Junji Hiraga

Malignant histiocytosis‐like B‐cell lymphoma, a distinct pathologic variant of intravascular lymphomatosis: a report of five cases and review of the literature

Malignant histiocytosis (MH)‐like B‐cell lymphoma (BCL) is a neoplastic proliferation of large B cells clinically characterized by fever, hepatosplenomegaly, haemophagocytosis and abnormal laboratory data, without lymphadenopathy or skin lesions. Interestingly, most cases have been reported in Asian patients, and it is unclear whether MH‐like BCL is biologically distinct from conventional large B‐cell lymphomas. We report five Japanese patients with MH‐like BCL. Biopsied specimens of bone marrow, liver and/or spleen showed infiltration of neoplastic B cells accompanied by haemophagocytosing histiocytes. Lymphoma cells were positive for CD19, CD20 and HLA‐DR surface antigens, and negative for CD5 and CD10. In four cases elevated serum levels of interleukin (IL)‐6 and the soluble IL‐2 receptor isoform were noted, but not IL‐1β, IL‐2 or tumour necrosis factor‐α. Autopsies of two cases were pathologically diagnosed as intravascular lymphomatosis (IVL). Based on these observations, the current and nine previous cases reported as MH‐like BCL in Japan were re‐evaluated. They appear to form a peculiar variant of IVL, characterized by bone marrow involvement at presentation, haemophagocytic syndrome, and a rapidly aggressive clinical course, but rarely neurological complications or skin lesions. This variant may merit separate consideration because of the problems posed in the initial diagnosis and therapeutic approaches.

Epigenetic Regulation of CD20 Protein Expression in a Novel B-Cell Lymphoma Cell Line, RRBL1, Established from a Patient Treated Repeatedly with Rituximab-Containing Chemotherapy

Rituximab is a chimeric monoclonal antibody to the surface antigen CD20 and has provided better outcomes against CD20+ B-cell lymphomas than chemotherapy with conventional antitumor drugs alone. Treatment with rituximab poses a considerable problem, however, because of CD20- tumor transformation and subsequent disease progression. We have established a CD20- lymphoma cell line, RRBL1, from a diffuse large B-cell lymphoma with CD20- transformation from CD20+ follicular lymphoma after treatment with rituximab. RRBL1 was CD10+, CD19+, and CD20- by flow cytometry. CD20 expression was not detected by immunohistochemistry. Immunoblotting with whole RRBL1 cell lysate showed a very faint CD20 band only with longer exposures. The level of CD20 messenger RNA (mRNA) expression detected by quantitative reverse transcriptase-polymerase chain reaction analysis was almost 100 times lower than that in CD20+ lymphoma cells. When we treated RRBL1 cells with trichostatin A, an epigenetic drug that modulates histone-acetylation status, we detected dramatically increased CD20 mRNA and protein expression, suggesting that epigenetic mechanisms may explain the CD20- phenotype in RRBL1 cells. Thus, RRBL1 may be useful not only for analyses of mechanisms for the absence of CD20 expression in vitro but also for exploration of therapies against CD20- B-cell malignancies in vivo.

A Case of Interstitial Pneumonia Induced by Rituximab Therapy

Rituximab, a chimeric human/murine monoclonal antibody directed against CD20, is an effective therapy for B-cell type non-Hodgkin’s lymphoma. Most of the adverse responses are mild to moderate nonhematological toxicities, which include fever, chill, or skin reactions. Severe respiratory adverse events have been infrequent [1,2]. Recently, Burton et al reported 2 interstitial pneumonia (IP) cases related to rituximab therapy [3]. IP is rarely induced by rituximab, and its etiological roles are still unclear. Here, we report an IP case related to rituximab therapy, for which we investigated its etiology as well as possible. An 80-year-old man with a diagnosis of diffuse large Bcell lymphoma was treated with rituximab and cyclophosphamide, vincristine, and prednisolone chemotherapy (RCOP). No severe adverse effects were observed during the first course, and after 3 weeks, he was given a second course. With the second rituximab infusion, he had a grade 2 fever and a systemic skin rash. The infusion reaction was abated with hydroxycortisone, and COP therapy was scheduled. Ten days after the second rituximab infusion, the patient complained of general fatigue and had a high fever.A chest x-ray revealed bilateral ground-glass opacities, and a high-resolution computed tomography scan of the chest confirmed the diagnosis of interstitial pneumonia. As respiratory insufficiency advanced, he needed noninvasive positive-pressure ventilation. He was treated with methylprednisolone pulse therapy after a bronchoalveolar lavage (BAL) examination. He made a dramatic recovery, corticosteroid was tapered, and he was discharged after 3 weeks. No pathogens were detected in the patient’s blood, sputum, or BAL fluids, and results for Pneumocystis carini and Mycobacterium tuberculosis were also negative. Serum antibodies did not indicate the presence of viruses, chlamydia A Case of Interstitial Pneumonia Induced by Rituximab Therapy

Clinical significance of nuclear non‐phosphorylated beta‐catenin in acute myeloid leukaemia and myelodysplastic syndrome

Wnt signaling activates the canonical pathway and induces the accumulation of non‐phosphorylated beta‐catenin (NPBC) in the nucleus. Although this pathway plays an important role in the maintenance of haematopoietic stem cells as well as in oncogenesis, the significance of nuclear NPBC remains unclear in malignant haematopoiesis. This study examined the expression of nuclear NPBC in bone marrow specimens from 54 and 44 patients with de novo acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS), respectively. On immunohistochemistry with an anti‐NPBC antibody, the nuclei were positively stained in 22 and 18 of AML and MDS specimens, respectively. Staining of nuclear NPBC was associated with AML subtypes (M6 and M7), low complete remission (CR) rate, and poor prognosis. Nuclear NPBC was also associated with a high score when using the International Prognostic Scoring System (IPSS) for MDS and with −7/−7q and complex karyotypes. These findings suggest that in situ detection of nuclear NPBC by immunohistochemistry could provide new insights into the pathogenesis and prognosis of AML and MDS.

Escape mechanisms from antibody therapy to lymphoma cells: downregulation of CD20 mRNA by recruitment of the HDAC complex and not by DNA methylation.

Although rituximab is a critical monoclonal antibody therapy for CD20-positive B-cell lymphomas, rituximab resistance showing a CD20-negative phenotypic change has been a considerable clinical problem. Here we demonstrate that CD20 mRNA and protein expression is repressed by recruitment of a histone deacetylase protein complex to the MS4A1 (CD20) gene promoter in CD20-negative transformed cells after treatment with rituximab. CD20 mRNA and protein expression were stimulated by decitabine (5-Aza-dC) in CD20-negative transformed cells, and was enhanced by trichostation A (TSA). Immunoblotting indicated that DNMT1 expression was first downregulated 1 day after treatment with 5-Aza-dC, but IRF4 and Pu.1, the transcriptional regulators of MS4A1, were still expressed with or without 5-Aza-dC. Interestingly, CpG methylation of the MS4A1 promoter was not observed in CD20-negative transformed cells without 5-Aza-dC. A chromatin immunoprecipitation (ChIP) assay indicated that the Sin3A-HDAC1 co-repressor complex was recruited to the promoter and dissociated from the promoter with 5-Aza-dC and TSA, resulting in histone acetylation. Under these conditions, IRF4 and Pu.1 were continually recruited to the promoter with or without 5-Aza-dC and TSA. These results suggest that recruitment of the Sin3A-HDAC1 complex is related to downregulation of CD20 expression in CD20-negative B-cells after treatment with rituximab.

Loss of O6-methylguanine-DNA methyltransferase protein expression is a favorable prognostic marker in diffuse large B-cell lymphoma

Although aberrant promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) is a favorable prognostic marker in patients with diffuse large B-cell lymphoma (DLBCL), MGMT protein expression has not been thoroughly examined. The aim of this study was to evaluate the clinical implication of MGMT protein expression and its correlation with promoter hypermethylation of the gene. We investigated MGMT protein expression by immunohistochemical analysis of 63 DLBCL patients who received cyclophosphamide as part of multidrug regimens. In addition, promoter methylation of the MGMT gene was analyzed by a methylation-specific polymerase chain reaction assay, and correlations with chemotherapeutic effect and prognosis were statistically evaluated. Immunohistochemical assay results for MGMT protein were negative in 30.2% of patients with newly diagnosed DLBCL. Immunostaining results were closely correlated with the methylation status of the promoter. Promoter DNA methylation of the gene was not detected in 34 (81.0%) of 42 tumor samples determined to be MGMT-positive DLBCL by immunostaining and was detected in 15 (88.2%) of 17 cases of MGMT-negative DLBCL. Overall survival (OS) and disease-free survival (DFS) rates were significantly higher in MGMT-negative patients than in MGMT-positive patients (5-year OS, 81.3% versus 56.6% [P = .0375]; 5-year DFS, 66.3% versus 39.9% [P = .0121]). The combined rate for complete response (CR) plus unconfirmed CR was significantly higher in MGMT-negative patients (15/19, 79.0%) than in MGMT-positive patients (25/44, 56.8%) (P = .0488). A multivariate analysis showed that absence of MGMT expression was an independent prognostic factor for OS (relative risk, 4.09; P = .0258). Lack of MGMT protein expression is associated with aberrant promoter DNA methylation and appears to be a useful marker for predicting the survival of DLBCL patients.

Promoter Hypermethylation of the DNA-Repair Gene O 6 - Methylguanine—DNA Methyltransferase and p53 Mutation in Diffuse Large B-cell Lymphoma

The gene for the DNA-repair enzyme O6-methylguanine—DNA methyltransferase (MGMT), which is closely related with cellular sensitivity to alkylating agents, is inactivated by promoter hypermethylation in several human cancers, including malignant lymphoma. Promoter hypermethylation of the MGMT gene is a favorable prognostic factor in diffuse large B-cell lymphoma (DLBCL). Although inactivation of the MGMT gene is closely related to p53 gene mutations in several cancers, the relationship between p53 gene mutation and MGMT inactivation in malignant lymphoma has not been thoroughly examined. We studied the correlation between MGMT hypermethylation and p53 mutation in DLBCL and their impacts on patient prognosis. In a retrospective cohort study, we used a methylation-specific polymerase chain reaction technique to analyze the methylation status of the promoter region of the MGMT gene in 116 DLBCL patients who received cyclophosphamide as part of multidrug combination chemotherapies. Denaturing high-performance liquid chromatography and direct sequencing were used to search for p53 gene mutations in exons 5 through 9 in 96 of the 116 samples. Disease-free survival and overall survival were estimated by the Kaplan-Meier method. Multivariate survival analyses were performed with the Cox proportional hazards model. Forty-five (38.8%) of 116 DLBCL patients showed MGMT promoter hypermethylation.The presence of MGMT hypermethylation was associated with better overall survival (P = .036). MGMT promoter hypermethylation was a prognostic factor that was independent of established prognostic factors, such as age, disease stage, serum lactic dehydrogenase level, and the number of extranodal disease sites (hazard ratio, 2.43; 95% confidence interval, 1.28-4.61; P = .007). p53 mutations were detected in 19 (19.8%) of 96 patients and were identified as a risk factor in the complete remission rate and overall survival (P = .0040, and P = .027, respectively).A correlation between MGMT hypermethylation and p53 mutation or p53 G:C-to-A:T mutation was not observed (P = .88, and P = .31, respectively). MGMT promoter hypermethylation and p53 mutation are useful prognostic markers in DLBCL. The impact of MGMT inactivation on p53 mutation in DLBCL is unclear.

Prognostic analysis of aberrant somatic hypermutation of RhoH gene in diffuse large B cell lymphoma

Prognostic analysis of aberrant somatic hypermutation of RhoH gene in diffuse large B cell lymphoma

Retention of CD34+ CML stem/progenitor cells during imatinib treatment and rapid decline after treatment with second-generation BCR–ABL inhibitors

ABL-tyrosine kinase inhibitor (TKI), imatinib (IM) is suggested to be effective for proliferating leukemic cells, not quiescent chronic myeloid leukemia (CML) stem cells.1, 2, 3 Recent clinical trials suggest that CML treatment can be improved with more potent BCR–ABL inhibition during the second generation of ABL-tyrosine kinase inhibitors (2nd-TKIs) such as dasatinib and nilotinib.4 To comparatively examine the effects of IM and 2nd-TKIs on the BCR–ABL-positive hematopoietic stem cells (HSC) and progenitors, we investigated 47 CML-chronic phase cases using methods previously reported with FACSAria and quantitative real-time-PCR of BCR–ABL among each sorted population: total mononuclear cells, HSC/Thy-1+, HSC/Thy-1−, common myeloid progenitors, granulocyte macrophage progenitors and megakaryocyte erythroid progenitors (detailed in Supplementary Materials and Methods).5, 6 By using this method in the HSC population, more than 30% of cells are supposed to have stem cell potential, probably as long-term culture-initiating cells.7

Successful immunosuppressive and iron chelation therapy for a severe aplastic anemia patient undergoing hemodialysis due to chronic renal failure

Immunosuppressive treatment using antithymocytic globulin (ATG) is the treatment of choice for severe aplastic anemia patients [1, 2]. Iron chelation therapy is also effective for patients with frequent blood transfusions [3–5]. However, the effects of these treatments on hemodialysis patients have not been reported. Here, we present a case of severe aplastic anemia on hemodialysis, treated with immunosuppressive and iron chelation therapy. A 49-year-old male presented in December 2007 with fever and pancytopenia. He required regular hemodialysis following history of chronic renal failure 25 years earlier, and had also been treated with recombinant epoetin beta. Laboratory studies revealed a white blood cell count of 1.2 9 10/ll, neutrophil cell count of 468/ll, platelet count of 0.9 9 10/ll, hemoglobin concentration of 5.4 g/dl, and reticulocytes at only 0.9 9 10/ll. Bone marrow biopsy revealed severe hypoplasia. He was diagnosed with severe aplastic anemia, and required frequent transfusions of PC and RBC to maintain a 2 9 10/ll platelet level and 8.0 g/dl hemoglobin concentration. He was given the antibiotic, meropenem, for febrile neutropenia, which rapidly controlled his fever. The same drug, epoetin beta, was continued for treatment. Cyclosporin A and granulocyte colony-stimulating factor (G-CSF) were given as the first therapy for severe aplastic anemia, but his bone marrow remained hypoplastic. Horse ATG was added at a dose of 15 mg/kg for 5 days. Methylprednisolone was used at a dose of 2 mg/kg/day for 5 days, and then tapered off. The only adverse event was a grade 2 allergic reaction. His hematopoietic system recovered slightly, and G-CSF was lowered, but transfusion frequency could not be reduced. An anabolic steroid was added, but yielded no improvement. Then, 8 months after the first ATG, a second rabbit ATG was given at a dose of 5 mg/kg for 5 days. Methylprednisolone was given concomitantly, and no significant adverse event was seen, except an acute allergic reaction. Total red-blood cell transfusion volume was over 80 Japanese units, so deferasirox was given. Bone marrow biopsy revealed a moderate number of positive granules on iron staining, which were not observed at the initial examination. The initial dose was 10 mg/kg/day, due to renal failure, but the serum ferritin level remained above 3000 ng/ml. The dose was increased to 20 mg/kg/day. Six months later, the serum ferritin level was below 1000 ng/ml. Transfusion frequency was reduced. One year later after the second ATG therapy, he became transfusionfree (Fig. 1). Bone marrow biopsy revealed the recovery of hematopoiesis, but on iron staining, a moderate number of positive granules were still observed. During this course of treatments, he continued dialysis three times per week in an open space, and bacterial infection was not a threat. J. Hiraga (&) R. Sakemura S. Mizuno Department of Hematology, Toyota Memorial Hospital, 1-1 Heiwa-cho, Toyota 471-8513, Japan e-mail: junji_hiraga@mail.toyota.co.jp