Molecular targeted treatment--new treatment strategy for patients with chronic myeloid leukemia

Imatinib mesylate is a new drug that can inhibit the tyrosine kinase activity of Bcr-Abl, the receptors for platelet-derived growth factor receptor(PDGF) and stem cell factor, or c-kit. Chronic myeloid leukemia (CML) is distinguished by the presence of a reciprocal translocation between chromosomes 9 and 22 that results in a shortened chromosome 22, termed the Philadelphia(Ph) chromosome. As a result of the translocation, a fusion gene called the Bcr-Abl gene is created from two normal cellular genes, encoding a chimeric Bcr-Abl protein with a deregulated tyrosine kinase activity. The expression of Bcr-Abl tyrosine kinase has been shown to be necessary and sufficient for the transformed phenotype of CML cells. Imatinib can block the kinase activity of Bcr-Abl, thus inhibiting the proliferation of Ph-positive progenitors, and has shown activity against all phases of CML, though responses are most substantial and durable in patients in the chronic phase. An international phase III study which compared the efficacy of imatinib with that of interferon alpha combined with low-dose cytarabine in newly diagnosed chronic-phase CML showed the rate of major cytogenetic response at 24 months was 90%, including 82% of complete cytogenetic response. These results indicated that imatinib was superior to interferon-containing treatment as a first-line therapy. More than 10,000 patients worldwide, including those in Japan, have been treated with imatinib in clinical trials, and a lot of information has been accumulated on the use of this drug. The aim of this article is to review the use of this drug and the practical management of patients with chronic myeloid leukemia.     

Chronic myeloid leukemia and allogeneic natural killer cells: a surprising dialogue

Chronic myeloid leukemia (CML) is a clonal multilineage myeloproliferative disease of stem cell origin characterized by the presence of the Bcr/Abl oncoprotein, a constitutively active tyrosine kinase. The actual treatment of CML patients in chronic phase is the specific abl kinase inhibitor imatinib mesylate that induces 90% of cytogenetic responses in early-phase patients. However, resistance in long-term treated patients occurs and the allogeneic stem cell transplantation remains the only curative treatment in resistant patients. Despite recent reports outlining the role of allogeneic natural killer (NK) cells as potent antileukemic effectors, the mechanisms controlling the leukemic target recognition and lysis by activated NK cells have not been well identified. The authors’ experimental data obtained on appropriate cellular models identify diverse mechanisms that could explain the increased NK-cell susceptibility of Bcr/Abl targets to NK-mediated lysis. They further delineate unexpected effects of the inhibition of the tyrosine kinase activity on the cross-talk between NK and CML leukemic cells. The consequences of such discoveries are discussed in the context of combined treatments with antikinases as well as adoptive cellular therapy approaches in myeloid leukemia patients.     

Chronic myeloid leukemia: "Archetype" of the impact of targeted therapies

Chronic myeloid leukemia (CML) is a chronic blood disorder characterized by a reciprocal translocation between chromosomes 9 and 22, leading to the creation of a chimeric gene encoding the BCR-ABL fusion protein with a constitutive tyrosine kinase activity. Although long known as a disease with an inexorable progression to acute leukemia, CML history has been significantly improved by the use of imatinib, a tyrosine kinase inhibitor. Imatinib has revolutionized the treatment of CML by transforming it from an invariably fatal disease to a chronic but manageable condition. In fact, the discovery of this class of targeted therapy had an impact not only on the survival of CML patients but also on other scientific and medical fields. This review illustrates the impact of imatinib, the first example of tyrosine kinase inhibitors on the treatment of CML, on the treatment of other cancers, the impact on health systems and on the scientific research in general.     

Hämatologische Nebenwirkungen der Tyrosinkinaseinhibition mit Imatinib

Imatinib (STI571, Gleevec/Glivec) and other small-molecule tyrosine kinase inhibitors are highly effective in the treatment of chronic myeloid leukemia (CML), gastrointestinal stromal tumors and, for example, eosinophilia-associated chronic myeloproliferative disorders. This molecularly targeted approach disrupts abnormal tyrosine kinase dependent signalling pathways, thus providing a preferred treatment option for selected neoplastic disorders with activating mutations of Abelson-, Abl-related-, Kit-, and platelet-derived growth factor receptor A and B genes. Loss of response to imatinib may be due to an acquired resistance of emerging mutant tumor cell clones.Therapy is generally well tolerated. However, toxicities including edema, skin rashes, fatigue, nausea and myelosuppression have been reported. Philadelphia/Bcr-Abl-negative clonal chromosomal abnormalities may develop. Bone marrow trephines obtained from CML patients in complete remission with prolonged pancytopenia secondary to imatinib generally show marrow hypoplasia. Morphological features may be in keeping with either aplastic anemia or myelodysplasia developing in Philadelphia-negative hematopoiesis. Single or multilineage myelodysplasia may be accompanied by an excess of blasts and rarely evolves into acute leukemia in CML patients. Severe adverse hematological effects of imatinib are extremely rare.Current questions involve the molecular mechanisms of hematological side effects of tyrosine kinase inhibitors with special regard to the emergence of distinct aberrant clones.     

Dasatinib

Dasatinib is approved for the treatment of chronic myeloid leukemia (CML) in patients with resistance or intolerance to imatinib. This article reviews pharmacokinetic, pharmacodynamic, and clinical data on dasatinib, and highlights some of the most important issues that need to be addressed. Imatinib and dasatinib both target the tyrosine kinase activity of the BCR/ABL oncogenic fusion protein. In terms of pharmacodynamics, the two agents differ in several ways: (i) dasatinib is >300-fold more potent than imatinib in inhibiting BCR/ABL activity; (ii) inhibition profiles on other tyrosine kinases differ between imatinib and dasatinib; and (iii) dasatinib has other peculiar effects on the leukemogenic signaling, including activation of p38 mitogen-activated protein kinase (MAPK) and reduction of the apoptotic-inactive form of the BCL2-associated agonist of cell death (BAD) protein. Recent pharmacodynamic data suggested combination therapy with dasatinib and signaling inhibitors (e.g. flavopiridol, farnesyl transferase inhibitors, or histone deacetylase inhibitors) may be beneficial. In contrast to other tyrosine kinase inhibitors (TKIs), dasatinib has a reduced half-life and no active metabolites. In a randomized, open-label, phase III trial, dasatinib 100 mg once daily demonstrated similar efficacy and a better tolerability profile than 70 mg twice daily. This unexpected result has been confirmed in recent studies, in which a dose of dasatinib 100 mg once daily was sufficient to trigger apoptosis in leukemic cells. Furthermore, cytogenetic responses correlate with BCR/ABL inhibition. Data suggest dasatinib 100 mg once daily achieves oncogenic shock and chronic inhibition of BCR/ABL activity, suggesting that in the future, pulse therapy with TKIs may be an option in some specific patients with CML.     

Current and emerging tests for the laboratory monitoring of chronic myeloid leukaemia and related disorders

Chronic myeloid leukaemia (CML) is a molecularly defined disease. The BCR-ABL fusion occurs in all cases of classical CML and leukaemic cells express a constitutively activated BCR-ABL tyrosine kinase. Other fusion oncogenes involving tyrosine kinases, including ABL and PDGFRA/B, have been identified, and are associated with leukaemic syndromes that may resemble CML. The discovery and treatment of these related disorders has been facilitated by our detailed understanding of CML. Imatinib mesylate has significantly improved the outcome of patients with CML, but there remains a significant minority of chronic phase CML patients for whom the response to treatment with standard dose imatinib is suboptimal. Cytogenetic and molecular monitoring of the response to treatment provides important prognostic information. Achievement of a major molecular response (MMR) in chronic phase patients treated de novo with imatinib confers near 100% freedom from progression to advanced phase, and MMR is now an important goal of therapy. Standardisation of BCR-ABL molecular monitoring is under way and should enable the accurate and reproducible identification of MMR in laboratories around the world. Point mutations in the kinase domain of BCR-ABL are the most common cause of acquired resistance to imatinib treatment. The susceptibility of a mutation to imatinib, nilotinib, or dasatinib may help to guide changes in therapy in a patient with resistance. In addition to these established methods of monitoring, there are new tests in development that may assist in determining prognosis and optimising therapy. Among patients receiving the same dose of imatinib, the plasma level of imatinib shows considerable inter-patient variation, and there is emerging evidence that higher levels may be associated with improved response to treatment. The intracellular concentration of imatinib also shows considerable variation, most likely related to differences in influx and efflux transport mechanisms. We discuss how these established and emerging assays might be used to optimise the treatment of CML patients.     

The combination therapy of imatinib and dasatinib achieves long-term molecular response in two imatinib-resistant and dasatinibintolerant patients with advanced chronic myeloid leukemia

For patients with chronic myeloid leukemia (CML) failing imatinib therapy, second-generation tyrosine kinase inhibitors (TKIs) are recommended. Here, we describe two patients with advanced CML who failed imatinib therapy and did not tolerate the recommended dose of dasatinib, but then achieved a major molecular response with the combination of imatinib and dasatinib with no significant extramedullary toxicity. Our observations suggest that combination of TKIs may provide an additive/synergistic antileukemic effect.     

Imatinib mesylate (STI 571)--a new oral target therapy for chronic myelogenous leukemia (CML)

The publication provides an up-to-date review of the significance of cytogenetic abnormalities in chronic myelogenous leukemia (CML) and the development of a promising agent with specific molecular target against tyrosine kinase, product of the BCR-ABL fusion gene, namely imatinib mesylate (STI 571, Glivec). The publication summarizes the achieved results with this compound in the chronic phase CML (in patients resistant to interferon and in newly diagnosed patients) further in patients in the accelerated phase and in blast crisis and in patients in relapse after allogeneic stem cells transplantations for CML. The results in Ph+ acute lymphoblastic leukemia are also presented. The mechanisms of resistance to imatinib mesylate and the possibilities how to overcome or circumvent it are mentioned (escalation of the dosage, combination of imatinib with some other treatment modalities as immunotherapy, interferon or convention chemotherapy and development of new drugs).     

Analysis of gene expression profiles in an imatinib-resistant cell line,KCL22/SR

The BCR/ABL tyrosine kinase inhibitor, imatinib, has shown substantial effects in blast crises of chronic myelogenous leukemia. However, most patients relapse after an initial clinical response, indicating that drug resistance is a major problem for patients being treated with imatinib. In this study, we generated a new imatinib-resistant BCR/ABL-positive cell line, KCL22/SR. The 50% inhibitory concentration of imatinib was 11-fold higher in KCL22/SR than in the imatinib-sensitive parental cell line, KCL22. However, KCL22/SR showed no mutations in the BCR/ABL gene and no increase in the levels of BCR/ABL protein and P-glycoprotein. Furthermore, the level of phosphorylated BCR/ABL protein was suppressed by imatinib treatment, suggesting that mechanisms independent of BCR/ABL signaling are involved in the imatinib resistance in KCL22/SR cells. DNA microarray analyses demonstrated that the signal transduction-related molecules, RAS p21 protein activator and RhoA, which could affect Ras signaling, and a surface tumor antigen, L6, were upregulated, while c-Myb and activin A receptor were downregulated in KCL22/SR cells. Furthermore, imatinib treatment significantly suppressed the level of phosphorylated p44/42 in KCL22 cells but not in KCL22/SR cells, even when BCR/ABL was inhibited by imatinib. These results suggest that various mechanisms, including disturbance of Ras-mitogen-activated protein kinase signaling, are involved in imatinib resistance.     

Tyrosine kinase activity associated with the CD7 antigen: correlation with regulation of T cell integrin function

Rapid up-regulation of the functional activity of integrin adhesion receptors is a hallmark of T cell activation. Monoclonal antibody engagement of the CD7 antigen on human T cells results in an increase in β1 and β2 integrin-mediated adhesion within minutes. This suggests that CD7 is capable of transducing intracellular signals, and is consistent with other indirect studies implicating CD7 as a signaling receptor on T cells. In this report, we have explored the intracellular mechanism by which CD7 modulates integrin functional activity. First, CD7-mediated up-regulation of T cell adhesion was found to be unique when compared to phorbol ester stimulation and CD3/T cell receptor cross-linking, based on differences in the kinetics of activation-dependent integrin-mediated adhesion and lack of increase in CD2 functional activity. Second, up-regulation of integrin activity mediated by CD7 cross-linking was completely inhibited by the tyrosine kinase inhibitor herbimycin A. Third, antiphosphotyrosine immunoblotting demonstrated that antibody engagement of CD7 results in a rapid but transient increase in tyrosine phosphorylation in human T cells. Finally, CD7 immunoprecipitates contain in vitro kinase activity, as demonstrated by phosphorylation of a predominant band of 80 kDa and multiple other bands. Phosphoamino acid analysis of the 80-kDa substrate revealed phosphorylation on tyrosine as well as serine and threonine residues. Together, our results suggest that CD7 is associated with tyrosine kinase activity and that this tyrosine kinase activity correlates with the ability of CD7 to regulate T cell integrin functional activity.     

Random aneuploidy in CML patients at diagnosis and under imatinib treatment

Chronic myeloid leukemia (CML) is characterized by the presence of a BCR-ABL fusion gene, which is the result of a reciprocal translocation between chromosomes 9 and 22, and is cytogenetically visible as a shortened chromosome 22 (Philadelphia). Research during the past two decades has established that BCR-ABL is probably the pathogenetic pathway leading to CML, and that constitutive tyrosine kinase activity is central to BCR-ABL capacity to transform hematopoietic cells in vitro and in vivo. The tyrosine kinase inhibitor imatinib mesylate was introduced into the treatment regimen for CML in 1998. During the last few years, reports on chromosomal changes during imatinib treatment have been described. In this study, we evaluated the random aneuploidy rate with chromosomes 9 and 18 in bone marrow from treated and untreated patients. We found higher aneuploidy rates in both treated and untreated patients compared to the control group. In three patients who were treated with imatinib mesylate for more than 1.5 years, triploidy also appeared in some nuclei. To our knowledge, this is the first report on new chromosomal changes such as random aneuploidy and triploidy under imatinib treatment, but more studies are needed to investigate the long-term effect of the imatinib treatment on genetic instability.     

The Mechanism of Mononuclear Cell Infiltration in Oral Lichen Planus: The Role of Cytokines Released from Keratinocytes

To clarify the pathogenesis of oral lichen planus (OLP), we investigated the roles of keratinocytes (KC) in mononuclear cell infiltration. When peripheral blood mononuclear cells (PBMC) obtained from healthy donors were cultured in the presence of culture supernatants of KC separated from the noninflamed gingivae (Nor-KC) and cheek mucosae of patients with OLP (OLP-KC), the number of migrated PBMC across monolayered human umbilical vein endothelial cells (HUVEC) were increased to about 1.3-fold and 1.5-fold of the control level, respectively, with increases of the expression of CD11a, CD11b, CD18, and CD49d on PBMC and intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial–leukocyte adhesion molecule-1 on HUVEC. The number of migrated PBMC was reduced to about 60% of the control level by pretreatment of PBMC with anti-CD11a or anti-CD18 MAb and reduced to about 70% by pretreatment of HUVEC with anti-CD54 MAb. The pretreatment of PBMC with genistein, H-7, wortmannin, or exoenzyme C3 decreased the migrated PBMC by about 70 to 90%. In agreement with these results, the culture supernatants of OLP-KC up-regulated tyrosine phosphorylation of 62-kDa, 70-kDa, and 102-kDa proteins, phosphatidylinositol-3 kinase, and protein kinase C activities and activated Rho protein level more so than did those of Nor-KC. Additionally, actin reorganization with the formation of membrane ruffles and lamellipodia was distinctly induced by the culture supernatants of OLP-KC. These results indicate that cytokines generated by KC transduce their signals in PBMC, up-regulating the expression of cell surface adhesion molecules and migration activity with reorganization of actin filaments.     

Crosstalk between BCR/ABL and RNAi

Chronic myeloid leukemia (CML) is a malignant disease of progenitor myeloid cells caused by chromosomal translocation that results in the forming of diminutive Philadephia chromosome that harbors BCR/ABL fusion oncogene. The product of this oncogene, a tyrosine kinase, alters several important regulatory pathways related to cell growth and differentiation thus leading to cancer transformation. Major form of CML therapy is based on tyrosine kinase inhibitors, first of all imatinib (IM). Some patients develop resistance to IM in the course of treatment. In the process of leukemogenesis the activity of miRNAs – one of groups of RNAs involved in RNA interference (RNAi) – is altered. Signatures of altered miRNAs activity may serve as a prognostic factor in the development and therapy of several diseases. Moreover, other group of RNAs involved in RNAi – siRNA – might be valuable addition to array of specific therapeutics targeted the BCR/ABL kinase.     

BCR-ABL kinase domain mutations in tyrosine kinase inhibitors-naïve and -exposed Southeast Asian chronic myeloid leukemia patients

BCR-ABL kinase domain (KD) mutation is the main mechanism associated with resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients. This study targeted a large cohort of CML (n = 171) comprising 80 naïve CML cases without prior TKI exposure as well as 91 cases undergoing 1st generation (imatinib) and/or 2nd generation (nilotinib/dasatinib) TKI therapy. KD mutations were analyzed by denaturing high performance liquid chromatography followed by direct sequencing. Twenty-one types of mutations were found in 37 patients including 13 known mutations and 8 previously unidentified mutations. Thirty cases had a single mutation while 7 cases had multiple mutations. Twenty-three percent of patients receiving first-line imatinib, 69% of imatinib-resistant patients receiving 2nd generation TKI, and 75% of advanced phase patients treated with front-line 2nd generation TKI had KD mutations. Interestingly, 9% of TKI-naïve CML cases were also discovered to carry the KD mutations including 5 novel variants. Patients who received hydroxyurea had a 2-fold increase in KD mutations as compared to newly diagnosed patients but they still had a lower mutation frequency than TKI-exposed cases. Mutations in the naïve cases were mainly localized in the C-helix domain and SH3 contact site whereas in exposed cases predominantly in the drug contact site, P-loop, and catalytic domain. T315I resistant mutation was identified only in TKI-exposed cases. In conclusion, several known and novel BCR-ABL KD mutations were discovered in the TKI-naïve and -exposed Southeast Asian CML patients, supporting the concept that naturally occurring KD mutations were present in leukemic cells prior to drug exposure. T315I resistant mutation was completely undetectable in this naïve Southeast Asian cohort; its incidence, however, increases with drug exposure.     

Adhesion molecule expression patterns indicate activation and recruitment of CD4+ T cells from the lymph node to the peripheral blood of early cutaneous leishmaniasis patients

Adhesion molecules play a crucial role in cell migration and recruitment. Expression of adhesion molecules that preferentially address cells to inflammatory sites is a critical event in the formation and maintenance of leishmaniasis lesions. In this work, we analyzed the expression of CD11a, CD11b and CD62L, adhesion molecules involved in cell activation and circulation, in CD4+ and CD8+ T cells from peripheral blood and lymph nodes of patients with early cutaneous leishmaniasis. The percentage of expression of CD62L, CD11a and CD11b in total lymphocytes was lower in lymph nodes as compared to peripheral blood. Moreover, differences in adhesion molecule expression between blood and lymph nodes were more striking in CD4+ than CD8+ T cells. Stimulation of PBMC from leishmaniasis patients with soluble Leishmania antigens (SLA) lead to the expansion of CD4+CD62Lhigh cells, CD4+CD11b+ cells and to an increase in the intensity of expression of CD11a in CD4+, but not CD8+ T cells. Our data suggest that early activation events that occur in the lymph nodes of patients recently infected with Leishmania lead to changes in T cell adhesion molecule expression, favoring migration to the periphery and increasing the likelihood of further recruitment to lesion sites.     

Monitoring twenty-six chronic myeloid leukemia patients by BCR-ABL mRNA level in bone marrow:a single hospital experience

Chronic myeloid leukemia (CML) is caused by the BCR-ABL oncogene. The Philadelphia chromosome (Ph) from a reciprocal translocation, t(9;22) (q34;q11) causes a fusion gene, BCR-ABL, that encodes a constitutively active tyrosine kinase. Treatment of CML by imatinib is effective to control the tyrosyl phosphorylation of the protein related to the cell signaling. BCR-ABL mRNA is overexpressed in the minimal residual disease (MRD), known as an early sign of relapse. Between December 2005 and June 2008, we measured BCR-ABL mRNA levels in the bone marrow (BM) from patients by quantitative real-time polymerase chain reaction (RQ-PCR) in Aomori Prefectural Central Hospital. Eighty-six samples from 26 patients were collected. Among the 26 CML patients, 11 patients (42%) were in the pretreatment group. Seven (64%) of the 11 patients achieved complete molecular response (CMR). In the post-treatment group consisting of the remaining 15 patients, 9 (60%) patients achieved CMR. The patients receiving imatinib at a dose over 300 mg per day required 13 (6-77) months [median (range)] to achieve CMR. On the other hand, the patients receiving a dose below 300 mg per day required 29.5 (11-84) months [median (range)]. When BCR-ABL mRNA was detected during the treatment course of patients with CMR, careful observation of BCR-ABL mRNA was useful for tracking the clinical course of patients. In conclusion, the BCR-ABL mRNA level was useful for monitoring the clinical course in 26 patients with CML.