Shinya Fujisawa

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.

Clinicopathological and prognostic characteristics of CD56‐negative multiple myeloma

Summary. We analysed CD56 expression in 70 patients with multiple myeloma (MM) to determine its clinicopathological and prognostic significance. Fifty‐five (79%) patients were CD56+. CD56– patients (n = 15) had higher β2 microglobulin levels and a higher incidence of extramedullary disease, Bence Jones protein, renal insufficiency and thrombocytopenia than CD56+ patients. Their myelomas more frequently had a plasmablastic morphology. Overall survival was significantly lower in CD56– than CD56+ patients (22 vs 63 months, P = 0·0002). We conclude that CD56– MM is a discrete entity associated with more aggressive disease. The higher incidence of plasmablastic cases suggested that CD56– MM may develop from a less mature plasma cell than CD56+ MM.

Arsenic trioxide therapy for relapsed or refractory Japanese patients with acute promyelocytic leukemia: need for careful electrocardiogram monitoring.

Recent studies have shown that arsenic trioxide (As2O3) can induce complete remission in patients with acute promyelocytic leukemia (APL). We tested the efficacy and safety of As2O3 for the treatment of patients with APL who had relapsed from or become refractory to all-trans retinoic acid (ATRA) and conventional chemotherapy in a prospective study. As2O3 at a dose of 0.15 mg/kg was administered until the date of bone marrow remission to a maximum of 60 days. In patients who achieved complete remission (CR), one additional course of As2O3 was administered using the same dose for 25 days. Of 14 patients, 11 (78%) achieved CR. Six of 10 patients who achieved CR showed disappearance of PML-RARα transcript by RT-PCR assay. The duration of As2O3-induced CR ranged from 4 to 22 months (median, 8 months) at a median follow-up of 17 months. Adverse events included 13 electrocardiogram abnormalities (13 QTc prolongation, eight ventricular premature contraction, four nonsustained ventricular tachycardia and two paroxysmal supraventricular tachycardia), seven nausea and vomiting, four pruritus, three peripheral neuropathy, three fluid retention and one APL differentiation syndrome. Four patients received antiarrhythmic agents. Hyperleukocytosis developed in five patients and in three cytotoxic drugs were necessary. Other adverse events were relatively mild. As2O3 treatment is effective and relatively safe in relapsed or refectory patients with APL. Cardiac toxicities in patients with QTc prolongation should be carefully monitored.

The FOXM1 transcriptional factor promotes the proliferation of leukemia cells through modulation of cell cycle progression in acute myeloid leukemia

FOXM1 is an important cell cycle regulator and regulates cell proliferation. In addition, FOXM1 has been reported to contribute to oncogenesis in various cancers. However, it is not clearly understood how FOXM1 contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of FOXM1 in AML cells. The FOXM1 messenger RNA (mRNA) expressed in AML cell lines was predominantly the FOXM1B isoform, and its levels were significantly higher than in normal high aldehyde dehydrogenase activity (ALDH(hi)) cells. Reduction of FOXM1 expression in AML cells inhibited cell proliferation compared with control cells, through induction of G(2)/M cell cycle arrest, a decrease in the protein expression of Aurora kinase B, Survivin, Cyclin B1, S-phase kinase-associated protein 2 and Cdc25B and an increase in the protein expression of p21(Cip1) and p27(Kip1). FOXM1 messenger RNA (mRNA) was overexpressed in all 127 AML clinical specimens tested (n = 21, 56, 32 and 18 for M1, M2, M4 and M5 subtypes, respectively). Compared with normal ALDH(hi) cells, FOXM1 gene expression was 1.65- to 2.26-fold higher in AML cells. Moreover, the FOXM1 protein was more strongly expressed in AML-derived ALDH(hi) cells compared with normal ALDH(hi) cells. In addition, depletion of FOXM1 reduced colony formation of AML-derived ALDH(hi) cells due to inhibition of Cdc25B and Cyclin B1 expression. In summary, we found that FOXM1B mRNA is predominantly expressed in AML cells and that aberrant expression of FOXM1 induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of FOXM1 expression represents an attractive target for AML therapy.

Disease-related potential of mutations in transcriptional cofactors CREB-binding protein and p300 in leukemias.

CREB-binding protein (CBP) and highly related p300 protein are transcriptional co-activators that play an essential role in chromatin remodeling through histone acetyltransferase activity and interaction with other transcriptional regulators. In this study, various hematological malignancies, including nine cell lines and 45 clinical samples (32 acute myeloid leukemias (AML), nine acute lymphoblastic leukemias (ALL), two cases of myelodysplastic syndrome (MDS), one multiple myeloma, and one chronic myelogenous leukemia in blast crisis), were examined to ask whether mutation of the CBP and p300 genes could be involved in leukemogenesis. The answer was approached by employing the reverse transcription-polymerase chain reaction and single-strand conformation polymorphism (RT-PCR/SSCP) technique and subsequent sequence analysis. A T-lymphoblastic cell line, CEM had an in-frame 21-base-pair deletion within the bromodomain of its p300 cDNA. Genomic DNA analysis revealed aberrant splicing caused by mutation of the acceptor site of intron 17 from ag to gg, which should interfere with catalytic step II of the pre-mRNA splicing reaction. In 1 MDS patient, a missense mutation was detected, which caused a replacement from Ser to Gly at codon 507 of p300. This is the first report of CBP/p300 mutations in leukemias, which might be relatively rare but nonetheless contribute to pathogenesis in some fraction of cases.

Depletion of Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatases 1 and 2 by Bcr-Abl Promotes Chronic Myelogenous Leukemia Cell Proliferation through Continuous Phosphorylation of Akt Isoforms

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However, the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study, we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2, which dephosphorylated Ser-473 on Akt1, -2, and -3, resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; colony-forming unit-granulocyte, macrophage; and burst-forming unit-erythroid, treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells, whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus, Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1, -2, and -3 via phosphorylation on Ser-473, resulting in the proliferation of CML cells.

Transcriptional Repression of Cdc25B by IER5 Inhibits the Proliferation of Leukemic Progenitor Cells through NF-YB and p300 in Acute Myeloid Leukemia

The immediately-early response gene 5 (IER5) has been reported to be induced by γ-ray irradiation and to play a role in the induction of cell death caused by radiation. We previously identified IER5 as one of the 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP)-induced transcriptional responses in AML cells, using microarrays that encompassed the entire human genome. However, the biochemical pathway and mechanisms of IER5 function in regulation of the cell cycle remain unclear. In this study, we investigated the involvement of IER5 in the cell cycle and in cell proliferation of acute myeloid leukemia (AML) cells. We found that the over-expression of IER5 in AML cell lines and in AML-derived ALDHhi (High Aldehyde Dehydrogenase activity)/CD34+ cells inhibited their proliferation compared to control cells, through induction of G2/M cell cycle arrest and a decrease in Cdc25B expression. Moreover, the over-expression of IER5 reduced colony formation of AML-derived ALDHhi/CD34+ cells due to a decrease in Cdc25B expression. In addition, over-expression of Cdc25B restored TMPP inhibitory effects on colony formation in IER5-suppressed AML-derived ALDHhi/CD34+ cells. Furthermore, the IER5 reduced Cdc25B mRNA expression through direct binding to Cdc25B promoter and mediated its transcriptional attenuation through NF-YB and p300 transcriptinal factors. In summary, we found that transcriptional repression mediated by IER5 regulates Cdc25B expression levels via the release of NF-YB and p300 in AML-derived ALDHhi/CD34+ cells, resulting in inhibition of AML progenitor cell proliferation through modulation of cell cycle. Thus, the induction of IER5 expression represents an attractive target for AML therapy.

A variant transcript, e1a3, of the minor BCR–ABL fusion gene in acute lymphoblastic leukemia: case report and review of the literature

We report a rare case of adult Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) with an e1a3 fusion transcript. A 25-year-old female consulted our hospital for leukocytosis and thrombocytopenia. She was diagnosed with Ph-positive precursor B cell ALL. The patient’s BCR–ABL fusion gene showed the e1a3 transcript. She received bone marrow transplantation (BMT) in the first complete remission (CR). However, the disease relapsed 4 months later, and she received a second BMT in the second CR, which caused lethal venoocculusive disease. The duration of the total clinical course was 18 months. We established a new cell line from the patient’s leukemic cells at the time of relapse, which is very rare and useful for study as an atypical Ph-positive ALL model. The literature on Ph-positive leukemia lacking ABL exon 2 was also reviewed.

CD5^+ Diffuse Large B-Cell Lymphoma with c-myc/IgH Rearrangement Presenting as Primary Effusion Lymphoma

We report an instructive case of diffuse large B-cell lymphoma presenting as acute heart failure. A 69-year-old human immunodeficiency virus-negative man was admitted to our hospital for general fatigue. A computed tomographic scan of the chest and abdomen showed pericardial effusion, but there was no evidence of tumor masses, lymph node enlargement, or hepatosplenomegaly. During the chemotherapy, increased lactate dehydrogenase and pleural effusion appeared. The tumor cells in the effusion showed positivity for CD5, CD19, CD20, κ chain, and Bcl-2 and negativity for CD10 and CD23.The chromosomes showed t(8;14)(q24;q32) with c-myc/immunoglobulin (Ig)H rearrangement, and the MIB-1 index was not high (60%). Neither human herpes virus 8 nor Epstein-Barr virus DNA was detected in the cells by polymerase chain reaction. The response to chemotherapy was very poor, and the patient died 4 months after the diagnosis. A spectrum of the symptoms of CD5+ lymphoma encompasses pericardial effusion and also can accompany c-myc/IgH rearrangement.