ALBERT

Description: Imatinib mesylate (Glivec; Novartis, Basel, Switzerland), which is a selective inhibitor of the Bcr/Abl tyrosine kinase, is now widely used for the treatment of Ph-positive leukemia based on the remarkable efficacy. Treatment with imatinib mesylate is generally well tolerated, and the risk for severe adverse effects is low. Hepatic toxicity is less common and usually resolves with interruption of imatinib therapy. On the other hand, although it is well known that HBV reactivations are observed in cancer patients with chronic HBV infection during chemotherapies, imatinib mesylate-induced HBV reactivation has not been reported yet. Here, we report the first case complicated by fatal fulminant HBV reactivation during imatinib mesylate treatment for CML. A 54-year-old man was diagnosed as CML on October 2003. At that time, HBsAg, HBeAb, and HBcAb were positive, whereas HBsAb was negative. HCV Ab and HCV RNA were also positive. However, hepatic examinations revealed normal findings, except slightly elevated levels of AST (50 IU/L). On November 2003, imatinib mesylate was started at the dose of 400mg/day p.o. and continued without any hepatic adverse effects. In contrast, the patient was suffered from neutropenia (grade 2) and lymphocytopenia (grade 2) on December 2003. Although dose of imatinib mesylate was reduced (300mg/day), lymphocytopenia continued. Since May 6, 2004, the patient complained general fatigue. On May 11, AST, ALT, and total bilirubin were 125 IU/L, 95 IU/L, and 0.7 mg/dl, respectively. At that time, bone marrow cytogenetics using a FISH of Bcr/Abl detected 2.0% fusion gene. Then, hepatic function deteriorated despite imatinib mesylate administration was stopped. On May 27, the patient had severe hepatic dysfunction with high AST, ALT, and total bilirubin (2098 IU/L, 1574 IU/L, and 5.6 mg/dl, respectively). In addition, prolonged prothrombin time (13.6%) and deterioration of consciousness were observed. HBV DNA polymerase was extremely increased (〉 20000 cpm; normal range:

Description: It is unclear how a paroxysmal nocturnal hemoglobinuria (PNH) clone expands and bone marrow failure (BMF) occurs in PNH patients, although an immunologic mechanism by human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTLs) has been suggested. It has been also reported that immunization with HLA-binding peptides of Wilms’ tumor gene (WT1) in hematopoietic cells induces a WT1 peptide-specific CTL response, and WT1 RNA is highly expressed in BM mononuclear cells (MNCs) in PNH patients (Shichishima T et al., Blood, 2002). In this study, to clarify some roles of WT1 peptide-specific and HLA-restricted CTLs, the frequencies of peripheral blood (PB) WT1 peptide-specific and HLA-A*2402-restricted CTLs by flow cytometric tetramer analysis and WT1 peptide-stimulated interferon (IFN)-γ-producing MNCs by enzyme-linked immunospot assay in 5 PNH patients with the HLA-A*2402 allele were examined. We also investigated cytotoxicity of WT1 peptide-specific and HLA-A*2402-restricted CTL clone (TAK-1) cells on BM MNCs by 51Cr-releasing assay, colony forming-unit granulocyte-macrophage colony formation of CD34+CD59+ and CD34+CD59− cells, and CD59 expression in viable 7AAD−CD34+ cells by flow cytometry in those patients, and expression of IFN-γ in TAK-1 cells by flow cytometry, after co-incubation of BM cells from them with TAK-1 cells. As controls, 8 healthy volunteers (HV) with the HLA-A*2402 allele and 2 PNH patients and HV without the allele were examined. We found that the frequencies of PB WT1 peptide-specific and HLA-A*2402-restricted CD8+ cells (p

Description: To investigate erythropoiesis in paroxysmal nocturnal hemoglobinuria (PNH), we studied the expression of glycosylphosphatidylinositol (GPI)-anchored membrane proteins on circulating erythrocytes and erythroblasts obtained by erythropoietic cell culture in nine patients with this disease. One-color and two-color flow cytometric analyses were performed using monoclonal antibodies for decay-accelerating factor (DAF ) and/or CD59/membrane attack complex-inhibitory factor (MACIF). In addition, terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL) analysis was performed to assess apoptosis of erythroblasts from six patients. On flow cytometric analysis, cases 1 to 6 had positive and negative erythrocyte populations, case 7 intermediate and negative populations, case 8 positive, intermediate, and negative populations, and case 9 a single double-negative population. In addition, cases 1 to 6 and 8 had positive, intermediate, and negative erythroblast populations, while cases 7 and 9 had intermediate and negative populations. The percentage of double-negative erythrocytes showed a significant correlation with that of double-negative erythroblasts (r = .741, P 〈 .05). In seven of nine patients, more erythroblasts than erythrocytes were negative for the two membrane proteins. Also, some patients with an intermediate population of erythrocytes did not necessarily show an increase of PNH II erythroblasts. Apoptosis of PNH erythroblasts was also detected, but the percentage of apoptotic cells in PNH patients showed no difference from that in healthy volunteers. These findings suggest that the final phenotype of mature erythrocytes in PNH is determined during maturation from erythroblasts to erythrocytes by the disappearance or persistence of PNH II erythroblasts. In addition, PNH erythroblasts in vitro may be partly lost by apoptosis, but apoptosis does not play an important role in determining GPI-linked protein expression.

Description: To investigate erythropoiesis in paroxysmal nocturnal hemoglobinuria (PNH), we studied the expression of glycosylphosphatidylinositol (GPI)-anchored membrane proteins on circulating erythrocytes and erythroblasts obtained by erythropoietic cell culture in nine patients with this disease. One-color and two-color flow cytometric analyses were performed using monoclonal antibodies for decay-accelerating factor (DAF ) and/or CD59/membrane attack complex-inhibitory factor (MACIF). In addition, terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end-labeling (TUNEL) analysis was performed to assess apoptosis of erythroblasts from six patients. On flow cytometric analysis, cases 1 to 6 had positive and negative erythrocyte populations, case 7 intermediate and negative populations, case 8 positive, intermediate, and negative populations, and case 9 a single double-negative population. In addition, cases 1 to 6 and 8 had positive, intermediate, and negative erythroblast populations, while cases 7 and 9 had intermediate and negative populations. The percentage of double-negative erythrocytes showed a significant correlation with that of double-negative erythroblasts (r = .741, P 〈 .05). In seven of nine patients, more erythroblasts than erythrocytes were negative for the two membrane proteins. Also, some patients with an intermediate population of erythrocytes did not necessarily show an increase of PNH II erythroblasts. Apoptosis of PNH erythroblasts was also detected, but the percentage of apoptotic cells in PNH patients showed no difference from that in healthy volunteers. These findings suggest that the final phenotype of mature erythrocytes in PNH is determined during maturation from erythroblasts to erythrocytes by the disappearance or persistence of PNH II erythroblasts. In addition, PNH erythroblasts in vitro may be partly lost by apoptosis, but apoptosis does not play an important role in determining GPI-linked protein expression.

Description: PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome. It is considered that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) and interferon-γ (IFN-γ) contribute to hypoplastic bone marrow of these disorders. In addition, PNH is an acquired clonal disorder of the hematopoietic stem cell. Recently, it has been reported that analysis of T cell-antigen receptor (TCR)-Vβ repertoires, especially TCR-Vβ CDR3 (complementarity- determining region 3) spectrotypes, is an effective tool to study immunologic mechanisms by CTLs in pathophysiology of PNH (Karadimitris et al, Blood, 2000; Kook et al, Blood, 2002; Risitano et al, Blood, 2002). In the present study, we investigated 21 kinds of TCR-Vβ repertoires by flow cytometry in CD4 and CD8 lymphocytes from 5 PNH patients and a healthy volunteer and the TCR-Vβ CDR3 spectrotypes using polymerase chain reaction assay in CD4 and CD8 lymphocytes from 3 of 5 PNH patients and the control. We also quantitated intracellular IFN-γ in CD4 and CD8 lymphocytes from 5 PNH patients and the control according to the method by Sloand et al (Blood, 2002). We found no specific TCR-Vβ repertoires in CD4 and CD8 lymphocytes from PNH patients compared with the control. The TCR-Vβ repertoires with relative increase of CD4 or CD8 lymphocytes (over 10 of ratio of the proportion of each TCR-Vβ repertoire in a PNH patient/the proportion of the same TCR-Vβ repertoire in a healthy volunteer) were 13.6 or 4 and 22 in Case 1, 3 and 11 or 1 in Case 2, 3 and 13.6 or 3 in Case 3, 5.3 and 7.2 or 2, 3, 7, and 18 in Case 4, and 4, 5.2, 13.6, 16, and 23 or 1 and 14 in Case 5, respectively. TCR-Vβ CDR3 spectrotyping showed that in CD4 lymphocytes most CDR3 patterns were chiefly polyclonal, except for one oligoclonal (Case 1) and one monoclonal (Case 3) patterns of TCR-Vβ25; in CD8 lymphocytes most CDR3 consisted of polyclonal, oligoclonal, and/or monoclonal patterns, suggesting the possibility that CD8 lymphocytes recognize much more antigens of abnormal cells, probably including PNH clones, than CD4 lymphocytes. Unfortunately, we found the same patterns as described above in CD8 lymphocytes from the control, although CD4 lymphocytes from the control presented only polyclonal pattern of CDR3. Quantitative analyses of IFN-γ showed that index values of IFN-γ in CD4 and CD8 lymphocytes from PNH patients were higher than those from the control. However, we did not find any significant correlations between the spectrotypes of TCR-Vβ CDR3 and the index values of IFN-γ in PNH patients, suggesting that TCR-Vβ repertoires with monoclonal and oligoclonal CDR3 patterns do not necessarily produce much IFN-γ. In conclusion, our findings suggest that TCR-Vβ CDR3 spectrotyping is more effective tool to resolve some immune mechanisms of pathophysiology in PNH, especially by auto-reactive CTLs.

Description: To define the phosphatidylinositol glycan-class A (PIG-A) gene abnormality in precursor cells and the changes of expression of glycosylphosphatidylinositol-anchored protein and contribution of paroxysmal nocturnal hemoglobinuria (PNH) clones with PIG-A gene abnormalities among various cell lineages during differentiation and maturation, we investigated CD59 expression on bone marrow CD34+ cells and peripheral granulocytes from 3 patients with PNH and the PIG-A gene abnormalities in the CD59−, CD59+/−, and CD59+ populations by nucleotide sequence analyses. We also performed clonogeneic assays of CD34+CD59+ and CD34+CD59− cells from 2 of the patients and examined the PIG-A gene abnormalities in the cultured cells. In case 1, the CD34+ cells and granulocytes consisted of CD59− and CD59+ populations and CD59−, CD59+/−, and CD59+populations, respectively. Sequence analyses indicated that mutation 1-2 was in the CD59+/− granulocyte population (20 of 20) and the CD34+CD59− population (2 of 38). In cases 2 and 3, the CD34+ cells and granulocytes consisted of CD59+ and CD59− cells. Sequence analyses in case 3 showed that mutation 3-2 was not in CD34+CD59− cells and was present in the CD59− granulocyte population. However, PIG-A gene analysis of cultured CD34+CD59− cells showed that they had the mutation. This analysis also revealed that there were some other mutations, which were not found in CD34+CD59− cells and CD59− or CD59+/− granulocytes in vivo, and that sometimes they were distributed specifically among different cell lineages. In conclusion, our findings suggest that PNH clones might contribute qualitatively and quantitatively differentially to specific blood cell lineages during differentiation and maturation of hematopoietic stem cells.

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is one of the bone marrow failure syndromes, including aplastic anemia (AA) and myelodysplastic syndromes (MDS). Recently, the International PNH Interest Group proposed that evidence of a population of erythrocytes and granulocytes deficient in glycosylphosphatidylinositol (GPI) proteins and assessment of hemolytic parameters, including haptoglobin concentration, are important as minimal essential diagnostic criteria of PNH and that less than 1.0% GPI-deficient erythrocytes and granulocytes identifies subclinical PNH from classic PNH (Parker C et al, Blood, 2005). To know whether haptoglobin can be a hallmark which expects the occurrence of classic PNH during the clinical course in AA and MDS patients, we examined the expressions of CD59 on erythrocytes and granulocytes by flow cytometry and relationship between proportions of negative populations of them and various clinical parameters, including haptoglobin concentrations, in Japanese patients with AA (n=23; M:F=11:12; 50.5 ± 19.1 years), PNH (n=28; M:F=14:14; 42.7 ± 16.1 years), and MDS (n=29; M:F=20:9; 66.1 ± 13.9 years). Less than 20 mg/dl of haptoglobin were judged as significant decrease. Flow cytometry showed that the proportions of CD59− erythrocytes (38.11 ± 35.49%) and granulocytes (52.57 ± 42.39%) from PNH patients were significantly higher than those from AA and MDS patients and healthy individuals (n=21; M:F=12:9; 41.3 ± 12.2 years). The values of serum asparatate aminotransferase (AST) and lactate dehydrogenase (LDH) were significantly higher in PNH patients (54.9 ± 53.1U/l and 1035 ± 1052 U/l, respectively) than AA (20.8 ± 9.1 U/l and 205.8 ± 45.0 U/l, respectively) and MDS (22.4 ± 16.9 U/l and 217.7 ± 64.0 U/l, respectively) patients. In contrast, the concentrations of serum haptoglobin were significantly lower in PNH patients (12.9 ± 27.6 mg/dl) than AA (84.6 ± 81.9 mg/dl) and MDS (77.7 ± 47.3 mg/dl) patients. When comparing PNH patients (n=9; minimal PNH) with less than 5% of CD59− erythrocytes with those (n=19; bulky PNH) with over 5% of CD59− erythrocytes, the values of AST (70.7 ± 58.2U/l) and LDH (1021 ± 1083U/l) of the latter were significantly higher than those of the former (21.4 ± 6.2U/l and 220.3 ± 45.1U/l, respectively), but the concentrations of haptoglobin were similar between the latter (11.8 ± 28.9mg/dl) and the former (15.8 ± 26.0mg/dl). In addition, all the PNH patients, but not AA and MDS patients, with over 1% of CD59− erythrocytes had significant decrease of haptoglobin concentration, suggesting that low concentrations of serum haptoglobin may predict occurrence of classic PNH during the clinical course in AA and MDS patients. In conclusion, over 1% of CD59− erythrocytes in PNH patients certainly cause clinical hemolysis and serum haptoglobin is a useful marker which can predict the occurrence of classic PNH.

Description: PNH is an acquired hematologic disorder which is characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia (AA) and myelodysplastic syndrome (MDS). It is well known that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) contribute to pathophysiology of these disorders. In fact, some reports (Maciejewski et al, Blood, 2001; Shichishima et al, Blood, 2002) showed that HLA-DR*1501 is related with clinical pathophysiology of PNH. In this study, to clarify significance of CD8+ CTLs in pathophysiology of PNH, we investigated HLA class I (A and B) alleles in Japanese patients with PNH (female: male=7:17), AA (female: male=14:15), and MDS (female: male=6:16) by high-resolution method using polymerase-chain reaction after obtaining informed consent and approval from the institutional Human Research Committee. Mean age ± standard deviation of PNH, AA, and MDS patients was 52 ± 16, 54 ± 20, and 59 ± 18, respectively. HLA genotyping showed that the frequency of HLA-A*0206 allele in PNH patients (22.9%) was significantly different from those in 309 unrelated Japanese individuals (Saito et al, Tissue Antigens, 2000) (7.7%; p

Description: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder affecting all hematopoietic lineages, which lack glycosylphosphatidylinositol (GPI)-anchored membrane proteins due to somatic mutations in the phosphatidylinositol glycan-class A gene, and is one disorder of bone marrow failure (BMF) syndromes. Autoreactive T lymphocytes are implicated in some of the immune mechanisms involved in PNH. In fact, we reported recently that the HLA-DRB1*1501 allele and HLA-A*0206 allele is frequent and is related to grading of hemolysis, respectively, in PNH patients (Shichishima T et al, Blood, 2002 and Haematologica, 2006, respectively). However, some characteristics of CD4+ and CD8+ T lymphocytes, including GPI-negative CD4+ and CD8+ T lymphocytes, in PNH patients remain unknown. To know some characteristics of CD4+ and CD8+ T lymphocytes with and without expressions of GPI proteins in PNH, we examined preferential variable beta chain (Vβ) repertoires of the T-cell receptor (TCR) and expressions of interferon-γ (IFN-γ) by flow cytometry and the TCR Vβ complementarity-determining region 3 (CDR3) spectratypes by genetic methods at the same time in CD4+CD59+, CD4+CD59−, CD8+CD59+, and/or CD8+CD59− T lymphocytes from 10 Japanese patients, including 6 and 4 with the HLA-DRB1*1501 allele and HLA-A*0206 allele, respectively, and from 5 age-matched healthy individuals. In the analyses of TCR Vβ repertoires, over-expressed TCR Vβ subfamilies were found in any T lymphocytes subsets from all the patients. We found significantly higher numbers (mean ± standard deviation; 1.9 ± 1.2) of over-expressed TCR Vβ subfamilies in CD8+CD59+ T lymphocytes from PNH patients compared with those (0 ± 0, p