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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.

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.

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOAmutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOAmutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOAmutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss- and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOAmutations in human cancers.

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.

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3‐mediated main signal and a CD28‐mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100‐kDa protein (pp100) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3‐kinase, Valosin‐containing protein (VCP), Nucleolin, Gab2 (Grb2‐associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3‐mediated main signal.

AbstractObjective:Growth hormone (GH) has been reported to have a potent effect on the immune system. However, the detailed mechanism of the effect of GH on the immune system has not yet been clarified. This study was designed to investigate the nature of this mechanism. Methods:In the present study, we investigated the effects of GH on the susceptibility of both human CEM/C7 lymphocytes and human IM-9 lymphocytes to Fas-induced apoptosis. Results:Both cell lines expressed GH receptor mRNA. GH rescued Fas-induced suppression of [3H]-thymidine incorporation into each cell line. GH prevented Fas-induced apoptosis in each cell line without changing Fas antigen expression. We next investigated the mechanisms of the prevention of Fas-induced apoptosis, by focusing on intracellular molecules related to the apoptotic signal. Bcl-2 expression was increased by GH treatment in both CEM/C7 and IM-9 lymphocytes. GH also downregulated caspase-3 expression and inhibited activation of caspase-3 in both cell lines. Conclusion:These findings suggest that GH regulates the human immune system through inhibition of Fas-induced apoptosis in activated T and B lymphocytes.Copyright © 2002 S. Karger AG, Basel

A variety of growth factor receptors induce the tyrosine phosphorylation of a nonreceptor protein-tyrosine kinase Tec as well as that of a Tec-binding protein of 62 kDa. Given the similarity in properties between this 62-kDa protein and p62Dok-1, the possibility that these two proteins are identical was investigated. Overexpression of a constitutively active form of Tec in a pro-B cell line induced the hyperphosphorylation of endogenous Dok-1. Tec also associated with Dok-1 in a phosphorylation-dependent manner in 293 cells. Tec mediated marked phosphorylation of Dok-1 both in vivoand in vitro, and this effect required both the Tec homology and Src homology 2 domains of Tec in addition to its kinase activity. Expression of Dok-1 in 293 cells induced inhibition of Ras activity, suggesting that Dok-1 is a negative regulator of Ras. In the immature B cell line Ramos, cross-linking of the B cell antigen receptor (BCR) resulted in tyrosine phosphorylation of Dok-1, and this effect was markedly inhibited by expression of dominant negative mutants of Tec. Furthermore, overexpression of Dok-1 inhibited activation of the c-fospromoter induced by stimulation of the BCR. These results suggest that Tec is an important mediator of signaling from the BCR to Dok-1.

CD38 is a transmembrane molecule whose expression varies during hematopoietic cell differentiation. We used stroma-supported cultures of human myeloid cells to assess the effects of CD38 ligation on myeloid differentiation. In 8 experiments with CD34+cells purified from normal bone marrow or cord blood, flow cytometry used with antibodies to CD34 and myeloperoxidase (MPO) identified 4 cell populations after 7 days of culture. Addition of anti-CD38 (T16) to the cultures induced a profound reduction of the most mature (CD34−MPO++) cell population, which includes promyelocytes, myelocytes and metamyelocytes; mean (± SD) cell recovery was 12.8% ± 9.8% of that in parallel cultures with an isotype-matched control antibody. The suppressive effect of CD38 ligation on phenotypically more immature normal cells was inconsistent but generally less pronounced. Recovery of CD34++MPO− cells was 63.3% ± 24.4%, recovery of CD34[+/−]MPO− cells was 95.3% ± 35.1%, and recovery of CD34−MPO+cells was 42.0% ± 18.7% of that in control cultures. However, anti-CD38 suppressed recovery of cells obtained from 6 patients with CD38+ acute myeloid leukemia; after 7-day cultures, cell recovery was 25.2% ± 21.7% of that in control cultures. Cell recovery was also reduced by F(ab′)2 or Fab fragments of anti-CD38. CD38 ligation dramatically suppressed recovery of murine 32D myeloid cells transfected with human CD38 and cocultured with stroma (3.8% ± 7.3%; n = 7). CD38 ligation of CD38 + 32D cells also induced cell aggregation, tyrosine kinase activity, and Ca++ influx. We conclude that CD38 mediates signals that culminate in suppression of myeloid cell growth and survival.

The tyrosine kinase Tec belongs to a new group of structurally related nonreceptor tyrosine kinases that also includes Btk and Itk. Previous studies have suggested that these kinases have lineage-specific roles, with Tec being involved mainly in the regulation of cytokine-mediated myeloid cell growth and differentiation. In this study, we investigated expression and activation of Tec in human B-lymphoid cell lines representing different stages of B-cell maturation, including pro-B (RS4;11, 380, REH), pre-B (NALM6), and mature B (Ramos, and one Epstein-Barr virus [EBV]-transformed lymphoblastoid line) cells. Like Btk, Tec protein was expressed in all B-cell lines tested. Tec was also highly expressed in two EBV-transformed lymphoblastoid lines derived from patients with X-linked agammaglobulinemia (XLA) lacking Btk expression, as well as in tonsillar lymphoid cells. In surface immunoglobulin-positive B cells (Ramos), ligation of the B-cell antigen receptor (BCR) with anti-IgM antibodies caused marked tyrosine phosphorylation of Tec and increased Tec tyrosine kinase activity. Likewise, cross-linking of CD19 with a monoclonal antibody in BCR-negative pro-B (RS4;11, 380) and pre-B (NALM6) cells induced Tec tyrosine phosphorylation and increased Tec autophosphorylation, as well as Btk activation. Tyrosine phosphorylation of Tec, but not of Btk, was detectable in RS4;11 cells after CD38 ligation, suggesting that these kinases are regulated differently. We conclude that Tec is expressed and can be stimulated throughout human B-cell differentiation, implying that this tyrosine kinase plays a role in B-cell development and activation.

The signaling pathways that arrest the cell cycle and trigger cell death are only partially known. Dimerization of CD38, a 45-kD transmembrane type II glycoprotein highly expressed in immature B cells, inhibits cell growth and causes apoptosis in normal and leukemic B-cell progenitors, but the molecular mechanisms underlying these cellular responses are unknown. In the present study, we found that CD38 ligation in the immature B-cell lines 380, REH, and RS4;11 caused rapid tyrosine phosphorylation of the protein product of the proto-oncogene c-cbl.Dimerization of CD38 was accompanied by the association of cblwith the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K), resulting in markedly increased PI 3-K activity in antiphosphotyrosine and anti-cblimmunoprecipitates. Wortmannin and LY294002, two potent inhibitors of PI 3-K, rescued immature B cells from CD38-mediated growth suppression. This effect was observed not only in model B-cell lines, but also in cultures of leukemic lymphoblasts from patients, and in normal bone marrow B-cell progenitors as well. Concentrations of inhibitors that reversed cellular responses to CD38 significantly decreased PI 3-K activity. By contrast, rapamycin, a p70 S6-kinase inhibitor, did not rescue immature B cells from CD38-mediated suppression. These results suggest that PI 3-K activity is essential for CD38-mediated inhibition of lymphopoiesis and that cbland PI 3-K are regulatory molecules whose activation can result in suppression of cell proliferation and apoptosis in immature lymphoid cells.

No relevant conflicts of interest to declare.

Myelodysplastic syndromes (MDS) have a risk of progression to acute myeloid leukemia (AML), but the deterioration mechanisms of MDS and the alteration points still remain to be elucidated. We previously established a myelodysplastic cell line, MDS92 from the bone marrow of an MDS patient, and after a long-term interleukin(IL)-3-containing culture of MDS92, five blastic sublines including MDS-L were isolated. From MDS-L, we obtained two sublines, MDS-L-2007 and MDS-LGF after culture in the presence and absence of IL-3, respectively. To investigate the mechanism of leukemic evolution, we applied a next-generation sequencing (NGS) to the series of cell lines for comprehensive, comparative exome analyses, and searched for the origin of mutations by ultra-deep target sequencing of the original patient bone marrow.

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.

No relevant conflicts of interest to declare.

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.

The survival of patients with adult T-cell leukemia (ATL) has been improved by the introduction of anti-CCR4 monoclonal antibody and the expanding use of allogeneic hematopoietic stem cell transplantation. However, not all patients benefit from these treatment modalities, warranting development of a novel therapeutic strategy. CD38, a cell surface ectoenzyme that functions as cyclic ADP ribose hydrolase, is an attractive target of chimeric antigen receptor (CAR) therapy for lymphoid neoplasms because it is widely expressed on the cells of B- or T-lymphoid malignancies. We have previously demonstrated the prominent cytotoxicity of T cells engineered to express an anti-CD38-CAR against B-lymphoma cells and myeloma cells expressing CD38. To expand the applicability of anti-CD38-CAR against ATL cells that usually express undetectable or low levels of CD38, notably, we were successfully able to induce cell surface CD38 expression in HTLV-1-infected cell lines with all-trans retinoic acid (ATRA) (Yoshida T, et al. 2013 ASH Meeting). In ATL cells freshly isolated obtained from the patients, we were able to induce CD38 with ATRA in 60-80% of the cells; the remaining cells survived under the anti-CD38-CAR treatment. We hereby report our attempts in improving the efficacy of anti-CD38-CAR T cells against ATL cells from the patients through the expression of CD38 enhanced with the entry of agents, which are clinically used. Firstly, we investigated whether ATL cells from patients could be transduced with anti-CD38-CAR and what is the efficiency of transduction into T cells in our settings. ATL cells (CD4+CD25+GFP+) transduced with retroviral vector were little detected. CD4-CD25-GFP+T cells alone were detected in our transduction methods. Transduction efficiency was over 40%. To increase the expression of CD38 on ATL cells, we took notice of the CD38 gene upstream region that contains binding sites for interferon regulatory factor-1 (IRF-1) and peroxisome proliferator-activated receptor (PPAR). We thus investigated whether IFN-α, IFN-γ or troglitazone, which is a PPAR-α and -γ agonist, could enhance CD38 expression in ATL cell lines (MT-4, Su9T, ED, and S1T cells), which are negative for CD38. IFN-α and IFN-γ efficiently enhanced CD38 expression in MT-4 cells in a dose-dependent manner but not in Su9T, ED, and S1T cells. As little as 2.5U/ml of IFN-α induced CD38 expression in MT-4 cells for 18 hours in vitro (>95% at positivity of CD38). 10-25% increase in CD38 expression was observed in ED cells with 125-250 pM troglitazone after 18 hours of treatment, but not in MT-4, Su9T, and S1T cells. Prolonged exposure to troglitazone was toxic to cells. Combined treatment with 10nM ATRA and IFN-α, which induced higher expression of CD38 than IFN-γ, synergistically enhanced CD38 expression of ATL cells from the patients (>90%at positivity of CD38). We next co-cultured ATL cells form three patients with T cells transduced with mock or anti-CD38-CAR in the presence of both ATRA and IFN-α at effector (E): target (T) ratio of 1: 2 for 3 days. The treatment eradicated more than 95% of these ATL cells, demonstrating that ATL cells can be eliminated by T cells harboring anti-CD38-CAR in the presence of ATRA and IFN-α, which is actively used for ATL patients. CD38 targeting therapy is a feasible method, because an anti-CD38 antibody, daratuzumab, has been used to treat plasma cell myeloma. The safety regarding the clinical use of T cells bearing anti-CD38-CAR still needs to be established. As CAR therapy reportedly causes cytokine storm and can potentially be lethal, we envision an inducible immunotherapy with CAR to be a preferred modality with increased efficacy and safety. Our results provide a rationale for a novel therapeutic strategy involving T cells carrying anti-CD38-CAR in combination with ATRA and IFN-α for patients with ATL.

No relevant conflicts of interest to declare.

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.

Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma (PTCL). ATL is initiated by immortalization of T-cells by human T-lymphotropic virus type I (HTLV-1) infection during early infancy, followed by an accumulation of multiple genetic hits to develop leukemia. However, little has been known about these genetic hits. Recently, frequent somatic mutations in RHOA, DNMT3A, IDH2and TET2have been reported in angioimmunoblastic T cell lymphoma (AITL) and PTCL not otherwise specified (PTCL-NOS), which are characterized by follicular helper T-cell phenotypes. Most of the RHOAmutations in AITL invariably cause an identical amino acid change (Gly17Val), which was shown to inhibit wild-type RHOA function in a dominant-negative manner. In addition, RHOAmutations have been also identified in gastric cancer and Burkitt lymphoma, with mutational hotspots including Tyr42 and Arg5. Interestingly, some of them were reported as working in a gain-of function manner.

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.

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.

No relevant conflicts of interest to declare.

Patients with adult T-cell leukemia and lymphoma (ATLL) often succumb to death even though multi-anti-cancer drugs are used. Thus, it is essential for establishing a novel therapeutic strategy for ATLL. We have previously developed a chimeric antigen receptor against CD38 (anti-CD38-CAR) and showed powerful cytotoxicity of anti-CD38-CAR to B-cell lymphoma cells as well as to myeloma cells expressing CD38. Unfortunately, as CD38 is poorly expressed on the cell surface of ATLL cells, it is required to induce CD38 to apply our anti-CD38-CAR. Here, we investigated cytotoxicity of T cells transduced with anti-CD38-CAR against ATLL cell lines and cells obtained from ATLL patients through CD38 induction by all-trans retinoic acid (ATRA), which is clinically available for acute promyelocytic leukemia. We evaluated an effect of ATRA on cytotoxicity of T cells bearing anti-CD38-CAR against ATLL cells through flow cytometry. We firstly confirmed the expression of anti-CD38-CAR on human T cells retrovirally transduced (10-70%). Then, secondly, we prepared ATLL cell lines (MT-2, MT-4, S1T, Hut102, and Su9T: >95%, <5%, <5%, 15%, and <5% at CD38 expression, respectively). We co-incubated CD38-specific T cells with ATLL cell line cells for 3 days. MT-2 cells were entirely abrogated by T cells harboring anti-CD38-CAR. However, others were restrictedly succumbed to death after 3-day co-culture with T cells carrying anti-CD38-CAR. Next, we investigated whether ATRA could enhance CD38 expression on the cell surface of ATLL cell lines and exert a cytotoxicity of T cells with anti-CD38-CAR. Intriguingly, even 10nM of ATRA augmented CD38 expression in MT-4, S1T, and Hut102 cells (>80%), but not in Su9T cells. Co-culture experiments in the presence of ATRA showed that MT-4, S1T, and Hut102 but Su9T cells were efficiently eliminated by T cells bearing anti-CD38-CAR, leading to a positive correlation of cytotoxicity with CD38 expression level. We tested whether ATLL cells obtained from 3 patients were disrupted by T cells bearing anti-CD38-CAR. CD38 was expressed in the cells from patients at a variety of expression ratio (0-30%). Intriguingly, CD38 expression was significantly enhanced in ATLL cells from 2 of 3 individual patients with ATRA (>50%). And resultantly, T cells bearing anti-CD38-CAR exerted more powerful cytotoxicity against ATLL cells with CD38 enhanced by ATRA (cytotoxicity of T cells with anti-CD38-CAR in CD38-positive ATLL fraction: >90%). ATRA exerted enhancing effect on the cytotoxicity of T cells bearing anti-CD38-CAR against ATLL cells through the augmentation of CD38 expression. These results may provide us a rationale for novel clinical settings of T cells carrying anti-CD38-CAR on patients with ATLL using ATRA.

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.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive form of peripheral T-cell lymphoma (PTCL), which is etiologically associated with human T-lymphotropic virus type I (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize T-cells, there is a long latency period of ∼50 years prior to the onset of ATL, suggesting that HTLV-1 infection alone may not be sufficient for the development of ATL, but additional acquired genetic hits that occur in immortalized T-cells during the later life are essential for its pathogenesis. However, little has been known about those genetic hits that are involved in the pathogenesis of ATL.

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.

Abstract 2697

No relevant conflicts of interest to declare.

With the widespread use of highly active antiretroviral therapy (HAART), the prognosis of HIV infected subjects has been improved, even if with AIDS-related lymphoma. Burkitt lymphoma progresses very rapidly, but highly intensive chemotherapy (e.g. CODOX-M/IVAC, hyper-CVAD/MA) has been shown to be a promising strategy. In this time, AIDS-related lymphoma is treated with similarly to non AIDS lymphoma, nevertheless it is not clear whether the highly intensive regimens are feasible and beneficial for AIDS-related Burkitt or not. We conducted nationwide retrospective survey to clarify the clinical outcomes of AIDS-related Burkitt lymphoma, especially in context with the usage of rituximab.All AIDS-related Burkitt lymphoma newly diagnosed at center hospitals for HIV/AIDS in Japan during the period 2002–2010 were serially registered. The chart review was performed for all identified patients (pts) to obtain the following information; age, PS, CD4 count, previous AIDS, clinical stage, disease site, chemotherapy regimen, tumor response, and clinical outcomes. The effects of treatment and the clinical variables on overall survival (OS) were assessed.We identified 33 pts; the median age was 41 (range, 26–70 years) and the male gender accounted for 97% of the pts (32/33). Twenty-three pts (70%) had history of AIDS and the median CD4 count was 205/mm3 (range, 3–488/mm3). Twenty-nine (88%) were diagnosed in advanced stage (III/IV), with bone marrow (BM) involvement in 16 (48%) and central nervous system (CNS) infiltration in 7 (21%). Nineteen (58%) were treated with rituximab-contain regimen and 14 (42%) were not. As chemotherapy regimen, 6 (18%) were treated with CODOX-M/IVAC, 22 (67%) with hyper-CVAD/MA and 5 (15%) with other regimens. Of 19 pts treated with rituximab-contain regimen, none were treated with CODOX-M/IVAC. Response at the end of treatment among 32 assessable pts was as follows: complete response (CR): 24 (73%); partial response (PR): 2 (6%); stable disease (SD): 1 (3%); progression disease (PD): 5 (15%). The median follow-up was 17 months. Two-year OS of total pts was 68.1%. There was no significant difference between chemotherapy regimens with/without rituximab (p =0.367). Two-year OS was 66.7% in CODOX-M/IVAC and 71.6% in hyper-CVAD/MA (p =0.724). There was no significant difference in OS by BM involvement and CNS infiltration, respectively. There were a few pts with treatment-related death.The favorable overall outcomes of AIDS-related Burkitt lymphoma were shown in this study. Highly intensive chemotherapy regimens were feasible and brought high remission rate and long OS. The addition of rituximab to highly intensive chemotherapy has not shown to be beneficial for AIDS-related Burkitt lymphoma. We now conduct prospective a clinical trial to optimize the treatment strategy for AIDS-related Burkitt lymphoma.No relevant conflicts of interest to declare.

Abstract 1629