Objective. Although a number of reports have documented a significantly increased incidence of HLA-DR15 in aplastic anemia (AA), the exact role of HLA-DR15 in the immune mechanisms of AA remains unclear. We herein clarify the difference between DRB1*1501 and DRB1*1502, the 2 DRB1 alleles which determine the presentation of HLA-DR15, in the pathophysiology of AA. Materials and Methods. We investigated the relationships of the patients’ HLA-DRB1 allele with both the presence of a small population of CD55CD59 (PNH-type) blood cells and the response to antithymocyte globulin (ATG) plus cyclosporine (CsA) therapy in 140 Japanese AA patients. Results. Of the 30 different DRB1 alleles, only DRB1*1501 (33.6% vs. 12.8%, Pc<0.01) and DRB1*1502 (43.6% vs. 24.4%, Pc<0.01) displayed significantly higher frequencies among the AA patients than among a control. AA patients possessing HLA-DR15 tended to be old, and especially, the frequency of DRB1*1502 in patients ≥40 years old (52.4%) was markedly higher than that in those <40 years old (16.2%, Pc<0.01). Only DRB1*1501 was significantly associated with the presence of a small population of PNH-type cells and it also showed a good response to ATG plus CsA therapy in a univariate analysis. A multivariate analysis showed only the presence of a small population of PNH-type cells to be a significant factor associated with a good response to the immunosuppressive therapy (P<0.01). Conclusion. Although both DRB1*1501 and DRB1*1502 contribute to the development of AA, the methods of contribution differ between the two alleles.

Pure red cell aplasia (PRCA) is characterized by normocytic normochromic anemia associated with reticulocytopenia and erythroid hypoplasia in otherwise normal bone marrow. The acquired chronic PRCA may present as a primary hematological disorder in the absence of any other diseases or develop associated with certain conditions such as thymoma, lymphoproliferative disorders, infections, collagen vascular diseases, or after exposure to a wide variety of drugs. Idiopathic PRCA and secondary PRCA not responding to the treatment of the underlying diseases are generally treated by immunosuppressive therapy and the most patients require long-term treatment. Glucocorticoids, cyclosporine and cyclophosphamide are commonly used for initial treatment as monotherapy or combination therapy and majority of the patients respond to immunosuppression. However, most patients treated with these medications relapse during tapering of the treatment. Because of the extreme rarity of this disease, the long-term outcome of chronic PRCA following immunosuppression remains unclear and the adverse risk factors for survival have to be elucidated.

Immunosuppressive therapy has been employed as the initial treatment for acquired chronic pure red cell aplasia (PRCA), such as idiopathic, thymoma-associated, or large granular lymphocyte (LGL) leukaemia-associated PRCA, which is thought to be immune-mediated. To explore the overall long-term outcome following immunosuppression and to identify the risk factors for death in these disorders, we conducted nationwide surveys in Japan 2004 and 2006, and identified a total of 185 patients with acquired chronic PRCA, including 72 idiopathic, 41 thymoma-associated and 14 LGL leukaemia-associated cases of PRCA for whom data was available. The present study evaluated 127 patients with these three subsets of PRCA. The median overall survival has not yet been reached in idiopathic PRCA. The estimated median overall survival times in patients with thymoma-associated and LGL leukaemia-associated PRCA were 142·1 and 147·8 months, respectively. Twenty-two deaths were reported, and the response to induction therapy and relapse of anaemia were found to be associated with death. The major causes of death were infection in seven patients and organ failure in another seven patients. The results suggest that maintenance therapy and the management of infectious complications are crucial for improving the prognosis of chronic PRCA.

Myelodysplastic syndromes (MDS) are heterogeneous clonal disorders characterized by cytopenias that arise due to ineffective haematopoiesis and morphological dysplasia and carry an increased risk of incident acute myeloid leukaemia. The pathogenesis of marrow dysfunction in MDS is multifactorial and consistent with a multistep model and may lead to heterogeneity of MDS. We investigated the proteome profile of circulating neutrophils purified from patients with refractory cytopenia with multilineage dysplasia (RCMD) to identify proteins that have a role in the pathogenesis. Using 2-dimensional difference gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we found that peroxiredoxin 2 (PRDX2), a member of the peroxiredoxin family that regulates reactive oxygen species, was markedly upregulated in neutrophils of RCMD patients compared to healthy donors. Increased PRDX2 expression in the neutrophils of RCMD patients was confirmed using quantitative reverse transcription polymerase chain reaction, immunoblotting and immunocytochemical analysis. In addition, white blood cell and neutrophil counts in RCMD patients correlated inversely with the PRDX2 expression of. Oxidative stress is a known factor involved in the pathogenesis of MDS, and PRDX2 is associated with tumourigenesis of several solid tumours. Accordingly, our results suggest that PRDX2 may perform an important function in the pathogeneis of RCMD.

A study to evaluate WT1 mRNA expression levels in peripheral blood (PB) and bone marrow aspirate (BM) was conducted in 172 patients, including 115 with myelodysplastic syndromes (MDS), in Japan. The level of WT1 mRNA expression was evaluated according to the French-American-British (FAB) and World Health Organization (WHO) classifications (2001, 2008) and using the International Prognostic Scoring System and the WHO Prognostic Scoring System scales. WT1 mRNA expression levels in PB and BM were well correlated (r = 0.85), and they tended to increase with disease stage progression and in those at higher risk of leukemic transformation. WT1 mRNA expression can be a useful marker for the diagnosis and risk evaluation of MDS.

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.

Prednisone is the most common first-line treatment for adult primary immune thrombocytopenia (ITP). However, the best initial therapeutic approach is still a matter of debate. Prior studies have shown that high-dose dexamethasone (HD-DXM) produces a high sustained efficacy not achieved by conventional prednisone therapy. However, the definition of response widely differs between individual reports, and this heterogeneity makes comparison of the efficacy difficult. The aim of our study was to compare the therapeutic outcomes of a conventional dose of prednisone with HD-DXM for adult ITP patients as initial therapy. Thirty patients treated with prednisone and 22 patients treated HD-DXM were retrospectively analyzed. No significant differences between the HD-DXM and prednisone groups were observed for the rates of complete response (68% vs. 70%) and response (18% vs. 17%). However, 1 year probability of sustained response was significantly greater in the HD-DXM group than in the prednisone group (78% vs. 38%; P = 0.008). No adverse events necessitating discontinuation of treatment were observed in either group. Our retrospective analysis showed that initial treatment with HD-DXM produced longer response duration compared to a conventional dose of prednisone. Randomized clinical trials are warranted to establish the optimal initial steroid therapy for adult ITP.

Intracranial involvement of relapsed Hodgkin lymphoma (HL) is quite rare and its prognosis is very poor. We report a patient with relapsed HL with central nervous system (CNS) involvement after autologous stem cell transplantation successfully treated with allogeneic bone marrow transplantation with reduced intensity conditioning regimen. A standard therapy for relapsed CNS HL has not yet established. To the best of our knowledge, this is the first case report describing allogeneic stem cell transplantation for relapsed CNS HL in an elderly patient. Our results in this case suggest that allogeneic stem cell transplantation could be a useful therapeutic option in relapsed CNS HL patients, if their CNS lesions are controlled before stem cell transplantation.

Many patients with POEMS syndrome have osteosclerotic plasmacytoma. Radiation therapy is useful for patients who have localized lesions, although chemotherapy is necessary for patients who have widespread lesions. Thus, evaluation of these lesions is important to determine the therapeutic strategy. We evaluated the activities of lesions in two patients with POEMS syndrome by (18)F-FDG positron emission tomography (PET)/computed tomography (CT) scan. In the first patient, PET/CT scan revealed osteosclerotic lesions, which were not detected by Ga-scintigraphy or plain X-ray. It also detected residual disease activity and relapse. In the second patient, lymph node involvement was suggested by (18)F-FDG uptake, and plasmacytoma was confirmed by subsequent biopsy. In the extramedullary lesions of this case, FDG uptake was as marked as in myeloma, whereas bone lesion was only detectable by CT scan. In POEMS syndrome, the PET and CT are complementary, and the combined PET/CT scan is considered to be very useful for evaluation of involved lesions.

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.

A 69-year-old woman with essential thrombocythemia (ET) developed giant ecchymosis, and she was admitted to hospital. Marked anemia (Hb 8.1 g/dl) accompanied by a prolonged activated partial thromboplastin time (89.6 s) was observed, and she received red blood cells (RBC) and fresh frozen plasma (FFP). On day 2 after admission, consciousness disturbance suddenly occurred, whereas computed tomography of the brain showed no evidence of bleeding. As the ecchymosis progressed, she developed shock. Although RBC and FFP transfusions were administered, she developed multi-organ failure and died 48 h after admission. Low factor VIII activity (<1%) accompanied by factor VIII inhibitor (17 Bethesda units) was found after her death. An autopsy revealed cerebral infarction without cerebral herniation. To date, acquired hemophilia A accompanying ET has been described in only one other patient. Although acquired factor VIII inhibitor is a rare disease, it should be tested for in ET patients with marked hemorrhagic tendency.

We describe a patient with refractory immune thrombocytopenic purpura (ITP) in whom both intravenous immunoglobulin (IVIg) and thrombopoietin-receptor (TPO-R) agonists failed to increase the platelet count sufficiently to perform a splenectomy. However, IVIg in combination with romiplostim rapidly increased the platelet count, thus facilitating splenectomy. A 72-year-old man was referred for thrombocytopenia in March 2009. Physical examination indicated petechiae and purpura over his entire body. A complete blood count showed severe thrombocytopenia (platelet count 12 × 109/l), normal numbers of red and white blood cells, and no fragmented erythrocytes. No coagulation abnormalities were observed. Blood chemistry analysis showed no renal or liver dysfunction. Tests for antinuclear antibodies (ANA) and anti-β2-glycoprotein I antibody were negative. Anti-platelet antibody screening was positive and an elevated platelet-associated IgG level of 346 ng/107 cells was observed. No hepatitis virus or human immunodeficiency virus was detected. Helicobacter pylori infection was negative based on a urea breath test. Bone marrow examination indicated no abnormalities except for the presence of megakaryocytic hyperplasia. These findings led to the diagnosis of ITP. Treatment consisted of two courses of high-dose dexamethasone (40 mg/d for 4 d) and prednisolone (1 mg/kg per day initially, then tapered), but this treatment proved ineffective. Therefore, we decided to perform splenectomy. The patient received IVIg (400 mg/kg per day) for 5 d; however, his platelet count was insufficient to safely perform splenectomy. In September 2009, rituximab (375 mg/m2 per day) was initiated as a second-line therapy but was discontinued due to the development of sepsis (Fig 1A). Thereafter, danazol, methenolone, and diaphenylsulfone were administered in addition to low-dose prednisolone. However, his platelet count fluctuated between 3 and 9 × 109/l, and the severe bleeding tendency continued (data not shown). In January 2011, the novel TPO-R agonists, eltrombopag and romiplostim, were approved for use in Japan. In March 2011, we attempted to increase the platelet count prior to performing splenectomy by administering eltrombopag with low-dose prednisolone. Eltrombopag was well tolerated and was continued for 16 weeks at a dose of 25 mg/d, but the platelet count did not increase above 17 × 109/l. Subsequently, romiplostim treatment was initiated in August 2011. Romiplostim was administered subcutaneously at an initial dose of 1 μg/kg per week, which was gradually increased, raising the platelet count to 20–50 × 109/l. When the dose of romiplostim was increased to 4 μg/kg, the patient developed arthralgia, which improved when the dose was reduced to 2 μg/kg. In spite of the long-term treatment with romiplostim over 12 weeks, the platelet count remained too low to perform splenectomy. Therefore, IVIg (400 mg/kg per day for 5 d), although previously proved to be ineffective for this patient as a single agent, was re-administered in combination with romiplostim (2 μg/kg). As shown in Fig 1B, a rapid increase in the platelet count to 150 × 109/l was observed. Subsequently, successful laparoscopic splenectomy was performed at 10 d after IVIg and romiplostim administration. Nine days post-splenectomy, the pateints' platelet count further increased, to 589 × 109/l and then gradually decreased to normal values within 1 month (Fig 1B). At 4 months post-splenectomy, his platelet count remained stable (platelet count 162 × 109/l). Primary ITP is an autoimmune disorder characterized by autoantibody-induced platelet destruction, leading to a low peripheral blood platelet count (Cooper & Bussel, 2006). In addition, antiplatelet antibodies have been shown to suppress megakaryocyte production in vitro (McMillan et al, 2004). Recent observations suggested that endogenous thrombopoietin (TPO) levels in ITP are lower than anticipated, and the resulting reduced platelet production may be involved in the pathogenesis of ITP (Aledort et al, 2004). The evidence-based practice guideline for ITP (Neunert et al, 2011), recommends that corticosteroids should be the standard initial first-line treatment (IVIg is used with corticosteroids when a more rapid increase in platelet count is required) and splenectomy is the main second-line therapy for ITP patients who fail to respond sufficiently to first-line therapy. TPO-R agonists are recommended for patients at risk of bleeding who relapse following splenectomy or who have a contraindication to splenectomy (Neunert et al, 2011). IVIg inhibits autoantibody-mediated platelet destruction by blocking the Fc receptors on phagocytic reticuloendothelial cells of the spleen (Bierling & Godeau, 2005). IVIg is generally used for increasing platelet count prior to performing splenectomy, and up to 80% of patients respond to this treatment. The second generation TPO-R agonists, eltrombopag and romiplostim, have been reported to yield high response rates (>80%) in patients with refractory ITP (Kuter et al, 2008; Cheng et al, 2011). TPO-R agonists are administered as an alternative agent to increase platelet count prior to splenectomy. In this present case, splenectomy was not indicated because IVIg or TPO-R agonists had no or only a modest effect on the platelet count. However, subsequent combination therapy of romiplostim with IVIg rapidly increased platelet count and facilitated splenectomy. The development of thrombocytopenia in ITP might be associated with increased platelet destruction and insufficient platelet production. Corticosteroid and IVIg therapy reduces platelet destruction, whereas TPO-R agonists increase platelet production. The reason for the modest or no effect induced by monotherapy with either IVIg or TPO-R agonist in this patient is unclear, but a likely explanation is that both an increase in platelet destruction and a reduction in platelet production evidently resulted in severe thrombocytopenia in this patient. Therefore, the combination therapy may have indicated increased efficacy by reducing platelet destruction and stimulating platelet production. Splenectomy remains the only treatment that provides sustained remission in a high proportion of ITP patients. The present case report suggests that combination therapy with romiplostim and IVIg is a promising pre-splenectomy strategy for refractory ITP patients who fail to respond to single use of IVIg or romiplostim. The full validation of this strategy will require further investigation in a larger number of cases.