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Optimising chronic myeloid leukaemia therapy in the face of resistance to tyrosine kinase inhibitors – A synthesis of clinical and laboratory data
Blood Reviews, 1, 24, pages 1 - 9
The introduction of imatinib, a tyrosine kinase inhibitor (TKI) that targets the BCR-ABL protein, has revolutionised the treatment of chronic myeloid leukaemia (CML), producing high rates of response that have been durable in many patients. However, because of intrinsic or acquired mechanisms of imatinib resistance, in addition to the persistence of leukaemic stem cells that are resistant to imatinib-induced apoptosis, imatinib treatment does not appear to be curative. Cytogenetic and molecular monitoring enable the identification of patients showing signs of treatment failure and can be used to guide choices regarding subsequent therapeutic options, including imatinib dose escalation, treatment with a secondary TKI or, in selected cases, allogeneic stem cell transplant (allo-SCT). Although these alternative therapies may overcome imatinib resistance, long-term remission or cure from CML is likely to require development of novel interventions that effectively eliminate CML stem cells (Ph+HSC).
Keywords: Leukaemia, Myeloid, Chronic, Stem cells, Drug resistance, Protein kinase inhibitors.
Chronic myeloid leukaemia (CML) is a haemopoietic stem cell cancer that arises following a reciprocal genetic translocation that produces the Philadelphia (Ph) chromosome. This translocation fuses the ABL tyrosine kinase gene from chromosome 9 with the BCR gene on chromosome 22 causing the transcription of a constitutively active oncogenic tyrosine kinase: BCR-ABL.
CML is normally a triphasic disease. The great majority of cases are diagnosed during an initial chronic phase (CP) characterised by increased myeloproliferation and the accumulation of mature myeloid cells over several years. If untreated, CML inevitably progresses to an accelerated phase (AP) and/or blast phase (BP). Patients who progress to the advanced stages have a poor prognosis and the only known curative therapy for CML is allogeneic stem cell transplant (allo-SCT). However, lack of donor availability, high levels of associated morbidity and mortality (particularly in older patients) and the availability of effective drug therapies, limit its widespread use. The treatment of CML has been revolutionised by the advent of specific tyrosine kinase inhibitors (TKI) that target the BCR-ABL oncoprotein. While the majority of newly diagnosed patients undergoing TKI therapy will achieve lasting disease control with minimal toxicity, a significant minority are intolerant, refractory or develop resistance to these agents.
By providing current preclinical and clinical data, the purpose of this review is to discuss the concepts of response and resistance to TKI therapy in CML with relevance to optimising day-to-day patient management.
Imatinib was the first TKI used in the treatment of CML and radically changed the natural history of the disease. In a phase III trial of interferon (IFN) and imatinib (IRIS; International Randomized study of Interferon and STI-571) in newly diagnosed patients with CML in CP, 98% of patients who received imatinib as an initial therapy achieved a complete haematologic response (CHR; normalisation of peripheral blood counts) and 82% achieved a complete cytogenetic response (CCyR; complete absence of Ph+ cells in a minimum of 20 scored metaphases). At 60 months, the estimated overall survival (OS) rate was 89%. Importantly, only an estimated 6% of all patients progressed to AP or BP. 1 Patients with CML who receive initial imatinib therapy after progressing to AP or BP also respond to imatinib; in phase II studies of patients in advanced phases of CML imatinib induced a CHR lasting at least 4 weeks in 34% of patients in AP and 8% of patients in BP and CCyR rates were 17% and 7%, respectively. Estimated 12-month progression-free survival (PFS) and OS rates for patients in AP were 59% and 74%. Patients in BP survived for a median of approximately 7 months.2 and 3
Prognostic benefits associated with imatinib treatment responses
Numerous studies have suggested that patient outcomes in CML correlate with both the depth and speed of response to treatment.1, 4, 5, 6, 7, and 8 Achievement of a cytogenetic response is clearly associated with improved outcome. In a phase II study of 454 patients receiving imatinib after becoming refractory or intolerant to IFN, the achievement of a cytogenetic response prior to 12 months significantly correlated with PFS, with estimated 6-year PFS rates of 84% in patients achieving a partial cytogenetic response (PCyR; 1–35% Ph+ metaphases) or better at 12 months, versus 36% in those that failed to reach this level of response. Overall survival at 6 years correlated with the degree of cytogenetic response at 12 months: 92% for patients in CCyR, 89% for those in PCyR, and 63% for those failing to achieve at least a PCyR at 12 months. 5
The importance of the time taken to achieve CCyR has been debated; in the IRIS study those patients achieving a CCyR after 12 months had a significantly lower risk of progression to AP/BP than patients without a CCyR. However, early achievement of a CCyR at 6 months compared to 18 months did not significantly affect OS or event free survival (EFS) – defined in the IRIS study as death, progression or loss of haematologic or cytogenetic response. Iacobucci et al. reported similar PFS and OS rates in patients achieving CCyR regardless of the time taken however the likelihood of reaching CCyR diminished with time. 9 Similarly, a recent retrospective study by Quintas-Cardama et al. reported that for patients failing to achieve a CCyR at 3, 6 and 12 months the risk of progression increased and the likelihood of achieving CCyR diminished. 10
Quantitative PCR techniques are used to monitor molecular evidence of residual disease. A major molecular response (MMR) is defined as a 3 log reduction (to approximately 0.1%) in BCR-ABL transcript relative to a comparator gene and represents a disease burden of approximately 10-fold less than a CCyR. Despite this greater reduction in disease burden the evidence for additional survival benefit from achieving MMR over and above CCyR is less clear.
Early reduction in BCR-ABL transcript levels correlates with eventual achievement of a cytogenetic response. 8 Quintas-Cardama et al. reported that patients with a BCR-ABL level of >10% at 3 months were less likely to achieve a CCyR at 12 months and were more likely to progress. 10 MMR has been associated with response stability and a reduction in disease progression. In the study above, those with a BCR-ABL level of >1% at three months were at significantly greater risk of progression compared to those with a level ⩽1%. In the IRIS study, 57% of those in CCyR achieved a MMR at 1 year and these patients were shown to have a slightly better PFS as compared to those in CCyR but failing to achieve MMR (100% versus 95% 2 year PFS). 11 Cortes et al. reported a cohort of CML-CP patients who achieved CCyR with imatinib over a median follow-up of 31 months. In this study, only 5% of those with MMR lost cytogenetic response compared to 37% who did not achieve this level of response. 12 In contrast other studies have found that achievement of MMR does not significantly improve prognosis over and above a CCyR.4 and 6
Defining response and estimating resistance during imatinib treatment
Resistance and intolerance to imatinib represent major clinical concerns. An estimated 16% of IRIS patients had primary (intrinsic) resistance to imatinib at 12 months (failure to achieve at least a PCyR) and 24% had primary resistance after 18 months (failure to achieve a CCyR). 13 An estimated 16% of patients relapsed within years 1–3 because of secondary (acquired) resistance. 1 The incidence of resistance and relapse is higher in patients with more advanced CML. In patients achieving CCyR the majority still have detectable levels of BCR-ABL transcripts, indicating the persistence of minimal residual disease. 11
Because responses to imatinib vary among patients with CML, the European LeukemiaNet (ELN) proposed definitions of treatment failure and suboptimal response based on haematologic, cytogenetic and molecular observations at fixed time points or ‘milestones’. The objective was to identify patients who are not deriving optimal benefit from imatinib for whom alternative treatment strategies should be considered ( Fig. 1 ). 14 These criteria were recently validated. Patients classified as ‘failure’ according to ELN recommendations had lower survival, lower PFS and lower cytogenetic responses than other patients. Furthermore, the prognosis of those whose response was classified as ‘sub-optimal’ at 6 and 12 months was more akin to those with overt treatment failure as opposed to optimal responders lending further weight to the concept that an early, robust response to TKI is associated with the best possible outcome. 15
Clinical trials in CML have typically reported best response level achieved at any time during follow-up. These cumulative response figures do not reveal those patients who achieve and subsequently lose a response. It has been suggested that a more informative indicator of long-term benefit would be to report on responses maintained at particular landmark time-points, i.e. 3, 6, 12 and 18 months.4 and 6 Based on this method of analysis, the 5-year probability of patients with newly diagnosed CML-CP being in cytogenetic remission with imatinib is considerably lower at 62.7%. 4
There have been strenuous efforts to elucidate the biological basis of imatinib resistance, identify patients at risk of disease progression and develop alternative therapeutic strategies. These mechanisms can be broadly divided into BCR-ABL-dependent mechanisms and BCR-ABL-independent mechanisms and are summarized in Fig. 2 .
BCR-ABL-dependent mechanisms of resistance
Amplification of the BCR-ABL fusion gene has been associated with resistance to imatinib therapy in CML. Multiple copies of the BCR-ABL gene were detected in leukaemic cell nuclei of 3 of 11 patients with CML-BP or Ph+ acute lymphoblastic leukaemia who had acquired resistance to imatinib. 16 However, in a study of 66 imatinib-resistant CML patients (35 of whom were in BC; 16 in AP; 13 in CP and 2 with Ph+ ALL) only 2 from 32 were demonstrated to have BCR-ABL amplification by FISH (a further 7 developed a second Ph chromosome). 17
Another cause of secondary imatinib resistance is the development of point mutations in the BCR-ABL kinase domain (KD). Imatinib binds to the ATP binding site of the inactive conformation of BCR-ABL. Broadly speaking, point mutations afford resistance by blocking this interaction, either directly, through mutation of residues critical for imatinib binding, or indirectly, by stabilising the protein in the active conformation (P-loop mutations) or rendering it unable to adopt the inactive conformation (A-loop mutations). These mutations arise because the BCR-ABL gene is affected by genomic instability resulting from its own kinase product, mutagenic reactive oxygen species and possibly compounded by compromised DNA repair mechanisms. 18
Many mutations have now been described and there is considerable variability in the frequency and degree of in vitro imatinib resistance conferred, ranging from mild insensitivity to virtually complete resistance to therapeutically achievable levels of all available TKIs in the case of the T315I mutant. Point mutations affecting BCR-ABL KD have been detected in 35–90% of patients with imatinib resistance and are increasingly common with advanced stage disease.7, 17, 19, 20, 21, and 22 Despite this, the contribution of KD mutations to clinical TKI resistance and prognosis is not currently clear. KD mutations have been detected both in patients maintaining a stable response to imatinib and in imatinib-naïve patients.7, 17, 19, 20, 21, 23, and 24
There is controversy surrounding the prognostic significance of certain mutations, such as those within the P loop and T315I.19, 21, 22, and 25 It has also been suggested that KD mutations may affect prognosis only when considered as a time-dependent variable. 20 Most of the clinical information regarding prevalence and behaviour of KD mutations is derived from studies within imatinib resistant patients and it has therefore been difficult to determine the extent to which kinase mutations are causative. Khorashad et al. addressed this question by systematically performing BCR-ABL mutation screens on 319 patients treated with imatinib irrespective of response status over a 5 year period. The cumulative incidence of mutations was 13.9%. The presence of a KD mutation was shown to predict loss of CCyR, disease progression and shorter PFS. This effect was dependent on the type of mutation and the context; the most prevalent mutant (M244V present in 18.4% of those detected) conferred no deleterious consequences, whereas other mutations were predictive of loss of CCyR and disease progression in those developing secondary resistance. 26
Variations in the intracellular transportation of imatinib have been proposed as possible mechanisms of resistance. Two members of the ATP-binding cassette (ABC) family of transporters, ABCB1 (MDR-1) and ABCG2 are involved in imatinib transport. ABCB1 gene overexpression confers resistance to imatinib in leukaemia cell line models, 27 although clinical studies have failed to find a similar association.28 and 29 Imatinib is transported into cells by the human organic cation transporter 1 (hOCT1) and recent studies have shown that treated patients with low expression or activity of hOCT1 are less able to achieve cytogenetic or molecular remission on standard (400 mg) dose imatinib.28 and 29 Many of these patients responded well to dose escalation. 30 Pharmacokinetic and pharmacodynamic considerations are also important. Imatinib is orally administered, relying on gastrointestinal absorption that can be increased by concurrent food intake or reduced by gastrointestinal disease. Following absorption, it is extensively metabolised by the liver cytochrome system (predominantly CYP3A4) and therefore subject to a large list of possible drug interactions. Failure to comply with therapy will clearly affect plasma levels and accumulating evidence suggests this is a significant problem.31, 32, and 33
These factors are important because higher plasma trough imatinib levels are associated with better disease responses in terms of achievement of CCyR and MMR. 34 An analysis of IRIS patients by Larson et al., demonstrated that the rate of CCyR at 5 years was 83% for patients with the lowest plasma concentrations compared with 93% for patients with the highest plasma concentrations and after 2 years, MMR rates were 63% and 86%, respectively. 35
BCR-ABL-independent mechanisms of resistance
Autocrine or paracrine factors could contribute to resistance. Studies have demonstrated that interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) are produced within primitive (CD34+) cells from patients with CML-CP. Both of these cytokines stimulate cellular proliferation in an autocrine manner and protect cells from imatinib-induced apoptosis. 36 Although quiescent CD34+/BCR-ABL+ stem cells do not express IL-3 or G-CSF mRNA, transcripts of both reappear when the cells spontaneously re-enter the cell cycle, 37 supporting a model of clonal expansion in which IL-3 activation contributes to the proliferation of the most primitive neoplastic cells.
Resistance may be mediated in part through overexpression of other tyrosine kinases such as the Src-family kinases (SFKs). BCR-ABL+ CML cells cultured in the presence of imatinib or obtained from patients with imatinib-resistant CML have been shown to have increased expression and activity of the SFKs LYN and HCK. 38 SFKs are involved in the regulation of cell survival and proliferation and their activation can support the anti-apoptotic functions of BCR-ABL. 39 In a recent study, over-expression of LYN and HCK kinases in CML cells derived from imatinib-resistant patients, were not suppressed by imatinib treatment; suggesting that SFK activation could potentially be associated with imatinib resistance. 40 Currently however, clinical data regarding these kinases in CML is lacking.
Clonal evolution is the occurrence of additional chromosome abnormalities in CML cells, which may drive disease progression. The most common defects in CML cells are isochromosome 17q (20%), trisomy 8 (34%) and duplication of the Ph+ chromosome (38%).41 and 42 These abnormalities are associated with specific genetic consequences; isochromosome 17 with loss of 17p and therefore p53, trisomy 8 causing over expression of MYC and duplication of Ph+ resulting in higher BCR-ABL expression. The presence of clonal evolution correlates with a decreased response to imatinib. 43
Quiescent leukaemic stem cells (Ph+HSC) which are functionally primitive (i.e. give rise to Ph+ progeny in secondary recipients), can be isolated from the blood and bone marrow of patients with untreated CML-CP. 44 These Ph+HSC comprise <1% of the total stem/progenitor cell population. 45 In vitro studies have demonstrated that imatinib has an anti-proliferative effect on Ph+HSC, causing them to enter reversible cell cycle arrest and accumulate. 45 Non-proliferating Ph+HSC are resistant to imatinib-induced apoptosis, despite achieving similar intracellular levels of imatinib to more mature CML cells. 46 Furthermore, recent evidence suggests that, at least in vitro, TKI treatment induces autophagy as a critical survival mechanism in Ph+HSC from patients with CP or BP CML. 47 Therefore, even in patients who are in complete molecular remission, i.e. with undetectable BCR-ABL transcripts following TKI therapy, there is likely to be a residual, highly resistant population of CML cells with the capacity to repopulate the disease.
Monitoring response and resistance
Current recommendations for monitoring signs of primary and secondary resistance were outlined in the recently updated National Comprehensive Cancer Network (NCCN) guidelines. These suggest that BCR-ABL transcript levels should be measured by QRT-PCR every 3 months (monthly if a rising BCR-ABL transcript level is detected), and bone marrow cytogenetics should be performed at 6 and 12 months from initiation of therapy. When a patient achieves a CCyR, cytogenetic analysis should be considered every 12–18 months to check for clonal evolution. Screening for mutations is appropriate in patients with CML-CP who experience inadequate initial responses to imatinib therapy or who experience any loss of response.
Plasma imatinib concentrations can be measured using liquid chromatography and tandem mass spectrometry to assess bioavailability. This measurement has been utilised to investigate potential drug interactions, suspected non-compliance, sub-optimal response to imatinib and in patients experiencing unusually severe side-effects.
Secondary treatment options
Alternative treatment strategies may be considered following treatment failure (including any loss of response), suboptimal response, or intolerance. After initial treatment with imatinib 400 mg/d for newly diagnosed CML-CP, subsequent therapeutic options include imatinib dose escalation (to 600 or 800 mg/d, where tolerated), secondary TKIs dasatinib and nilotinib, allo-SCT, or clinical trial with an investigational agent.
Imatinib dose escalation
The rationale for imatinib dose escalation is that the resistance conferred by imatinib dependent mechanisms may not be complete, e.g. BCR-ABL amplification may respond to higher imatinib concentrations; many of the BCR-ABL KD mutations are partially sensitive and reduced intracellular concentrations of imatinib caused by variations in cellular influx and efflux may be overcome by higher dosing. Furthermore, there is clinical evidence from dose escalation in initial phase I studies as well as in advanced phase CML that suggests additional efficacy at higher doses.
The effect of dose escalation has been investigated in a study of 54 patients with CML-CP who received imatinib doses of 800 or 600 mg daily. Of 20 treated for haematologic resistance or relapse, 65% achieved a haematologic response (HR), but only two had any evidence of cytogenetic response (one PCyR and one minor CyR). Among 34 patients treated for cytogenetic resistance or relapse, 56% achieved a CCyR or PCyR. 48 Kantarjian et al. reported the results of dose escalation from the IRIS study demonstrating a median dose of over 600 mg and a EFS of 89% at 36 months following intervention. 49 However, other reports suggest that responses to increased doses of imatinib are often transient and that patients with a poor initial response to standard doses have a low chance of responding to a higher dose.50, 51, and 52 Recently Jabbour et al. published a report on the long term follow-up of 84 patients who had dose escalation for treatment failure on standard dose imatinib. CCyR was achieved in 40% of patients overall and in 73% of those who had previously achieved at least a minor CyR with imatinib prior to failure. These responses were found to be relatively durable with 88% of patients reacquiring at least a major cytogenetic response (MCyR) and maintaining this for 2 years. Those that did not achieve at least an initial minor CyR to standard dose imatinib derived no benefit from imatinib escalation. 53
Dasatinib and nilotinib
Dasatinib is a dual SRC/ABL inhibitor with activity against BCR-ABL. Dasatinib binds to both the inactive and active states of ABL and is 325-fold more potent than imatinib. 54 Efficacy of 70 mg twice-daily dasatinib in imatinib resistant and intolerant patients has been investigated in a series of open label phase II trials known as START (SRC/ABL Tyrosine kinase inhibition Activity Research Trials). START-C evaluated the efficacy of dasatinib in imatinib resistant or intolerant CP patients. MCyR was reached in 52% of the resistant cohort and 80% of the intolerant cohort. These responses were long lasting with over 97% of patients achieving MCyR maintaining this response at a median follow-up of 15.2 months and with PFS and OS at 24 months of 80 and 94%. 55 START-A, -B and -L looked at the efficacy of dasatinib in AP, myeloid and lymphoid BP CML, respectively, and indicated that dasatinib was effective in imatinib resistant or intolerant patients with advanced phase CML.56, 57, 58, and 59
Nilotinib is a tyrosine kinase inhibitor with activity against BCR-ABL, KIT, PDGFR and ephrin receptor kinase. It is an analogue of imatinib and exerts its anti-leukaemic activity in a similar fashion, i.e. it binds to and stabilises the inactive conformation of ABL. However, because the topologic fit to ABL is improved, nilotinib has approximately 30-fold greater potency. 54 A series of phase II open label studies of 400 mg twice-daily nilotinib in imatinib resistant or intolerant CML patients in CP, AP and BP have been performed. 321 CP patients with imatinib intolerance or resistance received nilotinib 400 mg twice-daily; overall MCyR rate was 63% in the intolerant group and 56% in the resistant group. Again these responses were durable with 84% remaining in MCyR for 18 months.60 and 61 Estimated OS was reported as 91% at 18 months. 62 Equivalent trials have been performed in advanced phase CML indicating that nilotinib is clinically effective in AP and BP.
Thus both agents have demonstrated efficacy in the setting of imatinib resistance or intolerance in all disease phases. There is no data directly comparing these two agents and the pivotal trials of dasatinib and nilotinib had different inclusion criteria making comparison difficult; in the dasatinib trials intolerance was accepted as being purely the occurrence of intolerable toxicity whereas in the nilotinib trials there was a requirement for lack of response to imatinib in addition to intolerance. The most recent findings from clinical trials with dasatinib and nilotinib are summarised in Table 1 .55, 62, 63, 64, 65, and 66
|Median treatment duration (months)||CHR (%)||MCyR (%)||CCyR (%)||MMR (%)|
|Chronic (n = 387)||NR (⩾24 months of follow-up)||91||62 (55) d||53 (44) d||47|
|Accelerated (n = 174)||13.5 a||50||40||33||NR|
|Myeloid blast (n = 109)||3.4 a||26||34||27||NR|
|Lymphoid blast (n = 48)||29||52||46||NR|
|Chronic (n = 321)||15.5 b||77 c||58 (56) d||42 (39) d||NR|
|Accelerated (n = 136)||8.4 b||30||32||19||NR|
|Myeloid blast (n = 105)||2.8 b||11||38||29||NR|
|Lymphoid blast (n = 31)||13||48||32||NR|
a Treatment duration.
b Treatment exposure.
c Patients not in CHR at baseline.
d Considering resistant patients only.
CCyR, complete cytogenetic response; CHR, complete haematologic response; MCyR, major cytogenetic response; MMR, major molecular response; NR, not reported.
Choice of second-line therapy
Appropriate monitoring of response and mutational analysis may help guide second-line treatment strategies. Treatment failure as defined by the ELN response criteria or disease relapse on standard dose imatinib should be met with an investigation of potential causes and consideration of an alternative treatment plan. In the case of primary resistance or progression a second-line TKI may induce disease control but this is unlikely to be durable and therefore stem cell transplantation should be considered in appropriate patients. Patients with myelosuppression on TKI therapy can often be supported with growth factors and blood transfusions. In the case of patients failing to achieve response milestones or losing a response; the ELN suggest imatinib dose escalation or the use of an alternate TKI as second line with the possibility of a transplant procedure in appropriate non-responding patients. At this stage mutational analysis may provide useful information; as it will determine the presence of a significant mutant clone and provide information regarding the relative sensitivity of this clone to available alternate therapies thereby enabling a rational therapeutic decision. The presence of the T315I mutant indicates that TKI therapy is no longer appropriate and should be withdrawn, whereas patients with weakly resistant BCR-ABL kinase mutants, e.g. M351T, may benefit from imatinib dose escalation or switching to a second-line TKI. In vitro, dasatinib and nilotinib inhibited all BCR-ABL mutants tested except T315I. However, nilotinib had lower potency against certain highly imatinib-resistant mutations occurring in the P-loop region (Y253F/H, E255K/V) and dasatinib had lower potency against mutations occurring at amino acid F317.54, 67, and 68 Clinical study findings have mirrored these in vitro findings. For example, no CCyRs were observed in patients treated with nilotinib who had mutations in amino acids Y253, E255, T315, or F35960 and 61 and these mutations were frequently identified in patients with clinical nilotinib failure. 69 Resistance to dasatinib has been associated with mutations at residues T315, F317 and V299L and these mutations are often seen in patients developing dasatinib failure. 70 It must be noted however that the number of patients with KD mutations reported in the large dasatinib and nilotinib trials is less than 50% and the number with a mutation conferring more than low level resistance or specifically favouring one TKI over another smaller still.58 and 60
The best management of patients falling into the category “sub-optimal response” is not as clearly defined and is an area of current investigation. This is a heterogeneous group of patients: those not achieving an early haematologic response; those failing to reach a PCyR or CCyR at 6 or 12 months, respectively; those lacking a MMR at 18 months; or those losing a MMR at any time. While the prognostic effects of satisfactory cytogenetic responses are well documented, the effects of molecular responses are less so. Marin et al. found that those with early sub-optimal responses (i.e. those defined by failure to meet haematologic or cytogenetic criteria; lack of CHR at 3 months, lack of PCyR at 6 months and lack of CCyR at 12 months) had similar PFS to those with treatment failure as defined by the ELN, whereas those with sub-optimal molecular responses (at 18 months) did not. 15 It is not yet known whether an earlier switch in these patients would improve long term outcome.
The other reason for considering second-line therapy is the development of significant intolerance. The most commonly observed toxicities with TKI therapy are class specific, i.e. are observed with all TKI agents, and include myelosuppression, nausea, diarrhoea, fluid retention syndromes and rash. Many of these are amenable to standard supportive therapy. Other toxicities are specifically related to individual agents and there is little non-haematologic cross intolerance between them. Very significant rates of pleural effusion have been reported in trials of dasatinib. 71 This prompted a recent phase III dose-optimisation study which demonstrated that rates of pleural effusion could be significantly reduced without apparent loss of efficacy if the initial dosing schedule was reduced from 70 mg twice daily to 100 mg once daily. 72 Higher rates of common toxicity criteria (CTC) grade 1–3 bleeding complications have been observed in patients using dasatinib compared to imatinib and nilotinib. This is due to inhibition of platelet function, as well as thrombocytopenia. Hepatic toxicity has been reported in patients treated with imatinib or dasatinib, although grade 3/4 events occurred in <3%.1 and 56 Among nilotinib-treated patients, a grade 3/4 elevation in bilirubin (predominantly unconjugated) was reported in 14% and a grade 3/4 lipase elevation was reported in 9%. 73 Unconjugated, non-progressive hyperbilirubinaemia associated with nilotinib occurs due to polymorphism within the UGT1A1 gene and is considered to be benign. 74 Both nilotinib and dasatinib affect cardiac conduction by prolonging the QT interval and care should be taken to maintain an electrolyte balance within the normal range and avoid medications that may prolong the QT. Nilotinib in particular carries a warning regarding its use in patients with congenital long QT syndromes.
On the whole, where there is no reduction in sensitivity due to mutation (in about 50% cases) there is little to choose between either agent in terms of efficacy. The differing side effect profiles may lead one agent to be more suitable taking into account patient co-morbidities, e.g. nilotinib for those with significant respiratory compromise or bleeding diatheses and dasatinib in patients with liver dysfunction, pancreatitis and cardiac conduction abnormalities.
Therapeutic implications of CML stem cells
Despite the increased potency of second-line TKIs and their ability to target specific BCR-ABL mutations, the issue of residual disease and relapse still exists in a substantial number of patients. One reason for this is the continued presence of reversibly quiescent Ph+HSC in these patients. Targeting and eliminating this population may therefore be required for long term remission or cure. It is known that there are very high levels of BCR-ABL in Ph+HSC, and although TKIs inhibit BCR-ABL activity in these cells, the inhibition is not complete. As a result, it is not certain whether Ph+HSC are ‘addicted’ to BCR-ABL activity for survival and whether, therefore, more potent BCR-ABL inhibition would actually kill them.75 and 76 We do know that quiescent Ph+HSC are resistant to imatinib-induced apoptosis, 77 with studies also indicating that the increased potency of nilotinib and dasatinib does not result in increased Ph+HSC apoptosis in vitro.75, 76, and 78
As CML progresses, the level of BCR-ABL transcripts and frequency of chromosomal abnormalities and mutagenesis all increase. This is because of failed genome surveillance, deficiencies in DNA repair, telomere shortening, and abnormalities in tumour suppressor genes. 79 Although TKIs do not kill CML stem cells, they reduce their rate of proliferation. In the absence of DNA replication, the chances of DNA damage and disrepair are low. As a result, early and prolonged intervention with TKIs in CP should minimise the risk of disease progression. Mathematical models have been designed to investigate the curative potential of TKI inhibition. In one such model there is a moderate but continuing decline in Ph+HSC, ultimately leading to their eradication at about 20 years post therapy initiation. 80 In this model, the process could be accelerated by combinations of agents that stimulate proliferation of Ph+HSC. 81 A second model refutes the curative potential of TKIs and suggests that while TKI therapy potently inhibits Ph+HSC differentiation it does not deplete the Ph+HSC fraction and therefore, when treatment is discontinued, the leukaemic cell load will rapidly return to pre-treatment levels. 82 Additionally, continued TKI therapy may select for resistant mutants that could eventually outgrow drug-insensitive leukaemic cells.
Seeking a cure
As it appears unlikely that TKI therapy represents a curative strategy for CML due to the persistence of Ph+HSC, strenuous efforts have been made to identify agents capable of selectively targeting these cells. A myriad of approaches are therefore under investigation. The following section discusses promising examples of possible cancer stem cell therapies, without intending to be exhaustive.
In the development of a leukaemic stem cell therapy, one approach has been to combine TKIs with agents that either antagonise the anti-proliferative effect seen with a TKI or that may have a synergistic action by targeting other signalling pathways. For example, growth factor stimulation may enhance the cycling of quiescent Ph+HSC. Indeed, high concentrations of growth factors antagonise the anti-proliferative effect of TKIs and significantly reduce the number of residual non-dividing cells remaining after imatinib treatment. 83 Pre-treatment with bryostatin-1 (an inhibitor of protein kinase C isoenzymes) prior to imatinib effectively antagonised the anti-proliferative effect of imatinib, allowing greater efficacy against the non-cycling population. 84 Other drugs which enhance the toxicity of imatinib against CML CD34+ cells include lonafarnib (a farnesyl transferase inhibitor; FTI), 17-AAG, Ara-C and LY294002. However, only lonafarnib had effects on the Ph+HSC population. 85 Another study demonstrated that BMS-214662, a cytotoxic FTI, acted alone or in combination with imatinib or dasatinib to selectively induce apoptosis in proliferating and quiescent Ph+HSC. This compound was equally effective in cell lines harbouring unmutated or mutated BCR-ABL including the T315I mutation. 86
Recently, a great deal of interest has focused on the fundamental stem cell property of self-renewal and its control in Ph+HSC. Retroviral transfection of BCR-ABL into murine haemopoietic stem cells (HSC) is sufficient to cause a CML-like condition in transplanted mice however it is not capable of conferring self-renewal to committed myeloid progenitors. 87 It has been suggested that CML develops only if BCR-ABL is generated in cells with a high capacity for self-renewal and CML-CP arises due to clonal expansion of Ph+ HSC. 88 Self-renewal behaviour is normally tightly regulated by a network of signals arising from the stem cell niche that includes input from embryonic morphogenic pathways such as the Hedgehog (Hh) and the Wnt signalling pathways. Recent in vitro and murine research has suggested that elements of these pathways are upregulated in Ph+HSC as compared to non-CML HSC. Furthermore, abrogation of these signals in murine model systems, either through pharmacological inhibition or genetic manipulation, diminishes the Ph+HSC fraction, appears to retard leukaemia progression and reduces the capacity for these cells to recapitulate the disease in transplanted hosts.89 and 90 Additionally, progression of CML appears to be associated with increased activity of the Wnt/β catenin signalling pathway and reacquisition of self-renewal activity in committed myeloid progenitor cells that would not normally be expected have this faculty. 91 Specific inhibitors of key elements of these pathways have been developed and are in early clinical trials in solid malignancies.
Another promising approach has been to target putative stem cell survival mechanisms. For example, autophagy, a process whereby cells can breakdown internal structures and recycle the components for energy, has been shown to be active in Ph+HSC during TKI treatment and may represent a potential rescue pathway. In vitro studies in primary CP CML cells demonstrated that inhibition of autophagy with chloroquine, in combination with TKI, resulted in significantly higher rates of apoptosis of Ph+HSC than with either agent alone. Clinical studies are now planned aiming to completely eradicate the Ph+HSC population in CML patients using this combination. 47
Immunotherapy and tumour vaccination represent other important mechanisms for targeting the Ph+HSC compartment. CML cells over-express tumour specific antigens (Ag), e.g. BCR-ABL, and tumour associated Ag (PR-1, PRAME or Wilms tumour 1 [WT1]) and the use of peptide vaccinations to induce immunological recognition of these Ag has produced promising results, particularly in those in a minimal residual disease state.92 and 93 Research along these lines has led to the inception of a clinical study of peptide vaccination, aiming to eradicate the remaining CML cells in stable imatinib-treated CP CML patients. Finally, allo-SCT is the quintessential immunotherapy; acting through a graft versus leukaemia effect exerted by donor T-cells on minor histocompatibility Ag on leukaemic cells, that effectively targets the Ph+HSC population. Allo-SCT remains the only curative strategy in CML and continues to play a significant role in CML management – particularly for suitable patients failing TKI therapy and for those with evidence of disease progression or highly resistant KD mutations. However, in practice, factors such as age, treatment related mortality and donor availability limit its utility.
TKIs are highly effective in managing and controlling CML. Treatment with imatinib results in a sustainable reduction in disease burden and early and durable responses are associated with prognostic benefits. For the 30% of patients who do not respond adequately or relapse on imatinib, dasatinib and nilotinib are effective treatment options that can overcome most mechanisms of imatinib resistance. However, because of the persistence of insensitive Ph+HSC, any cure for CML is likely to depend upon an increased understanding of the biology of leukaemic stem cells and the development of therapies that target this cell population.
- 1. Standard dose imatinib is highly effective in controlling CML in the majority of patients leading to increased survival and reduced disease progression.
- 2. Regular cytogenetic and molecular monitoring enables identification of the 30% of patients that do not respond optimally or fail standard dose imatinib allowing consideration of alternative therapeutic strategies which include allo-SCT, secondary TKI or imatinib dose escalation.
- 3. Compliance with TKI therapy is increasingly being recognised as a clinical concern.
- 4. In the absence of ‘problem’ mutations, there appears to be little difference in efficacy between nilotinib and dasatinib with both capable of inducing prolonged cytogenetic remission in imatinib resistant patients.
- 5. Even patients responding optimally to TKI exist in a minimal residual disease state with a persistent population of Ph+HSC that are capable of causing disease recrudescence. TKI therapy does not effectively target this population; necessitating long term disease monitoring and indefinite TKI therapy to maintain disease control.
- 1. Investigating disease persistence.
- a. What governs quiescence in Ph+HSC?
- b. What mediates the antiproliferative effect of TKI therapy?
- c. Are Ph+HSC entirely dependent on BCR-ABL for survival?
- 2. Investigating possible mechanisms for selectively targeting and eradicating the Ph+HSC population.
- 3. Further defining optimal treatment strategies for patients with sub-optimal responses to imatinib.
- 4. Investigating discontinuation of TKI therapy in stable patients with no molecular evidence of disease.
Conflict of interest statement
Tessa Holyoake has received research funding and honoraria from Novartis and Bristol-Myers Squibb.
The authors take full responsibility for the content of this publication, and confirm that it reflects their viewpoint and medical expertise. StemScientific, funded by Bristol-Myers Squibb, provided writing and editing support. Bristol-Myers Squibb did not influence the content of the manuscript, nor did the authors receive financial compensation for authoring the manuscript.
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