Masamitsu Karasawa

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.

Hair dye use and occupational exposure to organic solvents as risk factors for myelodysplastic syndrome

To investigate the relationships of personal hair dye use and environmental factors to myelodysplastic syndromes (MDS), we conducted a case-control study in Japan. A total of 111 MDS cases and 830 controls randomly selected from the residents in the same prefecture of cases using telephone directories responded to a health questionnaire. The odds ratio (OR) for ever having used hair dye was 1.99 (95% confidence interval (CI) 1.17-3.38) and there were statistically significant trends in risk with increasing duration and number of hair dye use. Occupational exposure to organic solvents was marginally associated with the risk of MDS (OR = 1.99; 95% CI 0.97-4.10).

Long-term responses and outcomes following immunosuppressive therapy in large granular lymphocyte leukemia-associated pure red cell aplasia: a Nationwide Cohort Study in Japan for the PRCA Collaborative Study Group

This report describes long-term responses following immunosuppressive therapy in large granular lymphocyte leukemia-associated pure red cell aplasia. Large granular lymphocyte leukemia-associated pure red cell aplasia accounts for a significant portion of secondary pure red cell aplasia cases. However, because of its rarity, long-term responses and relapse rates after immunosuppressive therapy are largely unknown. We conducted a nationwide survey in Japan and collected 185 evaluable patients. Fourteen patients with large granular lymphocyte leukemia-associated pure red cell aplasia were evaluated. Cyclophosphamide, cyclosporine A and prednisolone produced remissions in 6/8, 1/4 and 0/2 patients respectively. Seven and 5 patients were maintained on cyclophosphamide or cyclosporine A respectively. Two patients relapsed after stopping cyclophosphamide, and 2 patients relapsed during maintenance therapy with cyclosporine A. The median relapse-free survival in the cyclophosphamide - and the cyclosporine A groups was 53 and 123 months respectively. Large granular lymphocyte leukemia-associated pure red cell aplasia showed a good response to either cyclophosphamide or cyclosporine A. Most patients continued to receive maintenance therapy and it remains uncertain whether cyclophosphamide or cyclosporine A can induce a maintenance-free hematologic response in large granular lymphocyte leukemia-associated pure red cell aplasia.

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.

Immunological abnormalities in splenic marginal zone cell lymphoma

The clinical features of patients with splenic marginal zone cell lymphoma (SMZCL) have rarely been reported. In the present study, immunological abnormalities, particularly hematological abnormalities, observed in SMZCL were described. Autoimmune hemolytic anemia, immune thrombocytopenia, and appearance of lupus anticoagulant were observed in 2 of 3 patients with SMZCL. Other abnormal data including monoclonal gammopathy and cold agglutinin were also observed in 2 of the 3 patients. Immunological abnormalities may be characteristic complications in patients with SMZCL and must be followed carefully, since they may be a reliable marker of this type of lymphoma activity. Am. J. Hematol. 56:173–178, 1997.

Prevalence and Clinical Characteristics of Acute Myeloid Leukemia Associated with Disseminated Intravascular Coagulation

Disseminated intravascular coagulation (DIC) is one of the important complications to develop in patients with acute myeloid leukemia (AML). While acute promyelocytic leukemia (APL) is a strong risk factor for DIC, other clinical features have not been fully defined. We retrospectively analyzed 161 consecutive adult patients with de novo non-APL AML. DIC was diagnosed in 52 patients (32%); 28 patients at diagnosis and 24 soon after the initiation of induction chemotherapy. Leukocyte counts, C-reactive protein, and lactate dehydrogenase were significantly higher in the DIC+ group. Negative expressions of CD13, CD19, CD34, and HLA-DR were more prevalent in the DIC+ group. On multivariate logistic-regression analysis, variables that were independently associated with the development of DIC were high C-reactive protein, high leukocyte count, negative expressions of CD13 and HLA-DR, and cytogenetics with a normal karyotype or 11q23 abnormality. Although DIC is considered to be associated with serious morbidity and occasional mortality, we did not find any significant differences in the complete remission rate, overall or disease-free survival between DIC+ and DIC- groups. This study is the first to define the clinical characteristics associated with DIC in patients with non-APL AML, but exactly how and when DIC should be treated remains to be determined.

Improvement of extrathymic T cell type of large granular lymphocyte (LGL) leukemia by cyclosporin A: the serum level of Fas ligand is a marker of LGL leukemia activity

Abstract: We report a case of γδ T‐cell‐type large granular lymphocyte (LGL) leukemia (CD3+,CD8+, CD57+,TCR γδ+), which was accompanied by pure red cell aplasia, neutropenia and thrombocytosis. Southern blotting analysis of the T‐cell receptor β gene showed the germline configuration, but clonal TCR J γ rearrangements were identified. These granular lymphocytes demonstrated non‐major histocompatibility complex‐restricted cytotoxicitity. The serum‐soluble FasL (sFasL) concentration of this patient was very high, whereas the serum levels of tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), interleukin‐1 beta (IL‐1β), interleukin‐2 (IL‐2) and thrombopoietin were normal. After treatment with cyclosporin A, anemia and thrombocytosis were improved, and LGL and the elevated sFasL concentration decreased. These observations suggested that FasL may have played a role in the establishment of the clinical symptoms of this patient and could be useful as an indicator of disease activity.

JAK2-V617F mutation analysis of granulocytes and platelets from patients with chronic myeloproliferative disorders: advantage of studying platelets

There have been conflicting reports over the JAK2‐V617F mutation status of platelets in chronic myeloproliferative diseases (CMPDs). The aim of this study was to analyse JAK2‐V617F status, not only in granulocytes but also in platelets. The JAK2‐V617F mutation was analysed in both granulocytes and platelets in 115 patients with CMPDs using direct sequencing. JAK2‐V617F was detected in granulocytes from 71 of those patients, all 71 of whom also had platelet JAK2‐V617F expression. The remaining 44 patients showed negative JAK2‐V617F expression on granulocytes, but positive JAK2‐V617F expression was detected on the platelets from nine of the 33 essential thrombocythaemia (ET) patients, one of the eight polycythaemia vera patients, and two of the three primary myelofibrosis patients. When ET patients were divided into three groups according to granulocyte and platelet JAK2‐V617F status (both‐positive, platelets‐only positive and both‐negative), the both‐positive and platelets‐only positive groups shared the clinical features of higher white blood cell count and frequent thrombosis. These results suggest that analysis of platelets is a more sensitive approach for detecting JAK2‐V617F in CMPD patients than analysis of granulocytes. They also suggest that previous reports of the incidence of JAK2‐V617F in CMPD patients, obtained using only analysis of granulocytes, could be underestimations.