Adult T-cell Leukemia/lymphoma Samples Research Articles

Development of a novel multiplex digital PCR-based method for the detection of HTLV-1 proviral deletion

The human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, integrates into host DNA and causes adult T-cell leukemia/lymphoma (ATL) in some individuals. Two types of defective proviruses, Type 1 and Type 2, are often observed in ATL cells. Here, we developed a 3-plex digital PCR (dPCR) method to detect HTLV-1 proviral deletions by comparing the ratios of copy numbers quantified using specific primer-probes for the LTR, pol, and pX regions. We analyzed HTLV-1-positive asymptomatic carriers (ACs) and AC samples at high risk for developing ATL due to high proviral load (ATL high-risk (HR) ACs) using dPCR. Deletions were identified in 11.8 % (4/34, all Type 1) of ACs and 33.3 % (7/21, Type 1:1, Type 2:6) of ATL HR ACs. dPCR analysis revealed that in three ATL samples, all exhibited Type 1 defective characteristics, and two showed extremely low ratios in the pol region. Clonality analysis of these two samples revealed high monoclonality, indicating monoclonal expansion of ATL cells with defective proviruses. These findings demonstrate that our method effectively detects defective proviruses in both ACs and ATL, providing a valuable tool for understanding the genomic characteristics of proviruses in these conditions.

Abstract 3965: A cytolytic effector CD8+ T cell response is associated with remission in adult T-cell leukemia/lymphoma

Abstract Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a rare blood cancer that develops in 3-5% of human T-lymphotropic virus-1 (HTLV1) carriers when the combination of viral oncoproteins and somatic mutations lead to malignant transformation of HTLV1 infected CD4+ T cells. ATLL has the worst overall survival among peripheral T cell lymphomas and remains a fatal disease despite efforts to improve outcomes over the last 35 years. First-line combination chemotherapy rarely achieves a durable response and two immune-modulating treatments (α-CCR4 and α-PD-1 monoclonal antibodies) failed in clinical trials. These findings highlight an urgent need to improve our understanding of the functional state of host immunity in ATLL patients. A host immune response against HTLV1 is pivotal in preventing ATLL development. Malignant ATLL cells can escape immune surveillance by preventing antigen presentation and engaging immune checkpoints. The prospect of an ATLL clone inducing systemic suppression was suggested in the literature but has not been directly examined. Furthermore, HTLV1-specific CD8+ cytotoxic T lymphocytes (CTLs) play an important role during viral latency, yet it remains unknown if they play a clinically relevant role during ATLL treatment. Methodology: In this work, we hypothesized that malignant ATLL cells will exhibit a suppressive immune phenotype and that effective therapeutic interventions will augment anti-ATLL CTLs to promote durable remission. We implemented an immune profiling approach to analyze PBMC samples from ATLL patients. Specifically, we developed a powerful 30-marker spectral flow panel to discriminate malignant ATLL cells from non-transformed lymphocytes (T, B, and NK cells) and monitor the expression of key transcription factors and markers associated with proliferation, cytolysis, and exhaustion. We analyzed diagnostic samples from the Montefiore-Einstein ATLL Biobank and samples collected during a phase 2 clinical trial aimed to assess the efficacy of a promising Belinostat/Zidovudine/Interferon-α combination therapy against ATLL (NCT02737046). Results: Our findings reveal that malignant ATLL cells exhibit an immune phenotype associated with robust immunosuppression and yet distinct from other suppressive immune cells (i.e., regulatory T cells). Additionally, we discovered that ATLL patient samples contained a unique population of phenotypically exhausted CD8+ T cells, the proportion of which was drastically reduced in patients under remission. Remarkably, a decrease in this exhausted subset correlated with the expansion of highly cytolytic CD8+ CTLs. Conclusion: Altogether, our findings suggest that ATLL cells may drive CD8+ CTLs into exhaustion to achieve immune evasion and that a robust anti-ATLL CTL response actively contributes to a remission state. Citation Format: Erik Guillen, Eric Liu, Yanhua Wang, Aditi Shastri, Alejandro Sica, Amit Verma, Xingxing Zhang, Juan Carlos Ramos, Hilda Ye, Gregoire Lauvau. A cytolytic effector CD8+ T cell response is associated with remission in adult T-cell leukemia/lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3965.

MiR-150 in HTLV-1 infection and T-cell transformation.

Human T-cell leukemia virus-1 (HTLV-1) is a retrovirus that persistently infects CD4+ T-cells, and is the causative agent of adult T-cell leukemia/lymphoma (ATLL), tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM) and several inflammatory diseases. T-cell transformation by HTLV-1 is driven by multiple interactions between viral regulatory proteins and host cell pathways that govern cell proliferation and survival. Studies performed over the last decade have revealed alterations in the expression of many microRNAs in HTLV-1-infected cells and ATLL cells, and have identified several microRNA targets with roles in the viral life cycle and host cell turnover. This review centers on miR-150-5p, a microRNA whose expression is temporally regulated during lymphocyte development and altered in several hematological malignancies. The levels of miR-150-5p are reduced in many HTLV-1-transformed- and ATLL-derived cell lines. Experiments in these cell lines showed that downregulation of miR-150-5p results in activation of the transcription factor STAT1, which is a direct target of the miRNA. However, data on miR-150-5p levels in freshly isolated ATLL samples are suggestive of its upregulation compared to controls. These apparently puzzling findings highlight the need for more in-depth studies of the role of miR-150-5p in HTLV-1 infection and pathogenesis based on knowledge of miR-150-5p-target mRNA interactions and mechanisms regulating its function in normal leukocytes and hematologic neoplasms.

Signaling factors potentially associated to the pathogenesis of Adult T-cell leukemia /lymphoma: A network-analysis and novel findings assessment

Adult T-cell leukemia/lymphoma (ATLL) is a human T-cell leukemia virus (HTLV) type 1-associated disease of TCD4+ cell transformation. Despite extensive studies on ATLL development and progression, the fundamental processes of HTLV-1 oncogenicity are yet to be understood. This study aimed to integrate high-throughput microarray datasets to find novel genes involved in the mechanism of ATLL progression. For this purpose, five microarray datasets were downloaded from the Gene Expression Omnibus database and then profoundly analyzed. Differentially expressed genes and miRNAs were determined using the MetaDE package in the R software and the GEO2R web tool. The STRING database was utilized to construct the protein-protein interaction network and explore hub genes. Gene ontology and pathway enrichment analysis were carried out by employing the EnrichR web tool. Furthermore, flow cytometry was employed to assess the CD4/CD8 ratio, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to confirm the high-throughput data analysis results. Four miRNAs, including hsa-mir-146, hsa-mir-451, hsa-mir-31, and hsa-mir-125, were among the statistically significant differentially expressed miRNAs between healthy individuals and ATLL patients. Moreover, 924 differentially expressed genes were identified between normal and ATLL samples. Further network analysis highlighted 59 hub genes mainly regulating pathways implicated in viral interferences, immunological processes, cancer, and apoptosis pathways. Among the identified hub genes, RhoA and PRKACB were most considerable in the high-throughput analysis and were further validated by qRT-PCR. The RhoA and PRKACB expression were significantly down-regulated in ATLL patients compared to asymptomatic carriers (p<0.0001 and p=0.004) and healthy subjects (p=0.043 and p=0.002). Therefore, these corresponding miRNAs and proteins could be targeted for diagnosis purposes and designing effective treatments.

Notable Patterns in the Genomic Landscape of Adult T-Cell Leukemia/Lymphoma Encountered in HTLV-1 Endemic Western World Regions

Background: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy with dismal prognosis and associated with clonal T-cell expansion driven by Human T-Lymphotropic Virus 1 (HTLV-1) infection. Comprehensive genomic studies in Japan have identified recurrent alterations affecting TCR-NF-kB signaling (i.e. PRKCB, PLCG1, CARD11, VAV1, and IRF4), T-cell trafficking pathways (i.e. CCR4 and CCR7), and the tumor suppressor genes CDKN2A and TP53. HTLV-1 endemic regions include Africa, the Caribbean, and South America in addition to Japan. Retrospective studies from the Western population have reported distinctive features from the Japanese cohort, e.g. younger age, more common lymphomatous presentation, and worse outcomes. Our group sought to evaluate the unique molecular features of ATLL in a large cohort of patients from the Caribbean and South America.Methods: We performed a multimodal genomic study on specimens from 169 patients encountered in the United States (Miami), Peru, Brazil, France, and Spain. Data types included Oncoscan/Copy Number Variation (CNV) data for 129 patients, RNA-seq data for 97 patients, and whole exome sequencing (WES) data for 125 patients. Patients were ethnically classified based upon single nucleotide polymorphisms, under 3 main groups: African (n=80), native American (n= 32), or South Asian/Islander (n=12). 46 specimens without WES data could not be ethnically classified. Somatic variants were called using Mutect. Putative driver mutations were identified by frequency-based criteria. CNV significance was determined using GISTIC2.0. Data were compared to whole exome and targeted sequencing data published by Kataoka et al.Results: Our cohort replicated trends reported in Japanese datasets but included several distinctive findings. South American and Caribbean patients had fewer mutations in CCR4 and CD58. Three putative tumor suppressors not previously implicated in ATLL were identified based on recurrent damaging mutations. These included ANKRD11 (n=3), DGKZ (n=3), and PTPN6 (n=3). Both ANKRD11 and PTPN6 were only mutated in Afro-Caribbean patients with aggressive (acute and lymphomatous) cases. CNV analysis revealed ANKRD11 deletions in a significant portion of cases (n=16).As previously reported, STAT3 mutations were more common in indolent subtypes. IRF4 mutations (n=14) or amplifications (n=19) were only observed in aggressive ATLL subtypes. L70V was the most common IRF4 variant (n=5). Among Japanese samples, K59R mutations were seen twice as often as L70V mutations. Both K59R and L70V are located within the IRF4 DNA binding domain. In samples from patients with disease relapse (N = 10), IRF4 was the only gene mutated significantly more often (p = 0.03). 3 of 10 patients who were previously treated with interferon (IFN)-based therapy relapsed with new IRF4 mutations (L70V n=2, and K59R n=1), suggesting IRF4 may be associated with IFN resistance.A total of 11 patients in the Western cohort had FOXO3 mutations including R177W (n=7) and D199N (n=3) variants. These only occurred in Afro-Caribbean patients with aggressive subtypes. R177W is located within the FOXO3DNA binding domain, suggesting that dysregulation of its transcriptional targets may contribute to disease. In the Japanese cohort, only one patient had a FOXO3 mutation, (D199N). The majority of primary ATLL samples analyzed by Western Blot showed significantly reduced or no expression of FOXO3, or ANKRD11.Conclusion: The genomic landscape of ATLL encountered in patients from South America and the Caribbean resembles that of ATLL in Japanese patients. However, our study identified novel variants and tumor suppressor genes not previously implicated in ATLL that differ from the Japanese population. Furthermore, we correlate our genomic analysis with clinical findings to implicate ATLL driver genes in IFN resistance and disease prognosis. Functional Studies to determine the prognostic and functional roles of the gene alterations we identified in ATLL are ongoing. DisclosuresGru: StemLine: Honoraria, Research Funding, Speakers Bureau; CRISPT Therapeutics: Research Funding; Innate Pharma: Research Funding.

Comprehensive high-throughput meta-analysis of differentially expressed microRNAs in transcriptomic datasets reveals significant disruption of MAPK/JNK signal transduction pathway in Adult T-cell leukemia/lymphoma

BackgroundHuman T-lymphotropic virus 1 (HTLV-1) infection may lead to the development of Adult T-cell leukemia/lymphoma (ATLL). To further elucidate the pathophysiology of this aggressive CD4+ T-cell malignancy, we have performed an integrated systems biology approach to analyze previous transcriptome datasets focusing on differentially expressed miRNAs (DEMs) in peripheral blood of ATLL patients.MethodsDatasets GSE28626, GSE31629, GSE11577 were used to identify ATLL-specific DEM signatures. The target genes of each identified miRNA were obtained to construct a protein-protein interactions network using STRING database. The target gene hubs were subjected to further analysis to demonstrate significantly enriched gene ontology terms and signaling pathways. Quantitative reverse transcription Polymerase Chain Reaction (RTqPCR) was performed on major genes in certain pathways identified by network analysis to highlight gene expression alterations.ResultsHigh-throughput in silico analysis revealed 9 DEMs hsa-let-7a, hsa-let-7g, hsa-mir-181b, hsa-mir-26b, hsa-mir-30c, hsa-mir-186, hsa-mir-10a, hsa-mir-30b, and hsa-let-7f between ATLL patients and healthy donors. Further analysis revealed the first 5 of DEMs were directly associated with previously identified pathways in the pathogenesis of HTLV-1. Network analysis demonstrated the involvement of target gene hubs in several signaling cascades, mainly in the MAPK pathway. RT-qPCR on human ATLL samples showed significant upregulation of EVI1, MKP1, PTPRR, and JNK gene vs healthy donors in MAPK/JNK pathway.DiscussionThe results highlighted the functional impact of a subset dysregulated microRNAs in ATLL on cellular gene expression and signal transduction pathways. Further studies are needed to identify novel biomarkers to obtain a comprehensive mapping of deregulated biological pathways in ATLL.

Highly Sensitive Viral RNA-Seq Is a Powerful Tool to Analyze the Viral Transcripts in HTLV-1-Infected Individuals

IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is a retrovirus which causes two major pathologies in infected individuals, Adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There are tens of thousands of different infected clones in asymptomatic HTLV-1 carriers (AC) and each clone can be identified by the viral integration site. Monoclonal integration of HTLV-1 provirus is observed in the host genomic DNA in ATL cells, suggesting that these originate from a single virus-infected clone. It is well known that Tax and HBZ, encoded in the sense and antisense strands of HTLV-1 provirus, respectively, have the ability to induce oncogenesis. The question is how a certain infected clone, among various different ones, becomes a malignant clone. One hypothesis is that a malignant clone may express viral gene transcripts, associated with oncogenesis, more abundantly than a non-malignant clone. Another hypothesis is that HTLV-1 integration may induce an abnormal transcript in the host cell, such as the aberrant expression of the host gene nearby the integration site or the generation of a chimeric transcript between the virus and the host. To analyze HTLV-1 transcript or explore the presence of virus-host chimeric transcripts efficiently, we aimed to establish a method with higher resolution than the conventional RNA-seq by using DNA probe targeting for the entire HTLV-1 sequence. And then, we applied the protocol to characterize the pattern of transcription from HTLV-1 provirus and analyzed virus-host chimeric transcripts in patients' samples.MethodsTo check the efficiency and possible bias of the highly sensitive viral RNA-seq analysis, we analyzed viral transcript of an HTLV-1-infected clone, TBX-4B. Subsequently, mRNA-seq was performed with PBMCs from 6 AC, 4 HAM/TSP, and 5 ATL patients. After cDNA libraries synthesis, HTLV-1 sequences were enriched by the probe-based capture method as we reported previously for DNA-seq (Miyazato et al, Sci Rep 2016). The enriched libraries were sequenced in an Illumina NextSeq device.ResultsWe analyzed HTLV-1 transcript of a virus-infected T-cell clone both before and after the enrichment by viral-specific DNA probes. The proportion of viral sequences within the total number of reads was significantly increased from 0.05% to 80.62% (1600-fold) by the enrichment method. We found that most of the viral regions were significantly and similarly enriched by the additional enrichment step, suggesting the DNA probe-based enrichment of HTLV-1 sequences is applicable to RNA-seq. Next, we applied the protocol to analyze PBMC samples from HTLV-1-infected individuals. The results showed that the level of anti-sense HTLV-1 transcription was significantly higher than that of the sense transcription, not only in ATL patients, which is consistent with previous reports by other groups, but also in AC and HAM/TSP patients. Average proportion of sense and antisense signals were 3.42% and 96.46%, respectively. Even after the enrichment step, tax expression, a sense viral transcript, was undetectable in most of the samples except for one AC and one ATL patient, while an anti-sense transcript HBZ was detectable in all infected individuals. There was no significant difference in the expression level of HBZ between different clinical states. We identified more abundant virus-host chimeric transcripts in ATL samples than in HAM/TSP patients or AC. In ATL cases, HTLV-1 generated chimeric transcripts with the following host genes, HSD17B11, COLEC12, AHSA2, EEF2, TAOK1, KIAA0100, and KIF1B . The presence of abundant chimeric transcripts was validated by RT-PCR.DiscussionWe have established a highly sensitive viral RNA-seq for HTLV-1 transcript analysis. That significantly increased the sensitivity of detection of proviral and chimeric transcripts between virus and the host genome. The result was not consistent with the hypothesis that a malignant clone may express viral gene transcripts more abundantly than a non-malignant clone. Thus, the presence of chimeric transcripts could play a role in cell proliferation and help us reveal the mechanism of clonal selection for HTLV-1-infected cells. We are now going to analyze virus-host chimeric transcripts detected by the high sensitive RNA-seq in more patients' samples to clarify their association with oncogenesis. [Display omitted] DisclosuresYamano:Daiichi Sankyo: Research Funding; Ono Pharma: Research Funding. Utsunomiya:Celgene: Honoraria; Novartis Pharma: Honoraria; Roche Diagnostics: Honoraria; Japan Blood Products Organization: Honoraria; Daiichi Sankyo: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria; Chugai Pharma: Honoraria; Bristol-Myers Squibb: Honoraria; Siemens: Honoraria; Nippon Shinyaku: Honoraria; HUYA Bioscience International: Honoraria; Pfizer: Honoraria.

Deciphering microRNA-mRNA regulatory network in adult T-cell leukemia/lymphoma; the battle between oncogenes and anti-oncogenes.

Adult T-cell leukemia/lymphoma (ATLL) is virus-caused cancer that originates from the infection by human T-cell leukemia virus type 1. ATLL dysregulates various biological pathways related to the viral infection and cancer progression through the dysexpression of miRNAs and mRNAs. In this study, the potential regulatory subnetworks were constructed aiming to shed light on the pathogenesis mechanism of ATLL. For this purpose, two mRNA and one miRNA expression datasets were firstly downloaded from the GEO database. Next, the differentially expressed genes and miRNAs (DEGs and DE-miRNAs, respectively), as well as differentially co-expressed gene pairs (DCGs), were determined. Afterward, common DEGs and DCGs targeted by experimentally validated DE-miRNAs were explored. The oncogenic and anti-oncogenic miRNA-mRNA regulatory subnetworks were then generated. The expression levels of four genes and two miRNAs were examined in the blood samples by qRT-PCR. The members of three oncogenic/anti-oncogenic subnetworks were generally enriched in immune, virus, and cancer-related pathways. Among them, FZD6, THBS4, SIRT1, CPNE3, miR-142-3p, and miR-451a were further validated by real-time PCR. The significant up-regulation of FZD6, THBS4, and miR-451a as well as down-regulation of CPNE3, SIRT1, and miR-142-3p were found in ATLL samples than normal samples. The identified oncogenic/anti-oncogenic subnetworks are pieces of the pathogenesis puzzle of ATLL. The ultimate winner is probably an oncogenic network that determines the final fate of the disease. The identified genes and miRNAs are proposed as novel prognostic biomarkers for ATLL.

Dissecting Multicellular Ecosystems of HTLV-1 Infection and ATL By Multi-Omics Single Cell Analysis

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. Within malignant cells, we identified a fraction of cycling cells present across most ATL samples. This fraction showed an enhanced T-cell activation markers, such as CD25 and HLA-DR, and their frequency was increased in aggressive subtypes. On the other hand, in HTLV-1-infected carriers, HTLV-1-infected CD4+ T cells contained a small population of malignant-like cells showing clonal expansion. The degree of clonal expansion was significantly correlated with HTLV-1 viral load in PBMCs. These results clarify the heterogeneity within HTLV-1-infected cells and ATL malignant cells, pointing to its relevance during ATL initiation and progression. We also observed dynamic changes of the immune microenvironment in ATL. Although the relative frequencies of other cell types remained almost the same or reduced, only myeloid cells were increased in ATL patients compared with in HTLV-1-infected carriers. Re-clustering of myeloid cells identified a novel cluster of monocytes expressing FCGR1A, encoding CD64, a biomarker of IFN-stimulated gene levels. Transcriptome analysis revealed increased IFN signaling and decreased TNFa in myeloid cells from ATL patients compared with HTLV-1-infected carriers. Similar expression signatures changes were also observed in various immune cell types, such as B, CD8+ T, and NK cells, in ATL patients. In addition, substantial changes of surface marker proteins were also found in ATL patients. Particularly, T-cell activation markers, such as HLA-DR, and inhibitory immune checkpoint molecules, such as PD-1 and TIM-3, were up-regulated in CD8+ T cells from ATL patients. A co-culture experiment of ATL cell lines with PBMCs from healthy volunteers demonstrated that ATL cells induced immune-phenotypic changes of myeloid and CD8+ T cells, similar to those observed in ATL patient by our single-cell analysis, confirming the role of ATL cells in the modulation of the immune system. Taken together, the composition and function of immune microenvironment is dramatically altered in ATL patients, which may contribute to immunosuppression and disease progression in ATL. In summary, our multi-omics single-cell analysis comprehensively dissects the cellular and molecular architecture in HTLV-1-infected carriers and ATL patients. In particular, our approach clearly defines HTLV-1-infected cells by the expression of HTLV-1-related genes, leading to the detailed characterization of HTLV-1-infected cells and elucidation of their difference from ATL malignant cells. These findings will help to devise novel diagnostic and therapeutic strategies for HTLV-1-related disorders. Disclosures 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 &amp; 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.

Abstract IA07: Molecular classification of T-cell lymphomas

Abstract Peripheral T-cell lymphomas (PTCLs) represent a clinically, histologically, and molecularly heterogeneous group of non-Hodgkin lymphomas derived from mature post-thymic T cells. Among them, the most common in Western countries is PTCL, not otherwise specified (NOS), accounting for approximately 30% of all PTCLs. Combining whole-exome and deep targeted-capture sequencing of 133 cases, we delineated the entire picture of genetic alterations in PTCL, NOS. Of note is the identification of a previously undescribed molecular subtype characterized by TP53 and/or CDKN2A mutations and deletions in PTCL, NOS without showing a T follicular helper cell phenotype. This subtype exhibited different prognosis and unique genetic features, including extensive chromosomal instability, which preferentially affected molecules involved in immune escape and transcriptional regulation. Among PTCLs, the most common entity in Japan is adult T-cell leukemia/lymphoma (ATL), which is an aggressive peripheral T-cell lymphoma associated with human T-cell leukemia virus type-1 (HTLV-1) infection. We previously carried out an integrated molecular study, in which whole-exome, transcriptome, and targeted resequencing, as well as array-based copy number analysis, were performed. We found recurrent genetic alterations in T-cell receptor/NF-κB signaling, T-cell trafficking, and other T cell-related pathways as well as immunosurveillance. Although our previous study discovered many driver mutations and copy number alterations, the whole-genome landscape of ATL still remains elusive. To this end, we have recently performed whole-genome sequencing (WGS) of 155 ATL samples, with a median depth of 95-fold for tumor samples. WGS presented a substantially different overview of driver alterations compared with WES: most driver genes were affected by more than one classes of somatic alterations, including structural variations (SV) and noncoding mutations. We integrated these genetic drivers using non-negative factorization clustering and identified several molecular subsets with distinct clinical features, including a leukemic subtype characterized by PLCG1 and STAT3 mutations. In addition, we newly identified several putative driver SVs and noncoding mutations (such as NOTCH1 truncations and mutations involving NFKBIZ 3′-untraslated region). Several types of SVs were linked to distinct alterations, suggesting their etiology, including associations of TP53 disruption with inversions and translocations, and inactivation of immune-related molecules with deletions. Our WGS analysis not only identifies novel somatic alterations, but also extends the overview of ATL genome, which can lead to future improvement of patient management. Taken together, our findings provide novel insights into genetic and molecular heterogeneity in PTCLs, which should help to devise a novel molecular classification and to exploit a new therapeutic strategy for these malignancies. Citation Format: Keisuke Kataoka. Molecular classification of T-cell lymphomas [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA07.

Reversal of CYLD phosphorylation as a novel therapeutic approach for adult T-cell leukemia/lymphoma (ATLL)

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T cells associated with chronic infection by human T-cell lymphotropic virus type-1 (HTLV-1). ATLL patients with aggressive subtypes have dismal outcomes. We demonstrate that ATLL cells co-opt an early checkpoint within the tumor necrosis factor receptor 1 (TNFR1) pathway, resulting in survival advantage. This early checkpoint revolves around an interaction between the deubiquitinase CYLD and its target RIPK1. The status of RIPK1 K63-ubiquitination determines cell fate by creating either a prosurvival signal (ubiquitinated RIPK1) or a death signal (deubiquitinated RIPK1). In primary ATLL samples and in cell line models, an increased baseline level of CYLD phosphorylation was observed. We therefore tested the hypothesis that this modification of CYLD, which has been reported to inhibit its deubiquitinating function, leads to increased RIPK1 ubiquitination and thus provides a prosurvival signal to ATLL cells. CYLD phosphorylation can be pharmacologically reversed by IKK inhibitors, specifically by TBK1/IKKε and IKKβ inhibitors (MRT67307 and TPCA). Both of the IKK sub-families can phosphorylate CYLD, and the combination of MRT67307 and TPCA have a marked effect in reducing CYLD phosphorylation and triggering cell death. ATLL cells overexpressing a kinase-inactive TBK1 (TBK1-K38A) demonstrate lower CYLD phosphorylation and subsequently reduced proliferation. IKK blockade reactivates CYLD, as evidenced by the reduction in RIPK1 ubiquitination, which leads to the association of RIPK1 with the death-inducing signaling complex (DISC) to trigger cell death. In the absence of CYLD, RIPK1 ubiquitination remains elevated following IKK blockade and it does not associate with the DISC. SMAC mimetics can similarly disrupt CYLD phosphorylation and lead to ATLL cell death through reduction of RIPK1 ubiquitination, which is CYLD dependent. These results identify CYLD as a crucial regulator of ATLL survival and point to its role as a potential novel target for pharmacologic modification in this disease.

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

Mutational Intratumor Heterogeneity is a Complex and Early Event in the Development of Adult T-cell Leukemia/Lymphoma

The clonal architecture of tumors plays a vital role in their pathogenesis and invasiveness; however, it is not yet clear how this clonality contributes to different malignancies. In this study we sought to address mutational intratumor heterogeneity (ITH) in adult T-cell leukemia/lymphoma (ATL). ATL is a malignancy with an incompletely understood molecular pathogenesis caused by infection with human T-cell leukemia virus type-1 (HTLV-1). To determine the clonal structure through tumor genetic diversity profiles, we investigated 142 whole-exome sequencing data of tumor and matched normal samples from 71 ATL patients. Based on SciClone analysis, the ATL samples showed a wide spectrum of modes over clonal/subclonal frequencies ranging from one to nine clusters. The average number of clusters was six across samples, but the number of clusters differed among different samples. Of these ATL samples, 94% had more than two clusters. Aggressive ATL cases had slightly more clonal clusters than indolent types, indicating the presence of ITH during earlier stages of disease. The known significantly mutated genes in ATL were frequently clustered together and possibly coexisted in the same clone. IRF4, CCR4, TP53, and PLCG1 mutations were almost clustered in subclones with a moderate variant allele frequency (VAF), whereas HLA-B, CARD11, and NOTCH1 mutations were clustered in subclones with lower VAFs. Taken together, these results show that ATL displays a high degree of ITH and a complex subclonal structure. Our findings suggest that clonal/subclonal architecture might be a useful measure for prognostic purposes and personalized assessment of the therapeutic response.

Targeting the HTLV-I-Regulated BATF3/IRF4 Transcriptional Network in Adult T-Cell Leukemia/Lymphoma

After neonatal HTLV-I infection through breast feeding, approximately 5% of HTLV-I carriers eventually develop Adult T-Cell Leukemia/Lymphoma (ATLL) with a latency of ~50 years, suggesting that acquired genetic and epigenetic changes in cellular genes act in concert with HTLV-I to initiate and maintain oncogenic transformation. We and others have recently utilized next generation sequencing technology to identify mutated genes that could be pivotal in the pathogenesis of ATLL. However, due to the complexity of genomic/epigenetic alteration in the ATLL genome, the identification of indispensable genes for proliferation and/or survival of ATLL cells remains a formidable challenge.To discover essential regulatory networks that are required for the proliferation and survival of ATLL cells, we performed a pooled shRNA screen in 8 ATLL cell lines using a library enriched for shRNAs targeting lymphoid regulatory factors and discovered that two BATF3 shRNAs and one IRF4 shRNA were highly toxic for all ATLL lines, but had little if any effect in other T cell and B cell lines. It is recently shown that a transcriptional complex of Irf4 and Batf binds to AP1-IRF composite (AICE) DNA motifs and plays key roles in the differentiation and function of certain mouse helper T cell subsets. A close paralogue of Batf, Batf3, is an indispensable transcription factor in a mouse dendritic cell subset, but also appears to play a redundant role with Batf in the differentiation of TH2 cells and can substitute for Batf in Batf knockout T cells. Our observations from shRNA screening suggested that IRF4 and BATF3 may cooperate to drive a transcriptional program that is essential for ATLL viability.We next used genome-wide chromatin precipitation (ChIP-seq) to identify the loci that are bound by BATF3 and IRF4. The set of binding peaks and the associated genes in IRF4 and BATF3 ChIP-seq intersected significantly. By integrating the ChIP-seq and gene expression profiling data of shBATF3- and shIRF4-ATLL cells, we defined a set of 68 BATF3-IRF4 direct target genes. Gene set enrichment analysis using gene expression profiling data from primary T cell lymphomas demonstrated that BATF3-IRF4 direct target genes were significantly enriched among genes that are more highly expressed in ATLL than in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), suggesting that the BATF3 and IRF4 cooperatively regulate transcription in primary ATLL cells.HBZ is unique among HTLV-I viral proteins in being maintained in expression in all ATLL cases, suggesting that it may help maintain the malignant phenotype. Given that BATF3 and IRF4 are essential regulators in ATLL, we hypothesized possible relationship between HBZ and BATF3-IRF4 complex. We defined HBZ direct target genes by integrating the ChIP-seq and gene expression profiling data of HBZ-knockout ATLL cell lines by CRISPR/Cas9. Notably we discovered that BATF3 was among these. BATF3 mRNA and protein expression decreased following HBZ inactivation.The above considerations suggested that pharmacologic inhibition of the BATF3-IRF4 regulatory network might be a means to attack the HBZ oncogenic program therapeutically. ChIP-seq analysis of two enhancer marks, H3K27ac and BRD4, identified super-enhancers at the BATF3 locus in two ATLL cell lines. The small molecule JQ1 prevents the BET-protein BRD4 from interacting with chromatin, which is required for the function of super-enhancers. JQ1 treatment reduced BATF3 mRNA and protein levels in all ATLL lines tested, correlating with the eviction of BRD4 from the BATF3 super-enhancer. MYC mRNA and protein expression was also broadly downmodulated by JQ1. JQ1 treatment was consistently toxic for all ATLL cell lines tested at dose ranges that killed cell line models of T-ALL and DLBCL, which are known to rely on BET-proteins. In a dose-dependent manner, JQ1 also reduced the viability of primary ATLL samples and downregulated their expression BATF3 and MYC mRNA. Finally, we treated mouse xenograft models of ATLL with the BET-protein inhibitor CPI-203, a JQ1 analog with superior bioavailability in mice. In two different xenograft models, we observed significant tumor regression or growth inhibition, without evidence of systemic toxicity. Our study demonstrates that the HTLV-I virus exploits a regulatory module that can potentially be attacked therapeutically with BET protein inhibitors. DisclosuresYu:Celgene Corporation: Employment.

Enhancer profiling identifies critical cancer genes and characterizes cell identity in adult T-cell leukemia

AbstractA number of studies have recently demonstrated that super-enhancers, which are large cluster of enhancers typically marked by a high level of acetylation of histone H3 lysine 27 and mediator bindings, are frequently associated with genes that control and define cell identity during normal development. Super-enhancers are also often enriched at cancer genes in various malignancies. The identification of such enhancers would pinpoint critical factors that directly contribute to pathogenesis. In this study, we performed enhancer profiling using primary leukemia samples from adult T-cell leukemia/lymphoma (ATL), which is a genetically heterogeneous intractable cancer. Super-enhancers were enriched at genes involved in the T-cell activation pathway, including IL2RA/CD25, CD30, and FYN, in both ATL and normal mature T cells, which reflected the origin of the leukemic cells. Super-enhancers were found at several known cancer gene loci, including CCR4, PIK3R1, and TP73, in multiple ATL samples, but not in normal mature T cells, which implicated those genes in ATL pathogenesis. A small-molecule CDK7 inhibitor, THZ1, efficiently inhibited cell growth, induced apoptosis, and downregulated the expression of super-enhancer–associated genes in ATL cells. Furthermore, enhancer profiling combined with gene expression analysis identified a previously uncharacterized gene, TIAM2, that was associated with super-enhancers in all ATL samples, but not in normal T cells. Knockdown of TIAM2 induced apoptosis in ATL cell lines, whereas overexpression of this gene promoted cell growth. Our study provides a novel strategy for identifying critical cancer genes.

Abstract 5487: Identification of super-enhancer-associated cancer genes provides novel therapeutic targets in adult T-cell leukemia/lymphoma

Abstract Super-enhancers are often enriched at cancer genes in various malignancies. Identification of such enhancers would pinpoint critical factors that directly contribute to pathogenesis. Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive lymphoproliferative disorder that arises from CD4+ mature T-lymphocytes. A recent genome-wide sequencing study revealed a large number of genetic and chromosomal abnormalities. However, there is still a lack of functional evidence for these genetic abnormalities in ATL pathogenesis due to the large mutational burden and the considerable genetic heterogeneity. Here, we performed super-enhancer profiling combined with gene expression analysis in primary ATL samples and cell lines. Super-enhancers are frequently enriched at genes involved in T-cell activation and T-cell receptor signaling pathway in both ATL and normal CD4+ T-cells, reflecting the origin of leukemia cells. Super-enhancers at the CCR4 gene, a known therapeutic target in ATL, were observed in all ATL cases. Additionally, we identified previously-uncharacterized genes that were highly activated in ATL cells and required for cell growth. A small-molecule inhibitor of CDK7 efficiently downregulated the expression of these genes and inhibited cell growth. Our study provides a novel strategy to identify critical cancer genes in cancer. Note: This abstract was not presented at the meeting. Citation Format: Takaomi Sanda, Regina WJ Wong, Phuong TN Cao, Wei Zhong Leong, Alice WY Yam, Tinghu Zhang, Shinsuke Iida, Takashi Okamoto, Ryuzo Ueda, Nathanael S. Gray, Takashi Ishida. Identification of super-enhancer-associated cancer genes provides novel therapeutic targets in adult T-cell leukemia/lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5487. doi:10.1158/1538-7445.AM2017-5487

Identification of Super-Enhancer-Associated Cancer Genes Provides Novel Therapeutic Targets in Adult T-Cell Leukemia/Lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive lymphoproliferative disorder that arises from mature T-lymphocytes. The onset of this disease is preceded by a long period of latency of 30 to 50 years, indicating that multiple cellular oncogenic mechanisms after HTLV-1 infection are involved in its pathogenesis. Notably, the recent genome-wide sequencing study revealed that a large number of genetic and chromosomal abnormalities are found in ATL. However, there is still a lack of functional evidence of those genetic abnormalities in the ATL pathogenesis due to a large mutation burden per sample and a high genetic heterogeneity across different ATL samples.Super-enhancers are clusters of enhancers typically marked by a high level of acetylation of histone H3 lysine 27 (H3K27Ac) using ChIP-seq analysis. Recently, rapidly accumulating evidences have shown that super-enhancers are often enriched at cancer genes in various malignancies. Therefore, the identification of such enhancers would pinpoint crucial genes, on which cancer/leukemia cells depend on in the acquisition of hallmark capabilities in cancer.Here, we performed a super-enhancer profiling combined with gene expression analysis followed by a loss-of-function RNA interference (RNAi) screen in ATL cells. By H3K27Ac ChIP-seq, we found that super-enhancers are frequently enriched at the genes involved in T-cell activation and T-cell signaling pathway, including CD2, IL2RA/CD25 and TNFRSF8/CD30, both in ATL and normal mature T-cells, confirming the origin of ATL cells. RNA Polymerase II (Pol II) metagene analysis demonstrated that super-enhancer-associated genes showed higher Pol II signal at the transcriptional start site (TSS) to transcriptional end site (TES) than the typical enhancer-associated genes. These super-enhancer-associated genes are sensitive to the THZ1 small-molecule CDK7 inhibitor treatment. Additionally, by a RNAi screen, we identified several genes including CCR4, which are highly activated in primary ATL samples and are required for the growth of ATL cell line. THZ1 treatment efficiently downregulated the expression of these genes and resulted in cell growth inhibition. Individual knockdown analysis demonstrated that a loss of each of these proteins significantly blocked the growth of multiple ATL cell lines but not T-ALL cells.Taken together, our study identified critical cancer genes in ATL based on a super-enhancer profiling. This study is the first to utilize a combinatorial approach of super-enhancer profiling, gene analysis after THZ1 treatment and functional RNAi screening to identify genes that are aberrantly activated and required in cancer. DisclosuresIida:Celgene: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding. Ueda:Kyowa Hakko Kirin: Research Funding; Mundipharma KK: Consultancy. Ishida:Bayer Pharma AG: Research Funding; Kyowa Hakko Kirin, Co., Ltd.: Honoraria, Research Funding; Celgene KK: Research Funding.

TIM-3 expression in lymphoma cells predicts chemoresistance in patients with adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL), an aggressive type of malignant lymphoma, is highly resistant to chemotherapy. However, the detailed mechanisms of the chemoresistance of ATLL have never been elucidated. We previously demonstrated that direct cell-cell interaction between macrophages and lymphoma cells was significantly associated with lymphoma progression in patients with ATLL. The present study aimed to further analyze the effects of cell-cell interaction between macrophages and ATLL cells by means of cell culture studies and immunohistochemical analysis using human ATLL samples. It was found that direct co-culture with macrophages induced chemoresistance in the ATLL ATN-1 cell line, but not in other cell lines, including TL-Mor, ED and ATL-2S. It was also found that expression of the T cell Ig and mucin domain-containing molecule-3 (TIM-3) was induced in ATN-1 cells by their long-term co-culture with macrophages. TIM-3 gene transfection induced chemoresistance in the ATN-1 cells. Immunostaining of ATLL tissues showed TIM-3 expression in 25 out of 58 ATLL cases. Although TIM-3 expression was not associated with overall survival or T classification, it was associated with resistance to chemotherapy. TIM-3 expression is therefore considered to be a marker for predicting the efficacy of chemotherapy, and TIM-3-associated signals may be a therapeutic target for patients with ATLL.

Frequent Activating Somatic Alterations in T-Cell Receptor / NF-κb Signaling in Adult T-Cell Leukemia/Lymphoma

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.A striking feature of driver lesions in ATL was their strong enrichment in the components of T-cell receptor (TCR) / NF-κB pathway. Accounting for more than 90% of ATL cases, these lesions were characterized by the predominance of activating alterations, including hotspot missense mutations in PLCG1 (36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), IRF4 (14%) and FYN (4%). Among these, most frequently mutated was PLCG1, which encodes phospholipase C γ1 (PLCγ1), a key regulator of the proximal TCR signaling. Besides the S345F and S520F mutations recently reported in cutaneous T-cell lymphoma, we identified an additional hotspot mutations (R48W, E1163K, and D1165H).The second most frequently mutated gene was PRKCB, encoding a member of the protein kinase C (PKC) family of proteins (PKCβ), a pivotal signaling molecule downstream of PLCγ. The frequent mutations of PKCβ were unexpected, because it is PKCθ that has been implicated in TCR signaling, whereas PKCβ has been more focused in the context of B-cell receptor signaling. Approximately 93% of the PRKCB mutations were confined to the catalytic domain with a prominent hotspot at D427, suggesting gain-of-function nature of these mutations. Consistent with this, when transduced with the D427N PKCβ mutant, HEK293T and/or Jurkat cells showed increased membrane translocation after PMA/Ionomycin-stimulation, enhanced IKK phosphorylation and p65 nuclear translocation, and augmented NF-κB transcription, compared to wild-type PKCβ-transduced cells. Thus, these PRKCB mutations are the first activating mutations of this family identified in human cancers.Downstream to PKC lies CARD11, a scaffolding protein required for antigen receptor-induced NF-κB activation. Although previously reported in B-cell lymphomas, CARD11 mutations were more common in ATL (24%). In B-cell lymphomas, mutations are largely limited to the coiled-coil (CC) domain, whereas in ATL, they were clustered not only within the CC domain, but also within the PKC-responsive inhibitory domain, showing a prominent mutational hotspot at E626. The inhibitory domain has been implicated in autoinhibition, whose deletion leads to constitutive activation of CARD11. Intriguingly, WGS identified small intragenic deletions confined to this domain (exons 14-17) in 4 cases (8%) without canonical mutations, and RNA-seq confirmed the skipping of the corresponding exons in these cases. Remarkably, CARD11 mutation significantly co-occurred with PRKCBmutations, suggesting potential functional synergism between these lesions. Actually, overexpression of wild-type CARD11 induced NF-κB activation, which was further augmented by E626K mutation. Similarly, when both CARD11 (E626K) and PRKCB (D427N) mutants were co-expressed, more enhanced NF-κB activation was observed.RNA-seq and follow-up RT-PCR screening also identified novel gene fusions in TCR / NF-κB pathway: five CTLA4-CD28 and three ICOS-CD28 fusions were observed in seven (7%) of the 105 cases examined, of whom one patient carried both chimeric fusions. WGS revealed tandem duplications of 2q33.2 segments containing CD28, CTLA4, and ICOS, compatible with the corresponding fusion transcripts. B7/CD28 co-signaling molecules, including CD28, CTLA4, and ICOS co-receptors, play pivotal roles in positive and negative regulations of TCR signaling. All the predicted chimeric proteins had the cytoplasmic part of CD28, and are expected to be expressed under the control of the regulatory element of CTLA4 or ICOS, likely leading to prolonged expression of CD28 co-stimulator.Our findings suggest that deregulated TCR / NF-κB pathway caused by genetic alterations is a hallmark of ATL pathogenesis. The predominance of gain-of-function mutations in this pathway offers good opportunities for exploiting these mutations for the targets of novel drugs to better manage patients. DisclosuresTobinai: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.

Prognostic Relevance of Integrated Genetic Profiling in Adult T-Cell Leukemia/Lymphoma

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.We analyzed a total of 361 ATL samples, including acute (n = 192), lymphoma (n = 66), chronic (n = 89), and smoldering (n = 14) subtypes, for recurrent mutations and CNAs. Each subtype had characteristic genetic/epigenetic features, suggesting a distinct molecular pathogenesis therein. Aggressive (acute and lymphoma) subtypes were characterized by a higher number of mutations and CNAs including focal amplifications/deletions, hyperploid status, and CIMP phenotype, compared with indolent (chronic and smoldering) tumors. Two mutations (TP53 and IRF4) and eight focal deletions involving 1p13 (CD58), 6p21 (HLA-B), 9p21 (CDKN2A), 10p11 (CCDC7), 13q32 (GPR183), 16q23 (WWOX), 17p13 (TP53), and 19q13 (CEBPA), were more common in aggressive ATL than in indolent ATL. In contrast, showing a similar mutational distribution to those found in large granular lymphocytic leukemia, STAT3 mutations were characteristic of the indolent diseases. Gene set enrichment analysis of RNA-seq data showed a significant enrichment of MYC pathway and genes regulating cell cycle and DNA repair in upregulated genes in aggressive ATL.Next, we assessed the impact of mutations and CNVs on prognosis among 215 ATL cases, for which survival data were available. In the entire cohort, mutation in CCR4 and IRF4, focal amplification in 9p24 (CD274) and 14q32 (BCL11B), and focal deletion in 9p21 (CDKN2A) were found to be significant predictors of poor overall survival, after adjustment for disease subtype and age. Multivariate analysis revealed that disease subtype (aggressive vs. indolent) was the most significant predictor of clinical outcome in ATL. Subsequent multivariate analysis according to disease subtype showed that within the patients with aggressive ATL, older age (≥ 70 years), CCR4 mutations, and 9p24 amplification were independently associated with an adverse outcome. Based on the number of the risk factors they owned, patients with aggressive ATL were classified into three categories showing marked difference in 3-year overall survival (OS) (P < 0.001): those with no risk factors (OS, 32%), with one risk factor (18%), and with two or more (0%). Among the patients with indolent ATL, we found IRF4 and TP53 mutations, 9p24 amplification, and deletions in 9p21 and 10p11 were independently associated with reduced survival. Interestingly, these alterations, except for 9p24 amplification, were also identified as genes more frequent in aggressive ATL. More importantly, based on these risk factors, the patients with indolent ATL can be classified into two categories showing very different prognostic profiles: patients with no risk factors (OS, 89%) and those with one or more risk factors (21%) (P < 0.001, HR = 16.8, 95% CI:5.4-52.5), suggesting that patients with indolent ATL having a genetic feature of the aggressive subtypes might genetically and biologically represent a distinct subset, which should be better managed as having an aggressive disease. Among these poor prognostic factors, 9p24 amplification and CCR4 mutation are especially interesting, because these lesions might be plausible targets of available agents, including anti-PD1/PD-L1 and anti-CCR4 antibodies.In conclusion, based on the comprehensive genetic profiling, we demonstrated that the known subtypes of ATL can be further classified into genetically and biologically distinct subsets of tumors characterized by discrete sets of genetic lesions and substantially different prognosis. Our results suggest that molecular profiling can improve the prediction of prognosis in ATL patients and better guide therapy. DisclosuresTobinai: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.