ABCB1 haplotype is associated with major molecular response in chronic myeloid leukemia patients treated with standard-dose of imatinib

Background: Imatinib mesylate (IM) is a selective tyrosine kinase inhibitor used for treating chronic myeloid leukemia (CML). IM has high efficacy, however some individuals develop a resistance due to impaired bio-availability. Polymorphisms in genes encoding membrane transporters such as ABCB1 have been associated with differences in protein expression and function that influence the response to several drugs. Aim: To investigate the relationship of ABCB1 polymorphisms with markers of response to IM in patients with CML Methods: One hundred eighteen CML patients initially treated with a standard dose of IM (400 mg/day) for 18 months were selected at two health centers in Sao Paulo City, Brazil. The response criteria were based on the European LeukemiaNet recommendations. ABCB1 polymorphisms c.1236C>T (rs1128503), c.3435C>T (rs1045642) and c.2677G>T/A (rs2032582) were evaluated by PCR-RFLP. Results: ABCB1 polymorphisms were not related with a risk for CML in this sample population (p<0.05). In the CML group, frequencies of ABCB1 SNPs were similar between responder and non-responder patients (p>0.05). In the responder group, the frequency of ABCB11236CT/2677GT/3435CT haplotype was higher in patients with major molecular response (MMR) (51.7%) than in patients without MMR (8.3%, p = 0.010). Furthermore, carriers of this haplotype had increased the probability of reaching the MMR compared with the non-carriers (OR: 11.8; 95% CI: 1.43-97.3, p = 0.022). Conclusions: The ABCB1 1236CT/2677GT/3435CT haplotype is positively associated with the major molecular response to IM in CML patients. (C) 2011 Elsevier Inc. All rights reserved.

Dasatinib Overrides Imatinib Resistance Mediated by the F359I Residue Mutation in Two Patients with Chronic Myeloid Leukemia

Despite the beneficial effects of imatinib mesylate, some patients may either not respond or respond suboptimally. Here, we report two chronic myelogenous leukemia patients; one had a suboptimal response according to European LeukemiaNet criteria (a major molecular response was not achieved after 18 months of standard-dose imatinib therapy) and the other had failure with a standard dose of imatinib. At the time of the suboptimal response in patient 1 and the failure in patient 2, we were able to detect the F359I mutation in the BCR-ABL tyrosine kinase domain using DNA sequencing in both patients. Therefore, it was decided to change the therapeutic regimen to dasatinib at a dose of 100 mg once daily in both patients. This change resulted in the achievement of complete cytogenetic remission in patient 1 after 4 months and a major molecular response within 2 and 3 months in both patients. Detection of the F359I mutation in our two cases likely explains the suboptimal response to imatinib in case 1 and the failure in case 2. This implies that in such cases dasatinib should be considered to effectively suppress the mutated clones. Copyright (C) 2011 S. Karger AG, Basel

The Aurora A and B kinases are up-regulated in bone marrow-derived chronic lymphocytic leukemia cells and represent potential therapeutic targets

Background The malignant B cells in chronic lymphocytic leukemia receive signals from the bone marrow and lymph node microenvironments which regulate their survival and proliferation. Characterization of these signals and the pathways that propagate them to the interior of the cell is important for the identification of novel potential targets for therapeutic intervention. Design and Methods We compared the gene expression profiles of chronic lymphocytic leukemia B cells purified from bone marrow and peripheral blood to identify genes that are induced by the bone marrow microenvironment. Two of the differentially expressed genes were further studied in cell culture experiments and in an animal model to determine whether they could represent appropriate therapeutic targets in chronic lymphocytic leukemia. Results Functional classification analysis revealed that the majority of differentially expressed genes belong to gene ontology categories related to cell cycle and mitosis. Significantly up-regulated genes in bone marrow-derived tumor cells included important cell cycle regulators, such as Aurora A and B, survivin and CDK6. Down-regulation of Aurora A and B by RNA interference inhibited proliferation of chronic lymphocytic leukemia-derived cell lines and induced low levels of apoptosis. A similar effect was observed with the Aurora kinase inhibitor VX-680 in primary chronic lymphocytic leukemia cells that were induced to proliferate by CpG-oligonucleotides and interleukin-2. Moreover, VX-680 significantly blocked leukemia growth in a mouse model of chronic lymphocytic leukemia. Conclusions Aurora A and B are up-regulated in proliferating chronic lymphocytic leukemia cells and represent potential therapeutic targets in this disease.

A new dic(7;12)(p12.21;p12.2) and i(12)(q10) during the lymphoid blast crisis of patient with Ph plus chronic myeloid leukemia

Chronic myelogenous leukemia (CML) is a common myeloproliferative disease that is characterized by the clonal expansion of marrow stem cells, and is associated with the Philadelphia chromosome. As the disease progresses, additional chromosome abnormalities may arise. The prognostic impact of secondary chromosomal abnormalities in CML is complex, heterogeneous, and sometimes related to previous treatment. Here, we describe a CML patient in lymphoid blast crisis associated with a new chromosomal abnormality identified, dic(7;12)(p12.21;p12.2) and i(12)(q10) using classical cytogenetics and spectral karyotype analysis. To the best of our knowledge, this is the first report of t(7;12)(p11.1;q11.1) and i(12)(q10) in a CML patient with lymphoid evolution.

Molecular measurement of BCR-ABL transcript variations in chronic myeloid leukemia patients in cytogenetic remission

Abstract Background The monitoring of BCR-ABL transcript levels by real-time quantitative polymerase chain reaction (RT-qPCR) has become important to assess minimal residual disease (MRD) and standard of care in the treatment of chronic myeloid leukemia (CML). In this study, we performed a prospective, sequential analysis using RT-qPCR monitoring of BCR-ABL gene rearrangements in blood samples from 91 CML patients in chronic phase (CP) who achieved complete cytogenetic remission (CCyR) and major molecular remission (MMR) throughout imatinib treatment. Methods The absolute level of BCR-ABL transcript from peripheral blood was serially measured every 4 to 12 weeks by RT-qPCR. Only level variations > 0.5%, according to the international scale, was considered positive. Sequential cytogenetic analysis was also performed in bone marrow samples from all patients using standard protocols. Results Based on sequential analysis of BCR-ABL transcripts, the 91 patients were divided into three categories: (A) 57 (62.6%) had no variation on sequential analysis; (B) 30 (32.9%) had a single positive variation result obtained in a single sample; and (C) 4 (4.39%) had variations of BCR-ABL transcripts in at least two consecutive samples. Of the 34 patients who had elevated levels of transcripts (group B and C), 19 (55.8%) had a < 1% of BCR-ABL/BCR ratio, 13 (38.2%) patients had a 1% to 10% increase and 2 patients had a >10% increase of RT-qPCR. The last two patients had lost a CCyR, and none of them showed mutations in the ABL gene. Transient cytogenetic alterations in Ph-negative cells were observed in five (5.5%) patients, and none of whom lost CCyR. Conclusions Despite an increase levels of BCR-ABL/BCR ratio variations by RT-qPCR, the majority of CML patients with MMR remained in CCyR. Thus, such single variations should neither be considered predictive of subsequent failure and nor an indication for altering imatinib dose or switching to second generation therapy. Changing of imatinib on the basis of BCR-ABL/BCR% sustained increase and mutational studies is a prudent approach for preserving other therapeutic options in imatinib-resistant patients.

A SMO inhibitor drug reduces the progenitor cell's maintenance in the Drosophila hematopoietic organ: a novel model to study Chronic Myelogenous Leukemia relapse

The chronic myeloid leukemia complexity and the difficulties of disease eradication have recently led to the development of drugs which, together with the inhibitors of TK, could eliminate leukemia stem cells preventing the occurrence of relapses in patients undergoing transplantation. The Hedgehog (Hh) signaling pathway positively regulates the self-renewal and the maintenance of leukemic stem cells and not, and this function is evolutionarily conserved. Using Drosophila as a model, we studied the efficacy of the SMO inhibitor drug that inhibit the human protein Smoothened (SMO). SMO is a crucial component in the signal transduction of Hh and its blockade in mammals leads to a reduction in the disease induction. Here we show that administration of the SMO inhibitor to animals has a specific effect directed against the Drosophila ortholog protein, causing loss of quiescence and hematopoietic precursors mobilization. The SMO inhibitor induces in L3 larvae the appearance of melanotic nodules generated as response by Drosophila immune system to the increase of its hemocytes. The same phenotype is induced even by the dsRNA:SMO specific expression in hematopoietic precursors of the lymph gland. The drug action is also confirmed at cellular level. The study of molecular markers has allowed us to demonstrate that SMO inhibitor leads to a reduction of the quiescent precursors and to an increase of the differentiated cells. Moreover administering the inhibitor to heterozygous for a null allele of Smo, we observe a significant increase in the phenotype penetrance compared to administration to wild type animals. This helps to confirm the specific effect of the drug itself. These data taken together indicate that the study of inhibitors of Smo in Drosophila can represent a useful way to dissect their action mechanism at the molecular-genetic level in order to collect information applicable to the studies of the disease in humans.

Clinical outcome and biological characteristics of Chronic Myeloid Leukemia patients treated with nilotinib front-line

The present work reports the outcome of the GIMEMA CML WP study CML0811, an independent trial investigating nilotinib as front-line treatment in chronic phase chronic myeloid leukemia (CML). Moreover, the results of the proteomic analysis of the CD34+ cells collected at CML diagnosis, compared to the counterpart from healthy donors, are reported. Our study confirmed that nilotinib is highly effective in the prevention of the progression to accelerated/blast phase, a condition that today is still associated with high mortality rates. Despite the relatively short follow-up, cardiovascular issues, particularly atherosclerotic adverse events (AE), have emerged, and the frequency of these AEs may counterbalance the anti-leukemic efficacy. The deep molecular response rates in our study compare favorably to those obtained with imatinib, in historic cohorts, and confirm the findings of the Company-sponsored ENESTnd study. Considering the increasing rates of deep MR over time we observed, a significant proportion of patients will be candidate to treatment discontinuation in the next years, with higher probability of remaining disease-free in the long term. The presence of the additional and complex changes we found at the proteomic level in CML CD34+ cells should be taken into account for the investigation on novel targeted therapies, aimed at the eradication of the disease.

Efficacy of rituximab and cladribine in patients with chronic lymphocytic leukemia and feasibility of stem cell mobilization: a prospective multicenter phase II trial (protocol SAKK 34/02)

This phase II trial investigated rituximab and cladribine in chronic lymphocytic leukemia. Four induction cycles, comprising cladribine (0.1 mg/kg/day days 1-5, cycles 1-4) and rituximab (375 mg/m(2) day 1, cycles 2-4), were given every 28 days. Stem cell mobilization (rituximab 375 mg/m(2) days 1 and 8; cyclophosphamide 4 g/m(2) day 2; and granulocyte colony-stimulating factor 10 microg/kg/day, from day 4) was performed in responders. Of 42 patients, nine achieved complete remission (CR), 15 very good partial remission, and two nodular partial remission (overall response rate 62%). Stem cell mobilization and harvesting (> or = 2 x 10(6) stem cells/kg body weight) were successful in 12 of 20 patients. Rituximab infusion-related adverse events were moderate. The main grade 3/4 adverse events during induction were neutropenia and lymphocytopenia. Rituximab plus cladribine was effective; however, the CR rate was modest and stem cell harvest was impaired in a large number of responding patients.

Chronic myelogenous leukemia maintains specific CD8(+) T cells through IL-7 signaling

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease of hematopoietic stem cells. The disease progresses after several years from an initial chronic phase to a blast phase. Leukemia-specific T cells are regularly detected in CML patients and may be involved in the immunological control of the disease. Here, we analyzed the role of leukemia-specific CD8(+) T cells in CML disease control and the mechanism that maintains CD8(+) T-cell immunosurveillance in a retroviral-induced murine CML model. To study antigen-specific immune responses, the glycoprotein of the lymphocytic choriomeningitis virus was used as model leukemia antigen. Leukemia-specific CTL activity was detectable in vivo in CML mice and depletion of CD8(+) T cells rapidly led to disease progression. CML-specific CTL were characterized by the expression of the IL-7 receptor -chain. In addition, leukemia cells produced IL-7 that was crucial for the maintenance of leukemia-specific CTL and for disease control. Therefore, CML cells maintain the specific CD8(+) T-cell-mediated immune control by IL-7 secretion. This results in prolonged control of disease and probably contributes to the characteristic chronic phase of the disease.

CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression

Chronic myelogenous leukemia (CML) results from a chromosomal translocation in hematopoietic stem or early progenitor cells that gives rise to the oncogenic BCR/ABL fusion protein. Clinically, CML has a chronic phase that eventually evolves into an accelerated stage and blast crisis. A CML-specific immune response is thought to contribute to the control of disease. Whether the immune system can also promote disease progression is not known. In the present study, we investigated the possibility that the TNF receptor family member CD27 is present on leukemia stem cells (LSCs) and mediates effects of the immune system on CML. In a mouse model of CML, BCR/ABL+ LSCs and leukemia progenitor cells were found to express CD27. Binding of CD27 by its ligand, CD70, increased expression of Wnt target genes in LSCs by enhancing nuclear localization of active β-catenin and TRAF2- and NCK-interacting kinase (TNIK). This resulted in increased proliferation and differentiation of LSCs. Blocking CD27 signaling in LSCs delayed disease progression and prolonged survival. Furthermore, CD27 was expressed on CML stem/progenitor cells in the bone marrow of CML patients, and CD27 signaling promoted growth of BCR/ABL+ human leukemia cells by activating the Wnt pathway. Since expression of CD70 is limited to activated lymphocytes and dendritic cells, our results reveal a mechanism by which adaptive immunity contributes to leukemia progression. In addition, targeting CD27 on LSCs may represent an attractive therapeutic approach to blocking the Wnt/β-catenin pathway in CML.

CLLU1 expression distinguishes chronic lymphocytic leukemia from other mature B-cell neoplasms

The distinction of CLL from other mature B-cell neoplasms, especially from leukemic forms of mantle cell lymphoma or splenic marginal zone lymphoma, can be difficult but has important prognostic and therapeutic implications. We measured CLLU1 (CLL upregulated gene1) mRNA by qPCR and found a highly significant difference between CLL and other lymphoid neoplasms (AUC 0.96, 95%CI 0.93-0.99). Based on our cut-off values we can predict CLL and other mature B-cell neoplasms with high probability (PPV 99% and 94%). Analysis of CLLU1 expression is a rapid and reliable tool that may facilitate the diagnosis of mature B-cell neoplasms especially in inconclusive cases.

Early molecular and cytogenetic response is predictive for long-term progression-free and overall survival in chronic myeloid leukemia (CML)

In the face of competing first-line treatment options for CML, early prediction of prognosis on imatinib is desirable to assure favorable survival or otherwise consider the use of a second-generation tyrosine kinase inhibitor (TKI). A total of 1303 newly diagnosed imatinib-treated patients (pts) were investigated to correlate molecular and cytogenetic response at 3 and 6 months with progression-free and overall survival (PFS, OS). The persistence of BCR-ABL transcript levels >10% according to the international scale (BCR-ABL(IS)) at 3 months separated a high-risk group (28% of pts; 5-year OS: 87%) from a group with >1-10% BCR-ABL(IS) (41% of pts; 5-year OS: 94%; P=0.012) and from a group with 1% BCR-ABL(IS) (31% of pts; 5-year OS: 97%; P=0.004). Cytogenetics identified high-risk pts by >35% Philadelphia chromosome-positive metaphases (Ph+, 27% of pts; 5-year OS: 87%) compared with 35% Ph+ (73% of pts; 5-year OS: 95%; P=0.036). At 6 months, >1% BCR-ABL(IS) (37% of pts; 5-year OS: 89%) was associated with inferior survival compared with 1% (63% of pts; 5-year OS: 97%; P<0.001) and correspondingly >0% Ph+ (34% of pts; 5-year OS: 91%) compared with 0% Ph+ (66% of pts; 5-year OS: 97%; P=0.015). Treatment optimization is recommended for pts missing these landmarks.

Drug treatment is superior to allografting as first-line therapy in chronic myeloid leukemia

Early allogeneic hematopoietic stem cell transplantation (HSCT) has been proposed as primary treatment modality for patients with chronic myeloid leukemia (CML). This concept has been challenged by transplantation mortality and improved drug therapy. In a randomized study, primary HSCT and best available drug treatment (IFN based) were compared in newly diagnosed chronic phase CML patients. Assignment to treatment strategy was by genetic randomization according to availability of a matched related donor. Evaluation followed the intention-to-treat principle. Six hundred and twenty one patients with chronic phase CML were stratified for eligibility for HSCT. Three hundred and fifty four patients (62% male; median age, 40 years; range, 11-59 years) were eligible and randomized. One hundred and thirty five patients (38%) had a matched related donor, of whom 123 (91%) received a transplant within a median of 10 months (range, 2-106 months) from diagnosis. Two hundred and nineteen patients (62%) had no related donor and received best available drug treatment. With an observation time up to 11.2 years (median, 8.9 years), survival was superior for patients with drug treatment (P = .049), superiority being most pronounced in low-risk patients (P = .032). The general recommendation of HSCT as first-line treatment option in chronic phase CML can no longer be maintained. It should be replaced by a trial with modern drug treatment first.