Hideharu Muto

Somatic RHOA mutation in angioimmunoblastic T cell lymphoma

Angioimmunoblastic T cell lymphoma (AITL) is a distinct subtype of peripheral T cell lymphoma characterized by generalized lymphadenopathy and frequent autoimmune-like manifestations. Although frequent mutations in TET2, IDH2 and DNMT3A, which are common to various hematologic malignancies, have been identified in AITL, the molecular pathogenesis specific to this lymphoma subtype is unknown. Here we report somatic RHOA mutations encoding a p.Gly17Val alteration in 68% of AITL samples. Remarkably, all cases with the mutation encoding p.Gly17Val also had TET2 mutations. The RHOA mutation encoding p.Gly17Val was specifically identified in tumor cells, whereas TET2 mutations were found in both tumor cells and non-tumor hematopoietic cells. RHOA encodes a small GTPase that regulates diverse biological processes. We demonstrated that the Gly17Val RHOA mutant did not bind GTP and also inhibited wild-type RHOA function. Our findings suggest that impaired RHOA function in cooperation with preceding loss of TET2 function contributes to AITL-specific pathogenesis.

Reduced TET2 function leads to T-cell lymphoma with follicular helper T-cell-like features in mice

TET2 (Ten Eleven Translocation 2) is a dioxygenase that converts methylcytosine (mC) to hydroxymethylcytosine (hmC). TET2 loss-of-function mutations are highly frequent in subtypes of T-cell lymphoma that harbor follicular helper T (Tfh)-cell-like features, such as angioimmunoblastic T-cell lymphoma (30–83%) or peripheral T-cell lymphoma, not otherwise specified (10–49%), as well as myeloid malignancies. Here, we show that middle-aged Tet2 knockdown (Tet2gt/gt) mice exhibit Tfh-like cell overproduction in the spleen compared with control mice. The Tet2 knockdown mice eventually develop T-cell lymphoma with Tfh-like features after a long latency (median 67 weeks). Transcriptome analysis revealed that these lymphoma cells had Tfh-like gene expression patterns when compared with splenic CD4-positive cells of wild-type mice. The lymphoma cells showed lower hmC densities around the transcription start site (TSS) and higher mC densities at the regions of the TSS, gene body and CpG islands. These epigenetic changes, seen in Tet2 insufficiency-triggered lymphoma, possibly contributed to predated outgrowth of Tfh-like cells and subsequent lymphomagenesis. The mouse model described here suggests that TET2 mutations play a major role in the development of T-cell lymphoma with Tfh-like features in humans.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix–loop–helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL–AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL–AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL–AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL–AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL–AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Detection of the G17V RHOA mutation in angioimmunoblastic T-cell lymphoma and related lymphomas using quantitative allele-specific PCR.

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) are subtypes of T-cell lymphoma. Due to low tumor cell content and substantial reactive cell infiltration, these lymphomas are sometimes mistaken for other types of lymphomas or even non-neoplastic diseases. In addition, a significant proportion of PTCL-NOS cases reportedly exhibit features of AITL (AITL-like PTCL-NOS). Thus disagreement is common in distinguishing between AITL and PTCL-NOS. Using whole-exome and subsequent targeted sequencing, we recently identified G17V RHOA mutations in 60–70% of AITL and AITL-like PTCL-NOS cases but not in other hematologic cancers, including other T-cell malignancies. Here, we establish a sensitive detection method for the G17V RHOA mutation using a quantitative allele-specific polymerase chain reaction (qAS-PCR) assay. Mutated allele frequencies deduced from this approach were highly correlated with those determined by deep sequencing. This method could serve as a novel diagnostic tool for 60–70% of AITL and AITL-like PTCL-NOS.

BCL6 locus is hypermethylated in angioimmunoblastic T-cell lymphoma.

BCL6, a master transcription factor for differentiation of follicular helper T (TFH) cells, is highly expressed in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphomas (PTCL) containing tumor cells with TFH features. TET2, encoding an epigenetic regulator, is frequently mutated in AITL/PTCL. We previously reported that Tet2 knockdown mice developed T-cell lymphomas with TFH features. Hypermethylation of the Bcl6 locus followed by BCL6 upregulation was thought to be the key event for lymphoma development in mice. The mechanisms by which BCL6 expression is upregulated in human AITL/PTCL, however, have not been elucidated. Here, we investigated the impact of TET2 mutations on methylation of BCL6 locus in human AITL/PTCL samples. Hypermethylation of the BCL6 locus was more frequent in PTCL samples than B-cell lymphoma samples (PTCL vs B-cell lymphomas: 9/42 vs 0/35). PTCL samples with TET2 mutations were more frequently hypermethylated than those without TET2 mutations (PTCL with TET2 mutations vs without mutations: 6/22 vs 0/17). BCL6 expression in hypermethylated samples was higher than that in low methylated samples. Deregulated BCL6 expression caused by hypermethylation and TET2 mutations may result in skewed TFH differentiation and eventually contribute to AITL/PTCL development in patients, as well as lymphoma development in Tet2 knockdown mice.

A nationwide survey of co-occurrence of malignant lymphomas and myelodysplastic syndromes/myeloproliferative neoplasms

Dear Editor, Co-occurrence of multiple hematologic malignancies in distinct lineages has been reported as rare cases. The diseases occur simultaneously in some cases, while they occur sequentially in others. Combinations of diseases are different among the cases: B-linage malignancies and T/NK-lineage malignancies, Bor T/NK-lineage malignancies and myeloid malignancies, and so on. Among them, co-occurrence of mature Band T/NK-lineage malignancies comprises composite lymphomas, which constitute 1–4 % of lymphoma [1]. Co-occurrence of malignant lymphomas and myeloid malignancies has also been experienced [2–4]. However, less information is available. To investigate the actual distribution of patients who developed both malignant lymphomas and myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), or idiopathic cytopenia of undetermined significance (ICUS), we conducted a nationwide survey in Japan. In the primary survey, we sent a short questionnaire about the experience of simultaneous or sequential development of malignant lymphomas and MDS, MPN, or ICUS to the institutions throughout Japan, registered as clinical training centers for hematology and pediatric hematology. The sequential development of lymphoid malignancies followed by myeloid malignancies was excluded, because such cases are defined as therapy-related malignancies. The secondary survey was conducted focusing on the detailed clinical information. This study was approved by the ethics committee in the University of Tsukuba Hospital. Questionnaire was sent to the 491 institutes, of which 241 returned the answer. The secondary questionnaire was sent to the 39 institutes that reported experiences of the applicable patients. In total, 72 cases, having both malignant lymphomas and MDS/MPN/ICUS, were reported. Forty five cases simultaneously developed malignant lymphomas and myeloid malignancies, while 27 sequentially developed myeloid malignancies and then malignant lymphomas. Detailed information was available for 45 cases (simultaneous, 26 cases; sequential, 19 cases) (Table 1, Supplementary Table 1, 2). The median age of the patients was 69 years (simultaneous, 69 years; sequential 69 years) (Table 1). History of chemotherapy and/or radiotherapywas found in three out of 45 cases (simultaneous, 3/26 [11.5 %]; sequential, 0/19 [0 %]) (Table 1). The most frequent combination was diffuse large B cell lymphomas (DLBCL) and any subtypes of MDS, which was found in up to 15 out of 45 cases (33.3 %) (simultaneous, 8/26 [30.8 %]; sequential, 7/19 [36.8 %]) (Table 1, Supplementary Table 1, 2). The combination of follicular lymphoma (FL) and any subtypes of MDS was found in four out of 45 cases (8.9 %), whereas it was confined to the simultaneous cases (simultaneous, 4/26 [15.4 %]; sequential, 0/9 [0 %]) (Table 1, Supplementary Table 1, 2). Electronic supplementary material The online version of this article (doi:10.1007/s00277-016-2612-3) contains supplementary material, which is available to authorized users.

Quinupristin/dalfopristin and voriconazole controlled Staphylococcus epidermidis pneumonia and chronic necrotizing aspergillosis in a patient with severe lung degradation consequent to multiple treatments for Hodgkin's lymphoma

We report here a 34-year-old woman with complicated severe opportunistic pulmonary infection, who was treated with the newly developed antibiotics quinupristin/dalfopristin (QPR/DPR) and voriconazole. She had received repeated chemotherapy, irradiation of the left lung, autologous and allogeneic bone marrow transplantation (BMT), and segmentectomy of the base of the left lung as treatments for Hodgkins lymphoma. Although she had been in complete remission (CR), the structure of the left lung was severely degraded. Four years after achieving CR, she developed complicated life-threatening pulmonary infections with methicillin-resistant Staphylococcus epidermidis and Aspergillus niger during outpatient care. Chemotherapies with QPR/DPR for S. epidermidis pneumonia and voriconazole for chronic necrotizing pulmonary aspergillosis (CNPA) improved her symptoms rapidly without any major complications. QPR/DPR and voriconazole are considered effective for patients with life-threatening opportunistic pulmonary infections who have previously been treated with intensive regimens including radiotherapies to the lung.

Outcome of patients with acute undifferentiated leukemia after allogeneic hematopoietic stem cell transplantation

The World Health Organization (WHO) has categorized acute undifferentiated leukemia (AUL) as a rare subtype of acute leukemias of ambiguous lineage (ALAL). The prognosis of AUL is considered poor and it expresses no known lineage-specific marker [1,2]. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) can potentially cure various hematological malignancies, little is known about the transplant modality and outcomes of patients with AUL. Most published reports to date have analyzed the clinical characteristics and prognosis of AUL together with those of mixed-phenotypic acute leukemia (MPAL) although they have quite different clinical characteristics [3–5]. Therefore, these studies might not be particularly informative for patients with AUL. Here, we analyzed the clinical outcomes of 10 patients with AUL after allo-HSCT at our institution. We retrospectively reviewed 911 patients with acute leukemia who were admitted to our institution between April 2005 and March 2017. Consensus diagnostic criteria for AUL have not yet been established. Therefore, we defined AUL based on the WHO classification [2] as leukemic cells that were not positive for any lineage-specific markers (myeloid lineage: myeloperoxidase [cytochemistry, immunohistochemistry, or flow cytometry]; B cell lineage: CD19, CD79a, or cytoplasmic CD22; T cell lineage: cytoplasmic CD3 or surface CD3). Our institutional committee on research ethics approved the study (approval number: 1973), which proceeded according to the Declaration of Helsinki. Transplant procedures have been described in detail elsewhere [6]. Generally, myeloablative conditioning mainly included a total body irradiation (TBI) regimen (12Gy) in combination with cyclophosphamide (CY) at 120mg/kg, or a non-TBI regimen that included intravenous busulfan at 12.8mg/kg, and CY at 120mg/kg. The preparative regimen for the reduced-intensity procedure consisted of fludarabine (30mg/m for 6 d), melphalan (40mg/m for 2 d), and TBI (4 Gy). The patients were given intravenous infusion of donor hematopoietic stem cells on day 0. All patients received acute graft-versus-host disease (GVHD) prophylaxis with cyclosporine or tacrolimus and short-term methotrexate. Tacrolimus was used in cases involving either unrelated or human leukocyte antigen (HLA) mismatched transplantation. Engraftment in alloHSCT is defined as the first of three consecutive days with an absolute neutrophil count of 0.5 10/l or greater. The probability of overall survival (OS) was estimated using Kaplan–Meier product limit method. We calculated OS from the date of allo-HSCT to the last assessment for survivors. The cumulative incidence of non-relapse mortality (NRM), relapse and acute GVHD were evaluated using Gray’s method. For each estimation of the cumulative incidence of an event, death without event was defined as a competing risk. All statistical analyses were performed with EZR, a graphical user interface for R software (The R Foundation for Statistical Computing, version 2.13.0; www.r-project.org). Among the 911 patients with acute leukemia, AUL was classified in only 12 (1.3%). Among those, one patient refused to undergo allo-HSCT and another died early. Table 1 shows the characteristics of 10 patients with AUL who underwent allo-HSCT. The median age at the time of transplantation was 45 (range: 22–63) years. Seven (70.0%) and three (30.0%) patients were male and female, respectively. Marrow fibrosis was found in two (20.0%) patients, but extramedullary disease was not present in any patient. Cytochemical findings were negative for myeloperoxidase in all patients. Immunophenotyping revealed the common expression of CD34 (90.0%), HLADR (80.0%), and CD13 (60.0%), but the lineage-specific markers were absent in all patients. Cytogenetic studies of nine patients revealed chromosomal abnormalities in five (55.6%) of them. In five patients (cases 2, 4, 5, 9, and 10) with available data, polymerase chain reaction (PCR)

A 56-Year-Old Woman With Multiple Pulmonary Cysts and Severe Chest Pain

&NA; A 56‐year‐old woman presented to our hospital with a 4‐month history of worsening chest pain. She denied having any respiratory symptoms, such as dyspnea, sputum, cough, or hemoptysis, or any history of smoking or exposure to dusts. One year previously she had a vertebral fracture. There was no specific family history, including pulmonary or autoimmune diseases. Chest CT performed 3 years earlier showed multiple thin‐walled pulmonary cysts, although no further investigations were performed.

Diagnostic open brain biopsy following initial negative results of cerebrospinal fluid assessment for Toxoplasma

Cerebral toxoplasmosis is a rare but fatal complication after allogeneic hematopoietic stem cell transplantation (HSCT), and reaching an accurate antemortem diagnosis with pathological proof is difficult.1,2 We describe herein the clinical course of a patient with cerebral toxoplasmosis following allogeneic HSCT. The diagnosis was successfully made by aggressive open brain biopsy following initial negative results from cerebrospinal fluid (CSF) assessment for Toxoplasma. This article is protected by copyright. All rights reserved.