Shin Imamura

Marked upregulation of Survivin and Aurora-B kinase is associated with disease progression in the myelodysplastic syndromes.

Background Myelodysplastic syndromes are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Survivin is a member of the inhibitor of apoptosis family and suppresses apoptosis. Survivin also functions as a subunit of the chromosomal passenger complex for regulating mitosis with Aurora-B. Survivin and Aurora-B play an important role in maintaining genome stability. The aim of this study was to determine the role of Survivin and Aurora-B kinase in disease progression and prognosis of myelodysplastic syndromes. Design and Methods We evaluated the expression levels of these two genes in CD34+ cells prepared from 64 patients with myelodysplastic syndrome or leukemic blasts from 50 patients with de novo acute myeloid leukemia using quantitative real-time PCR. Results Survivin and Aurora-B expression levels were highly correlated with the type of myelodysplastic syndrome, were much higher in refractory anemia with excess blasts-1, refractory anemia with excess blasts-2, and secondary acute myeloid leukemia following myelodysplastic syndrome than in normal control, and increased during disease progression. There was a significant correlation between these expression levels and the International Prognostic Scoring System. Interestingly, these levels were remarkably higher in patients with secondary acute myeloid leukemia following myelodysplastic syndromes than in those with de novo acute myeloid leukemia. Conclusions This is the first report showing that high levels of Survivin and Aurora-B kinase expression in CD34+ cells are distinctive molecular features of high-risk myelodysplastic syndromes and secondary acute myeloid leukemia following myelodysplastic syndrome. Marked upregulation of Survivin and Aurora-B kinase may contribute to genetic instability and disease progression of myelodysplastic syndromes. Our data may explain why patients with high-risk myelodysplastic syndromes frequently show complex chromosomal abnormality.

Effect of PSC 833 on the cytotoxicity of idarubicin and idarubicinol in multidrug-resistant K562 cells

We examined the effect of PSC 833, a non-immunosuppressive cyclosporin analogue, on the cytotoxicity, accumulation and retention of idarubicin (IDA) and its 13-dihydro metabolite, idarubicinol (IDAol). P-glycoprotein (PGP)-overexpressing multidrug-resistant K562/D1-9 cells were used for these studies. PSC 833 had no effect on the cytotoxicity, intracellular accumulation, or retention of IDA and IDAol in the parent K562 cells. However, intracellular accumulation of IDA and IDAol in K562/D1-9 cells after a 60-min incubation was restored by 0.4 microM PSC 833 to 104% and 116%, respectively, of the level in parent K562 cells. The retention of IDA and IDAol in K562/D1-9 cells was also restored by 0.4 microM PSC 833. Consequently, 0.4 microM PSC 833 increased the sensitivity of K562/D1-9 cells to IDA and IDAol. The resistance index (RI) of IDA decreased from 20-fold to 4.0-fold, and the RI of IDAol decreased from 104-fold to 1.5-fold. These results suggest that the combination of IDA and PSC 833 may be effective in reversing PGP-mediated multidrug resistance in leukemia cells.

Reduced intensity allogeneic stem cell transplantation for systemic primary amyloidosis refractory to high-dose melphalan.

Complete elimination of the plasma cell dyscrasia is a rational therapeutic goal, as intercepting supply of precursor protein is a necessary condition for a major regression of amyloid deposits. High‐dose melphalan with autologous stem cell transplantation has shown the ability to induce complete hematological response (HR) along with recovery of organ dysfunction. However, the rate of HR with this treatment rarely exceeds 40%. We describe here the first known case of successful reduced intensity allogeneic stem cell transplantation (RIST) for a patient with primary amyloidosis complicated with nephrotic syndrome but without cardiac disease, who had obtained only partial HR by high‐dose melphalan with autologous stem cell transplantation. RIST may be feasible and be capable of achieving complete HR along with recovery from nephrotic syndrome with acceptable toxicity.

Idarubicin and idarubicinol are less affected by topoisomerase II-related multidrug resistance than is daunorubicin

We investigated the cytotoxicity and cellular pharmacology of idarubicin (IDA), idarubicinol (IDAol) and daunorubicin (DNR) in K562/VP-H2 cells, which show topoisomerase II-related multidrug resistance but do not overexpress P-glycoprotein. K562/VP-H2 cells were less resistant to IDA and IDAol than to DNR. There was no significant difference in the accumulation of each drug between K562 and K562/VP-H2 cells. The cleavage of DNA induced by each drug was decreased in K562/VP-H2 cells, however, the decrease in cleavage in K562/VP-H2 cells was less with IDA and IDAol than with DNR. These results suggest that IDA and IDAol have more cytotoxic potency than DNR in topoisomerase II-related multidrug-resistant leukemia cells.

Induction of Cell Differentiation by IMPDH Antisense Oligomer in HL-60 and K562 Human Leukemia Cell Lines

Inosine-5’-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) is the enzyme that catalyzes the formation of xanthosine-5’-monophosphate from inosine-5’-monophosphate (IMP). In the purine de novo synthetic pathway, IMPDH is positioned at the branch point in the synthesis of adenine and guanine nucleotides and is thus the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. Alterations in the activity of IMPDH and the levels of guanine nucleotides have been implicated in the regulation of cell growth and differentiation[1]. The addition of IMPDH inhibitors, such as mycophenolic acid and tiazofurin, to cultures induces terminal differentiation in a variety of human leukemia cells, including K562 erythroid leukemia cells, and HL-60 myeloid leukemia cells as well as human breast cancer cells and melanoma cells[2–4]. To verify the role of this enzyme in controlling differentiation, we determined the ability of specific IMPDH antisense oligomers to induce maturation in the K562 and HL-60 human leukemia cell lines. From these experiments, we concluded that a specific reduction in IMPDH results in inhibition of cell replication and induction of terminal differentiation in the two human leukemia cell lines.

Monitoring of intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in 1-beta-D-arabinofuranosylcytosine therapy at low and conventional doses.

1‐β‐D‐Arabinofuranosylcytosine (ara‐C) is used empirically at a low, conventional, or high dose. Ara‐C therapy may be optimal if it is directed by the clinical pharmacokinetics of the intracellular active metabolite of ara‐C, 1‐β‐D‐arabinofuranosylcytosine 5′‐triphosphate (ara‐CTP). However, ara‐CTP has seldom been monitored during low‐ and conventional‐dose ara‐C therapies because detection methods were insufficiently sensitive. Here, with the use of our newly established method (Cancer Res., 56, 1800‐1804 (1996), ara‐CTP was monitored in leukemic cells from acute myelog‐enous leukemia patients receiving low‐ or conventional‐dose ara‐C [subcutaneous ara‐C administration (10 mg/m2) (3 patients), continuous ara‐C infusion (20 or 70 mg/m2/24 h) (7 patients), 2‐h ara‐C infusion (70 mg/m2) (4 patients), and 2‐h infusion of N4‐behenoyl‐l‐β‐D‐arabinofuranosylcy‐tosine, a deaminase‐resistant ara‐C derivative (70 mg/m2) (6 patients)]. Ara‐CTP could be determined at levels under 1μM. There was a close correlation between the elimination half‐life values of the plasma ara‐C and the intracellular ara‐CTP. The presence of ara‐C in the plasma was important to maintain ara‐CTP. The continuous ara‐C and the 2‐h N4‐behenoyl‐l‐β‐D‐arabinofura‐nosylcytosine infusions maintained ara‐CTP and the plasma ara‐C longer than the subcutaneous ara‐C or the 2‐h ara‐C infusion. They also afforded relatively higher ara‐CTP concentrations, and consequently produced ara‐CTP more efficiently than the 2‐h ara‐C infusion. Different administration methods produced different quantities of ara‐CTP even at the same dose.

Close Correlation of 1‐β‐D‐Arabinofuranosylcytosine 5′‐Triphosphate, an Intracellular Active Metabolite, to the Therapeutic Efficacy of N4‐Behenoyl‐1‐β‐D‐arabinofuranosylcytosine Therapy for Acute Myelogenous Leukemia

N4‐Behenoyl‐l‐β‐D‐arabinofuranosylcytosine (BHAC), a prodrug of 1‐β ‐D‐arabinofuranosylcy‐tosine, is used effectively for the treatment of leukemia in Japan. BHAC therapy may be more effective if it is delivered in conjunction with monitoring of 1‐β ‐D‐arabinofuranosylcytosine 5′‐tri‐phosphate (ara‐CTP), the intracellular active metabolite of ara‐C derived from BHAC. However, previous monitoring methods for ara‐CTP were insufficiently sensitive. Here, using our new sensitive method, we evaluated the ara‐CTP pharmacokinetics in relation to the therapeutic response in 11 acute myelogenous leukemia patients who received a 2‐h infusion of BHAC (70 mg/m2) in combination remission induction therapy. ara‐CTP could be monitored at levels under 1 μM. BHAC maintained effective levels of plasma ara‐C and intracellular ara‐CTP for a longer time, even compared with historical values of high‐dose ara‐C. The area under the concentration‐time curve of ara‐CTP was significantly greater in the patients with complete remission than in the patients without response. This greater amount of ara‐CTP was attributed to the higher ara‐CTP concentrations achieved in the responding patients. There was no apparent difference of plasma ara‐C pharmacokinetics between the two groups. Thus, for the first tune, the ara‐CTP pharmacokinetics was evaluated in relation to the therapeutic effect of BHAC, and the importance of ara‐CTP was proven. Administration of optimal BHAC therapy may require monitoring of the ara‐CTP pharmacokinetics in each individual patient.

Successful treatment of acquired myelofibrosis with pure red cell aplasia by cyclosporine

I read with interest the paper by Fleischhack et al (1998) describing their experience with IDA-FLAG in the treatment of children with high-risk AML: 17/23 patients with refractory or relapsed disease entered remission and nine of these children remained in complete remission (CR) at a median follow-up of 17·5 months, seven following BMT or ABMT. These results are important because they confirm that durable second remissions may be achieved in a significant proportion of such children, and provide toxicity data for this regimen. However, the authors’ recommendation of this regimen in relapsed patients is premature for several reasons:

Pure red cell aplasia developing into myeloproliferation with myelodysplasia and subsequent leukemia after cyclosporin A therapy.

We describe a very rare case of a patient who presented with red cell aplasia that later developed into myeloproliferation with myelodysplasia and eventually leukemia. A 63-year-old man presented with anemia and reticulocytopenia in May 1997. A bone marrow examination revealed erythroid aplasia with normal production of myeloid cells and megakaryocytes with a normal karyotype. After the diagnosis of pure red cell aplasia was made, the patient was treated with prednisolone and then with cyclosporin A (CyA). Two weeks after the initiation of CyA treatment, the peripheral reticulocyte count began to increase with a regrowth of erythroid cells in the bone marrow. Meanwhile, the peripheral white blood cell and platelet counts also increased to more than 10,000/μL and 1,000,000/μL, respectively. Examination of a bone marrow aspirate in December 1997 revealed myelodysplastic changes with trisomy 8. Despite the discontinuation of CyA and the administration of 1-β-D-arabinofuranosylcytosine stearyl monophosphate, leukemia developed in August 1998. In September 1998, the patient died of sepsis during a neutropenic period that followed remission-induction therapy. In the mechanism of pathogenesis, CyA may induce upon pure red cell aplasia a secondary myeloproliferative disorder with myelodysplasia and leukemia. An alternative possibility is that CyA reduces autoimmune-mediated suppression of the underlying stem cell disorder and that the result of this reduction is the manifestation of myeloproliferation and leukemia.

Peripheral blood stem cell collection and transplantation using the Haemonetics Multi Component System

AIM To assess clinical usefulness of an intermittent-flow blood cell separator in peripheral blood stem cell (PBSC) collection and transplantation. RESULTS The Haemonetics Multi Component System (Multi) was used to collect PBSC (52 aphereses in 17 patients). The mean processing blood volume and the mean PBSC yield were 7407 ml and 2.16 x 10(6) CD34+ cells/kg, respectively. When CD34+ cells exceeded 0.3% of the peripheral WBC, more than 2.0 x 10(6) CD34+ cells/kg could be collected by a single apheresis. Eight patients underwent PBSC transplantation after high-dose chemotherapy. Hematopoietic recovery was achieved in a median period of 10 days. CONCLUSIONS (1) A single-arm, light-weight machine has sufficient capability to collect PBSC. (2) The percentage of CD34+ cells in the peripheral WBC is a good predictor of the CD34+ cell yield of the collection.