Kimio Morita

Clonality in chronic myeloproliferative disorders defined by X-chromosome linked probes: demonstration of heterogeneity in lineage involvement

Summary The restriction fragment length polymorphisms (RFLP) of the X‐chromosome phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyltransferase (HPRT) genes were used to study the clonal basis of the chronic myeloproliferative disorders (CMPD). Analyses were performed on granulocyte and T‐lymphocyte fractions obtained from 24 females; 13 had essential thrombocythaemia (ET), eight polycythaemia vera (PV) and three myelofibrosis with myeloid metaplasia (MMM). All 24 of these patients had monoclonal patterns of X‐inactivation in the granulocyte fraction. For the T‐lymphocyte fraction, non‐clonal patterns of Xinactivation were observed in 8/13 patients with ET, 7/8 with PV and 1/3 with MMM, while the remaining eight subjects were found to have monoclonal patterns of N inactivation. Our findings suggest that the majority of the CMPD in these patients originated from a relatively committed progenitor cell without the capacity to differentiate into T cells, and convincingly demonstrated heterogeneity of lineage involvement.

Clonality in myelodysplastic syndromes: demonstration of pluripotent stem cell origin using X‐linked restriction fragment length polymorphisms

Restriction fragment length polymorphisms (RFLP) of the X‐chromosome genes phosphoglycerate kinase (PGK) and hypoxanthine phorphoribosyltransferase (HPRT) were used to determine the clonal nature of myelodysplastic syndromes (MDS) in 22 patients. These included eight with refractory anaemia (RA), four with RA with ring sideroblasts (RARS), six with RA with an excess of blasts (RAEB), three with RAEB in transformation (RAEB‐T), and one with chronic myelomonocytic leukaemia (CMML). Monoclonal X‐inactivation patterns were observed in 19/22 patients. The remaining three cases, one each with RA, RARS and RAEB, were of polyclonal composition. Separated T‐lymphocyte and granulocyte fraction analyses in six patients of the former cases revealed that T‐lymphocyte as well as granulocyte fractions showed a monoclonal pattern of X‐inactivation. These results support the view that the majority of MDS arise from a pluripotent stem cell capable of myeloid and lymphoid differentiation.

Steroid‐refractory chronic idiopathic thrombocytopenic purpura associated with hepatitis C virus infection

Abstract: Objectives: Hepatitis C virus infection has often been suggested as a possible cause of various kinds of autoimmune diseases. The aim of this study was to determine the relationship between chronic idiopathic thrombocytopenic purpura (ITP) and hepatitis C virus infection and to characterize the clinical features of anti‐HCV antibody (HCVab) positive chronic ITP patients. Subjects and methods: We studied HCVab in 79 patients with chronic ITP (25 males, 54 females, mean age 42.3 yr, range 11–86 yr) using the third‐generation ELISA method. Results: HCVab was detected in 11 of the 79 patients (13.9%). Quantitative HCV‐RNA studies showed a high serum concentration of HCV‐RNA in these patients. The platelet counts in these 11 HCVab‐positive patients (Group 1) were lower than in the 68 HCVab‐negative patients (Group 2)[(2.6 ± 0.9) versus (4.9 ± 3.0) × 1010/L, respectively; p<0.02]. Significantly more patients in Group 1 required prednisolone therapy (10/11, 90.9%) than in Group 2 (31/68, 45.6%) (P < 0.005). The response rate to prednisolone treatment was significantly higher in Group 2 (19/31, 61.3%) than in Group 1(0/10, 0%) (P < 0.001). There was no difference in the response to splenectomy between Groups 1 (4/7, 57.1%) and 2 (3/5, 60%). Conclusion: Given these findings, we recommend that HCVab is measured upon diagnosis of chronic ITP, and that splenectomy is planned in patients with HCVab in the event that prednisolone treatment is ineffective.

A novel acute lymphoid leukaemia type BCR/ABL transcript in chronic myelogenous leukaemia

Using a reverse transcription‐polymerase chain reaction (RT‐PCR), we identified a patient with typical clinical features of chronic myelogenous leukaemia (CML) in the chronic phase who showed no amplification of the CML‐type BCR/ABL transcript. RT‐PCR with primers detecting the acute lymphoid leukaemia (ALL)‐type transcript disclosed a novel fragment co‐amplified with an ALL‐type fragment. Sequencing revealed the novel transcript to be a chimaeric mRNA produced by fusion of a segment of BCR exon 2 (e2) to ABL exon 2 (a2), with a 21 base‐pair insertion of ABL intron 1b sequence between them. This transcript has not been reported previously.

Detection of c-myc oncogene amplification in a CML blastic phase patient with double minute chromosomes

Double minute chromosomes (dmin) are relatively rare in leukemias. Cytogenetic analysis of blood cells from a woman with blastic phase chronic myelogenous leukemia (BC-CML) showed numerous dmin chromosomes and complex abnormalities including a Philadelphia (ph(1))-chromosome. Oncogene amplification in hematopoietic malignancies is also rare. Using PCR, we retrospectively investigated the extent of c-myc gene amplification in DNA extracted from stored blood smears from the patient. To qualify the PCR products, the beta-globin gene was used as the internal reference gene and it was co-amplified with the c-myc gene. The extent of amplified c-myc was about 6.8-fold. This finding suggests that the c-myc gene was amplified in dmin and that the gene amplification contributes to the progression to acute leukemia or rapid growth of leukemic cells.

The majority of T lymphocytes are polyclonal during the chronic phase of chronic myelogenous leukemia

To clarify the extent of cell lineage involvement in chronic myelogenous leukemia (CML), we investigated the bcr gene rearrangement and clonality using the X-chromosome-linked restriction fragment length polymorphism (RFLP) methylation method in T lymphocytes and granulocytes. We examined the granulocyte and T-cell fractions from the peripheral blood of seven female patients with CML during the chronic phase; patients were heterozygous for RFLPs at the phosphoglycerate kinase (PGK) or the hypoxanthine phosphoribosyltransferase (HPRT) gene. RFLP-methylation analysis of granulocytes demonstrated a monoclonal pattern in six of the seven patients and a rearrangedbcr gene in all seven patients. In contrast, T lymphocytes exhibited a polyclonal pattern in six cases; in one case, a faint band was observed following methyl-sensitive enzyme cleavage. Thebcr gene analysis in T lymphocytes showed the germline in every case. Our results indicate that the majority of T lymphocytes are polyclonal during the chronic phase of CML and confirm previous reports based on glucose-6-phosphate dehydrogenase, cytogenetic, andbcr rearrangement analyses.

Multiple Myeloma Presenting High Fever and High Serum Levels of Lactic Dehydrogenase, CRP, and Interleukin-6

Two myeloma patients presented high fever with no signs or data indicating infection at diagnosis or relapse. Both patients had plasmablastic myeloma, and serum levels of lactic dehydrogenase (LDH) and CRP were extremely high. Plasmablastic morphology, high LDH, and CRP were recognized as poor prognostic factors, indicating a fulminant phase of multiple myeloma. Interleukin‐6 (IL‐6) was only high in measured cytokines. We proposed that IL‐6 caused high fever and induced the fulminant phase in these 2 cases. Am. J. Hematol. 64:76–77, 2000.

A staging system for multiple myeloma based on the morphology of myeloma cells

Abstract: The morphology of myeloma cells is reported to be one of the prognostic factors in multiple myeloma (MM) patients. We analyzed the prognostic factors, including morphological classification, in 292 patients with MM in order to select poor‐risk patients who should be considered candidates for early intensive chemotherapy, including stem cell transplantation. Multivariate analysis was applied to 90 patients diagnosed between 1989 and 1996, because serum beta‐2‐microglobulin (β2M) has been measured regularly since 1989, and showed that serum albumin, serum β2M, and the morphology of myeloma cells predicted survival. According to these factors, patients were divided into 3 risk groups; a high‐risk group (14%), a intermediate‐risk group (46%) and a low‐risk group (40%). There were significant differences between survival times in these 3 groups (median survival: high‐risk, 16; intermediate‐risk, 22; and low‐risk, 44 months).

Clinical Outcome in Three Patients with Myelodysplastic Syndrome Showing Polyclonal Hematopoiesis

The clinical outcome of 3 myelodysplastic syndrome (MDS) patients with polyclonal hematopoiesis is reported. All patients were heterozygous for the phosphoglycerate kinase (PGK) gene. The presence of polyclonal hematopoiesis was determined by the X-chromosome-linked restriction fragment length polymorphism-methylation method using the PGK gene as a marker. The patients were initially diagnosed as having refractory anemia (RA), RA with ring sideroblasts (RARS), and RA with an excess of blasts (RAEB), respectively. Their pancytopenia persisted during the follow-up period of 11.4 years for the RA patient, 19.5 years for the RARS patient and 0.8 years for the RAEB patient. Although the RARS patient continues to be in good health, leukemic transformation occurred in the other 2 patients. A karyotype change from 46,XX to 45,XX,t(3;21),-7 was observed at the time of disease progression in the RA patient. The coexistence of a monoclonal MDS clone and normal bone marrow cells is thought to be the most probable reason for the polyclonal hematopoiesis of these patients.

Ki-1-Positive Large-Cell Anaplastic Lymphoma with Protean Manifestations Including Central Nervous System Involvement

A 26-year-old female with Ki-1-positive large-cell anaplastic lymphoma is reported. The neoplastic cells were phenotypically and genotypically of T cell origin. Initially, neoplastic cells invaded the skin and lymph nodes, and then invaded the sternal and vertebral bones, ribs and the iliopsoas muscle. Central nervous system (CNS) involvement with paraplegia, large tumor mass formation in her breast and pleural infiltration ensued in succession. This case illustrates the protean manifestations of Ki-1-positive large cell anaplastic lymphoma complicated with CNS involvement.