Akihito Shinohara

Long‐term ultra‐low‐dose acyclovir against varicella‐zoster virus reactivation after allogeneic hematopoietic stem cell transplantation

To evaluate the efficacy of long‐term prophylaxis with ultra‐low‐dose acyclovir against varicella‐zoster virus (VZV) reactivation, we analyzed the records of 242 Japanese adult patients who underwent allogeneic hematopoietic stem cell transplantation for the first time from 1995 to 2006 at our hospital. We started long‐term oral acyclovir at 200 mg/day in July 2001. Acyclovir was continued until the end of immunosuppressive therapy and at least 1 year after transplantation. Sixty‐six patients developed VZV reactivation at a median of 248 days after HSCT, with a cumulative incidence of 34.7%. Only one breakthrough reactivation occurred during long‐term acyclovir, which responded well to therapeutic dose of valacyclovir. The use of long‐term acyclovir was the only independent determinant that significantly decreased the overall incidence of VZV reactivation (20% vs. 50%, P < 0.0001). With this prophylaxis, visceral dissemination and serious complications other than post‐herpetic neuralgia was completely eliminated, and thereby need for hospitalization was significantly reduced (21% vs. 71%, P = 0.0034). Fifteen of the 57 patients who discontinued acyclovir developed VZV reactivation, with a cumulative incidence of 32.1%. VZV reactivation following discontinuation tended to occur in patients who were receiving immunosuppressive therapy at the cessation of acyclovir. These findings suggested that long‐term prophylaxis of ultra‐low‐dose acyclovir resulted in a successful prevention of severe VZV‐related symptoms and death, with a significantly decreased overall incidence of VZV reactivation. Prolongation of prophylactic acyclovir on profound immunosuppression might be important for thorough suppression of VZV reactivation. Am. J. Hematol., 2008.

AML1/RUNX1 functions as a cytoplasmic attenuator of NF-κB signaling in the repression of myeloid tumors

Functional deregulation of transcription factors has been found in many types of tumors. Transcription factor AML1/RUNX1 is one of the most frequent targets of chromosomal abnormalities in human leukemia and altered function of AML1 is closely associated with malignant transformation of hematopoietic cells. However, the molecular basis and therapeutic targets of AML1-related leukemia are still elusive. Here, we explored immediate target pathways of AML1 by in vitro synchronous inactivation in hematopoietic cells. We found that AML1 inhibits NF-κB signaling through interaction with IκB kinase complex in the cytoplasm. Remarkably, AML1 mutants found in myeloid tumors lack the ability to inhibit NF-κB signaling, and human cases with AML1-related leukemia exhibits distinctly activated NF-κB signaling. Furthermore, inhibition of NF-κB signaling in leukemic cells with mutated AML1 efficiently blocks their growth and development of leukemia. These findings reveal a novel role for AML1 as a cytoplasmic attenuator of NF-κB signaling and indicate that NF-κB signaling is one of the promising therapeutic targets of hematologic malignancies with AML1 abnormality.

Clinical features of late cytomegalovirus infection after hematopoietic stem cell transplantation

Late cytomegalovirus (CMV) disease beyond day 100 after hematopoietic stem cell transplantation (HSCT) has become an increasing problem after the introduction of preemptive ganciclovir (GCV) administration. To clarify the risk factors and outcome for late CMV reactivation and disease, we retrospectively analyzed the records of 101 Japanese adult patients who underwent allogeneic HSCT between 1998 and 2005 at our hospital. Fifty-one developed late positive CMV antigenemia, with a cumulative incidence of 53%. Recipient CMV seropositivity, the use of alemtuzumab, chronic GVHD, and high-dose steroids were significantly associated with late positive antigenemia. Eight patients developed late CMV disease, with a cumulative incidence of 8%, including retinitis and gastrointestinal disease. None progressed to a fatal disease. The use of alemtuzumab was identified as an independent significant risk factor for late CMV disease, although it was not associated with increased non-relapse mortality. Among the 51 patients with late positive antigenemia, 28 had consistently less than three positive cells, 25 of whom showed negative conversion without antiviral agents. In conclusion, late CMV antigenemia appeared to develop frequently, especially in patients with profound immune suppression; however, a fatal outcome could be prevented by optimal preemptive therapy. Low-level antigenemia may not require antiviral treatments.

T cell acute lymphoblastic leukemia arising from familial platelet disorder

Familial platelet disorder (FPD) is a rare autosomal dominant disorder which causes moderate thrombocytopenia with or without impaired platelet function. Patients have a propensity to develop acute myeloid leukemia (AML), and various types of second hits have been postulated in the evolution to AML. However, only a few cases of acute lymphoblastic leukemia (ALL) have been reported thus far. Here, we report a family of FPD with a germ-line hemi-allelic mutation R174X in the RUNX1 gene. The proband of the family developed AML and her son had ALL of the T cell lineage. The balanced translocation t(1;7)(p34.1;q22) was detected in the lymphoblasts from the patient with ALL. This translocation was not seen in any other affected members of the family or in the bone marrow sample of this patient in complete remission. Taken together, t(1;7)(p34.1;q22) is thought to be one of the somatic second hits that predisposes FPD to acute leukemia with T cell phenotype.

The shortest isoform of C/EBPβ, liver inhibitory protein (LIP), collaborates with Evi1 to induce AML in a mouse BMT model

Ecotropic viral integration site 1 (Evi1) is one of the master regulators in the development of acute myeloid leukemia (AML) and myelodysplastic syndrome. High expression of Evi1 is found in 10% of patients with AML and indicates a poor outcome. Several recent studies have indicated that Evi1 requires collaborative factors to induce AML. Therefore, the search for candidate factors that collaborate with Evi1 in leukemogenesis is one of the key issues in uncovering the mechanism of Evi1-related leukemia. Previously, we succeeded in making a mouse model of Evi1-related leukemia using a bone marrow transplantation (BMT) system. In the Evi1-induced leukemic cells, we identified frequent retroviral integrations near the CCAAT/enhancer-binding protein β (C/EBPβ) gene and overexpression of its protein. These findings imply that C/EBPβ is a candidate gene that collaborates with Evi1 in leukemogenesis. Cotransduction of Evi1 and the shortest isoform of C/EBPβ, liver inhibitory protein (LIP), induced AML with short latencies in a mouse BMT model. Overexpression of LIP alone also induced AML with longer latencies. However, excision of all 3 isoforms of C/EBPβ (LAP*/LAP/LIP) did not inhibit the development of Evi1-induced leukemia. Therefore, isoform-specific intervention that targets LIP is required when we consider C/EBPβ as a therapeutic target.

Intracellular Reactive Oxygen Species Mark and Influence the Megakaryocyte‐Erythrocyte Progenitor Fate of Common Myeloid Progenitors

While most studies regarding reactive oxygen species (ROS) focus on their deleterious biological effects, a growing body of evidence indicates the importance of ROS as critical mediators of several signaling pathways, including those involved in hematopoiesis. In this study, we show the critical role of ROS in lineage decision of myeloid progenitors. In megakaryocyte‐erythrocyte progenitor cells (MEP), intracellular ROS levels were found to be as low as those in hematopoietic stem cells (HSC). In contrast, remarkably high intracellular ROS levels were observed in granulocyte‐monocyte progenitor cells. Intracellular ROS levels in common myeloid progenitors (CMP) were inversely correlated with their MEP differentiation potential. Moreover, gene set enrichment analysis revealed that ROS‐low CMP showed gene expression patterns similar to those of MEP, indicating that intracellular ROS levels mark the fate of CMP. In in vitro assays, ROS significantly suppressed the generation of MEP and the formation of megakaryocyte‐erythrocyte colonies from CMP. In ROS‐high CMP, expression of colony‐stimulating factor one receptor (CSF1R) was highly upregulated, and its surface expression correlated with their granulocyte‐monocyte differentiation potential. Furthermore, ROS was found to induce the expression of CSF1R mRNA in a leukemia cell line. These data provide novel insights into the relationship between ROS and the hematopoietic differentiation system. Stem Cells 2014;32:548–557

Serial profile of vitamins and trace elements during the acute phase of allogeneic stem cell transplantation.

Currently, we utilize vitamins and trace elements formulations that are not prepared specifically for patients receiving hematopoietic stem cell transplantation (HSCT), and adequacy of this strategy has not been evaluated. We prospectively measured blood level of vitamins and trace elements in 15 patients once per week at 6 time points around the acute phase of allogeneic HSCT. We provided standard nutrition support, including administration of parenteral nutrition with vitamin and trace elements formulation in case of impairment of oral intake. Most patients had vitamin B1 deficiency from the start of preparative regimens. Vitamin C deficiency was prominent throughout the acute phase of HSCT and this was significantly associated with high inflammatory markers, C-reactive protein and ferritin. Remarkable vitamin K overload associated with administration of parenteral supplementation and ferritin overload caused by repeated transfusions was observed. Moderate deficiency of zinc was at least partially linked to gastrointestinal loss by diarrhea. We revealed several features of vitamin and trace element status in the acute phase of HSCT and provided a basis for attempts to improve the nutritional condition in HSCT recipients.

Positive impact of chronic graft-versus-host disease on the outcome of patients with de novo myelodysplastic syndrome after allogeneic hematopoietic cell transplantation: a single-center analysis of 115 patients.

To evaluate the impact of graft‐versus‐host disease (GVHD) and prognostic factors for patients with myelodysplastic syndrome (MDS) after allogeneic hematopoietic cell transplantation (allo‐HCT), we retrospectively reviewed 115 patients with MDS or acute myeloid leukemia with multilineage dysplasia (AML‐MLD) after allo‐HCT at our center. Eighty one patients received reduced‐intensity conditioning (RIC) regimens, whereas 34 received myeloablative conditioning regimens. Although the RIC group was significantly older and included more patients with poor cytogenetic risk, no difference in 4‐yr overall survival (OS) was seen between the two groups. In a multivariate analysis, covariates associated with a worse OS were the French‐American‐British stage of refractory anemia excess blasts in transformation/AML‐MLD at peak, poor cytogenetic risk, bone marrow blasts of 20% or higher at HCT and the absence of chronic GVHD (cGVHD). By using semi‐landmark analyses, we found that the presence of cGVHD significantly improved OS in high‐risk patients or the RIC group. However, there was no difference in OS between those with and without cGVHD among low‐risk MDS patients. These findings suggest that the graft‐versus‐leukemia effect may be more beneficial in high‐risk patients who do not receive intensive preparative regimens.

Early onset of acute lymphoblastic leukemia with MLL rearrangement after autologous stem cell transplantation for multiple myeloma

Dear Editor, We would like to present our experience with a patient with multiple myeloma (MM) who developed acute lymphoblastic leukemia (ALL) with mixed lineage leukemia gene (MLL) rearrangement only 1 year after administration of alkylating agents. A 61-year-old woman presented with fatigue and was introduced to our hospital in January 2007. Serum immunoelectrophoresis showed increased monoclonal IgD (115.0 mg/dL) and λ light chain, and urine immunoelectrophoresis showed increased monoclonal Bence–Jones protein (4,360 mg/g creatinine). The bone marrow (BM) was infiltrated by malignant plasma cells (50% of nucleated cells) and conventional G-banding cytogenetics revealed normal karyotype (46,XX [20/20]). A diagnosis of MM was made. After four cycles of high-dose dexamethasone, we mobilized and collected her peripheral blood stem cells using cyclophosphamide (4 g/m) and granulocyte-colonystimulating factor in July 2007. She underwent autologous stem cell transplantation (ASCT) in August 2007 with highdose melphalan (200 mg/m) as a conditioning regimen. She made recovery without severe complication. We evaluated the response of treatment as very good partial remission. In July 2008, although she had no symptom, investigation revealed pancytopenia. The BM was heavily infiltrated by blasts (94%) with the immunophenotype as B cell lymphoblasts: CD19+, CD79a+, κ−, l−. G-banding chromosomal cytogenetics revealed chromosomal abnormalities: 46,XX,t(1;11)(p32–34;q23) [4/6]. Fluorescence in situ hybridization (FISH) examination showed split signals of MLL gene. A diagnosis of B-ALL with MLL rearrangement was made. She was commenced on the JALSG 202-O protocol (Clinicaltrials.gov; study ID:NCT00131027) and achieved hematological complete remission. We performed IgH gene rearrangement studies on genomic DNA extracted from the BM aspirates at the time of diagnosis of MM and that of ALL. Southern blotting (SRL inc., Nakagyo-ku, Kyoto, Japan) test showed different monoclonal bands on DNA extracted from both BM aspirates (Fig. 1). From these findings, we concluded that her MM and ALL were derived from different clones. Lau et al. [1] reported a patient with MM that terminated in ALL. Lau indicated that two diseases arose from different clones by IgH gene rearrangement analysis. Cases of secondary ALL supposed to be Topoisomerase II inhibitor-related were previously reported [2], and Lau’s case was administered doxorubicin. Although we did not administer Topoisomerase II inhibitor, we detected chromosomal abnormality of 11q23 that is typically determined from cases of Topoisomerase II inhibitor-related leukemia [2]. To our knowledge, this is the first report describing alkylating-agent-related ALL with MLL rearrangement. Our hypothesis is that her lymphoid progenitor cells had initial instability or a mutation that cannot be detected by conventional G-banding test, and different types of DNA modification separately led to MM and ALL. We suppose modification leading to ALL was MLL rearrangement. This is consistent with the reports that MLL rearrangement is Ann Hematol (2009) 88:813–814 DOI 10.1007/s00277-008-0680-8

Paraneoplastic pemphigus occurring after bendamustine and rituximab therapy for relapsed follicular lymphoma

Dear Editor, Paraneoplastic pemphigus (PNP) is a rare autoimmune mucocutaneous disorder which is predominantly associated with lymphoproliferative disorders including non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, Castleman disease, and Waldenström macroglobulinemia [1]. PNP is characterized by painful mucosal erosion with frequent skin eruption, histological changes of acantholysis or lichenoid/interface dermatitis, and serum autoantibodies against plakin family proteins [2, 3]. Although the cause of PNP is not identified in most cases, previous literatures have indicated that PNP may be triggered by specific treatment modalities for lymphoid neoplasms. Patients with lymphoid malignancies were recurrently affected by PNP within several cycles of fludarabine-containing therapy [4–7] or after local radiotherapy [8–10]. Here, we describe the first case of PNP occurring after bendamustine and rituximab therapy for follicular lymphoma. A 77-year-old Japanese woman was diagnosed with follicular lymphoma and achieved complete remission after six cycles of rituximab, cyclophosphamide, vincristine, and prednisolone therapy. A follow-up computed tomography scan detected recurrence of lymphadenopathy at 16 months after completion of the immunochemotherapy. At the age of 80 years, salvage therapy was commenced with bendamustine (60 mg/ m, days 1–2) and rituximab (375 mg/m, day 1) of a 28-day cycle, which led to salient regression of lymphadenopathy. On day 23 of the second cycle, the patient noticed painful stomatitis and skin lesions with fever. Physical examination detected hemorrhagic crusts on the lips, oral erosions, blistering of the eyelids, and eruptions on the trunk (Fig. 1a). Repeated blood cultures did not show any evidence of bacterial infections. Initial diagnosis of cytomegalovirus (CMV) infection was made on the basis of a positive CMV pp65 antigenemia test result. While fever was improved by ganciclovir administration, stomatitis and skin rash remained almost stable. A skin biopsy from the trunk was therefore performed, and histopathological examination revealed suprabasal clefts with scattered necrotic keratinocytes (Fig. 1b). The Nikolsky sign (i.e., blistering after manual rubbing of the skin) was positive. Serum autoantibodies against desmoglein 3 were detected by chemiluminescent enzyme immunoassay. Taken together, the patient was diagnosed with PNP. T. Higo : F. Nakamura :A. Shinohara :H. Asano : M. Kurokawa (*) Department of Hematology and Oncology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp