persistent large spleen size Rapid increase in WBC or excessively high WBC at diagnosis Very large spleen at diagnosis *In these patients, the criteria for AP are not (yet) fulfilled, but findings and symptoms point to imminent AP. These patients may be regarded as potential candidates for higher doses of imatinib (600 mg daily) or a second generation TKI such as nilotinib, similar to AP patients. **Serum tryptase 15 ng/ml. ***BM fibrosis may also be regarded as sign of acceleration. 50 **** In several classification proposals including the WHO, 140 ACA at diagnosis is not an established sign of AP. ***** ELN guidelines: Baccarani et al and Abbreviations: CML, chronic myeloid leukemia; AP, accelerated phase; BM, bone marrow; ACA, additional chromosomal abnormality; PB, peripheral blood; ELN, European Leukemia Net; WBC, white blood cell count. Clinical Medicine Insights: Therapeutics 2010:2 367

4 Valent phase of disease, blast counts, basophils, spleen size, and cytopenia are included as prognostic variables. Most scores have been established for patients receiving conventional cytostatic drugs or interferon-alpha, and may also work for CML patients receiving TKI. Therefore, these scores should be applied at diagnosis in all patients. Frontline therapy (treatment-naïve patients) Imatinib is still considered standard frontline therapy in patients with CP, independent of patient-related factors. The initial standard dose in CP in adults is 400 mg per day p.o., independent of body weight. 12,65 In patients with AP and BP, the recommended dose of imatinib ranges between 600 and 800 mg daily. 12,17,18 In all patients and disease phases, the dose may be adjusted (recommended maximum: 800 mg/day) during treatment with imatinib, based on responses and the plasma trough level In particular, it has been described that a lower trough level of imatinib ( 650 µg/ml) is associated with a lower probability of achieving a complete cytogenetic response (CCyR). 67,68 In patients with a CCyR, life-long treatment is recommended, since (at least subsets of) neoplastic stem cells display intrinsic resistance An unresolved question is whether imatinib therapy can be stopped in the few patients who reach a complete molecular remission without detectable BCR/ABL (in BM cells) by nested PCR, which may be indicative of complete suppression or even eradication of most CML subclones. However, most CML patients with CCyR relapse after treatment interruption. 22,23 Whether this will also be the case in CP patients treated with nilotinib or dasatinib upfront remains at present unknown, but should be addressed soon in clinical trials. It also remains unknown whether and when nilotinib or/and dasatinib can be regarded standard first line drug therapy in CML. Based on the strong activity and superior clinical effects, it appears that at least in AP and BP, initial therapy with nilotinib or dasatinib, with or without consecutive HSCT, may be regarded a reasonable upfront approach, although a consensus recommendation is not yet available. In CP patients, response rates obtained with nilotinib and dasatinib as frontline therapy clearly exceed response rates obtained with imatinib. 69,70 In addition, responses to second generation TKI may occur more rapidly. 69,70 However, long term response data and long term side effect profiles for nilotinib and dasatinib are not available for all patients groups yet. If such evaluation shows a favourable outcome and no safety issues remain, these new TKI (one or both) must be regarded as standard frontline treatment in CML. Prognostication and Follow Up During the First 18 Months As CML stem cells are resistant towards imatinib and probably also to nilotinib and dasatinib, MRD monitoring is an essential approach in the follow up in all patients. Established assays for MRD monitoring in CML are cytogenetics (percent of Ph+ metaphases in the BM) and FISH (percent of Ph+ interphases) as well as assessment of BCR/ABL transcript levels by qpcr in the peripheral blood ,71 75 FISH is usually performed when results from conventional cytogenetics are unclear or unavailable. In addition, certain ACA/OCA-related abnormalities (example: deletion of derivative chromosome 9) are only detectable by FISH During the first year, the BCR/ABL transcript burden is quantified at least every 3 months, and cytogenetics at least every 3 6 months. After the first year, the frequency of investigations depends on the responses achieved. The response is suboptimal when a CCyR is not achieved within 12 months or/and a major molecular response (MMR, defined as a 3-log reduction of BCR/ABL mrna levels) is not achieved within 18 months. In patients with stable CCyR and completely suppressed BCR/ABL or almost completely suppressed transcript levels (4 log reduction), it may be appropriate to reduce the frequency of investigations to 6- or even 12-month intervals. In these patients a nested PCR can be performed to confirm the depth of remission. 76 In daily practice, the levels of BCR/ABL are determined by real-time PCR and are expressed as the ratio of BCR/ABL mrna relative to a control gene (in percent) such as ABL. When possible the result should be normalized using the international reference standard. 77,78 A forthcoming approach may be to quantify CML subclones bearing BCR/ABL mutants by mutationspecific PCR. 79,80 With this approach, clinically relevant mutants like T315I may be detected early 368 Clinical Medicine Insights: Therapeutics 2010:2

5 Management and therapy of CML in disease evolution, 79,80 which may have clinical implications and may assist in planning optimal treatment (examples: switch to alternative TKI or urgent search for HSCT donor). These assays must show sufficient precision and sensitivity to detect BCR/ABL mutants in small-sized subclones and the expansion of such subclones over time. Notably, the clinical impact of subclones that remain small over time is usually unknown (an exception may be T315I). The current recommendation is to screen for BCR/ABL mutants as soon as BCR/ABL transcripts increase substantially from baseline MRD levels in repeated tests. The most common approach to screen for BCR/ ABL mutations is direct sequencing of critical regions in the tyrosine kinase domain of the oncogene. Long-Term Follow-Up Assays and laboratory investigations in the monitoring and long-term follow-up depend on cytogenetic and molecular responses that can be achieved over time. In patients with stable continuous CCyR and a complete molecular response, qpcr analyses should be performed every 6 12 months and BM investigations in case of a suspected relapse. In all other patients, shorter time intervals are recommended, depending on the levels of BCR/ABL transcripts, the dynamics in changes of BCR/ABL transcript levels over time, cytogenetics, and the overall risk profile. In high risk patients, a BM investigation should be performed at least once a year. Diagnostic Approaches and Assays in Imatinib-resistant Patients Appropriate criteria for responses to imatinib and other TKI, including a suboptimal response and a treatment failure have been provided by the European Leukemia Net (ELN). 81,82 These recommendations should be applied in all patients. In those with suspected treatment failure and thus suspected drug resistance, all diagnostic tests including a complete (re)staging with re-biopsy of the BM (histology, morphology, chromosome analysis, FISH), BCR/ABL mrna levels, and analysis of BCR/ABL mutations should be performed. 81,82 In select patients (e.g. suspected compliance problem, suspected pharmacologic resistance) the imatinib trough level should be determined. Analyses should be performed in a step-wise manner. First, the levels of BCR/ABL in the peripheral blood are determined, BM investigations are performed, and a karyogram is obtained. In case of a marked increase in BCR/ABL, a screen for BCR/ABL mutations should be initiated. The karyogram should provide information about the percentage of Ph+ cells and the presence of ACA and OCA. When no BCR/ABL mutations are found, the imatinib trough level may reveal a compliance problem or pharmacologic resistance. 67,68 Management and Therapeutic Algorithm for Imatinib-resistant Patients Resistance against imatinib (or intolerance) is a major challenge in the management of CML. In a substantial number of patients, BCR/ABL mutations are detectable The presence and type of mutation predict responses to TKI and thus represent essential information for the treating physician ,85 87 An important aspect is that due to genetic instability and CML stem cell plasticity, several different subclones with different BCR/ ABL mutants may develop in the same patient over time. 21,84 86 Therefore, in these patients, multiple testing for BCR/ABL mutations may be required, i.e. when BCR/ABL remains high or again increases over time. Table 2 provides a summary of BCR/ABL mutations found in patients with imatinib-resistant CML. These mutations exhibit variable IC50 values for imatinib, nilotinib, and dasatinib when tested in vitro in a kinase assay or bioassay. 38,83,87,88 However, these IC50 values cannot be employed directly as basis for drug selection because all three drugs differ in pharmacologic and pharmacodynamic properties. 83,88 To address this issue, response profiles (estimates) corrected for pharmacological determinants (peak and trough and half life) have recently been proposed for these drugs and are now available (Table 2). 83,88 An intriguing example for a mismatch between in vitro responsiveness (comparative IC50 on a molar basis) and clinical responsiveness is the BCR/ABL mutant F317L. Although this mutant produces a higher in vitro IC50 for nilotinib (50 nm) than for dasatinib (7.5 nm), clinically, the mutant is more frequently resistant to dasatinib, but is usually responsive against nilotinib. 89,90 Clinical Medicine Insights: Therapeutics 2010:2 369

6 Valent Table 2. Predicted effectiveness (PE) of BCR/ABL inhibitors in CML patients bearing various imatinib-resistant BCR/ABL mutants.* Mutation PE Imatinib** Response with higher PE Dasatinib PE Nilotinib imatinib dose no (wt) +/- yes L248V - no + ++ G250E - no + + Q252H - yes + ++ Y253H - no + + Y253F - no + + E255K - no +/- +/- E255V - no + + D276G - n.k E279K - no + ++ V299L - n.k. +/- ++ F311L - no T315I - no - - F317L - no +/- + F317V +/- n.k. +/- + M351T - yes M351I - yes n.k. n.k. F359I - no ++ +/- F359V - n.k. ++ +/- V379I - yes + ++ L384M - n.k H396P - no H396R - no + ++ G398R - n.k F486S - no + ++ *Effectiveness was estimated from the available literature and from corrected data comparing in vitro IC50 values obtained with Ba/F3 cells and in vivo maximum drug concentrations (Cmax) measured in CML patients. 83,87,88 **CE with imatinib 400 mg daily. Abbreviations: n.k., not known; PE score: -, resistant, +/-, modestly sensitive; +, moderately sensitive; ++, very sensitive. In this regard it is noteworthy that the half life of dasatinib (3 4 hours) is much shorter compared to the half life of nilotinib (12 17 hours). In fact, although dasatinib exhibits major pro-apoptotic effects on CML cells even when applied for a short time period, 91,92 this single-shot effect apparently depends on a low IC50 value ( 5 nm). 83,88 Another important aspect is that resistance against imatinib may not only be caused by BCR/ABL mutations, but may also result from other factors, such as the genetic background, BCR/ABL gene amplifications, mutations in other genes, ACA, OCA, or an altered drug uptake ,66 After re-staging and after having explored patient adherence (compliance), the risk profile, and all treatment options, the first question is whether the patient may benefit from imatinib dose escalation. Factors that would argue for an attempt to increase the imatinib dose are good tolerability (no adverse side effects), absence of signs of AP or BP, absence of ACA/OCA, no signs for central nervous system (CNS) involvement, no imatinib-resistant BCR/ ABL mutations or a BCR/ABL mutant known to respond to higher doses of imatinib (Table 2). 81,88 In a group of patients, a low imatinib trough level ( 650 µg/ml) may be detected, leading to the conclusion the imatinib dose should be increased. 65,67,68 If this is not the case and all clinical and laboratory findings support the conclusion the patient would not benefit from imatinib dose escalation, an alternative 370 Clinical Medicine Insights: Therapeutics 2010:2

7 Management and therapy of CML therapy should be offered. 81,82 At that point, the most important questions are i) whether it is primary or secondary resistance, ii) whether BCR/ABL mutants known to respond to nilotinib or/and dasatinib are present, and iii) whether the patient is a candidate for allogeneic HSCT. 21,88,93,94 The following factors would argue for allogeneic HSCT: young age, absence of co-morbidity, donor available, good HLA-match, primary resistance, rapid disease progression, BCR/ABL T315I or other BCR/ABL mutants known to be resistant to all TKI, poor-prognosis-aca, or a substantial increase in blasts. A rapid increase in blasts may argue for HSCT, but may require debulking prior to HSCT, which can usually be performed using conventional chemotherapy (e.g. patient with BCR/ABL T315I or Ph-negative clone) or by using a novel TKI, if the mutant is responsive. 95,96 The following factors would argue for long-term treatment with dasatinib or nilotinib: patient is not eligible for HSCT (because of age, co-morbidity or other features) or is not prepared to take the risk, presence of BCR/ABL mutants that will respond well to one of these drugs, no relevant ACA/OCA, no BP and no signs of rapid progression (Table 3). A most important pro for continuous treatment with dasatinib or nilotinib is a complete response (CCyR, MMR) to these drugs after 3 and 6 months. Selection of Patients for Either Nilotinib or Dasatinib An important decision is which of the two available second-generation TKI (dasatinib or nilotinib) to use in case of resistance or intolerance to imatinib. This decision should primarily be based on the type of BCR/ABL mutation, presence of extramedullary Table 3. When dasatinib or nilotinib should be avoided, interrupted, or switched based on the occurrence of side effects.* Comorbidity/condition/event Recommendation for Dasatinib Nilotinib Arterial hypertension Be cautious Be cautious Arterial thrombosis/stenosis Be cautious Be cautious or switch Pulmonary hypertension Avoid or switch Be cautious Pleural effusions, non severe Diuretics + Steroids** Be cautious Pleural effusions, severe Switch or reduce dose+ Reduce dose+ Diuretics + Steroids** Diuretics + Steroids Other pulmonary disease Avoid or switch Be cautious Cardiac disease, non severe Be cautious or avoid/switch Be cautious Cardiac disease, severe Avoid or switch Be cautious Pericardial effusions Avoid or switch Interrupt + reduce Pancreatitis Be cautious Avoid or switch Severe edema formation Switch Switch, reduce Severe allergic reaction Switch Switch Bleeding tendency Be cautious Be cautious Severe bleeding event Avoid or switch*** Interrupt + reduce Infectious episodes Be cautious or switch Be cautious Severe atypical infections Avoid or switch**** Be cautious Severe cytopenia Switch or reduce dose Reduce dose *Drugs are also selected depending on the presence and type of BCR/ABL mutant(s). **Low dose glucocorticosteroids plus diuretics may lead to a complete or partial resolution of pleural effusions. 104 ***Severe bleeding events have been reported during therapy with dasatinib, 141 but may also occur during nilotinib, especially when severe thrombocytopenia develops in advanced disease. In these patients, TKI should be interrupted until platelets increase. ****In these patients, prophylactic antiinfective therapy should be considered, especially when the patient suffers from overt immunosuppression or an additional neutropenia. 106 Clinical Medicine Insights: Therapeutics 2010:2 371

8 Valent disease (particularly CNS involvement), presence of ACA, and presence of co-morbidity. Factors that would argue to start with dasatinib are i) BCR/ABL mutant with reasonable IC50 for dasatinib, ii) extramedullary disease especially CNS involvement, iii) a specific comorbidity (e.g. known pancreatitis that would argue against nilotinib), and iv) absence of cardiac or pulmonary disease (Table 3). Factors that would argue for nilotinib would be i) a BCR/ABL mutant with a reasonable IC50 for this drug, ii) absence of CNS involvement, and iii) presence of co-morbidity e.g. cardiac and/or a pulmonary disease that would argue against dasatinib, iv) a high risk for (atypical) infections e.g. pre-existing immunodeficiency (Table 3). Previous side effects of imatinib may not predict adverse reactions to nilotinib or dasatinib. Another important question is whether sequential treatment with all three TKI is an appropriate manoeuvre. Here, it should be mentioned that crossresistance against dasatinib and nilotinib has been described, but does not occur in all patients. Thus, it may be a reasonable approach to switch from one second-generation BCR/ABL TKI to another in case of resistance An important aspect is that subclones bearing drug-resistant BCR/ABL mutants can occur by selection at any time during treatment. In most cases these mutants may be pre-existent before start of therapy Therefore, it is important to screen repeatedly for BCR/ABL mutants in TKI-resistant patients and those in whom BCR/ABL levels increase in repeated tests during therapy (suspected or imminent resistance). In some of these patients, multiple drug resistant BCR/ABL mutants are selected and it may happen that combinations of TKI are required to suppress all relevant subclones bearing BCR/ABL mutations (unpublished observation). When the T315I mutant is detectable, it is not appropriate to continue TKI as single agents because the T315I mutation confers resistance against these drugs However, it may be appropriate to continue TKI (to suppress all other relevant subclones) in combination with agents acting on BCR/ABL T315I in these patients. Moreover, recent data suggest that nilotinib and dasatinib may exert unexpected in vitro synergistic effects on primary CML cells and Ba/F3 cells exhibiting BCR/ ABL T315I (unpublished observation). The standard start dose of dasatinib in imatinibresistant CML is 100 mg once daily. 102 In patients with rapidly progressing BP or highly aggressive disease, the dose can be escalated (with caution) to 140 mg daily. It is important to determine cardiac and pulmonary parameters carefully before and during treatment with dasatinib as side effects including pericardial and pleural effusions are quite common, especially when the drug is applied at 140 mg daily In addition, these patients may be at a higher risk to develop atypical infections and weight loss, especially in an advanced phase of CML. 106 Dasatinib is prescribed on a continuous basis unless the patient is prepared for HSCT or develops resistance, and the same holds true for nilotinib. Nilotinib is prescribed at 400 mg twice daily per os in patients with imatinib-resistant CML. 107,108 Side effects of nilotinib have also been described, especially an elevation in bilirubin and elevation in pancreatic enzymes. 107,108 A slight elevation in bilirubin is quite commonly seen, whereas severe side effects (overt pancreatitis) are rather uncommon. An unresolved question is whether the suppression of KIT and other key kinases by nilotinib and dasatinib may result in long term side effects. Likewise, nilotinib-treated patients (unpublished) and dasatinib-treated patients 106 often have undetectable levels of serum tryptase suggesting that not only CML basophils have been eliminated but also growth and function of normal mast cells are markedly suppressed. Special Clinical Situations in Imatinib-resistant Patients A CNS relapse may not only develop in patients with Ph+ ALL, but also in patients with CML, especially in bi-phenotypic or lymphoid BC, but sometimes even in patients with myeloid BC In most patients, a systemic relapse is also found, whereas a local isolated CNS relapse is uncommon In most cases, local and systemic treatment is started in parallel. Local therapy usually consists of irradiation and/or intrathecal cytostatic drugs like Ara-C, glucocorticoids, and MTX Regarding systemic therapy, it is important to know that imatinib does not cross the blood-brain barrier in sufficient quantities, whereas dasatinib reportedly enters the CNS quite effectively. 114,118 Therefore, patients with a CNS relapse should be considered for treatment with dasatinib. Since every 372 Clinical Medicine Insights: Therapeutics 2010:2

9 Management and therapy of CML CNS relapse must be regarded a high-risk situation, patients should always be considered for allogeneic HSCT, if possible. Another important situation is the occurrence of OCA, i.e. an additional chromosomal abnormality in a Ph-negative (sub)clone. 56, Such type of evolution may occur during imatinib, but may also happen in patients treated with second generation TKI Clinically, Ph-negative (sub)clones may be completely silent and may remain small without any tendency to transform into a myelodysplastic syndrome (MDS) or AML over months. In other cases, however, rapid transformation to frank MDS or AML is recorded. 56, For these patients, alternative or additional therapy has to be considered. In young patients who are fit and eligible, HSCT may be an appropriate therapy. In other patients, drug combinations might be considered, especially when it appears that the BCR/ABL+ portion of the disease is completely suppressed by the TKI. In these patients, the Ph-negative component may be treated as if no CML had been diagnosed (e.g. as secondary AML), and the Ph+ disease-portion as if no other (sub)clones were found. However, it has to be borne in mind that all these are (highly) experimental therapeutic manoeuvres, and no standard therapy is available for these patients (exception: HSCT). An interesting observation is that some patients may have a complete loss of CCyR with predominant Ph+ hematopoiesis, but completely normal and apparently stable blood counts. A BCR/ABL mutant is frequently found in these patients. This type of relapse may reflect a pre-leukemic phase in the development of CML, in which (a) BCR/ABL-bearing subclone(s) has/have replaced normal hematopoiesis and thus become(s) predominant (with or without BCR/ABL mutation), but additional hits are required for full transformation into an overt leukemia. 21 In these patients, it may be reasonable to wait for a short time and to consider treatment options without time pressure. However, it is clear that such a subclone has the potential to acquire additional hits, and can then progress to an overt leukemia after a variable and unpredictable latency period (weeks to several months: personal observation). The time-window between complete loss of CCyR/MMR and loss of CHR in these patients is unpredictable. Therefore, it is desirable to address such clinically silent molecular/ cytogenetic relapse, and to plan alternative therapy even if blood counts remain normal. 21 Resistance Against Dasatinib or/and Nilotinib Although many patients with imatinib-resistant CML can be kept in CCyR by dasatinib or nilotinib, relapses may occur. The situation in such a patient is quite difficult and treatment options are limited. Important questions to be asked then are: i) is the patient eligible and thus a candidate for allogeneic HSCT, ii) what BCR/ABL mutants are detectable and what BCR/ ABL mutants are currently suppressed (had been detected in the past) by the TKI administered thus far, iii) is the T315I mutant of BCR/ABL detectable, iv) is resistance based on other (BCR/ABL-independent) factors such as pharmacologic determinants. As mentioned above, it may be reasonable to switch from one TKI to the other based on testing for BCR/ABL mutations and clinically relevant effect-estimates (Table 2). However, it should be pointed out that the response of a subclone to either TKI (nilotinib or dasatinib) can never be predicted with certainty as subclone evolution involves multiple hits (most of them are unknown at present) and one or more of these hits may lead to an altered response of affected leukemic (stem) cells to these TKI. Another important aspect is that it may turn out that both second generation TKI will be required to suppress all relevant subclones in one patient (e.g. patient has both BCR/ABL F317L and BCR/ABL F359I). For these patients, the question may be as to whether and how to combine these two drugs as an experimental manoeuvre in case the patient is not eligible for HSCT. Interestingly, our preliminary in vitro data suggest that this drug combination (nilotinib + dasatinib) is highly effective in all BCR/ABL mutants and may even inhibit the growth of CML cells bearing the T315I-mutated variant of the oncogene (unpublished observation). Other drug combinations (e.g. interferon-alpha plus nilotinib) may also be considered for such patients. In patients receiving a third TKI or a drug combination, monitoring of all disease-related parameters is of importance and should include a repeated screen for additional BCR/ABL mutations as well as chromosome analysis, since ACA or OCA may also develop and may codetermine the clinical course and thus the prognosis in these patients. Clinical Medicine Insights: Therapeutics 2010:2 373

10 Valent Prevention and Management of Side Effects of Dasatinib and Nilotinib Nilotinib displays a favorable side effect profile, and is considered to have the potential to become standard frontline therapy in CML in the near future. However, in some patients, substantial side effects such as an increase in pancreatic enzymes have been reported. 107,108 Most side effects rapidly resolve after dose-reduction or treatment interruption. If this is not the case or side effects appear again, it is appropriate to switch to another TKI (dasatinib) if possible. A side effect of nilotinib with unknown clinical significance is the continuous complete suppression of wild type (wt) KIT tyrosine kinase, 126 and thus mast cell growth and function, which is evident clinically by a suppressed serum tryptase level (unpublished observation). Since mast cells are considered to be key regulators of (cardio)vascular repair mechanisms (by providing histamine, heparin, and uncomplexed tissue type plasminogen activator), current projects in our center focus on possible effects of nilotinib on cardiovascular parameters and cardiovascular events in long term-treated patients. A major side effect of dasatinib is the development of pleural and pericardial effusions. 101, It is important to consider this side effect and to perform repeated examinations (chest X ray, echocardiography) in long term-treated patients. Pleural and pericardial effusions are quite common in patients with advanced CML treated with dasatinib at 70 mg twice daily In patients treated with dasatinib at 100 mg once daily, side effects may be less common. 102 Nevertheless, even in these patients, severe pleural and pericardial effusions may develop (personal observation). An important aspect is that pleural effusions may develop more frequently in advanced, drug resistant CML, and often during or shortly after an infectious or allergic episode. 106 All these observations suggest that the immune system may be involved in effusion formation. Once diagnosed, pleural effusions are best managed by administering low dose glucocorticosteroids and diuretics. 101, If such co-medication remains ineffective, pleuracenthesis is performed and treatment has to be reconsidered, i.e. dasatinib has to be dose-reduced or stopped or switched to an alternative drug. Weight loss and infections as well as the development of skin tumors have also been reported in patients treated with dasatinib at 140 mg daily. 106 Unfortunately, even opportunistic infections were recorded. 106 These side effects may be explained by a transient suppression of various immune cells in these patients. 106 In fact, it has been described that dasatinib blocks T cell receptor-dependent activation of T lymphocytes and IgE-receptor-dependent activation of blood basophils in vitro and in vivo. 106, These effects are explained by the broad target spectrum of dasatinib that includes not only BCR/ABL and KIT, but also PDGFR, several Src kinases, and also Tec kinases like Btk Since these molecules are key kinases of the immune system, the effect of dasatinib on various immune cells was not too unexpected. Rather, it was unexpected to learn that most patients treated with dasatinib (even at 140 mg daily) do not develop severe infections even when treated over a longer time period. This is best explained by the short half life of dasatinib which implies that the effects of this drug on immune cells in vivo are mostly transient (only seen within the first 1 2 hours after drug intake), 106 especially when the drug is applied once daily. Cytopenias have been reported in patients treated with nilotinib and dasatinib. This side effect usually develops in patients with advanced CML and may simply result from the eradication of CML cells in patients in whom normal stem cells are markedly suppressed and thus need time to recover and to regenerate normal hematopoiesis. The general strategy is to continue nilotinib or dasatinib in these patients at the same dose, provided that i) a molecular response is obtained, ii) no severe event (major bleeding, septicemia) occurs, and iii) cytopenia is transient and resolves after several weeks. In these patients, TKIinduced cytopenia is managed by supportive therapy including transfusions if necessary. If no adequate response is obtained or/and cytopenia becomes lifethreatening, the TKI must be stopped. Combination Treatment Strategies and Novel Anti-CML Drugs An increasing number of novel targeted drugs potentially effective in CML, have been developed in recent years The most promising drugs may be multikinase inhibitors that are effective against most or all mutants of BCR/ABL. Some of these drugs also block BCR/ABL-downstream or BCR/ABL-independent 374 Clinical Medicine Insights: Therapeutics 2010:2

11 Management and therapy of CML signaling molecules ,136,137 Examples for such TKI are bosutinib (SKI-606), bafetinib (INNO-406), or the Aurora-Kinase inhibitors (Table 4) , Especially the novel multi-kinase inhibitors that also target BCR/ABL T315I, may represent ideal drugs and drug partners in CML. Most of these drugs are currently tested in clinical trials. A short overview of some of these emerging new drugs is shown in Table 4. For the future, it might be a logical approach to combine most potent targeted drugs early in the course of disease, in order to prevent selection and outgrowth of imatinib-resistant subclones and thus to increase the rate of continuous CCyR. 136,137 Another interesting approach may be to combine these targeted drugs with established agents such as interferon-alpha. Finally, it has to be pointed out that several of these drugs can be combined with a transplantation strategy. Likewise, in high risk patients (AP, BP, T315I mutant) who are young and have a suitable donor, HSCT may still be the primary choice of treatment, even if an initial response was obtained with a TKI. In this regard it should also be pointed out that HSCT remains the only established CML-stem cell-eradicating and thus curative approach for patients with CML. Table 4. Some of the novel emerging anti-cml agents and their targets. Original name Official name Major target(s) Inhibits growth of CML cells bearing BCR/ABL T315I AMN107 Nilotinib Abl,Kit,PDGFR,.. no BMS Dasatinib Abl,Src,Lyn,Btk,Kit,PDGFR,.. no SKI-606 Bosutinib Abl,Src,Lyn,.. no INNO-406 Bafetinib Abl,Lyn,wtKit,.. no AZD0530 Saracatinib Abl,Src,.. n.k. AP23464 Abl,Src,Kit,.. no AP23848 Abl,Src,Kit,.. no CGP76030 Abl,Src,.. n.k. PP1 Abl,Src,.. n.k. PD Abl,Src,PDGFR,FGFR,.. no TG Abl,Src,.. yes TG Abl,Src,.. yes ON Abl,PDGFR,Lyn,.. yes ON01910 Abl,PLK1,Src,Fyn,.. n.k. AP24534 Abl,Src,Lyn,FGFR,PDGFR,.. yes VX-680 Tozasertib Abl,AuK,.. yes VE-465 Abl,AuK,.. yes PHA Danusertib Abl,AuK,Ret,Trk-A,.. yes KW-2449 Abl,AuK,FGFR1,.. yes AT9283 Abl,AuK,JAK2,JAK3,.. yes XL228 Abl,AuK,Src,IGF1R,.. yes AS Abl,AuK,FLT3,JAK2,FGFR3,.. yes SGX Abl,.. yes DCC-2036 Abl,.. yes SGX393 Abl,FLT3,CSFR1,.. yes FTY720 Fingolimod PP2A-activation yes BAY Sorafenib Abl,BRAF,VEGFR,PDGFR,.. +/- Abbreviations: PDGFR, platelet derived growth factor receptor; AuK, aurora kinase; n.k., not known; FGFR, fibroblast growth factor receptor; VEGFR, vascular endothelial growth factor receptor. Clinical Medicine Insights: Therapeutics 2010:2 375

12 Valent A novel elegant but nontrivial approach may be to eradicate leukemic stem cells in CML by using novel targeted drugs and certain drug combinations. 21 In fact, leukemic stem cells in CML may display a unique composition of antigens, some of which are involved in the mechanism of intrinsic resistance. Interesting targets and target pathways in CML stem cells include certain surface molecules (IL-3 receptor alpha chain, MDR-1, CD33, CD44, or KIT = CD117), the PI3 kinase pathway, the Sonic Hedgehog, Wnt and the β-catenine pathway. Currently, targeted drugs are examined for their potential to induce apoptosis in CML stem cells in preclinical models, and to eradicate these cells if possible. Some of these agents are already applied in clinical trials in patients with TKI-resistant CML. Another approach is to prime CML stem cells (often quiescent cells) with cytokines such as IL-3 or G-CSF, or to overcome intrinsic resistance of CML stem cells by applying MDR-1 inhibitors together with TKI or combinations of TKI in order to increase intracellular drug concentrations. Whether these new agents and approaches swill indeed improve therapy by combining molecular targeting and CML stem cell-targeting concepts, remains to be elucidated. Concluding Remarks and Future Perspectives With the advent of novel targeted drugs and secondand third generation TKI, the prognosis in CML may improve significantly during the next few years. Although imatinib is still considered frontline therapy in freshly diagnosed CML, novel more potent BCR/ ABL inhibitors have been developed, and most of them are also active in imatinib-resistant CML. Two of these drugs, nilotinib and dasatinib, are approved for the treatment of imatinib-resistant or imatinibintolerant patients. Currently, both drugs are evaluated as frontline therapy in patients with freshly diagnosed CML. More inhibitors are currently being developed in preclinical studies and clinical trials. Some of these novel TKI-type inhibitors, like the Aurora kinase inhibitors, may even be effective against the T315I mutant of BCR/ABL. The hope for the future is that such novel drugs, when applied in combination, will further improve treatment outcome and survival in CML. For younger patients who are resistant to all available drugs and have a suitable donor, allogeneic HSCT remains a solid alternative treatment option. Disclosures This manuscript has been read and approved by the author. This paper is unique and is not under consideration by any other journal and has not been published elsewhere. P.V. received research grants from Novartis and Bristol-Myers Squibb as well as speaker s honorarium (company-sponsored symposia) from both companies. References 1. Nowell PC, Hungerford DA. 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