Akihiro Takeshita

Internal tandem duplication of FLT3 associated with leukocytosis in acute promyelocytic leukemia

FLT3 is a member of receptor tyrosine kinases expressed in leukemia cells, as well as in hematopoietic stem cells. Recently, a somatic alteration of the FLT3 gene was found in acute myeloid leukemia, as an internal tandem duplication (FLT3/ITD) which caused elongation of the juxtamembrane (JM) domain of FLT3. Here we characterized the FLT3/ITD and investigated its clinical significance in acute promyelocytic leukemia (APL). Seventy-four newly diagnosed patients with APL, who were treated with the same protocol in a multi-institutional study, were studied for the FLT3/ITD. Genomic and message sequences of the FLT3 gene were amplified by means of polymerase chain reaction (PCR), and elongated PCR products were sequenced. Fifteen patients (20.3%) had FLT3/ITD, all of which were transcribed in frame. Location of the duplicated fragments (six to 30 amino acids) varied from patient to patient. However, they always contained either Y591 or Y599, but the tyrosine kinase domain was not significantly affected. This finding implied that signal transduction of FLT3 is amplified by the duplication. Clinically, the presence of FLT3/ITD was related to high peripheral white blood cell counts as well as peripheral leukemia cell counts (P < 0.0001), high ldh level (P = 0.04), and low fibrinogen concentration (P = 0.04). These data suggest that FLT3/ITD plays a significant role in progression of APL.

Prolongation of the QT interval and ventricular tachycardia in patients treated with arsenic trioxide for acute promyelocytic leukemia.

Arsenic trioxide therapy has recently been found to be very effective in relapsed or refractory acute promyelocytic leukemia. It has resulted in complete remission in more than 52% of cases in China and the United States (1, 2). Shen and colleagues (1) have given the most detailed report of adverse events related to arsenic trioxide therapy. Although most of the patients in their study were critically ill, arsenic trioxide was relatively well tolerated. Updated analyses showed that nonlife-threatening cardiac toxicities related to arsenic trioxide occurred in 8 of 47 patients (3). Electrocardiographic abnormalities, such as QRS complex broadening, prolonged QT intervals, ST-segment depression, T-wave flattening, and multifocal ventricular tachycardia, have been reported in acute arsenic poisoning (4-6). Recently, Huang and colleagues (7) found that 1 patient developed complete atrioventricular block during arsenic trioxide therapy and required implantation of a permanent pacemaker. After observing a prolonged QT interval in the first patient with relapsed acute promyelocytic leukemia who received arsenic trioxide in our hospital, we used continuous monitoring to prospectively examine electrocardiograms and echocardiograms and determine the cardiac toxicities of arsenic trioxide in 8 patients with this disease. We observed prolonged QT intervals in all 8 patients and serious arrhythmias in 4 patients. Methods We used arsenic trioxide to treat 8 patients with acute promyelocytic leukemia who had relapse after extensive previous therapy with all-trans retinoic acid and chemotherapy, including anthracycline (Table). Arsenic trioxide was provided by PolaRx Biopharmaceuticals, Inc. (New York, New York). Our protocol, which was the same as that of a phase II study in the United States, was reviewed and approved by the institutional review board of Hamamatsu University School of Medicine in Hamamatsu, Japan. All patients gave written informed consent and were hospitalized while receiving arsenic trioxide (0.15 mg/kg of body weight), which was administered daily by 2-hour infusion for a maximum of 60 days. Treatment was discontinued if patients met conventional criteria for complete remission (cellular bone marrow aspirate with blasts 5%, absolute neutrophil count 1.5 109 cells/L, and platelet count 100 109 cells/L). Patients who achieved complete remission received one additional 25-day course of arsenic trioxide at the same dose between 3 and 6 weeks after induction therapy. Patients were continuously monitored with ambulatory electrocardiography while receiving arsenic trioxide, and standard 12-lead electrocardiography was performed at least once per week. The QT intervals were calculated in the weekly electrocardiograms, were expressed as the mean values in the 12-lead electrocardiograms, and were corrected by heart rates according to the Bazett formula (QTc interval=QT interval/R-R interval) (8). Table. Patient Characteristics and Prolongation of the QTc Interval during Arsenic Trioxide Therapy The funding source had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Results Five patients (63%) achieved complete remission, and 4 patients received the second course of arsenic trioxide as consolidation therapy. Long QTc intervals (>440 ms) had been noted in 4 of 8 patients before arsenic trioxide therapy. Prolonged QT intervals were observed in all patients during induction therapy with arsenic trioxide and in 3 of 4 patients during the second course of therapy after complete remission (Table). The PQ interval and QRS duration were not prolonged in any case. Ventricular premature contractions were seen during 8 of 12 courses of therapy. Four patients developed nonsustained monomorphic ventricular tachycardia ( 3 successive ventricular premature contractions that stopped spontaneously within 30 seconds) and received antiarrhythmic agents (mexiletine HCl and lidocaine HCl). No patients developed sustained ventricular tachycardia or polymorphic ventricular tachycardia. Patient 1 received arsenic trioxide therapy during his second relapse. The QTc interval was prolonged gradually until day 33 and reverted to the pretreatment level after arsenic trioxide was stopped on day 43. The patient had seven successive ventricular premature contractions on day 25 when the QTc interval was 474 milliseconds (Figure). Therefore, mexiletine HCl (150 mg/d) was administered from day 28 to day 148 during arsenic trioxide therapy. The second course of arsenic trioxide with prophylactic mexiletine HCl was started on day 114. Although similar prolonged QT intervals were seen, only isolated ventricular premature contractions, not ventricular tachycardia, were induced. Patient 3 received arsenic trioxide for 46 days as induction therapy during her second relapse. She had five successive ventricular premature contractions on day 5 and three on day 31. She was given amphotericin B and received potassium and calcium supplements because she had low-normal levels of serum potassium and calcium on day 5. The QTc interval was prolonged from 408 to 460 milliseconds until day 50 and decreased to 442 milliseconds on day 87, when the second course was started. The second course again caused a prolonged QT interval. Figure. Electrocardiographic tracing and QT intervals in patient 1 during arsenic trioxide therapy. Patient 6 had two acute myocardial infarctions before developing acute promyelocytic leukemia. He relapsed four times and received large doses of chemotherapy before receiving arsenic trioxide. The ejection fraction was low (0.45) before arsenic trioxide therapy began. The patient had been receiving mexiletine HCl, 300 mg/d, since his second myocardial infarction. On day 38, nonsustained ventricular tachycardia (27 successive beats) was noticed when the QTc interval was prolonged from 443 to 485 milliseconds. Arsenic trioxide was reduced to 0.1 mg/kg per day and was administered intermittently until day 92 with lidocaine HCl or verapamil HCl. However, the patient developed accelerated idioventricular rhythm on day 62 and nonsustained ventricular tachycardia on day 70. He did not achieve complete remission, and arsenic trioxide therapy was discontinued on day 92. Patient 7 had nonsustained ventricular tachycardia before arsenic trioxide therapy in her second relapse, and mexiletine HCl, 150 mg/d, was given prophylactically until day 54. Arsenic trioxide was not effective and was withdrawn on day 40. The QTc interval was prolonged from 448 to 479 milliseconds; however, no serious arrhythmias were induced. Patient 8, who had a second relapse, had four successive ventricular premature contractions on day 23 and day 24 when the QTc interval was 461 milliseconds. Arsenic trioxide was withdrawn on day 23, and mexiletine HCl, 300 mg/d, was given. Thereafter, no ventricular tachycardia developed and arsenic trioxide therapy was restarted on day 26. Complete remission occurred, and arsenic trioxide therapy was stopped on day 41. No patient developed echocardiographic abnormalities (such as contractile dysfunction, cardiac enlargement, or hypertrophy) except the patient with previous myocardial infarctions. Serum electrolyte levels were within normal limits in all patients during the study. Discussion We used continuous monitoring by ambulatory electrocardiography to show prolonged QT intervals in 8 patients receiving arsenic trioxide. Ventricular arrhythmias, including ventricular tachycardia, developed in 5 of 12 courses of therapy and were associated with prolonged QT intervals. Previous reports of cardiographic abnormalities in arsenic trioxide therapy have shown low-flat T-wave, sinus tachycardia, prolonged QT intervals, and atrioventricular blocks, but not ventricular arrhythmias (1, 2, 7). However, multifocal ventricular tachycardia and ventricular fibrillation have been reported in arsenic poisoning (4-6). The tachyarrhythmias in our study were not sustained ventricular tachycardia or torsade de pointes but nonsustained ventricular tachycardia, accelerated idioventricular rhythm, or paroxysmal supraventricular tachycardia. It is unknown why polymorphic ventricular tachycardias and torsade de pointes were not observed. We believe that spatial inhomogeneity (QT dispersion) or abnormal ventricular repolarization might also be related to the arrhythmias. Indeed, in some cases, prolonged QT intervals were accompanied by an increase in the QT dispersion with no change in the QRS duration. It remains unclear why arsenic prolongs the QT interval. The metal is known to affect the peripheral nervous system diffusely (9), and imbalance of the sympathetic nervous system may be involved. Arsenic also causes widespread damage in many organs by combining with sulfhydryl proteins (9). A direct effect of arsenic on the myocardium could also be involved. However, the evidence remains speculative and further study is needed. Because of its remarkable effectiveness, arsenic trioxide will continue to be widely used for relapsed or refractory acute promyelocytic leukemia. Since such patients have been heavily treated with chemotherapeutic agents, including anthracycline and all-trans retinoic acid, cardiac damage is likely to be universal before arsenic trioxide therapy begins. Arsenic trioxide thus might induce arrhythmia. In our study, although the number of patients was small, ventricular arrhythmias were observed, often through careful monitoring. Therefore, we believe that patients taking arsenic trioxide should have frequent electrocardiographic monitoring and, in particular, should be monitored carefully for serious arrhythmias when QT intervals are prolonged. Prophylactic antiarrhythmic drugs that do not prolong the QT interval should be used because previous reports showed an association between fatal ventricular tachycardias and prolonged QT intervals in arsenic intoxication (4-6). Elect

The induction of apoptosis and cell cycle arrest by arsenic trioxide in lymphoid neoplasms.

Arsenic trioxide (As2O3) has recently been shown to induce complete remission in acute promyelocytic leukemia (APL). As2O3 reportedly has dose-dependent dual effects on APL cells, triggering apoptosis at relatively high concentrations and inducing differentiation at lower concentrations. However, its effect is still controversial for other AML cells and hematological neoplasms. We studied the in vitro effect of As2O3 on lymphoid lineage cells: lymphoma cell lines, NOL-3, Raji and Daudi, a myeloma cell line, NOP-1, normal peripheral blood lymphocytes (PBL), non-Hodgkins lymphoma (NHL) cells and chronic lymphocytic leukemia (CLL) cells, and compared it with the effect on APL cell line, NB4, as well as other myeloid cell lines, HL-60 and NKM-1. As2O3 at a concentration of 1 μmol/l markedly inhibited both proliferation and viability of NB4, NOP-1, NOL-3 and NKM-1 cells, but it reduced only viability in normal PBL, CLL cells and NHL cells. As2O3 induced apoptosis and down-regulated bcl-2 expression in NB4, NOP-1 and NKM-1 cells. On the other hand, in HL-60, Raji and Daudi cells, 1 μmol/l As2O3 inhibited only the proliferation weakly, and neither induced apoptosis nor down-regulated bcl-2 expression, but arrested only cell cycle at G1 phase. As2O3 at a low concentration of 0.1 μmol/l had no effect on proliferation and viability of these cells except for NB4. These results showed that As2O3 exerted variable and definite effects on lymphoid cells and indicated that As2O3 might be clinically useful in lymphoid neoplasms such as malignant lymphoma and CLL.

Calicheamicin-conjugated humanized anti-CD33 monoclonal antibody (gemtuzumab zogamicin, CMA-676) shows cytocidal effect on CD33-positive leukemia cell lines, but is inactive on P-glycoprotein-expressing sublines

Calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, CMA-676, has recently been introduced to clinics as a promising drug to treat patients with acute myeloid leukemia (AML) in relapse. However, the mechanism of action of CMA-676 has not been well elucidated. The cytotoxic effect of CMA-676 on HL60, NOMO-1, NB4, NKM-1, K562, Daudi, and the multidrug-resistant sublines, NOMO-1/ADR and NB4/MDR, was investigated by cell cycle distribution and morphology. These studies were done by a video-microscopic system, DNA fragmentation, dye exclusion and 3H-thymidine uptake after analysis of CD33, CD34, P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein and lung-related protein on these cells. A dose-dependent, selective cytotoxic effect of CMA-676 was observed in cell lines that expressed CD33, and was dependent on the amount of CD33 and the proliferative speed of the cells. Sensitive cells were temporally arrested at the G2/M phase before undergoing morphological changes. CMA-676 is not effective on P-gp-expressing multidrug-resistant sublines compared with parental cell lines. MDR modifiers, MS209 and PSC833, restored the cytotoxic effect of CMA-676 in P-gp-expressing sublines. CMA-676 is a promising agent in the treatment of patients with AML that expresses CD33. The combined use of CMA-676 and MDR modifiers may increase the selective cytotoxic effect in multidrug-resistant AML.

Serum thrombopoietin levels in patients with chronic hepatitis and liver cirrhosis

OBJECTIVES:Thrombocytopenia is a common manifestation of cirrhosis. The aim of this study was to examine the relationship between serum thrombopoietin concentrations, circulating platelet levels, and the stage of hepatic fibrosis in patients with chronic viral hepatitis.METHODS:The study included 48 patients with chronic viral hepatitis (14 with stage 1 fibrosis; five with stage 2 fibrosis; three with stage 3 fibrosis; 26 with cirrhosis) and 30 healthy volunteers. Serum thrombopoietin levels were measured using an enzyme-linked immunosorbent assay. Spleen size, platelet counts, and prothrombin time were measured.RESULTS:Thrombopoietin levels of patients with fibrosis stage 1 (2.50 ± 1.60 fmol/ml) or stage 2 (1.89 ± 0.65) were significantly higher than those in patients with cirrhosis (1.21 ± 0.55) or healthy volunteers (1.26 ± 0.74). Mean platelet counts of patients with cirrhosis (8.0 ± 4.6 × 104/μl) were significantly lower than those with fibrosis stage 1 (18.6 ± 3.9) or stage 2 (16.0 ± 5.8), or healthy volunteers (24.5 ± 7.3). Patients with cirrhosis had larger spleens (30.9 ± 18.4 cm2) than those with fibrosis stage 1 (18.2 ± 6.4). Platelet counts showed a significant inverse relationship to spleen size (ρ=−0.51, p < 0.0005) and a significant positive relationship with thrombopoietin levels (ρ= 0.34, p < 0.02). Thrombopoietin levels were significantly correlated to prothrombin time (ρ= 0.45, p < 0.005).CONCLUSIONS:Serum thrombopoietin levels are elevated in patients with an early stage of chronic viral hepatitis. As the disease progresses from mild fibrosis to cirrhosis, decreased production of thrombopoietin may contribute to the further development of thrombocytopenia in cirrhosis.

Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients.

To clarify the cooperative roles of recurrently identified mutations and to establish a more precise risk classification system in acute myeloid leukemia (AML), we comprehensively analyzed mutations in 51 genes, as well as cytogenetics and 11 chimeric transcripts, in 197 adult patients with de novo AML who were registered in the Japan Adult Leukemia Study Group AML201 study. We identified a total of 505 mutations in 44 genes, while only five genes, FLT3, NPM1, CEBPA, DNMT3A and KIT, were mutated in more than 10% of the patients. Although several cooperative and exclusive mutation patterns were observed, the accumulated mutation number was higher in cytogenetically normal AML and lower in AML with RUNX1-RUNX1T1 and CBFB-MYH11, indicating a strong potential of these translocations for the initiation of AML. Furthermore, we evaluated the prognostic impacts of each sole mutation and the combinations of mutations and/or cytogenetics, and demonstrated that AML patients could be clearly stratified into five risk groups for overall survival by including the mutation status of DNMT3A, MLL-PTD and TP53 genes in the risk classification system of the European LeukemiaNet. These results indicate that the prognosis of AML could be stratified by the major mutation status in combination with cytogenetics.

Arsenic trioxide therapy in relapsed or refractory Japanese patients with acute promyelocytic leukemia: updated outcomes of the phase II study and postremission therapies.

Recently, arsenic trioxide (ATO) has been proved to induce complete remission (CR) at a high rate in patients with acute promyelocytic leukemia (APL).We prospectively investigated the safety and efficacy of ATO therapy in patients with relapsed and refractory APL and examined the duration of CR and the postremission therapies. Initially, 0.15 mg/kg ATO was administered until bone marrow remission to a maximum of 60 days. After the patient achieved CR, 1 additional ATO course at the same dosage was administered for 25 days. Of 34 patients, 31 (91%) achieved CR. PML-RARα messenger RNA was not detected in the bone marrow of 18 (72%) of the 25 patients evaluated by reverse transcriptase—polymerase chain reaction analysis. At a median follow-up of 30 months, the estimated 2-year overall survival rate was 56%, and the estimated 2-year event-free survival rate was 17%. During the ATO therapy, QTc prolongation was observed in most cases. Fifteen patients developed ventricular tachycardia, and 1 of them showed torsades de pointes. Other adverse events were nausea, water retention, APL differentiation syndrome, skin eruption, liver dysfunction, and peripheral neuropathy, all of which were quite tolerable. ATO therapy was remarkably effective for relapsed APL; however, postremission therapies were necessary to maintain a durable remission.

Granulocyte colony-stimulating factor receptor at various differentiation stages of normal and leukemic hematopoietic cells.

Granulocyte colony-stimulating factor (G-CSF) is the most important cytokine in granulopoiesis, and induces the proliferation and differentiation of normal bone marrow granulocytic precursors. The physiologic effect of G-CSF is mediated through binding to specific cell surface receptors for G-CSF. Using a newly-devised quantitative flow-cytometric assay, we analyzed the expression of G-CSF receptors on normal and leukemic hematopoietic cells. In normal donors, G-CSF receptors are widely expressed from CD34-positive immature bone marrow cells to mature peripheral granulocytes. The highest expression of G-CSF receptors is observed in peripheral granulocytes. In the bone marrow, the level of G-CSF receptors expression increases in the following order; CD34+ CD33- cells < CD34+ CD33+ cells < CD34- CD33+ cells, indicating that G-CSF receptors are expressed on myeloid cells from a very early stage of differentiation and that the level of expression increases with the progress of cell maturation. G-CSF receptors are also detected on blast cells of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). The number of cell surface receptors varies from patient to patient, and no clear correlation is observed between the expression of receptors and the leukemia subtype or the cell surface markers. In this respect, the clinical use of G-CSF should be carefully controlled in ALL as well as AML patients.

Simple and reliably sensitive diagnosis and monitoring of Philadelphia chromosome-positive cells in chronic myeloid leukemia by interphase fluorescence in situ hybridization of peripheral blood cells

Philadelphia (Ph) chromosome or the bcr/abl fusion gene is the hallmark of chronic myeloid leukemia (CML) and serves as a prognostic marker during its treatment. Its detection has been primarily done by karyotype analysis of bone marrow cells. The major limitation of the karyotypic technique is an absolute need for metaphases, often difficult to obtain in an appropriate number in patients under therapy. Fluorescence in situ hybridization (FISH) is a sensitive and quantitative method to detect the bcr/abl fusion gene in cells in both metaphase and interphase. Using M-bcr and abl probes, we performed the interphase FISH in the peripheral blood of 30 healthy volunteers and in 20 hematologically normal bone marrow samples. False-positive cells were detected in 2.7 ± 0.7% (mean ± standard deviation) and 2.3 ± 0.7% among 500 cells, respectively. Then we tested 31 patients with CML at various stages of disease on 50 occasions. Although there was a good correlation between the percentage of FISH-positive cells in the peripheral blood and that in the bone marrow (r = 0.977), between the percentage of FISH-positive cells in the peripheral blood and that of Ph chromosome in the bone marrow (r = 0.841), and between the percentage of FISH-positive cells and that of Ph chromosome in the bone marrow (r = 0.933), the limits of agreement in each group were not small, and thus the peripheral blood FISH test can not be interpreted as the same method with conventional karyotyping. Additionally, we could easily rule out CML in 15 individuals with leukocytosis without performing bone marrow aspiration. The present study indicates that FISH analysis in the peripheral blood is a simple and reliably sensitive test for the detection and quantitative monitoring of the M-bcr/abl fusion gene in CML in routine clinical practice, although this can not entirely replace karyotype analysis of bone marrow cells.

CMC-544 (inotuzumab ozogamicin) shows less effect on multidrug resistant cells: analyses in cell lines and cells from patients with B-cell chronic lymphocytic leukaemia and lymphoma

The effect of CMC‐544, a calicheamicin‐conjugated anti‐CD22 monoclonal antibody, was analysed in relation to CD22 and P‐glycoprotein (P‐gp) in B‐cell chronic lymphocytic leukaemia (CLL) and non‐Hodgkin lymphoma (NHL) in vitro. The cell lines used were CD22‐positive parental Daudi and Raji, and their P‐gp positive sublines, Daudi/MDR and Raji/MDR. Cells obtained from 19 patients with B‐cell CLL or NHL were also used. The effect of CMC‐544 was analysed by viable cell count, morphology, annexin‐V staining, and cell cycle distribution. A dose‐dependent, selective cytotoxic effect of CMC‐544 was observed in cell lines that expressed CD22. CMC‐544 was not effective on Daudi/MDR and Raji/MDR cells compared with their parental cells. The MDR modifiers, PSC833 and MS209, restored the cytotoxic effect of CMC‐544 in P‐gp‐expressing sublines. In clinical samples, the cytotoxic effect of CMC‐544 was inversely related to the amount of P‐gp (P = 0·003), and to intracellular rhodamine‐123 accumulation (P < 0·001). On the other hand, the effect positively correlated with the amount of CD22 (P = 0·010). The effect of CMC‐544 depends on the levels of CD22 and P‐gp. Our findings will help to predict the clinical effectiveness of this drug on these B‐cell malignancies, suggesting a beneficial effect with combined use of CMC‐544 and MDR modifiers.