Michiko Okada

A summary of cytogenetic studies on 534 cases of chronic myelocytic leukemia in Japan

Cytogenetic and clinical data on 534 patients with chronic myelocytic leukemia (CML) were collected from 10 institutions in Japan. The results of the analysis of the data were in substantial accord with those of the First International Workshop on Chromosomes in Leukemia and other published data, but certain differences were noted in the frequency of Philadelphia chromosome (Ph1)-negative cases, unusual and complex Ph1 translocations, and additional chromosome changes. Some of the findings are discussed with respect to the origin of unusual and complex Ph1 translocations, the relationship between chromosome abnormalities and survival, and geographic differences in chromosome abnormalities.

Myeloproliferative disorders terminating in acute megakaryoblastic leukemia with chromosome 3q26 abnormality

Two cases of myeloproliferative disorders terminating in acute megakaryoblastic leukemia are reported. One case began as primary myelofibrosis and the other as chronic myelogenous leukemia. Blast cells in the acute leukemic phase were identified as megakaryoblasts by the presence of platelet peroxidase. The clinical course is described, and the morphology, immunologic studies, and ultrastructure studies of the blast cells are reported. On cytogenetic analysis both cases had a translocation involving the No. 3 chromosome locus q26.2. The present data suggest that 3q26 may be associated with transformation of the megakaryocytic lineage.

Clinical and cytogenetic remission induced by interferon-α in a patient with chronic eosinophilic leukemia associated with a unique t(3;9;5) translocation

A patient with chronic eosinophilic leukemia and a unique complex chromosomal translocation 46,XY,t(3;9;5)(q25;q34;q33) who had elevated blood interleukin‐5 is reported. Complete cytogenetic remission was induced by interferon‐α after treatment failure with corticosteroids and cytotoxic drugs. Severe cardiopulmonary symptoms due to hypereosinophilia and thromboembolic complication improved dramatically in the first 6 months of interferon therapy. Since it is known that the gene encoding for interleukin‐5 resides on the long arm of chromosome 5, it may be possible that the chromosomal translocation in our patient resulted in overproduction of this cytokine, and our findings may be helpful for understanding the pathogenesis of this disorder. Am. J. Hematol. 58:137–141, 1998.

Chromosome banding studies in 106 cases of chronic myelogenous leukemia

SummaryChromosome banding studies performed on 106 cases of CML in Sapporo revealed that 101 (95.3%) were Ph1-positive, and 5 (4.7%) Ph1-negative, the latter including a case of juvenile type CML. Of the 101 Ph1-positive patients, 98 showed the standard type Ph1 translocation, t(9;22)(q34;q11), while the remaining 3 had a complex Ph1 translocation as represented by t(4;9;22), t(9;14;22), or t(9;10;15;19;22). There were 28 patients who showed other chromosome changes in addition to the Ph1 translocation. Trisomy 8, duplication of Ph1, isochromosome 17q, and trisomy 19 were most frequently involved in the additional changes, and 2 or more of them often participated in the major routes of karyotypic evolution. Other additional changes observed were 6 translocations, 4 partial deletions, 2 partial trisomies for 1q, trisomies 6, 7, 12, 15, and 21, a monosomy 5, a partial duplication of no. 9, a missing Y, and so on.The present cytogenetic findings were evaluated with respect to some of the clinical and therapeutic parameters.

Lack of nucleophosmin mutation in patients with myelodysplastic syndrome and acute myeloid leukemia with chromosome 5 abnormalities

Nucleophosmin (NPM1) gene exon 12 mutations are frequently present in patients with acute myeloid leukemia (AML) with normal karyotype. The NPM1 gene is located on chromosome 5q35, which is often affected in myeloid malignancies including myelodysplastic syndrome (MDS). This suggests that the NPM1 gene is a one of the target genes affected by chromosome 5 abnormalities and play a role in the development of MDS. It has not been clarified whether MPM1 mutations are present in patients with MDS and AML with chromosome 5 abnormalities. Therefore, we carried out a mutational analysis on the NPM1 gene exon 12. NPM1 mutations were not detected in the 28 patients with MDS and AML with chromosome 5 abnormalities.

A summary of cytogenetic, morphologic, and clinical data on t(8q−;21q+) and t(15q+;17q−) translocation leukemias in Japan

Cytogenetic, morphologic, and clinical data of 33 acute nonlymphocytic leukemia (ANLL) patients with t(8q - ;21q+) and 19 patients with acute promyelocytic leukemia (APL) were collected from seven laboratories in Japan. The latter class included 18 patients with t(15q + ;17q-) and one with a normal karyotype. The t(8q - ;21q+) and t(15q + ;17q-) translocations were each shown to be associated with a specific type of ANLL, namely, AML-M2 and APL-M3, respectively. No patient with APL had the M3 variant. The t(8q - ;21q+) translocation seems to be more common as an abnormality in ANLL in Japan as compared to findings in other countries. The high incidence of t(15q + ;17q-) among Japanese patients with APL indicated in this study, however, still awaits confirmation.

L265P Mutation of the MYD88 Gene Is Frequent in Waldenström’s Macroglobulinemia and Its Absence in Myeloma

L265P mutation in the MYD88 gene has recently been reported in Waldenström’s macroglobulinemia; however the incidence has been different according to the methods used. To determine the relevance and compare the incidence by different methods, we analyzed the L265P mutation in bone marrow mononuclear cells from lymphoid neoplasms. We first performed cloning and sequencing in 10 patients: 8 Waldenström’s macroglobulinemia; 1 non-IgM-secreting lymphoplasmacytic lymphoma; and 1 low grade B-cell lymphoma with monoclonal IgG protein. The L265P mutation was detected in only 1/8 Waldenström’s macroglobulinemia patients (2 of 9 clones). To confirm these results, direct sequencing was performed in the 10 patients and an additional 17 Waldenström’s macroglobulinemia patients and 1 lymphoplasmacytic lymphoma patient. Nine of 28 patients (7/25 Waldenström’s macroglobulinemia, 1/2 lymphoplasmacytic lymphoma, and B-cell lymphoma) harbored the mutation. We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma. Aberrant bands corresponding to the mutation were detected by BSiE1 digestion in 19/25 patients with Waldenström’s macroglobulinemia (76%), 1/2 lymphoplasmacytic lymphoma and B-cell lymphoma, but not in the 38 myeloma patients. The L265P mutation was more frequent in patients with Waldenström’s macroglobulinemia than in those with myeloma (p=1.3x10-10). The mutation was detected by allele-specific polymerase chain reaction in 18/25 Waldenström’s macroglobulinemia patients (72%). In the 25 Waldenström’s macroglobulinemia patients, the L265P was more frequently detected by BSiE1 digestion than by direct sequencing (p=5.3x10-4), and in males (15/16, 94%) than in females (4/9, 44%) (p=1.2x10-2). No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32). These results suggest that the L265P mutation is involved in the majority of Waldenström’s macroglobulinemia. BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.

Quantitative analysis of chromosomal G-bands in human hematopoietic disorders by methotrexate synchronization technique

Chromosomal G-bands in an improved morphology were demonstrated in bone marrow and unstimulated blood cultures from 12 patients with hematopoietic disorders and 1 with another disease. The procedure used was a cell synchronization technique involving methotrexate or cytosine-arabinoside and thymidine release. Quantitative analyses were made comparatively between synchronized and nonsynchronized (control) cultures in each patient. Mitotic indices varied with the individual. The average number of G-bands per haploid set increased statistically in 8 cases, and the count was often over 400. Improved chromosomal banding patterns in the synchronized preparation resulted in easy and precise identification of chromosome rearrangements in three patients with chronic myeloid leukemia (CML), two with acute myeloblastic leukemia (AML), and three with anemias. Normal cells were more frequently observed in synchronized overnight cultures than in control cultures from two patients with an admixture of normal and abnormal metaphases. There was a slight increase in a minor abnormal clone in the synchronized cultures from one patient. These findings indicate that the cell synchronization technique we use is not only useful in improving chromosome morphology, but is also efficient in detecting both normal and abnormal metaphases.

Microarray CGH analyses of chromosomal 20q deletions in patients with hematopoietic malignancies

The chromosomal abnormality del(20q) is mostly found in various myeloid disorders, including myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemia. Here, microarray comparative genomic hybridization (aCGH) analyses of 14 patients cytogenetically confirmed to carry the del(20q) aberration in their bone marrow demonstrated that all deletions were interstitial and both the proximal and distal breakpoints varied among individuals. The centromeric breakpoints were located in the 20q11.21-12 region, and the telomeric breakpoints, in the 20q13.13-13.33 region. The extent of the deletion ranged from 11.2 to 27.3 Mb, and the commonly deleted region (CDR) was estimated to be 7.2 Mb in size. Two commonly retained regions were present, the proximal region adjacent to the centromere (20q11.1-11.21) and a subtelomeric one (20q13.33). The CDR of our study was more distal than reported previously. Furthermore, in three patients fluorescence in situ hybridization (FISH) demonstrated that del(20q) cells were detected at a higher frequency in the karyotype analyses than by interphase FISH and aCGH analyses. As the size and breakpoints of del(20q) have been reported to vary among patients, the presence of one or more tumor suppressor genes in the CDR has been suggested. Our study will contribute to the identification of candidate tumor suppressor genes on 20q.

Aberrant methylation of the RIZ1 gene in myelodysplastic syndrome and acute myeloid leukemia.

We performed methylation specific PCR analysis on the RIZ1 promoter in MDS and AML. Methylation was detected in 17 of 34 MDS (50%) and 22 of 72 AML (31%) (p=0.053). Methylation was detected in eleven of 17 secondary AML from MDS (65%), and eleven of 55 de novo AML (20%) (p=0.0005). Bisulfite sequence revealed methylation at many CpG sites in the promoter. Decreased RIZ1 expression was accompanied by methylation in six of nine samples examined, while it was also observed in seven of 13 without methylation. Treatment of AML cells, that have RIZ1 methylation, with 5-Aza-dC, induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene is inactivated in MDS and AML in part by methylation, whereas another mechanism should be involved in others.