Kazutaka Kuriyama

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.

Induction therapy by frequent administration of doxorubicin with four other drugs, followed by intensive consolidation and maintenance therapy for adult acute lymphoblastic leukemia: the JALSG-ALL93 study.

In order to improve the disappointing prognosis of adult patients with acute lymphoblastic leukemia (ALL), we applied similar induction therapy as that used for acute myeloid leukemia (AML), ie frequent administration of doxorubicin (DOX). DOX 30 mg/m2 was administered from days 1 to 3 and from days 8 to 10 together with vincristine, prednisolone, cyclophosphamide and L-asparaginase, followed by three courses of consolidation and four courses of intensification. From December 1993 to February 1997, 285 untreated adult patients with de novo ALL were entered. Of 263 evaluable patients (age 15 to 59; median 31), 205 (78%) obtained complete remission (CR). At a median follow-up period of 63 months, the predicted 6-year overall survival (OS) rate of all patients was 33%, and disease-free survival (DFS) rate of CR patients was 30%, respectively. By multivariate analysis, favorable prognostic factors for the achievement of CR were age <40 and WBC <50 000/μl; for longer OS were age <30 and WBC <30 000/μl; and for longer DFS of CR patients were FAB L1 and ALT <50 IU/l. Among 229 patients who had adequate cytogenetic data, 51 (22%) had Philadelphia (Ph) chromosome. Ph-negative chromosome was a common favorable prognostic factor for CR, longer OS and DFS. DFS was not different between early sequential intensification (n = 48) and intermittent intensification (n = 43) during the maintenance phase. Among CR patients under 40 years old, the 6-year survival was not different between the allocated related allo-BMT group (34 patients) and the allocated chemotherapy group (108 patients). However, among patients with Ph-positive ALL, the survival of patients who actually received allo-BMT was superior to that of patients who received chemotherapy (P = 0.046).

Diagnostic significance of detecting pseudo‐Pelger‐Huet anomalies and micro‐megakaryocytes in myelodysplastic syndrome

To investigate the kind of morphological dysplastic changes important in the diagnosis of myelodysplastic syndrome (MDS), we examined 50 patients with MDS and 86 control subjects. Micromegakaryocytes and pseudo‐Pelger‐Huët anomalies were detected in 46 patients (92%) with MDS, and were also found in the differentiated types of acute myeloid leukaemia and chronic myeloid leukaemia, but not in other haematological disorders, or healthy subjects. We believe that evaluation of these changes would be helpful in diagnosing MDS.

Prognostic significance of the null genotype of glutathione S-transferase-T1 in patients with acute myeloid leukemia: increased early death after chemotherapy

We investigated the prognostic significance of genetic polymorphism in glutathione-S transferase mu 1 (GSTM1), glutathione-S transferase theta 1 (GSTT1), NAD(P)H:quinone oxidoreductase (NQO1) and myeloperoxidase (MPO), the products of which are associated with drug metabolism as well as with detoxication, in 193 patients with de novo acute myeloid leukemia (AML) other than M3. Of the patients, 64.2% were either homozygous or heterozygous for GSTT1 (GSTT1+), while 35.8% showed homozygous deletions of GSTT1 (GSTT1−). The GSTT1− group had a worse prognosis than the GSTT1+ group (P = 0.04), whereas other genotypes did not affect the outcome. Multivariate analysis revealed that GSTT1− was an independent prognostic factor for overall survival (relative risk: 1.53; P = 0.026) but not for disease-free survival of 140 patients who achieved complete remission (CR). The rate of early death after the initiation of chemotherapy was higher in the GSTT1− group than the GSTT1+ group (within 45 days after initial chemotherapy, P = 0.073; within 120 days, P = 0.028), whereas CR rates and relapse frequencies were similar. The null genotype of GSTT1 might be associated with increased toxicity after chemotherapy.

Prognostic value of p53 gene mutations and the product expression in de novo acute myeloid leukemia

Abstract: In acute myeloid leukemia (AML), p53 mutations are reportedly infrequent but associated with a poor prognosis. The majority of mutations are missense mutations, which generally lead to accumulation of nuclear p53 protein. However, the prognostic significance of the accumulation remains unknown in AML. In this study, we compared the prognostic value of p53 mutations versus accumulation of the product. p53 mutations were found in 9 (4.5%) of 200 patients with de novo AML. The p53 mutation detectable (mutation+) group had a worse prognosis (p=0.0009) than the mutation not detectable (mutation−) group. Multivariate analysis showed that the p53 mutation was an independent factor (p=0.005) for short overall survival as well as 60 yr or older (p=0.001) and unfavorable karyotypes (p=0.001). In 79 of the 200 patients, the expression of p53 was studied by immunocytochemistry (ICC) using anti‐p53 monoclonal antibody (DO‐7). All samples carrying missense mutations (N=6) were positive for ICC in over 15% of nuclei of each sample, chosen as the optimized cutoff value of p53 accumulation. Accumulation was thus found in 14 of the 79 patients. However, there was no prognostic difference according to the accumulation, because the mutation−/accumulation+ group (N=8) tended to have a good prognosis. These findings indicate that molecular detection of p53 mutations yields better prognostic information than ICC. In a subset of AML, p53 protein might be accumulated without mutation presumably due to upstream signals of p53.

Poor response to intensive chemotherapy in de novo acute myeloid leukaemia with trilineage myelodysplasia

Summary. The findings of morphologically dysplastic features in haemopoietic cells in de novo acute myeloid leukaemia (AML) has been named AML with trilineage myelodysplasia (AML/TMDS). We analysed the clinical data, karyotypes, and treatment outcomes of 230 de novo AML patients treated with the Japan Adult Leukaemia Study Group AML‐87 protocol. 40 (17%) patients had AML/TMDS. Platelet count was significantly higher (F=0·006) and bone marrow blasts were fewer (P=0·01) in the AML/TMDS group than in the AML without TMDS. Abnormal karyotype was shown in 12/30 patients (40%) analysed.

Dysmegakaryocytopoiesis in acute leukaemias: its predominance in myelomonocytic (M4) leukaemia and implication for poor response to chemotherapy

Megakaryocytopoiesis was morphologically investigated in 129 adults with de novo acute leukaemia. Three types were identified: type I (84 cases), no detectable megakaryocytes; type II (32 cases), quantitatively preserved megakaryocytes with normal morphology; type III (13 cases): quantitatively preserved megakaryocytes but with distinct dysplastic changes such as micromegakaryocytes and megakaryocytes with multiple small separated nuclei. Type III was found in M1 (one out of 21 cases), M2 (one out of 20 cases), M4 (eight out of 24 cases), M6 (two out of four cases) and hypoplastic leukaemia (one out of 13 cases). M3 cases were all classified into type I. Most of acute lymphoid leukaemia cases (21 cases) belonged to type II. Among AML cases, the complete remission (CR) rate by intensive chemotherapy with daunorubicin and cytosine arabinoside was significantly lower in type III (11%) than in types I (87%) and II (71%). Among M4 cases, CR rates in type III (14%) was also significantly lower than those in type I (75%) and II (100%). Thus, the present study indicates the importance of recognizing dysmegakaryocytopoiesis in AML for clariflcation of the heterogeneous biology or pathophysiology of acute leukaemias and formulation of an appropriate therapeutic strategy.

Comprehensive analysis of cooperative gene mutations between class I and class II in de novo acute myeloid leukemia

Acute myeloid leukemia (AML) has been thought to be the consequence of two broad complementation classes of mutations: class I and class II. However, overlap‐mutations between them or within the same class and the position of TP53 mutation are not fully analyzed. We comprehensively analyzed the FLT3, cKIT, N‐RAS, C/EBPA, AML1, MLL, NPM1, and TP53 mutations in 144 newly diagnosed de novo AML. We found 103 of 165 identified mutations were overlapped with other mutations, and most overlap‐mutations consisted of class I and class II mutations. Although overlap‐mutations within the same class were found in seven patients, five of them additionally had the other class mutation. These results suggest that most overlap‐mutations within the same class might be the consequence of acquiring an additional mutation after the completion both of class I and class II mutations. However, mutated genes overlapped with the same class were limited in N‐RAS, TP53, MLL‐PTD, and NPM1, suggesting the possibility that these irregular overlap‐mutations might cooperatively participate in the development of AML. Notably, TP53 mutation was overlapped with both class I and class II mutations, and associated with morphologic multilineage dysplasia and complex karyotype. The genotype consisting of complex karyotype and TP53 mutation was an unfavorable prognostic factor in entire AML patients, indicating this genotype generates a disease entity in de novo AML. These results collectively suggest that TP53 mutation might be a functionally distinguishable class of mutation.

Cytogenetic evidence for partially committed myeloid progenitor cell origin of chronic myelomonocytic leukaemia and juvenile chronic myeloid leukaemia: both granuloctye-macrophage precursors and erythroid precursors carry identical marker chromosome

Summary. We used a micromethod for cytogenetic analysis from single haematopoietic colonies to study two adults with chronic myelomonocytic leukaemia (CMML) and a boy with juvenile chronic myeloid leukaemia (JCML) carrying distinct chromosome abnormalities, 7q—, der(21), and trisomy 21. We wanted to know if both granulocyte‐macrophage (GM) and erythroid precursors are involved in the abnormal clone. In all three patients, their chromosome abnormality was seen in almost all metaphases obtained from GM‐colonies and erythroid bursts. Peripheral blood leucocytes stimulated with phytohaemagglutinin exhibited only normal karyotypes. Clones of B‐cell produced by Epstein‐Barr virus had only normal karyotypes in the CMML patients. These findings indicate that CMML and JCML are clonal haemopathies that originate in a partially committed myeloid progenitor cell.

Long-term efficacy of imatinib in a practical setting is correlated with imatinib trough concentration that is influenced by body size: a report by the Nagasaki CML Study Group

Imatinib has dramatically improved long-term survival of chronic myelogenous leukemia (CML) patients. To analyze its efficacy in a practical setting, we registered most of CML patients in Nagasaki Prefecture of Japan. Of these, 73 patients received imatinib as an initial therapy. The overall survival rate of these patients was 88.7% at 6 years, and the cumulative complete cytogenetic response rate was 82.5% at 18 months. These results are comparable with the data of other reports including the IRIS study; however, the administered imatinib dose was smaller in our study than that in other reports. To address these discrepancies, we measured the trough concentration of imatinib among 35 patients. Although 39% of the patients were administered less than 400 mg/day, the trough level was comparable to those of previous reports. The trough level of imatinib showed a significant relationship with its efficacy, and was clearly related to dose of imatinib administrated and dose of imatinib divided by body surface area (BSA). Considering the smaller BSA of Japanese patients as compared to those of foreign origin, the results suggest that a lower dose of imatinib could maintain enough trough level and provided excellent results for the treatment of CML in our registry.