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Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform–specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3′-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell–like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIClong isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform–specific inactivation of Cicselectively increased CD4+CD25+Foxp3+T cells in vivo. We also found recurrent (13%) 3′-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, RELtruncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell–like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal myeloproliferation, progressive bone marrow (BM) fibrosis, splenomegaly, and anemia. BM fibrosis was previously thought to be a reactive phenomenon induced by mesenchymal stromal cells that are stimulated by the overproduction of cytokines such as transforming growth factor (TGF)-ß1. However, the involvement of neoplastic fibrocytes in BM fibrosis was recently reported. In this study, we showed that the vast majority of collagen- and fibronectin-producing cells in the BM and spleens of Jak2V617F-induced myelofibrosis (MF) mice were fibrocytes derived from neoplastic hematopoietic cells. Neoplastic monocyte depletion eliminated collagen- and fibronectin-producing fibrocytes in BM and spleen, and ameliorated most characteristic MF features in Jak2V617F transgenic mice, including BM fibrosis, anemia, and splenomegaly, while had little effect on the elevated numbers of megakaryocytes and stem cells in BM, and leukothrombocytosis in peripheral blood. TGF-ß1, which was produced by hematopoietic cells including fibrocytes, promoted the differentiation of neoplastic monocytes to fibrocytes, and elevated plasma TGF-ß1 levels were normalized by monocyte depletion. Collectively, our data suggest that neoplastic fibrocytes are the major contributor to BM fibrosis in PMF, and TGF-ß1 is required for their differentiation.

Mutations in JAK2, myeloproliferative leukemia virus (MPL), or calreticulin (CALR) occur in hematopoietic stem cells (HSCs) and are detected in more than 80% of patients with myeloproliferative neoplasms (MPNs). They are thought to play a driver role in MPN pathogenesis via autosomal activation of the JAK-STAT signaling cascade. Mutant CALR binds to MPL, activates downstream MPL signaling cascades, and induces essential thrombocythemia in mice. However, embryonic lethality of Calr-deficient mice precludes determination of a role for CALR in hematopoiesis. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr-deficient mice. CALR deficiency had little effect on the leukocyte count, hemoglobin levels, or platelet count in peripheral blood. However, Calr-deficient mice showed some hematopoietic properties of MPN, including decreased erythropoiesis and increased myeloid progenitor cells in the bone marrow and extramedullary hematopoiesis in the spleen. Transplantation experiments revealed that Calrhaploinsufficiency promoted the self-renewal capacity of HSCs. We generated CALRdel52 mutant transgenic mice with Calrhaploinsufficiency as a model that mimics human MPN patients and found that Calrhaploinsufficiency restored the self-renewal capacity of HSCs damaged by CALRmutations. Only recipient mice transplanted with Lineage−Sca1+c-kit+cells harboring both CALRmutation and Calrhaploinsufficiency developed MPN in competitive conditions, showing that CALRhaploinsufficiency was necessary for the onset of CALR-mutated MPNs.

Mutations in JAK2, myeloproliferative leukemia virus (MPL), or calreticulin (CALR) occur in hematopoietic stem cells (HSCs) and are detected in more than 80% of patients with myeloproliferative neoplasms (MPNs). They are thought to play a driver role in MPN pathogenesis via autosomal activation of the JAK-STAT signaling cascade. Mutant CALR binds to MPL, activates downstream MPL signaling cascades, and induces essential thrombocythemia in mice. However, embryonic lethality of Calr-deficient mice precludes determination of a role for CALR in hematopoiesis. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr-deficient mice. CALR deficiency had little effect on the leukocyte count, hemoglobin levels, or platelet count in peripheral blood. However, Calr-deficient mice showed some hematopoietic properties of MPN, including decreased erythropoiesis and increased myeloid progenitor cells in the bone marrow and extramedullary hematopoiesis in the spleen. Transplantation experiments revealed that Calr haploinsufficiency promoted the self-renewal capacity of HSCs. We generated CALRdel52 mutant transgenic mice with Calr haploinsufficiency as a model that mimics human MPN patients and found that Calr haploinsufficiency restored the self-renewal capacity of HSCs damaged by CALR mutations. Only recipient mice transplanted with Lineage−Sca1+c-kit+ cells harboring both CALR mutation and Calr haploinsufficiency developed MPN in competitive conditions, showing that CALR haploinsufficiency was necessary for the onset of CALR-mutated MPNs.

Monocyte-derived fibrocytes play an important role in the progression of fibrosis in the skin, lungs, heart and kidney. However, the contribution of fibrocytes to liver fibrosis is unclear. The aim of this study was to investigate whether fibrocytes contributed to fibrosis progression in the livers of carbon tetrachloride (CCl4)-treated mice.

Interferon (IFN)-α exhibits antiviral and antiproliferative effects on normal and neoplastic cells. It is an effective treatment option for myeloproliferative neoplasms (MPNs). The intracellular signaling of IFN-α is triggered by binding of IFN-α to its specific receptors on the cell surface, followed by the activation of tyrosine kinase-2 (TYK2) and Janus kinase-1 (JAK1), both of which are receptor-associated Janus kinases (JAKs). Activated JAKs activate many signaling molecules, including signal transducers and activators of transcription (STATs). Antiviral activity of IFN-α was abrogated in Stat1deficient-embryonic fibroblasts, however, Tyk2deficient-cells were resistant to viral infection in the presence of IFN-α, indicating the essential role of STAT1 for the antiviral activity of IFN-α, and TYK2 is not required for that. The antiproliferative effect is another major activity of IFN-α, however, the precise antiproliferative mechanism of IFN-α is not completely understood. We assessed the effect of ropeginterferon-α-2b, a monopegylated IFN-α-2b, on MPN model mice ( Jak2V617F mice), and investigated the role of TYK2 in the antiproliferative effect of IFN-α.

Adult T-cell leukemia/lymphoma (ATL) is a heterogeneous group of peripheral T-cell malignancies characterized by human T-cell leukemia virus type-1 infection, whose genetic profile has recently been fully investigated. However, it is still poorly understood how these alterations affect clinical features and prognosis. We investigated the effects of genetic alterations commonly found in ATL on disease phenotypes and clinical outcomes, based on genotyping data obtained from 414 and 463 ATL patients using targeted-capture sequencing and single nucleotide polymorphism array karyotyping, respectively. Aggressive (acute/lymphoma) subtypes were associated with an increased burden of genetic and epigenetic alterations, higher frequencies of TP53 and IRF4 mutations, and many copy number alterations (CNAs), including PD-L1 amplifications and CDKN2A deletions, compared with indolent (chronic/smoldering) subtypes. By contrast, STAT3 mutations were more characteristic of indolent ATL. Higher numbers of somatic mutations and CNAs significantly correlated with worse survival. In a multivariate analysis incorporating both clinical factors and genetic alterations, the Japan Clinical Oncology Group prognostic index high-risk, older age, PRKCB mutations, and PD-L1 amplifications were independent poor prognostic factors in aggressive ATL. In indolent ATL, IRF4 mutations, PD-L1 amplifications, and CDKN2A deletions were significantly associated with shorter survival, although the chronic subtype with unfavorable clinical factors was only marginally significant. Thus, somatic alterations characterizing aggressive diseases predict worse prognosis in indolent ATL, among which PD-L1 amplifications are a strong genetic predictor in both aggressive and indolent ATL. ATL subtypes are further classified into molecularly distinct subsets with different prognosis. Genetic profiling might contribute to improved prognostication and management of ATL patients.

Adult T-cell leukemia/lymphoma (ATL) is a heterogeneous group of peripheral T-cell malignancies characterized by human T-cell leukemia virus type-1 infection, whose genetic profile has recently been fully investigated. However, it is still poorly understood how these alterations affect clinical features and prognosis. We investigated the effects of genetic alterations commonly found in ATL on disease phenotypes and clinical outcomes, based on genotyping data obtained from 414 and 463 ATL patients using targeted-capture sequencing and single nucleotide polymorphism array karyotyping, respectively. Aggressive (acute/lymphoma) subtypes were associated with an increased burden of genetic and epigenetic alterations, higher frequencies of TP53and IRF4mutations, and many copy number alterations (CNAs), including PD-L1amplifications and CDKN2Adeletions, compared with indolent (chronic/smoldering) subtypes. By contrast, STAT3mutations were more characteristic of indolent ATL. Higher numbers of somatic mutations and CNAs significantly correlated with worse survival. In a multivariate analysis incorporating both clinical factors and genetic alterations, the Japan Clinical Oncology Group prognostic index high-risk, older age, PRKCBmutations, and PD-L1amplifications were independent poor prognostic factors in aggressive ATL. In indolent ATL, IRF4mutations, PD-L1amplifications, and CDKN2Adeletions were significantly associated with shorter survival, although the chronic subtype with unfavorable clinical factors was only marginally significant. Thus, somatic alterations characterizing aggressive diseases predict worse prognosis in indolent ATL, among which PD-L1amplifications are a strong genetic predictor in both aggressive and indolent ATL. ATL subtypes are further classified into molecularly distinct subsets with different prognosis. Genetic profiling might contribute to improved prognostication and management of ATL patients.

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3′ region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3′-untranslated region (UTR). Disruption of the Pd-l1 3′-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3′-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

Myeloproliferative neoplasms (MPNs) are clinically characterized by the chronic overproduction of differentiated peripheral blood cells and the gradual expansion of malignant intramedullary/extramedullary hematopoiesis. In MPNs mutations in JAK2 MPLor CALRare detected mutually exclusive in more than 90% of cases [1,2]. Mutations in them lead to the abnormal activation of JAK/STAT signaling and the autonomous growth of differentiated cells therefore they are considered as “driver” gene mutations. In addition to the above driver gene mutations mutations in epigenetic regulators such as TET2 DNMT3A ASXL1 EZH2or IDH1/2are detected in about 5%–30% of cases respectively [3]. Mutations in TET2 DNMT3A EZH2or IDH1/2commonly confer the increased self-renewal capacity on normal hematopoietic stem cells (HSCs) but they do not lead to the autonomous growth of differentiated cells and only exhibit subtle clinical phenotypes [4,6–8,5]. It was unclear how mutations in such epigenetic regulators influenced abnormal HSCs with driver gene mutations how they influenced the disease phenotype or whether a single driver gene mutation was sufficient for the initiation of human MPNs. Therefore we focused on JAK2V617F and loss of TET2—the former as a representative of driver gene mutations and the latter as a representative of mutations in epigenetic regulators—and examined the influence of single or double mutations on HSCs (Lineage−Sca-1+c-Kit+cells (LSKs)) by functional analyses and microarray whole-genome expression analyses [9]. Gene expression profiling showed that the HSC fingerprint genes [10] was statistically equally enriched in TET2-knockdown-LSKs but negatively enriched in JAK2V617F–LSKs compared to that in wild-type-LSKs. Double-mutant-LSKs showed the same tendency as JAK2V617F–LSKs in terms of their HSC fingerprint genes but the expression of individual genes differed between the two groups. Among 245 HSC fingerprint genes 100 were more highly expressed in double-mutant-LSKs than in JAK2V617F–LSKs. These altered gene expressions might partly explain the mechanisms of initiation and progression of MPNs which was observed in the functional analyses [9]. Here we describe gene expression profiles deposited at the Gene Expression Omnibus (GEO) under the accession number GSE62302including experimental methods and quality control analyses.

Acquired mutations of JAK2and TET2are frequent in myeloproliferative neoplasms (MPNs). We examined the individual and cooperative effects of these mutations on MPN development. Recipients of JAK2V617F cells developed primary myelofibrosis–like features; the addition of loss of TET2 worsened this JAK2V617F-induced disease, causing prolonged leukocytosis, splenomegaly, extramedullary hematopoiesis, and modestly shorter survival. Double-mutant (JAK2V617F plus loss of TET2) myeloid cells were more likely to be in a proliferative state than JAK2V617F single-mutant myeloid cells. In a serial competitive transplantation assay, JAK2V617F cells resulted in decreased chimerism in the second recipients, which did not develop MPNs. In marked contrast, cooperation between JAK2V617F and loss of TET2 developed and maintained MPNs in the second recipients by compensating for impaired hematopoietic stem cell (HSC) functioning. In-vitro sequential colony formation assays also supported the observation that JAK2V617F did not maintain HSC functioning over the long-term, but concurrent loss of TET2 mutation restored it. Transcriptional profiling revealed that loss of TET2 affected the expression of many HSC signature genes. We conclude that loss of TET2 has two different roles in MPNs: disease accelerator and disease initiator and sustainer in combination with JAK2V617F.

The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.

Among various subtypes of malignant lymphomas, activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), mantle cell lymphoma (MCL), and adult T-cell leukemia/lymphoma (ATL) are clinically intractable as patients with these lymphomas carry a dismal prognosis, with long-term survival rates of 10-30%. Therefore, a novel therapeutic strategy is required to better manage patients with these malignancies. Recently, we and other investigators performed comprehensive genetic studies and revealed frequent genetic alterations in B and T cell antigen receptor signaling and NF-κB pathway, such as CD79A/B and CARD11 mutations in ABC-DLBCL and PLCG1, PRKCB, and CARD11 mutations in ATL, suggesting the biological relevance of this pathway.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma that develops in about 5% of human T-cell leukemia/lymphoma virus 1 (HTLV-1) carriers. In addition to viral oncogenes, namely taxand HTLV-1 bZIP factor(HBZ), gene mutations, highly enriched for T-cell receptor (TCR)-NF-kB signaling, should be involved in the development of ATL. Among gene mutations, mutation in CARD11, a cytoplasmic scaffolding protein required for TCR-induced NF-kB activation, was detected in 24% of ATL patients. Here we generated a mouse model for conditional expression of a human ATL-derived CARD11(E626K) gain-of-function mutant, and demonstrated CARD11 activation induced oligoclonal expansion of T-cell and infiltration to many organs. We also showed that expression of HBZ accelerated mutant CARD11-induced lymphoproliferative diseases.

Morishita: Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Mizutani:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Tozaki:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Arikawa:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company. Yoda:Chordia Therapeutics Inc.: Research Funding. Izutsu:Symbio: Research Funding; Solasia: Research Funding; Celgene: Research Funding; Chugai: Research Funding; Novartis: Research Funding; Ono Pharmaceutical: Research Funding; Bayer pharmaceuticals: Research Funding; Daiichi Sankyo: Research Funding; AstraZeneca: Research Funding; Eisai: Research Funding; Incyte: Research Funding; Abbvie pharmaceuticals: Research Funding; HUYA Japan: Research Funding; Sanofi: Research Funding; Janssen: Research Funding; Yakult: Research Funding. Minami:Bristol-Myers Squibb Company: Honoraria; Novartis Pharma KK: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria. Shimoda:Otsuka Pharmaceutical: Research Funding; Pfizer Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Merck & Co.: Research Funding; Astellas Pharma: Research Funding; AbbVie Inc.: Research Funding; PharmaEssentia Japan: Research Funding; Perseus Proteomics: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company: Honoraria; Novartis: Honoraria, Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding. Miyake:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Ogawa:KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding.

Kogure: Takeda Pharmaceutical Company Limited.: Honoraria. Nosaka:Kyowa Kirin Co.Ltd: Honoraria; Chugai pharmaceutical Co. Ltd: Honoraria; Novartis international AG: Honoraria; Celgene K.K: Honoraria; Eisai Co., Ltd: Honoraria; Merck Sharp & Dohme K.K.: Honoraria; Bristol-Myer Squibb: Honoraria. Imaizumi:Kyowa Kirin Co. Ltd.: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Eisai: Honoraria. Utsunomiya:Kyowa Kirin: Honoraria; Celgene: Honoraria. Shah:Celgene: Research Funding; BMS: Research Funding; Physicians Education Resource: Honoraria. Janakiram:Takeda, Fate, Nektar: Research Funding. Ramos:NIH: Research Funding. Takaori-Kondo:Astellas Pharma: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; Eisai: Research Funding; Nippon Shinyaku: Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; MSD: Honoraria. Miyazaki:Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Celgene: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Otsuka Pharmaceutical: Honoraria; Novartis Pharma KK: Honoraria; Astellas Pharma Inc.: Honoraria. Matsuoka:Chugai Pharmaceutical Co. Ltd: Research Funding; Bristol-Myers Squibb: Research Funding; Kyowa Kirin Co. Ltd.: Research Funding. Ishitsuka:Takeda: Other: Personal fees, Research Funding; mundiharma: Other: Personal fees; Taiho Pharmaceuticals: Other: Personal fees, Research Funding; Janssen Pharmaceuticals: Other: Personal fees; Novartis: Other: Personal fees; Pfizer: Other: Personal fees; Astellas Pharma: Other, Research Funding; Genzyme: Other; Sumitomo Dainippon Pharma: Other, Research Funding; Eisai: Other, Research Funding; Mochida: Other, Research Funding; Shire: Other; Otsuka Pharmaceutical: Other; Ono Pharmaceutical: Other, Research Funding; Teijin Pharma: Research Funding; MSD: Research Funding; Asahi kasei: Research Funding; Eli Lilly: Research Funding; Daiichi Sankyo: Other; Huya Japan: Other; Celgene: Other: Personal Fees; Kyowa Hakko Kirin: Other: Personal fees, Research Funding; BMS: Other: Personal fees; Chugai Pharmaceutical: Other: Personal fees, Research Funding. Ogawa:Asahi Genomics Co., Ltd.: Current equity holder in private company; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding. Shimoda:Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria; Shire plc: Honoraria; Celgene: Honoraria; Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Novartis: Honoraria, Research Funding. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company.

Sugiyama: Chordia Therapeutics, Japan.: Current Employment. Morishita:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Shimoda:Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Novartis: Honoraria, Research Funding; Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy with a dismal prognosis, caused by HTLV-1. Although our previous study, mainly using whole-exome sequencing and SNP array karyotyping, discovered many driver mutations and copy number alterations (CNAs), the whole-genome landscape of ATL still remains elusive.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell malignancy, caused by human T-cell leukemia virus type-1 (HTLV-1) infection. To elucidate immune microenvironment and heterogeneity of HTLV-1-infected normal and leukemic cells, we performed multi-omics single cell analysis, evaluating whole-transcriptome, 101 surface marker proteins, and T/B-cell receptor repertoires in the same single cells. We analyzed 236,192 peripheral blood mononuclear cells (PBMCs) from 31 ATL patients (35 samples including 4 sequential samples), 11 HTLV-1-infected carriers, and 4 healthy donors. In our analysis, expression of HTLV-1-related genes, such as HBZ, clearly identified a distinct cluster of HTLV-1-infected cells within non-malignant CD4+T cells. These cells are characterized by a CD45RO+CD62L-CD7-CCR4+CD25+CD73+memory/effector phenotype. By contrast, malignant ATL cells were segregated into different clusters across patients, suggestive of inter-tumor heterogeneity. Transcriptome analysis of CD4+T cells revealed up-regulation of interferon (IFN) responses and down-regulation of TNFa signaling in malignant ATL cells compared with HTLV-1-infected normal CD4+T cells. Likewise, sequential sample analysis showed that progression from indolent to aggressive disease enhanced IFN responses, suggesting a pivotal role of this pathway in the ATL pathogenesis. Surface marker protein analysis demonstrated that HTLV-1 infection up-regulated the expression of stimulatory and inhibitory immune checkpoint molecules (such as OX40 and TIGIT, respectively), which was further augmented by ATL progression.

Kogure: Takeda Pharmaceutical Company Limited.: Honoraria. Shimoda:Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company: Honoraria; Novartis: Honoraria, Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Shire plc: Honoraria; Celgene: Honoraria; Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding. Kataoka:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Takeda Pharmaceutical Company: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Genomics: Current equity holder in private company.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Mutations in the Calreticulin (CALR)gene were identified in cases of myeloproriferative neoplasms (MPNs), and various functions of the CALR mutant protein are being elucidated. On the other hand, few data are available on the role of CALR in the hematopoietic system. The knockout (KO) mice of Calrare impaired in expression of transcription factors necessary for cardiac development and are embryonic lethal. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific CalrKO mice.

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008).

Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell lymphoma that is caused by HTLV-1. The prognosis of acute and lymphomatous variants of ATLL is poor, ranging from 2 weeks to >1 year. Compared to other types of malignant lymphomas, the organ infiltration is frequently observed in ATLL (Yamada et al. Leuk Lymphoma 1997). We previously reported the landscape of genetic mutations in ATLL, and showed that various mutations occurred in the TCR-NFκB pathway in more than 90% of ATLL cases (Kataoka et al. Nat Genet 2015). These somatic mutations are thought to develop ATLL in combination with viral genes such as HTLV-1 bZIP factor (HBZ). Among them, mutations in TET2, an epigenetic regulator, was observed in about 10% of ATLL cases. Higher frequencies inTET2mutation was reported in other types of peripheral T-cell lymphoma (PTCL); it was observed in about 80% of angioimmunoblastic T-cell lymphoma (AITL) and in about 50% of PTCL, not otherwise specified. In PTCL, it has been reported that additional mutations in lymphoid progenitors derived from TET2mutated hematopoietic stem cells cause increased cell proliferation and anti-apoptosis, leading to the disease progression. In ATLL, the role of TET2mutation in disease progression is still unknown. In this study, we investigated the role of TET2mutation in ATLL using mouse model and acute and lymphomatous variant ATLL cohort.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

CALRexon9 frameshift mutations function as driver mutations in essential thrombocythemia (ET) and primary myelofibrosis patients with non-mutated JAK2or MPL. The mutations augment signal transducer and activator of transcription activity in the presence of MPL, induce increased cell proliferation and growth factor independence in cell lines, and cause ET-like myeloproliferative neoplasms (MPN) in mice.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Introduction

Calreticulin(CALR) exon 9 mutations were reported in about two-thirds of JAK2or MPLmutation negative ET and PMF patients. The mutations cause frameshifts that result in proteins with novel C-terminus.Retrovirus-mediated gene transfer into cell lines and mouse bone marrow (BM) cells is a common technique, but the expression level is very high compared to the physiological expression.We investigated the effects of physiological expression of mutant CALRusing CRISPR/Cas9 gene editing techniques for cell lines, and as for the mouse model, we generated a transgenic mice (TG) expressing human CALRdel52 mutant.

Tobinai: Gilead Sciences: Research Funding. Miyazaki:Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Tobinai: Gilead Sciences: Research Funding. Miyazaki:Kyowa-Kirin: Honoraria, Research Funding; Celgene Japan: Honoraria; Sumitomo Dainippon: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria.

Tobinai: Gilead Sciences: Research Funding. Miyazaki:Shin-bio: Honoraria; Chugai: Honoraria, Research Funding; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Adult T-cell leukemia/lymphoma (ATL) is a distinct subtype of peripheral T-cell neoplasms associated with human T-cell leukemia virus type-1 retrovirus. ATL includes a heterogeneous group of patients in terms of pathological and clinical features as well as prognosis, suggesting the presence of underlying molecular pathogenesis that could explain such heterogeneity among patients. Recently, we performed an integrated molecular analysis of a large number of ATL cases and delineated a comprehensive registry of gene mutations and other genetic/epigenetic lesions in ATL. In this study, we investigated possible correlations between these genetic/epigenetic lesions and clinical/pathological phenotypes in a large set of ATL patients, with a special focus on the impact of mutations and copy number alterations (CNAs) on clinical outcome.

Background:

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm of largely unknown genetic basis, which is associated with human T-cell leukemia virus type-1 (HTLV-1) infection. To delineate a genetic landscape of somatic alterations in ATL, we have performed an integrated genetic study, in which whole-genome/exome (WGS/WES) and transcriptome sequencing (RNA-seq) was performed for a cohort of 83 paired ATL samples, followed by extensive validation using targeted sequencing of detected mutations in 370 follow-up samples.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma, which is etiologically associated with human T-cell leukemia virus type 1 (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize human T cells, there is a long latency period of ~50 years before the onset of ATL, suggesting that HTLV-1 infection alone may be insufficient for the development of ATL, but additional acquired genetic events that accumulate during the later life are essential for the development of ATL. However, such somatic alterations underlying the pathogenesis of ATL have not been fully elucidated.

In primary myelofibrosis patients, somatic mutations such as JAK2V617F(JAKVF) and MPLW515 that activate JAK-STAT signaling are often seen. Small-molecule JAK2 inhibitors are effective for organomegaly and constitutional symptoms, but the drugs have little effect on BM fibrosis. To clarify the mechanism by which MPN cells with JAK2mutations cause BM fibrosis, we compared the gene expression patterns of Lin−Sca1+BM cells in JAK2VF transgenic mice (JAK2VF-TG), which develop myelofibrosis (MF), with that in WT mice. We found that TGFb1and HOXB4, the target genes of transcription factor USF1 were highly expressed. TGFβ1, which is secreted by hematopoietic cells, is essential for fibrotic development in a murine model of MF (Chagraoui et al. Blood 2002), and increased expression of HOXB4 enhances human megakaryocytic development (Zhong et al. BBRC 2010).

JAK2V617F (JAK2VF) is the most frequent mutation in myeloproliferative neoplasms (MPN), and its role has been demonstrated in mouse models. Actually, JAK2VF transgenic (JAK2VF Tg) mice generated by us induce lethal MPN (Shide et al. Leukemia 2008). Recently, mutations of epigenetic regulator such as TET2 are also frequently identified in MPN, and several TET2 knock out or knock down (TET2KD) mouse models are generated. We previously analyzed TET2KD mice (Ayu17-449) (Shide et al. Leukemia 2012). TET2KD fetal liver (FL) or bone marrow (BM) cells showed a growth advantage over Wt BM cells, with increased self-renewal capacity of hematopoietic stem cells; however TET2KD mice didn’t develop MPN, and its role in MPN remained unclear. To explore the role of TET2 deficiency in MPN harboring JAK2VF, we examined the cooperative effect, using these mutant mice.(1) Mice and collection of test cells. JAK2VF Tg mice (C57BL/6, Ly5.2) and TET2KD mice (Ayu17-449, C57BL/6, Ly5.2) were used. We crossed them, and collected JAK2Wt-TET2Wt (Wt-Wt), JAK2Wt-TET2KD (Wt-KD), JAK2VF-TET2Wt (VF-Wt), and JAK2VF-TET2KD (VF-KD) FL cells. (2) Non-competitive repopulation assay (NCRA). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) without competitor cells. Recipients were analyzed by complete blood counts, flow cytometry, colony-forming assay, colony-replating assay, pathology at 20-28 weeks post-transplantation, and overall survival. (3) Competitive repopulation assay (CRA) and serial BM transplantation (sBMT). FL cells (Ly5.2, 1x106 cells) were transplanted into lethally irradiated recipients (Ly5.1) with competitor Wt BM cells (Ly5.1, 5x106 cells), and sBMT was performed by 1x106 BM cells of the recipients at every 12 weeks post-transplantation. Recipients which were not selected as the donors were analyzed. (4) Analyses of adult mutant mice. Mice were bred in BDF1 background and analyzed at 20 or more weeks of age, as well as the recipients in NCRA. (5) Statistical analysis. Results were presented as means±S.D. Two-tailed Student’s t-test and log-rank test were used.In NCRA, both recipients transplanted with VF-Wt cells and VF-KD cells developed MPN with increase in WBC and Plt, decrease in Hb, fibrosis in BM and spleen, and extramedullary hematopoiesis (EMH) of lung and liver; and the latter developed more severe MPN and died earlier: VF-Wt (n=10) vs. VF-KD (n=10); WBC (x104/µl), 4.2±1.6 vs. 7.3±3.3 (p<0.05); peripheral blood (PB) myeloid cells (%), 59.6±9.7 vs. 71.9±8.2 (p<0.05); liver weight (g), 1.15±0.22 vs. 1.48±0.22 (p<0.01); spleen weight (g), 0.26±0.11 vs. 0.52±0.19 (p<0.01): VF-Wt (n=36) vs. VF-KD (n=30); mean survival time (weeks), 36 vs. 39 (p<0.05). In colony-forming assay, number of CFU-GM was more increased in VF-KD cells than VF-Wt cells: VF-Wt (n=9) vs. VF-KD (n=9); colonies/2x104 BM cells, 107±37 vs. 157±46 (p<0.05). In colony-replating assay, VF-Wt BM cells lost replating capacity by 3rd to 5th passage; VF-KD BM cells retained replating capacity beyond 5th passage: VF-Wt (n=9) vs. VF-KD (n=6); number of colonies in 4th passage, 5.9±6.8 vs. 896±613 (p<0.01). In CRA, all recipients transplanted with VF-Wt cells (n=9) or VF-KD cells (n=9) showed ≥ 70% test cell-derived PB chimerism, and developed MPN with fibrosis and EMH at 12 weeks. In 2nd BMT, 4/9 recipients transplanted with VF-Wt cells showed ≥ 35% PB chimerism at 12 weeks. Six recipients were analyzed at 12-16weeks, and no one (0/6) showed pathological findings of MPN. Whereas, 7/9 recipients transplanted with VF-KD cells showed ≥ 35% PB chimerism. Five recipients were analyzed, and 3/5 developed MPN with fibrosis and EMH: VF-Wt (n=6) vs. VF-KD (n=5); liver weight (g), 1.00±0.12 vs. 1.39±0.15 (p<0.002); spleen weight (g), 0.069±0.019 vs. 0.20±0.097 (p<0.05). In analyses of adult mutant mice, both VF-Wt mice and VF-KD mice developed MPN, and disease severities or colony-replating capacities are similar tendencies as those in transplantation model.TET2 deficiency increases severity of MPN harboring JAK2VF. TET2 deficiency enhances disease initiating potential of JAK2VF-MPN stem cells. TET2 deficiency is considered to be critical for both onset and progression of MPN harboring JAK2V617F.No relevant conflicts of interest to declare.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T cell neoplasm caused by human T-cell lymphotropic virus type I with very poor prognosis. The relationship between chemotherapy dose intensity and clinical outcome of ATL in clinical practice remains unclear.To elucidate the clinical characteristics and outcome of ATL patients, we retrospectively analyzed 118 patients diagnosed with ATL at 7 institutes in Miyazaki Prefecture, Japan from 2010 to 2012. There were 67 males and 51 females. The median age of the patients was 70 years (range 44–92). Subtypes included acute- (n=85) and lymphoma-type (n=33). One hundred one patients were treated with one of the below combination chemotherapy: (1) VCAP-AMP-VECP (LSG15); (2) CHOP (including pirarubicin (THP)-COP); and (3) non-LSG15, non-CHOP regimen. The prognostic value of the recently proposed prognostic index for ATL, namely ATL-PI (Katsuya et al. J Clin Oncol. 2012), was evaluated in this cohort. Relative dose intensity (RDI) during the first 12 weeks of therapy was calculated based on the standard regimen. Average RDI (ARDI) for LSG15 and CHOP was calculated.The median survival time (MST), 1- and 2-years overall survival (OS) rates of the entire cohort were 8.5 months, 35.3% and 23.0%, respectively. MSTs of patients less than 70 years and patients 70 years or older were 11.8 and 5.7 months, respectively (p=0.03). MSTs among all age groups for acute- and lymphoma- type were 8.3 and 10.0 months, respectively (p=0.445). Although ATL-PI could efficiently discriminate high-risk patients, it failed to separate the intermediate- and low-risk patients in this cohort. As almost all patients in this cohort were stage III or IV, ATL-PI was modified to exclude the Ann Arbor stage from variables. This modified ATL-PI could stratify our cohort into three distinct risk groups. MSTs were 3.9, 10.9, and 18.1 months for patients in high, intermediate, and low risk groups, respectively (P < 0.01).Among this cohort, 38 patients have received LSG15 and 47 patients received CHOP. Variables known to affect outcome were similar in both groups, except the age. MSTs were 11.5 and 8.1 months for patients treated with LSG15 and CHOP, respectively (p=0.206). As the median age of CHOP group is about 10 years greater than that of LSG15 group, we examined the MST in each therapy group according to the age. The MSTs for less than 70 years old were 11.7 and 21.9 months for patients treated with LSG15 and CHOP, respectively (p=0.311). Similarly, the MSTs for 70 years or older were 8.6 and 5.5 months for patients treated with LSG15 and CHOP, respectively (p=0.142). In practice, we conclude that CHOP is still a standard therapy for ATL.We next examined the RDI in each therapy groups and its relationship with OS. During the first 12 weeks, 73.7% of patients treated with LSG15 received ≥50% of planned DI, whereas 50.0% of patients treated with CHOP received ≥50% of planned DI. MSTs were significantly longer in patients treated with higher ARDI (≥50%) than that in patients with lower ARDI (<50%) (14.7 vs. 6.2 months for LSG15; p<0.01, 19.0 vs. 4.0 months for CHOP; p<0.01).Our modified ATL-PI excluding the Ann Arbor stage from variables in original ATL-PI may have prognostic value for patients with ATL. In the daily practice in Japan, no superiority of LSG15 compared to CHOP could be demonstrated both in young and elderly patient groups. Reduced ARDI in the first line chemotherapy for ATL is pervasive. Considering the significantly poor outcome of patients treated with ARDI less than 50%, changes in therapy with alternative strategy such as applying mogamulizumab, relatively early in the course of treatment, may improve outcome.No relevant conflicts of interest to declare.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T cell neoplasm caused by human T-cell lymphotropic virus type I with very poor prognosis. The relationship between chemotherapy dose intensity and clinical outcome of ATL in clinical practice remains unclear.

JAK2V617F (JAK2VF) is the most frequent mutation in myeloproliferative neoplasms (MPN), and its role has been demonstrated in mouse models. Actually, JAK2VF transgenic (JAK2VF Tg) mice generated by us induce lethal MPN (Shide et al. Leukemia 2008). Recently, mutations of epigenetic regulator such as TET2 are also frequently identified in MPN, and several TET2 knock out or knock down (TET2KD) mouse models are generated. We previously analyzed TET2KD mice (Ayu17-449) (Shide et al. Leukemia 2012). TET2KD fetal liver (FL) or bone marrow (BM) cells showed a growth advantage over Wt BM cells, with increased self-renewal capacity of hematopoietic stem cells; however TET2KD mice didn't develop MPN, and its role in MPN remained unclear. To explore the role of TET2 deficiency in MPN harboring JAK2VF, we examined the cooperative effect, using these mutant mice.

No relevant conflicts of interest to declare.