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Sustained deep molecular responses in patients switched to nilotinib due to persistent BCR-ABL1 on imatinib: final ENESTcmr randomized trial results

Although tyrosine kinase inhibitors (TKIs) efficiently cure chronic myeloid leukemia (CML), they can fail to eradicate CML stem cells (CML-SCs). The mechanisms responsible for CML-SC survival need to be understood for designing therapies. Several previous studies suggest that TKIs could modulate CML-SC quiescence. Unfortunately, CML-SCs are insufficiently available. Induced pluripotent stem cells (iPSCs) offer a promising alternative. In this work, we used iPSCs derived from CML patients (Ph+). Ph+ iPSC clones expressed lower levels of stemness markers than normal iPSCs. BCR-ABL1 was found to be involved in stemness regulation and ERK1/2 to have a key role in the signaling pathway. TKIs unexpectedly promoted stemness marker expression in Ph+ iPSC clones. Imatinib also retained quiescence and induced stemness gene expression in CML-SCs. Our results suggest that TKIs might have a role in residual disease and confirm the need for a targeted therapy different from TKIs that could overcome the stemness-promoting effect caused by TKIs. Interestingly, a similar pro-stemness effect was observed in normal iPSCs and hematopoietic SCs. These findings could help to explain CML resistance mechanisms and the teratogenic side-effects of TKIs in embryonic cells.

It is unknown, why only a minority of chronic myeloid leukemia (CML) patients sustains treatment free remission (TFR) after discontinuation of tyrosine kinase inhibitor (TKI) therapy in deep molecular remission (MR). Here we studied, whether expression of the T-cell inhibitory receptor (CTLA-4)-ligand CD86 (B7.2) on plasmacytoid dendritic cells (pDC) affects relapse risk after TKI cessation. CML patients in MR displayed significantly higher CD86+pDC frequencies than normal donors (P 95 CD86+pDC per 105 lymphocytes, but 70.0% (95% CI 59.3-78.3) for patients with 8 years) TKI exposure before discontinuation (HR 0.3, 95% CI 0.1-0.8; P=0.0263). High CD86+pDC counts significantly correlated with leukemia-specific CD8+ T-cell exhaustion (Spearman correlation: 0.74, 95%-CI: 0.21-0.92; P=0.0098). Our data demonstrate that CML patients with high CD86+pDC counts have a higher risk of relapse after TKI discontinuation.

Low incidence of peripheral arterial disease in patients receiving dasatinib in clinical trials

In the past decade, the treatment of chronic myeloid leukemia (CML) has undergone a drastic evolution. The discovery and success of imatinib and second-generation tyrosine kinase inhibitors have substantially increased the outcome for CML patients. The next step in medical and scientific research is to better understand the malignancy so as to eventually find a cure to eliminate all leukemic cells from patients. One of the key issues is about the resistance of the leukemic stem cells to tyrosine kinase inhibitors. Here, we briefly describe our current studies on CML resistance, and leukemic stem cell modeling and characterization.

Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naive CD56(bright) NK cells had decreased relapse-free survival. In addition, the TNF-alpha/IFN-gamma cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents. Funding Agencies|Nordic Cancer Union; Finnish Society of Hematology; Biomedicum Helsinki Foundation; Research Foundation of Blood Diseases in Finland; Academy of Finland; Finnish Cancer Organizations; Signe and Ane Gyllenberg Foundation; Finnish Cancer Institute; Doctoral Programme in Clinical Research in the University of Helsinki

The Evaluating Nilotinib Efficacy and Safety in Clinical Trials as First-Line Treatment (ENEST1st) study included 1089 patients with newly diagnosed chronic myeloid leukemia in chronic phase. The rate of deep molecular response (MR4 (BCR-ABL1less than or equal to0.01% on the International Scale or undetectable BCR-ABL1 with greater than or equal to10?000 ABL1 transcripts)) at 18 months was evaluated as the primary end point, with molecular responses monitored by the European Treatment and Outcome Study network of standardized laboratories. This analysis was conducted after all patients had completed 24 months of study treatment (80.9% of patients) or discontinued early. In patients with typical BCR-ABL1 transcripts and less than or equal to3 months of prior imatinib therapy, 38.4% (404/1052) achieved MR4 at 18 months. Six patients (0.6%) developed accelerated or blastic phase, and 13 (1.2%) died. The safety profile of nilotinib was consistent with that of previous studies, although the frequencies of some nilotinib-associated adverse events were lower (for example, rash, 21.4%). Ischemic cardiovascular events occurred in 6.0% of patients. Routine monitoring of lipid and glucose levels was not mandated in the protocol. These results support the use of frontline nilotinib, particularly when achievement of a deep molecular response (a prerequisite for attempting treatment-free remission in clinical trials) is a treatment goal.

Nilotinib is a highly selective Bcr-Abl inhibitor approved for imatinib-resistant chronic myeloid leukemia (CML). Nilotinib and dasatinib, a multi-targeted kinase inhibitor also approved for second-line therapy in CML, have different patterns of kinase selectivity, pharmacokinetics, and cell uptake and efflux properties, and thus patients may respond to one following failure of the other. An international phase II study of nilotinib was conducted in CML patients (39 chronic phase (CP), 21 accelerated phase (AP)) after failure of both imatinib and dasatinib. Median times from diagnosis of CP or AP to nilotinib therapy were 89 and 83 months, respectively. Complete hematological response and major cytogenetic response (MCyR) rates in CP were 79% and 43%, respectively. Of 17 evaluable patients with CML-AP, 5 (29%) had a confirmed hematological response and 2 (12%) a MCyR. The median time to progression has not yet been reached in CP patients. At 18 months 59% of patients were progression-free. Median overall survival for both populations has not been reached, and the estimated 18-month survival rate in CML-CP was 86% and that at 12 months for CML-AP was 80%. Nilotinib is an effective therapy in CML-CP and -AP following failure of both imatinib and dasatinib therapy.

The malignant phenotype of chronic myeloid leukemia (CML) is due to the abnormal tyrosine kinase activity of the BCR-ABL oncoprotein, which signals several downstream cell survival pathways, including phosphoinositide 3-kinase/AKT, signal transducer and activator of transcription 5 and extracellular signal-regulated kinase 1/2. In patients with CML, tyrosine kinase inhibitors (TKIs) are used to suppress the BCR-ABL tyrosine kinase, resulting in impressive response rates. However, resistance can occur, especially in acute-phase CML, through various mechanisms. Here, we show that the glucocorticoid-induced leucine zipper protein (GILZ) modulates imatinib and dasatinib resistance and suppresses tumor growth by inactivating the mammalian target of rapamycin complex-2 (mTORC2)/AKT signaling pathway. In mouse and human models, GILZ binds to mTORC2, but not to mTORC1, inhibiting phosphorylation of AKT (at Ser473) and activating FoxO3a-mediated transcription of the pro-apoptotic protein Bim; these results demonstrate that GILZ is a key inhibitor of the mTORC2 pathway. Furthermore, CD34(+) stem cells isolated from relapsing CML patients underwent apoptosis and showed inhibition of mTORC2 after incubation with glucocorticoids and imatinib. Our findings provide new mechanistic insights into the role of mTORC2 in BCR-ABL(+) cells and indicate that regulation by GILZ may influence TKI sensitivity.

Imatinib is the current standard of care in the treatment of chronic myeloid leukemia (CML), inducing durable responses and prolonged progression-free survival. However, plasma exposure to the drug from a given dosing regimen can vary widely among patients. Reasons for this may include incomplete adherence, intrinsic variations in the metabolism of imatinib, and drug-drug interactions. Data from two recent studies have shown a correlation between imatinib trough plasma concentration and clinical response, leading to suggestions that maintaining imatinib blood concentrations above approximately 1000 ng/ml might be associated with improved outcomes. In patients who do not respond as well as expected to initial imatinib treatment, measurement of trough plasma concentration could assist with decisions about whether to increase the dose. Blood-level testing may also be helpful in other clinical scenarios: for example, when poor adherence is suspected, adverse reactions are unusually severe, or there is a possible drug-drug interaction. Further work is required to confirm prospectively the link between imatinib plasma concentrations and response, and to define effective trough concentrations in different patient populations. However, based on the current data, imatinib blood-level testing seems to be a useful aid when making clinical decisions in CML.

Imatinib is an effective first-line therapy for chronic myelogenous leukemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. To overcome resistance, second-generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is more potent against BCR-ABL than imatinib, and is effective against many imatinib-resistant BCR-ABL mutants. In this study, an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both the drugs induced significant apoptosis in CD34+ cells from 36 CML bone marrow samples (P10(-4)), whereas CD34+ cells from BCR-ABL negative samples were unaffected. When the experiments were carried out in the presence of a cocktail of cytokines, nilotinib- but not imatinib-induced apoptosis was inhibited. This differential inhibition was confirmed on K562 cells. A blocking anti-CD117 antibody alleviated the antiapoptotic effect of cytokines against nilotinib. Moreover, using short hairpin RNA against BCR-ABL, we showed that K562 cells were not dependent on BCR-ABL signaling as long as the stem cell factor (SCF) receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals, and that c-KIT must be inhibited besides Bcr-Abl to allow apoptosis of CML cells.

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

The emergence of ABL point mutations is the most frequent cause for imatinib resistance in chronic myelogenous leukemia (CML) patients and can occur during any phase of the disease; however, their clinical impact remains controversial. In this study, we retrospectively analyzed the predictive impact of 94 BCR-ABL kinase domain mutations (18 T315I, 26 P-loop, 50 in other sites) found in 89 imatinib-resistant CML patients. At imatinib onset, 64% of patients (57/89) were in chronic phase (CP), 24% (21/89) in accelerated phase (AP) and 12% (11/89) in blastic phase (BP). T315I and P-loop mutations were preferentially discovered in accelerated phase of BP CML, and other types of mutations in CP (P=0.003). With a median follow-up of 39.2 months (6.3-67.2), since imatinib initiation, overall survival (OS) was significantly worse for P-loop (28.3 months) and for T315I (12.6 months), and not reached for other mutations (P=0.0004). For CP only, multivariate analysis demonstrated a worse OS for P-loop mutations (P=0.014), and a worse progression-free survival (PFS) for T315I mutations (P=0.014). Therefore, P-loop and T315I mutations selectively impair the outcome of imatinib-resistant CML patients, in contrast to other mutations, which may benefit from dose escalation of imatinib, able to improve or stabilize disease response.

International audience; In chronic myeloid leukemia (CML), evidence is supporting the role of VEGF in growth, and survival of leukemia cells. The evaluation of plasma VEGF levels in 403 CML patients randomized within SPIRIT study to received imatinib-400mg versus imatinib+cytarabine versus imatinib+interferon (IFN) versus imatinib-600mg demonstrated that VEGF is an independent factor of BCR-ABL burden. VEGF low levels at diagnosis were associated with a progression-free survival of 100% at 48 months. Under treatment, significant lowest levels were observed in imatinib+IFN arm. These results support the use of VEGF as a parameter to predict CML evolution and let us to speculate about antiangiogenic properties of IFN.

In a single institution, we have used recombinant interferon- (IFN-) to treat 116 newly diagnosed Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients and analyzed the predictive factors for response and survival. The patients whose median age was 50.3 years (range, 9 to 70) were administered IFN- (5 million units/m2/d) subcutaneously. The IFN- dose was subsequently adjusted to maintain the white blood cell and platelet counts between 1.5 and 5 × 109/L, 50 and 100 × 109/L, respectively. At diagnosis, the Sokal score was used to classify the patients into three groups: low (n = 57), intermediate (n = 42), and high risk (n = 16). A complete hematological response (CHR) was achieved in 93 cases (80.2%). Of the 116 patients, 113 were available for cytogenetic evaluation. Fifty patients (43%) achieved a major cytogenetic response (MCR) (=65% marrow Ph− cells), 37 of them having a complete cytogenetic response (CCR). The estimated 5-year survival of the 116 patients was 68% ± 11% (95% confidence interval [CI]) with a median follow-up of 42 months (range, 3 to 114) and 85% ± 11% (95% CI) with a median follow-up of 30.9 (range, 3 to 111) when patients were censored at the time of transplantation. Event-free survival at 5 years (adding death and transplant as event) was 46% ± 11% (95% CI). Using proportional hazards regression to study time-dependent variables, we confirmed that the most significant factor associated with survival was the cytogenetic response (MCR or CCR) (P < .0001). This factor was independent compared with the Sokal score and baseline variables used to calculate the Sokal score. Moreover, using either univariate or multivariate analysis, the achievement of CHR within 3 months was strongly correlated with MCR (P < .0001). Minimum cytogenetic response (mCR, ie, at least 5% of Ph− metaphases) at 3 months was also a significant predictive factor for MCR (P < .0001). These results show that IFN- can induce a high rate of hematological and cytogenetic response when administered in doses leading to myelosuppression. Factors such as the achievement of CHR and mCR within 3 months could be useful to identify early those patients who will not respond to IFN- and who need alternative treatments such as stem cell transplantation.

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34(+)-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34(+)-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP cells led to 83-100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in longterm culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.

An amino-acid switch in the BCR-ABL kinase domain modifies sensitivity to imatinib mesylate

From in vitro to in vivo: intracellular determination of imatinib and nilotinib may be related with clinical outcome

International audience; Imatinib, the anti-Abl tyrosine kinase inhibitor used as first-line therapy in chronic myeloid leukemia (CML), eliminates CML cells mainly by apoptosis and induces autophagy. Analysis of imatinib-treated K562 cells reveals a cell population with cell cycle arrest, p27 increase and senescence-associated beta galactosidase (SA-β-Gal) staining. Preventing apoptosis by caspase inhibition decreases annexin V-positive cells, caspase-3 cleavage and increases the SA-β-Gal-positive cell population. In addition, a concomitant increase of the cell cycle inhibitors p21 and p27 is detected emphasizing the senescent phenotype. Inhibition of apoptosis by targeting Bim expression or overexpression of Bcl2 potentiates senescence. The inhibition of autophagy by silencing the expression of the proteins ATG7 or Beclin-1 prevents the increase of SA-β-Gal staining in response to imatinib plus Z-Vad. In contrast, in apoptotic-deficient cells (Bim expression or overexpression of Bcl2), the inhibition of autophagy did not significantly modify the SA-β-Gal-positive cell population. Surprisingly, targeting autophagy by inhibiting ATG5 is accompanied by a strong SA-β-Gal staining, suggesting a specific inhibitory role on senescence. These results demonstrate that in addition to apoptosis and autophagy, imatinib induced senescence in K562 CML cells. Moreover, apoptosis is limiting the senescent response to imatinib, whereas autophagy seems to have an opposite role.

Imatinib mesylate is the sole BCR-ABL tyrosine kinase inhibitor approved as first-line treatment of accelerated-phase (AP) chronic myeloid leukemia (CML). Indication was based on the STI571 0109 study, in which imatinib favorably compared to historical treatments in patients failing prior therapies. The relevance of these results to currently newly diagnosed AP-CML patients remains unknown. We evaluated the benefit of imatinib in 42 newly diagnosed AP-CML patients. In all, 16 patients had hematological acceleration without chromosomal abnormalities in addition to the Philadelphia chromosome (ACAs; HEM-AP), 16 solely had ACAs (ACA-AP) and 10 had hematological acceleration plus ACAs (HEM-AP + ACA). Major cytogenetic responses were achieved in 93.7% of HEM-AP patients, 75% of patients with ACA-AP (P=NS) and 40% of patients with HEM-AP + ACA (P=0.0053). The 24-month failure-free survival rate was 87.5% in HEM-AP patients, 43.8% in ACA-AP patients and 15% in HEM-AP + ACA patients (P=0.022). The 24-month estimate of progression-free survival was 100% in HEM-AP patients, 92.8% in ACA-AP patients and 58.3% in HEM-AP + ACA patients (P=0.0052). In conclusion, frontline imatinib allows favorable outcomes in HEM-AP and ACA-AP patients but appears insufficient for patients with HEM-AP + ACA. Broader-target and/or more potent BCR-ABL tyrosine kinase inhibitors alone or in combination may be considered in this setting.

Acquired somatic mutations affecting TET oncogene family number 2 (TET2), isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and additional sex combs-like 1 (ASXL1) have been recently described in BCR–ABL-negative neoplasm, myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), MDS/MPN and acute myeloid leukemias (AMLs), including transformations of MDS or MPN.1 TET2 mutations are diverse (frameshift, nonsense and missense mutations) and do not cluster in a particular region of the protein. In contrast, mutations affecting IDH1/2 and ASXL1 are found in restricted regions.2, 3 None of these gene alterations are disease-specific and their incidence ranges from rare to 40%.1 Another important observation from recent related studies is that these mutations can coexist with other pathogenetically relevant mutations including JAK2V617F, sometimes before or emerging in a subclone.4 Therefore, and especially in view of the occurrence of these mutations across varying molecular profiles, their contribution to disease initiation, clonal evolution or blastic transformation is not clear. Also, the possibility of independently emerging multiple abnormal clones rather than monoclonal myeloproliferation is raised. In chronic myeloid leukemia (CML), BCR–ABL fusion is known to be the driver mutation. However, leukemogenesis is a complex process, and genomic heterogeneity of the chronic phase (CP) of the disease has been reported.5 This intrinsic heterogeneity at the molecular level could support a causative link with the varying response to treatment and disease progression. Furthermore, we still do not know for sure whether the BCR–ABL fusion is really the initiating lesion. In order to describe TET2, IDH1, IDH2 and ASXL1 mutation prevalence, to investigate whether these defects could represent an important event in CML initiation, and to define the relationships between acquisition of these mutations and CML transformation and/or imatinib (IM) resistance, here we report on a mutation analysis done on paired samples from CML at diagnosis (untreated), time of IM response and, when available, complete cytogenetic response (CCR) in 91 CML patients treated with IM as first line—most of them from the French SPIRIT trial—and presenting five profiles of IM response at the time of analysis: (1) 25 had CCR and major molecular response (MMR) at 12 months of IM; (2) 11 were in CCR but showed a Ph negative (Ph−) clonal evolution 12–24 months after IM (median 12 months); (3) 20 were in partial cytogenetic response (4 minor and 16 major), 3 of them with additional cytogenetic abnormalities (−Y, +8, +8) 18 months after IM and referred as primary resistant (R1); (4) 20 were in acute transformation (14 myeloid, 6 lymphoid) 4–72 months after onset of IM (median 12 months); and (5) 15 referred as secondary IM-resistant (R2) had relapse in CP (4 molecular relapse, 6 loss of CCR and 5 hematologic relapse). Sokal scores were homogeneously distributed among the five groups except for group 2, which did not have higher scores. Except for group 1, BCR–ABL residual disease evaluated by RQ-PCR remained high (median value 0.09%, according to the International Scale (IS)).

Imatinib dose escalation for chronic phase–chronic myelogenous leukaemia patients in primary suboptimal response to imatinib 400 mg daily standard therapy

International audience; Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.

Background and Objectives. This study was aimed at examining major cytogenetic response (MCR) as a valid predictor of the course of chronic myeloid leukemia (CML) and at assessing the survival of CML patients treated with interferon alpha (IFN) in dependence on the combination of MCR (yes or no) with the baseline risk group of the New CML score. MCR was defined as a reduction of Philadelphia chromosome-positive bone marrow cells to Design and Methods. Data from individual patients with a confirmed diagnosis of Philadelphia chromosome-positive CML treated with IFN were collected from 10 prospective studies in Europe and Japan. Stratified for baseline risk group, patients with a major cytogenetic response by 21 months after the start of therapy (n=171) were compared with patients achieving a minor response or less (n=487). Survival probabilities after the landmark at 21 months were compared by using the two-sided log-rank test. Results. MCR was a major predictor for low- and intermediate-risk patients (log-rank test, p Interpretation and Conclusions. Cytogenetic response per se is not a valid surrogate marker, as it is dependent on the baseline prognostic profile. The combination of risk group and cytogenetic response does, however, provide useful clinical information. The survival data presented here can serve as a benchmark for the assessment of the long-term effectiveness of imatinib.

Imatinib mesylate (STI571, Glivec, Gleevec) is a powerful inhibitor of the tyrosine kinase activity of Bcr-Abl, the oncoprotein responsible for chronic myeloid leukemia (CML). The drug shows great efficacy in chronic phase, but is less effective in maintaining hematologic remissions in blast crisis patients. Our group has previously described several cell lines made resistant to imatinib. We now examine the question of cross-resistance to other chemotherapeutic drugs used in CML. Four paired imatinib-sensitive/resistant CML cell lines were assessed by caspase-3 and MTS assays for their proliferative response to cytosine arabinoside (Ara-C), daunorubicin (DNR), homoharringtonine (HHT) and hydroxyurea (HU), either alone or in combination with imatinib. Primary blasts from advanced-stage CML patients refractory to imatinib therapy were studied by semi-solid media clonogenic assays. We found that these drugs are generally capable of major inhibition of proliferation of the CML cell lines, although differential responses to DNR and HHT were noted between some sensitive and resistant cell line pairs, implying that resistance to imatinib may confer a growth advantage under such conditions. The four drugs were also effective in preventing the formation of progenitor cell colonies from CML patients both before treatment with imatinib, and after relapse on the drug. Isobolographic analysis implied that these drugs will generally combine well with imatinib, and in some cases will be synergistic. We conclude that Ara-C, DNR or HHT, either alone or in combination with imatinib, are likely to be the best therapeutic alternatives in the management of patients who become resistant to imatinib monotherapy.

Aurora kinase overexpression has been observed in patients with hematologic malignancies. MK-0457, a pan-aurora kinase inhibitor that also inhibits the ABL T315I mutant, was evaluated to treat patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome (Ph+) acute lymphoblastic leukemia (ALL) with the T315I mutation. Adults with Ph+ chronic phase (CP)-, accelerated phase (AP)- or blast phase (BP)-CML, or ALL and documented BCR-ABL T315I mutation were treated with a 5-day continuous infusion of MK-0457 administered every 14 days at 40 mg/m(2)/h, 32 mg/m(2)/h or 24 mg/m(2)/h. Fifty-two patients (CP, n=15; AP, n=14; BP, n=11; Ph+ ALL, n=12) were treated. Overall, 8% of patients achieved major cytogenetic response; 6% achieved unconfirmed complete or partial response; 39% had no response. Two patients (CP CML) achieved complete hematologic response. No patients with advanced CML or Ph+ ALL achieved major hematologic response. The most common adverse event (AE) was neutropenia (50%). The most common grade 3/4 AEs were neutropenia (46%) and febrile neutropenia (35%). MK-0457 demonstrated minimal efficacy and only at higher, intolerable doses; lower doses were tolerated and no unexpected toxicities were observed. These data will assist in the development of future aurora kinase inhibitors and in the selection of appropriate target patient populations.

The leukemogenic property of BCR-ABL in chronic myeloid leukemia (CML) is critically dependent on its protein tyrosine kinase activity. STI571 inhibits the BCR-ABL kinase activity, the growth and the viability of BCR-ABL expressing cells. In this study, we report the apoptotic effect of STI571 in combination with daunorubicin (DNR) on peripheral blood mononuclear cells from 11 CML patients and four BCR-ABL-positive cell lines: AR230, LAMA84, K562 and KCL22. Primary blast cells were identified by flow cytometry on the basis of their low CD45 expression. Nucleus fragmentation, exposure of phosphatidylserines and decrease in mitochondrial membrane potential were measured using acridine orange, FITC-annexin V and DiOC6(3), respectively, to evaluate apoptosis. On cell lines, the effect of DNR was negligible, whereas STI571 induced 10 to 35% of apoptosis in 18 h. STI571 sensitized AR230, LAMA84 and K562 cells to DNR when apoptosis was measured at the mitochondrial and membrane but not the nuclear levels. On CML blast cells, phosphatidyl serine exposure was significantly induced by both DNR and STI571 and was higher when these drugs were used in combination (P < 0.0003). However, the effects of this drug combination were only additive and no sensitization of blast cells to DNR by STI571 was observed. Interestingly, sensitization was evidenced in CML but not normal lymphocytes. These results suggest that other mechanisms additional to Bcr-Abl tyrosine kinase activity could be responsible for DNR resistance, and further investigations are needed to understand its origin.

International audience; Flt3-ligand (FL) is a cytokine that is of paramount importance in the proliferation of primitive hematopoietic progenitors. In this study, we show that endothelial cells (EC) produce large amounts of soluble FL and express a membrane-bound form of the molecule. Bone marrow microvascular EC also produce FL, suggesting that EC are an important source of FL in the bone marrow. High concentrations of FL in EC supernatants contrast with its undetectable levels in long-term bone marrow cultures. A single mRNA for FL is detected, suggesting that soluble FL derives from the membrane-bound species by proteolytic release. FL mRNA is stable with a half-life of about 3 h. II-1alpha increases FL mRNA levels and membrane and soluble FL expression. Glucocorticoids, known inhibitors for many hematopoietic growth factors do not down-regulate the expression of FL. On the contrary, GC increase the expression of both species of FL. The neutralization of FL in cocultures EC/ hematopoietic progenitors results in an acceleration of the maturation of the progenitors. IFN-alpha, MIP-1 alpha and TGF-beta stimulate production of membrane-bound and soluble FL. This stimulation is essential to explain their modulatory effect on the generation of clonogenic cells in cocultures EC/hematopoietic progenitors. Leukemia (2000) 14, 153-162.

Nilotinib is a second-generation tyrosine kinase inhibitor, designed to specifically inhibit break-point cluster region (BCR)-Abelson (ABL) and developed to treat chronic myeloid leukemia (CML) in patients showing a resistance to imatinib. We previously demonstrated that nilotinib-induced apoptosis was reduced by stem cell factor (SCF) addition. Here, the SCF-activated survival pathway was investigated. BCR-ABL expression was accompanied by the activation of the SCF receptor: c-KIT. Nilotinib inhibited this activation that was restored by SCF binding. Parallel variations were observed for mammaliam target of rapamycin (mTOR) kinase and mTOR complex 1 substrate S6K. The inhibition of mTORC1 restored the response of BCR-ABL cell lines to nilotinib in the presence of SCF. PI3K inhibition restored nilotinib-induced apoptosis. On hematopoietic progenitors from CML patient's bone marrows, mTORC1 inhibition also restored nilotinib sensitivity in the presence of SCF, confirming its involvement in SCF-activated survival pathway. However, this pathway seems not to be involved in the nilotinib-induced resistance of the CML stem cell population. Conversely, PI3K inhibition sensitized both CML progenitors and stem cells to nilotinib, suggesting that, downstream PI3K, two different kinase pathways are activated in CML progenitor and stem cell populations.

7058 Background: IM 400 mg daily is the front-line treatment of CP CML, but provides only 50% major molecular responses (MMR) at 18 months (Mo). We designed a phase III randomized multicenter open-label prospective trial comparing IM 400 mg/d (n=159) with 3 experimental arms: IM 600 mg/d (n=160), IM 400 mg/d + s/c cytarabine (Ara-C), (20 mg/m2/d, d15–28 of 28-day cycles) (n=158) and IM 400 mg/d + s/c Peg-IFN2a (90 μg/wk) (n=159). Pts were allocated at a 1.1.1.1 ratio, stratified by Sokal risk groups. Molecular assessments were centralized and blinded. An interim analysis of 636 pts was planned based on an IS BCR-ABL/ABL ratio -3). At 18 Mo the cumulative OMR rates were 22% (IM-400), 28% (IM-600), 25% (IM-Ara-c), 43% (IM-PegIFN). Grade 3/4 neutropenia and/or thrombocytopenia occurred during the first year in 8% IM-400, 14% IM-600, 41% IM-Ara-C and 40% IM-PegIFN arms respectively. Grade 3/4 non-hematological toxicities occurred in 19% IM-400 (edemas, muscle cramps), 30% IM-600, 27% IM-Ara-C (diarrhea) and 31% IM-PegIFN pts (skin rashes, asthenia). Within the first 12 Mo, discontinuation of experimental treatment occurred in 8% IM-600, 39% Ara-C and 45% PegIFN pts. Conclusions: Although a significant number of pts reduced or stopped PegIFN within the first year, significant improvements in molecular response rates were observed in the IM-Peg IFN arm and may translate into survival benefit. [Table: see text] [Table: see text]

7084 Background: Imatinib (IM) has greatly improved survival in chronic myeloid leukemia (CML). However, IM must be continued for an indefinite period of time. A multicenter trial “Stop Imatinib” (STIM) was initiated in July 2007, in order to evaluate the persistence of complete molecular remission (CMR) after stopping IM and determine factors associated with CMR persistence. Methods: Inclusion criteria were IM treatment duration ≥3 years and sustained CMR, defined as BCR-ABL transcripts below a detection threshold of a 5-log reduction (undetectable by RQ-PCR) for ≥2 years. Molecular relapse was defined as RQ-PCR positivity confirmed on 2 successive occasions. In case of relapse, pts were rechallenged with IM at 400 mg daily. Molecular monitoring was performed according to international recommendations. Results: Sixty-nine pts were recruited among which 60 with a follow-up >1 month, including 22 males and 38 females. Median age was 62 (29–80), 31 pts had been treated with IFN prior to IM and 29 with IM frontline (de novo). Median follow-up was 5 months (1–16). Relapse occurred in 37 pts within 6 months of IM discontinuation. Only 1 pt relapsed after 6 months (M7). All patients in molecular relapse were sensitive to IM reintroduction. At last follow-up in December 2008, CMR was sustained in 8 pts at M14 and 3 pts at M16. No differences in terms of demography, duration of IM treatment and CMR duration were found between pts who relapsed and those who did not. At M9, the probability of persistent CMR was 46% (95% CI: 32–59%), 53% (95% CI: 33–69%) for previously IFN-treated pts and 39% (95% CI: 20–58%) for de novo pts (p = 0.54). A trend for a lower probability of relapse in low Sokal score pts was observed. Conclusions: Our data confirm that CMR can be long-lasting after discontinuation of IM. Using stringent criteria, it is possible to stop IM in patients with sustained CMR, even in those treated with IM as a single agent. Updated follow-up will be presented. No significant financial relationships to disclose.

18006 Background: The aim of this study was to evaluate whether or not a CMR could be induced by addition of zoledronic acid in patients with CCR during IM therapy. Indeed, this ras signaling inhib...

7027 Background: Variable responses to imatinib in the treatment of myelogenous leukemia (CML) are incompletely understood. Previous studies focused on cellular mechanisms of resistance to imatinib. Another hypothesis for variable response lies in pharmacokinetic variability that may reduce drug exposure in patients receiving imatinib. Methods: Using high- performance liquid chromatography-tandem mass spectrometry, we assessed trough plasma imatinib concentrations at steady state (Cmin) in more than 150 CML patients. Major molecular response (MMR) was defined as a 3 logarithm reduction of BCR-ABL transcripts, quantified from peripheral blood using Q-RTPCR. In addition, suboptimal response criteria defined by the European Leukemianet were used to increase doses of imatinib. Results: i) we assessed Cmin in 85 CML patients and demonstated a correlation with imatinib doses (median values 813, 1,135, 1,709 ng/ml for respectively 300,400, 600 mg/day). ii) for 68 patients after at least one year's treatment mean Cmin were significantly higher in the group with MMR (34 patients) than in the group without (1,452.1±649.1 ng per milliliter vs. 869.3±427.5 ng per milliliter, P No significant financial relationships to disclose.

Purpose: Major initial prognostic factors predicting chronic myeloid leukaemia (CML) response to imatinib mesylate (IM) therapy are represented by hema-tological status, Sokal score and duration of disease before IM. Before the era of IM, CML patients harbouring der(9) deletion classically had a worse outcome when compared to non deleted pts, but some preliminary studies suggest that this adverse factor might be reversed by IM. Additionally der(9) deletion seems to be more frequent in Variant Ph chromosome (vPh) patients (up to 30%) whereas its frequency is about 10% in classical Ph pts. We evaluated in this retrospective multicentric study the response to IM in vPh population and the possible impact of der(9) deletion on this response. 92 vPh patients treated by IM were selected from 11 hematology departments within the country. vPh chromosome was assessed by conventional karyotyping analysis and der(9) deletion by FISH. IM resistant patients were screened for ABL kinase site mutation by direct sequencing. Statistical analysis of survival and descriptive parameters were performed according to Kaplan Meyer method, log-rank test, Chi-2 and non parametric Kruskal Wallis tests. Results: among the vPh population 75% pts had a simple translocation and 25% had a more complex one (chromosomes 9, 22 and at least two other break-points). Initially 95% pts were in chronic phase (CP), 2.5% in accelerated phase (AP) and 2.5% in blastic crisis (BC).19 pts (33.3%) were scored in each Sokal score category (low, intermediate and high). At IM initiation: 83% were in CP (>35% Ph+ ), 8% were in major cytogenetic response (after interferon) and 9% were either in AP or in BC. 74 pts were screened for der(9) deletion, which was present in 16.2% of them. IM response and survival data were available for 53 pts with CP (with > 35% Ph+ ) at IM initiation. Median age of this CP population at diagnosis was 50.8 (19.5–79.4), median disease duration before IM initiation : 2 Mo (0–92.4), median follow-up after IM initiation : 24.8 Mo (6.1–69.9). 71.7% achieved RcyM including 67.9% in RcyC. 19 pts presented IM failure or suboptimal response (European LeukemiaNet consensus). 1 pt stopped IM for liver intolerance. We observed 9 (16.9%) primary resistance and 10 (18.9%) loss of response. 12% pts progressed towards AP/BC. 12 resistant pts were screened for ABL kinase site mutations : 3 of them harboured mutations. We found no difference for the overall survival between the populations with (n=9) and without (n=33) der(9q) deletions (median of survival not reached ). Respectively 1 pt (11%) and 3 pts (9%) died in these two cohorts. We found a trend for an increased rate of resistance in the der(9) deletion cohort (55.6% vs 30.3%) but the difference was not significative (p=0.16). We only found significant differences in the repartition of the Sokal score between these two cohorts with more frequent high scores for vPh patients (p=0.03). Resistance in vPh IM treated pts is high (35.8%) but the disease duration before IM was heterogenous. Der(9) deletions slightly increases the rate of IM resistance but we found no difference of survival between the two cohorts.