Adult T-cell Leukemia/lymphoma Cells Research Articles

A case of associated refractory acute graft-versus-host disease following umbilical cord blood transplantation in an adult T-cell leukemia/lymphoma patient pretreated with mogamulizumab

Adult T cell leukemia/lymphoma(ATLL)is a highly aggressive peripheral T-cell neoplasm associated with infection by a specific retrovirus, human T-cell lymphotropic virus type-I(HTLV-I). This hematological malignancy is resistant to conventional chemotherapy, and has a poor prognosis. Two previous multicenter trials for ATLL(Japan Clinical Oncology Group 9303 and 9801)evaluated the efficacy of dose-intensified multi-agent chemotherapy with vincristine, cyclophosphamide, doxorubicin, and prednisolone(VCAP), doxorubicin, ranimustine, and prednisolone(AMP), and vindesine, etoposide, carboplatin, and prednisolone(VECP)with granulocyte-colony stimulating factor(G-CSF)and intrathecal prophylaxis. However, these trials showed a median survival of only 13 months, not yielding satisfactory outcomes. Thus, a novel therapeutic modality is needed. A defucosylated humanized monoclonal antibody targeting CC chemokine receptor 4(CCR4), designated mogamulizumab, recently became available for this purpose. CCR4 is a chemokine receptor expressed on helper T cells, regulatory T cells(Tregs), and certain types of T-cell neoplasms, such as ATLL. With an increased binding affinity to the Fc-gamma receptor on effector cells, this drug markedly enhances antibody-dependent cell-mediated cytotoxicity(ADCC)against ATLL cells expressing CCR4. In a phase II clinical trial for relapsed ATLL, objective responses were noted in 13 of 26 evaluable patients, including eight complete responses. Although mogamulizumab is a promising therapeutic alternative for patients with relapsed/refractory ATLL, there is some apprehension regarding treating patients with mogamulizumab before allogeneic hematopoietic stem cell transplantation(allo-HSCT). The fear is that it might affect regulatory T cell(Treg)reconstitution because these cells also express CCR4. Fewer circulating Tregs cause severe graft-versus-host disease(GVHD)after allo-HSCT because these cells have an Case Report

Shedding by ADAM10 and ADAM17 is associated with progression of adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a retrovirus-associated mature T-cell leukemia/lymphoma. It is speculated that ATL is an age-related disease and some changes are involved in malignant transformation and monoclonal expansion of the HTLV-1-infected cells. We previously reported that HTLV-1-infected cells and ATL cells exhibit CD30 and soluble CD30 (sCD30) is elevated in the sera of patients with ATL. Recently, we also evaluated the levels of sCD30 in ATL patients underwent chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) to predict prognosis of ATL patients under 2 different clinical settings; before initiation therapy and before HSCT. Our results suggest that sCD30 may be a useful biomarker in HSCT therapy, because a high sCD30 level before HSCT was implicated in early death after HSCT. In addition, we report that sCD30 elevation was followed by acute crisis from chronic type of ATL. Previously, high levels of soluble proteins including cytokine receptor and membrane-binding protein were observed in patients with ATL. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 worked as sheddases of CD30 as well as the other proteins. ADAM10/17 also cleaved collagen and elastin which are structural proteins of tissues and may cause the tissue injury of important organs. It seems that ADAM10/17 plays a role as oncoproteins for tumorigensis in ATL.

Gain-of-function CCR4 mutations in adult T cell leukemia/lymphoma.

Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T cell lymphotropic virus type-I (HTLV-I) without curative treatment at present. To illuminate the pathogenesis of ATLL we performed whole transcriptome sequencing of purified ATLL patient samples and discovered recurrent somatic mutations in CCR4, encoding CC chemokine receptor 4. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330, or Y331 in the carboxy terminus. Functionally, the CCR4-Q330 nonsense isoform was gain-of-function because it increased cell migration toward the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation after receptor engagement by CCL22 in ATLL cells and conferred a growth advantage in long-term in vitro cultures. These findings implicate somatic gain-of-function CCR4 mutations in the pathogenesis of ATLL and suggest that inhibition of CCR4 signaling might have therapeutic potential in this refractory malignancy.

Comprehensive Analysis of Surface Antigens on Adult T-Cell Leukemia/Lymphoma (ATL) Cells and Search for ATL-Initiating Cell Markers

Adult T-cell leukemia/lymphoma (ATL) is highly aggressive malignancy caused by human T-cell leukemia virus type 1 (HTLV1). Intensive combination chemotherapy can initially reduce ATL cells, but relapses are common. Today many kinds of tumors are considered to be organized in a hierarchy of heterogeneous cell populations, with only a small proportion of cancer initiating cells (CICs) capable of sustaining tumor formation and growth. Even if most of tumor cells are killed, remaining chemo-resistant CICs can be causes of relapses. However, CICs of ATL have not been identified yet. Our first goal is to identify ATL-initiating cells using in-vitro and in-vivo models.Because co-culture of HTLV1-infected ATL cells and normal lymphocytes results in extraordinarily high HTLV1 integration into normal lymphocytes, which is not seen in the bodies of patients, purification of ATL cells is necessary for evaluation of tumor-specific proliferation. We reported that HTLV1-infected ATL cells could be specifically enriched in the CD4+CADM1+ fraction, while the HTLV-1 basic leucine zipper (HBZ) gene was not detected in CD4+CADM1- cells by PCR analysis. To purify ATL cells, CD4+CADM1+ gating was used.First, we performed comprehensive analysis of surface antigens specifically on ATL cells. Peripheral blood mononuclear cells (PBMCs) were isolated from typical acute-type ATL patients and healthy volunteers. Using flow cytometry and gating, surface expression of 105 antigens, including activated T-cell markers, cell adhesive molecules, and lineage markers, was analyzed. The expression on ATL cells was compared with other CD4-positive non-ATL lymphocytes. Statistical hierarchical clustering analysis of 10 sample groups showed that ATL cells were all grouped into the same cluster, and markers specific for acute-type ATL cells were therefore picked up as candidate markers.Second, CD4+CADM1+ cells were classified by differences in expression of those candidate markers and sorted using 12-color flow cytometry. Although growing primary ATL cells in vitro is very difficult, it was reported that only a small proportion of ATL cells can survive and proliferate on the murine MS-5 stromal cell line, which also allows the proliferation of some ALL and hematopoietic progenitor cells. We repeated these 14-days in-vitro assays and tried to identify the cells capable of surviving and proliferating on MS-5, which we consider to be ATL-initiating cells. Although most of picked-up markers didn’t have any impacts on survival and proliferation of ATL cells, several surface antigens affected the results. After confirmation and prioritization, CD25+ and CD71- fractions were finally picked up. Combining these two fractions, we found that CD71-CD25+CD4+CADM1+ cells could survive and proliferate on MS-5, while other CD4+CADM1+ cells died off in many cases. Even in a case where CD71-CD25+ cells accounted only for 1.1% among the CD4+CADM1+ fraction, CD71-CD25+ cells exclusively survived and proliferated in vitro (figure 1). Moreover, CD71-CD25+ cells could generate CD71+ and CD25- cells on MS-5. In serial culture models, the CD71-CD25+ cells could serially generate other CD4+CADM1+ cells in vitro.Then, we focused on the clinical course of actual ATL patients, and followed up the CD71-CD25+ ATL cells before and after chemotherapy. After chemotherapy, the frequency of CD71-CD25+ cells among CD4+CADM1+ cells became obviously higher, and they therefore seemed to be resistant to chemotherapy. In a case where robust reduction of ATL cells was achieved, more than 90% of remaining ATL cells were in the CD71-CD25+ fraction.Lastly, we compared the in-vivo proliferation of CD71-CD25+ ATL cells and other CD4+CADM1+ cells. Both cells were transplanted intravenously into neonatal NOD/Shi-scid/IL-2Rγnull (NOG) mice within 48 hours from birth. CD71-CD25+CD4+CADM1+ cells could proliferate in vivo while others couldn’t as far as we tried. Moreover, flow cytometric analysis of murine peripheral blood in 70 days after transplantation showed that CD71-CD25+CD4+CADM1+ cells could generate ATL cells with other phenotype seen in primary ATL patients.In summary, we successfully found that CD71-CD25+CD4+CADM1+ cells could generate other ATL cells in vitro and in vivo. This research suggested ATL-initiating cells could be in the CD71-CD25+CD4+CADM1+ fraction. [Display omitted] DisclosuresTojo:Novartis: Research Funding, Speakers Bureau.

Landscape of Genetic Alterations in Adult T-Cell Leukemia/Lymphoma

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.To obtain a complete registry of genetic alterations in ATL, we performed an integrated genetic study, in which whole-genome/exome and RNA sequencing (RNA-seq) was performed together with array-based methylation and genomic copy number analysis among a cohort of 50 paired ATL samples, followed by extensive validation using targeted deep sequencing of detected mutations in > 400 follow-up samples. Compared with other lymphoid malignancies, ATL cells carried higher numbers of mutations, copy number alterations, and rearrangements than in other lymphoid malignancies, suggesting the presence of global genomic instability in ATL. In addition to previously reported mutational targets in ATL (TP53,TCF8, and FAS) and known targets frequently mutated in other lymphoid malignancies (CARD11, GATA3, IRF4, POT1, and RHOA), we identified a variety of highly recurrent mutations affecting previously unknown mutational targets, many of which are involved in T-cell development, activation and migration, immunosurveillance, and transcriptional regulation. Molecular and functional analysis using human T-cell leukemia cell lines showed that some of these novel mutations actually augment T-cell receptor signaling, validating their biological significance in ATL. A comparison of mutations among disease subtypes revealed that several subtype-specific mutations, including TP53, CD58, IRF4 and TBL1XR1 mutations in acute and lymphoma types, and STAT3mutation in chronic and smoldering types, suggesting that different oncogenic mechanisms underlie different ATL subtypes. Furthermore, ATL cells had a distinct pattern of copy number changes and genomic rearrangements. Interestingly, their gene targets showed a significant overlap to mutational targets. Surprisingly, somatic focal deletions involving the 14q31.1 locus were observed in all the cases examined by whole-genome sequencing and therefore are thought to uniquely characterize ATL genomes, although their gene targets remained to be identified. Like other regions also frequently deleted in ATL, such as 7q31.1 and 1p21.3 loci, these deletions were thought to reflect high levels of genetic instability. Finally and conspicuously, pathway analysis revealed that multiple genes involved in the Tax interactome were systematically altered in ATL, although Tax itself underwent gene silencing in most cases. These data suggested that ATL cells can escape from cytotoxic T-lymphocytes by silencing immunogenic Tax expression, while developing alternative oncogenic mechanisms through acquiring somatic mutations or copy number alterations in the Tax-related pathway. Our findings suggest that deregulated T-cell functionalities caused by genetic alterations, especially those associated with HTLV-1 Tax oncoprotein, are central to ATL pathogenesis, and provide a novel clue to contrive new diagnostics and therapeutics for this intractable disease. DisclosuresNo relevant conflicts of interest to declare.

Cytotoxic T-Lymphocyte Analysis of Aggressive Types of Adult T-Cell Leukemia/Lymphoma Patients with Complete Remission after Intensive Combination Chemotherapy

Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-I). Most ATLL cells are CD4 and CCR4 positive, and CD8 negative. ATLL is classified into 4 clinical subtypes: smoldering, chronic, acute, and lymphoma. Acute and lymphoma type ATLLs, which are aggressive types, have a very poor prognosis. In Japan, aggressive ATLLs are commonly treated by intensive chemotherapies, such as VCAP-AMP-VECP, modified LSG15 therapy, and biweekly CHOP therapy. Recently, an anti-CCR4 monoclonal antibody (mogamulizumab) was approved for relapse and refractory ATLL in Japan. Mogamulizumab induces a highly potent antibody dependent cellular cytotoxicity, suggesting the importance of immuno-cell therapy for treatment of ATLL. We report here the extremely interesting results of aggressive ATLL patients with long-term survival and complete remission (CR) after activation of cellular immunity against ATLL cells following intensive chemotherapies.Patients and Methods: We retrospectively evaluated 46 cases of aggressive ATLLs diagnosed at the Nagasaki Genbaku Hospital between January 2001 and August 2011. Of these, 7 patients had long-term survival greater than 3 years with CR after intensive chemotherapies. Four of these 7 patients had human leukocyte antigen (HLA)-A02:01 or HLA-A24:02, and were investigated using anti-HTLV-I specific cytotoxic T-lymphocyte (CTL) analysis. The HTLV-I provirus load in peripheral blood was also analyzed.Results: Table 1 summarizes the characteristics of 7 aggressive type ATLL patients with long-term survival. Four patients were male and 3 were female. Six patients were classified as lymphoma type and 1 as acute type ATLL. The median age was 68 (range, 60–78) years. The median survival period from the onset of the disease was 111 (range, 36–165) months. In all 7 patients, the CD4/CD8 ratio reversed during, or shortly after, chemotherapy and CD8 predominance continued for more than 1 year (range, 13–165 months, median 24 months). Three patients had herpes virus infection during chemotherapy and reversal of the CD4/CD8 ratio appeared just after herpes virus infection in 2 of these patients. These observations suggested that HTLV-I specific CTLs were induced and contributed to the treatment of ATLL in these patients. An HTLV-I specific CTL analysis currently is available in patients with HLA-A02:01 and HLA-A24:02. Three of 7 aggressive ATLL patients with long-term survival and CR had HLA-A02:01 and 1 had HLA-A24:02. Therefore, HTLV-I specific CTL analysis and HTLV-I provirus load in the peripheral blood were performed in all 4 patients. Each patient was examined twice, once in 2012 and once in 2014. HTLV-I specific CTLs were detected in all patients (Table 2 and Figure 1). Although all patients maintained CR for, HTLV-I proviruses were detected in the peripheral blood in all patients (Table 2). This phenomenon was observed both in 2012 and in 2014 (Table 2 and Figure 1).Conclusions: The findings from this study suggest that HTLV-I specific CTLs can be induced in patients with aggressive types of ATLL. In patients having long survival with CR, these CTLs can contribute to treatment and may play a roll inhibiting the relapse of ATLL. The development of efficacious methods to induce HTLV-I specific CTLs in individual ATLL patients may lead to improved outcomes for aggressive types of ATLL.Table 1Summary of aggressive type ATLL patients with long-term survival and complete remissionPatientNo.GenderAge(year)Survival fromthe onset of ATLL (months)Duration ofCD4/CD8 reversal(months)Herpes virus infection1Male69165165+2Male6814096+3Female717165-4Male6012413+5Female6111118-6Male783613-7Female655624-Table 2A Summary of HTLV-I specific CTL analysis and HTLV-I provirus load in the peripheral bloodPatient No.HLA20122014CTLHTLV-I provirusCTLHTLV-I provirus(%)(copies/1000 cells)(%)(copies/1000 cells)1A02:010.1135.40.1361.92A02:010.7824.41.5614.75A02:011.067.11.3113.37A24:022.0726.43.6427.9 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Using HDAC Inhibitors to Eradicate Adult T-Cell Leukemia-Lymphoma

Adult T-cell leukemia-lymphoma (ATLL) is an aggressive malignancy with a poor prognosis caused by HTLV-I, which is endemic in Japan, the Caribbean, and South America. ATLL cannot be cured with conventional chemotherapy thus urging the development of new therapeutic strategies. Histone deacetyalse (HDAC) inhibitors are broadly active anti-neoplastic agents, which have shown efficacy in T-cell lymphomas. At our institution we encounter a relatively high number of ATLL cases as compared to other regions in the U.S. We have conducted a pilot trial using AZT/interferon-α(IFNα) plus the HDAC inhibitor valproic acid (VPA) during maintenance therapy in acute (leukemia-type) ATLL. We hypothesized that VPA would reactivate HTLV-I leading to a cytotoxic T-cell immune response followed by reduction of residual blood circulating ATLL clones that normally persist during AZT/IFNα therapy despite its suppressive effects. Supporting this notion, the addition of VPA to AZT/IFNα resulted in reduction of HTLV-I proviral loads in treated patients and a sustained molecular response in one subject who is still alive >4.5 years later. More recently, we have tested newly available and more potent HDAC inhibitors using fresh primary leukemic ATLL cells and low-passage cell lines. Through HDAC inhibition, these agents reactivated HTLV-I Tax gene expression and induced apoptosis in a dose-dependent manner. Belinostat, which is currently FDA-approved for the treatment of relapsed or refractory peripheral T-cell lymphoma, augmented ATLL cell death when added to AZT/IFNα at nanomolar concentrations. Therefore, we have proposed a new pilot clinical trial using belinostat for the treatment of ATLL during post-induction therapy. The role of HDAC inhibitors in ATLL, pre-clinical laboratory studies involving these agents, and clinical trial design will be discussed at the meeting DisclosuresOff Label Use: The use of HDAC inhibitors in adult T-cell Leukemia-lymphoma.

Anchorage-dependent multicellular aggregate formation induces CD44 high cancer stem cell-like ATL cells in an NF-κB- and vimentin-dependent manner

Adult T-cell leukemia/lymphoma (ATL) is an intractable T-cell malignancy accompanied by massive invasion of lymphoma cells into various tissues. We demonstrate here that ATL cells cultured on a layer of epithelial-like feeder cells form anchorage-dependent multicellular aggregates (Ad-MCAs) and that a fraction of MCA-forming ATL cells acquire CD44 high cancer stem cell-like phenotypes. ATL cells forming Ad-MCAs displayed extracellular microvesicles with enhanced expression of CD44v9 at cell synapses, augmented expression of multidrug resistance protein 1, and increased NF-κB activity. Blockade of the NF-κB pathway dramatically reduced Ad-MCA formation by ATL cells and the emergence of CD44 high ATL cells, but left a considerable number of ATL cells adhering to the feeder layer. Disruption of vimentin cytoskeleton by treatment with withaferin A, a natural steroidal lactone, suppressed not only the adhesion of ATL cells to the feeder layer but also subsequent Ad-MCA formation by ATL cells, suggesting the involvement of vimentin in anchoring ATL cells to the feeder layer. Ad-MCA formation by ATL cells on a layer of epithelial-like feeder cells may mimic critical events that occur in metastatic colonization.

Heat shock protein 90 inhibitor NVP-AUY922 exerts potent activity against adult T-cell leukemia-lymphoma cells.

Adult T-cell leukemia–lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Heat shock protein 90 (HSP90) is involved in folding and functions as a chaperone for multiple client proteins, many of which are important in tumorigenesis. In this study, we examined NVP-AUY922 (AUY922), a second generation isoxazole-based non-geldanamycin HSP90 inhibitor, and confirmed its effects on survival of ATL-related cell lines. Analysis using FACS revealed that AUY922 induced cell-cycle arrest and apoptosis; it also inhibited the growth of primary ATL cells, but not of normal PBMCs. AUY922 caused strong upregulation of HSP70, a surrogate marker of HSP90 inhibition, and a dose-dependent decrease in HSP90 client proteins associated with cell survival, proliferation, and cell cycle in the G1 phase, including phospho-Akt, Akt, IKKα, IKKβ, IKKγ, Cdk4, Cdk6, and survivin. Interestingly, AUY922 induced downregulation of the proviral integration site for Moloney murine leukemia virus (PIM) in ATL cells. The PIM family (PIM-1, -2, -3) is made up of oncogenes that encode a serine/threonine protein kinase family. As PIM kinases have multiple functions involved in cell proliferation, survival, differentiation, apoptosis, and tumorigenesis, their downregulation could play an important role in AUY922-induced death of ATL cells. In fact, SGI-1776, a pan-PIM kinase inhibitor, successfully inhibited the growth of primary ATL cells as well as ATL-related cell lines. Our findings suggest that AUY922 is an effective therapeutic agent for ATL, and PIM kinases may be a novel therapeutic target.

HTLV-1 bZIP factor HBZ promotes cell proliferation and genetic instability by activating OncomiRs.

Viruses disrupt the host cell microRNA (miRNA) network to facilitate their replication. Human T-cell leukemia virus type I (HTLV-1) replication relies on the clonal expansion of its host CD4(+) and CD8(+) T cells, yet this virus causes adult T-cell leukemia/lymphoma (ATLL) that typically has a CD4(+) phenotype. The viral oncoprotein Tax, which is rarely expressed in ATLL cells, has long been recognized for its involvement in tumor initiation by promoting cell proliferation, genetic instability, and miRNA dysregulation. Meanwhile, HBZ is expressed in both untransformed infected cells and ATLL cells and is involved in sustaining cell proliferation and silencing virus expression. Here, we show that an HBZ-miRNA axis promotes cell proliferation and genetic instability, as indicated by comet assays that showed increased numbers of DNA-strand breaks. Expression profiling of miRNA revealed that infected CD4(+) cells, but not CD8(+) T cells, overexpressed oncogenic miRNAs, including miR17 and miR21. HBZ activated these miRNAs via a posttranscriptional mechanism. These effects were alleviated by knocking down miR21 or miR17 and by ectopic expression of OBFC2A, a DNA-damage factor that is downregulated by miR17 and miR21 in HTLV-1-infected CD4(+) T cells. These findings extend the oncogenic potential of HBZ and suggest that viral expression might be involved in the remarkable genetic instability of ATLL cells.

WS3-3 - Possible Proposal of an Extranodal Primary Gastric Variant of Lymphoma Type Atl

Adult T-cell leukemia-lymphoma (ATL) is a mature T-cell malignancy caused by human T-cell leukemia virus type-I (HTLV-1) and has been classified into four clinical subtypes: acute, lymphoma, chronic, and smoldering. This classification is very useful when deciding on a treatment strategy at the time of diagnosis. Although ATL is generally thought to be a T-cell non-Hodgkin's lymphoma with frequent leukemic manifestations, this classification does not include extranodal lymphoma in ATL. Extranodal ATL in which lesions are located in organs such as the stomach, nasopharhynx, skin and so forth are rare. Although primary gastric ATL is included in the category of acute ATL, we have noted several cases in which the prognosis was much better than is usual with acute ATL. In order to more precisely define primary extranodal ATL, we analyzed patients with primary gastric ATL.The clinicopathological features of six patients (four male, two female; median age 64 years with a range of 46 - 75 years) with localized lesions in the stomach were analyzed and compared. Two patients experienced regional lymph node swelling, and no patients had leukemic manifestations (ATL cells in peripheral blood: ≤ 1%) or hypercalcemia. Serum LDH level was elevated in only one case. Monoclonal integration of HTLV-1 proviral DNA was detected in gastric lesions by Southern blotting in all three cases analyzed. No patients received allo-HSCT. Median overall survival time (OS) after chemotherapy in these six cases was 66 months (3 - 216 months), which is longer than is typically reported for acute type ATL. In conclusion, these cases with localized ATL with relatively good prognosis possibly represent a new clinical subtype of ATL, an extranodal primary gastric variant of the lymphoma type of ATL.This work was supported by a grant (H23-ganrinsho-ippan-022) from the Ministry of Health, Labor and Welfare in Japan.

Quantification of adult T-cell leukemia/lymphoma cells using simple four-color flow cytometry.

The absolute number of adult T-cell leukemia/lymphoma (ATL) cells in peripheral blood is an essential indicator to evaluate disease status. However, microscopically counting ATL cells based on morphology requires experience and tends to be inaccurate due to the rarity of ATL. Based on our research showing that acute-type ATL cells are specifically enriched in the CD4+/CD7- (CD7N) fraction, a new analytical method to accurately quantify ATL cells was established using an internal bead standard and simple four-color flow cytometry. This method was verified by comparison with microscopic examination of 49 peripheral blood samples and used to follow up patients. A strong correlation was observed between the number of CD7N cells measured by flow cytometry and the number of abnormal lymphocytes measured microscopically by experienced technicians [Pearson's R, 0.963; Spearman's rho, 0.921; intercorrelation coefficient, 0.962]. The linear regression coefficient was close to 1 (β=1.013). Our method could detect 1 cell/μL, and the limit of quantitation was between 2.9 and 9.8 cells/μL. The frequency of CD7N cells among CD4+ cells changed during chemotherapy, which reflected differences between chemosensitive and chemoresistant cases. Kaplan-Meier analysis with a log-rank test showed that patients with decreased CD7N proportion after chemotherapy had significantly longer disease-specific survival (p=0.003). Our newly established method quantified tumor cells in patients with acute-type ATL. Furthermore, this method was useful for assessing the efficacy of chemotherapy, and the change of the CD7N proportion could be more important to predict prognosis.

Effect of anti‐CCR4 monoclonal antibody (mogamulizumab) on adult T‐cell leukemia–lymphoma: Cutaneous adverse reactions may predict the prognosis

Adult T-cell leukemia-lymphoma (ATL) is one of the most malignant lymphomas with poor prognosis. ATL cells express CC chemokine receptor 4 (CCR4) and mogamulizumab, a monoclonal antibody against CCR4 that exhibits very strong cytotoxicity for ATL cells via antibody-dependent cellular cytotoxicity. Although its effect is dramatic in ATL, serious adverse reactions such as Stevens-Johnson syndrome have been reported. However, these eruptions can appear as therapeutic signs of mogamulizumab. We evaluated the effectiveness of mogamulizumab in five acute-type ATL patients. Peripheral blood (PB) and lymph nodes (LN) were affected in three and four patients, respectively. In PB, complete response (CR) was obtained in all three patients and partial response (PR) was recorded in LN of one patient. In skin lesions, four of five patients manifested CR; in two, the lesions worsened after the start of mogamulizumab treatment and subsequently improved. In these lesions, CD4(+) 8(-) 25(+) ATL cells were replaced by CD3(+) 8(+) cytotoxic T cells. Cutaneous adverse reactions (CAR) developed in two patients with CR; they did not show a relapse of ATL over the course of 9 months. Our findings suggest that mogamulizumab should be continued and surface marker evaluation should be performed even in patients whose skin lesions show aggravation, and that CAR may be a marker for a favorable prognosis.

Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers

Constitutive phosphatidylinositol 3-kinase (PI3K)-AKT activation has a causal role in adult T-cell leukaemia-lymphoma (ATLL) and other cancers. ATLL cells do not harbour genetic alterations in PTEN and PI3KCA but express high levels of PTEN that is highly phosphorylated at its C-terminal tail. Here we report a mechanism for the N-myc downstream-regulated gene 2 (NDRG2)-dependent regulation of PTEN phosphatase activity via the dephosphorylation of PTEN at the Ser380, Thr382 and Thr383 cluster within the C-terminal tail. We show that NDRG2 is a PTEN-binding protein that recruits protein phosphatase 2A (PP2A) to PTEN. The expression of NDRG2 is frequently downregulated in ATLL, resulting in enhanced phosphorylation of PTEN at the Ser380/Thr382/Thr383 cluster and enhanced activation of the PI3K-AKT pathway. Given the high incidence of T-cell lymphoma and other cancers in NDRG2-deficient mice, PI3K-AKT activation via enhanced PTEN phosphorylation may be critical for the development of cancer.

Prognostic impact of microRNA-145 down-regulation in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive tumor caused by human T-cell leukemia virus type 1. MicroRNAs (miRNAs) are closely involved in the development and progression of various tumors. Here we investigated the dysregulation of miRNAs in ATL and its clinical significance. Studies using miRNA arrays and subsequent real-time reverse transcription polymerase chain reaction showed that, in the 9 ATL cell lines examined, 1 miRNA was consistently up-regulated, whereas another 3 were consistently down-regulated, compared with normal CD4-positive lymphocytes. Next, we analyzed the prognostic impact of these 4 miRNAs in patients with aggressive-type ATL (n = 40). Of the 4 dysregulated miRNAs selected, 3 (miR-130b higher expression, miR-145 lower expression, and miR-223 lower expression) were significantly associated with a worsened overall patient survival. We found that expressions of these 3 miRNAs were correlated with each other. To clarify which of the 3 had the most significant impact on overall survival, we performed a multivariate prognostic analysis that included these 3 miRNAs, and only miR-145 lower expression was selected as an independent risk factor (P = .0005). When overexpressed in an ATL cell line in vitro, miR-145 specifically inhibited tumor cell growth. In conclusion, our study suggests that miR-145 down-regulation provides a growth advantage in ATL and is highly associated with a worsened prognosis for patients with ALT. Hence, miR-145 may be a useful prognostic marker and a potential therapeutic target for ATL.

What is the Role of Soluble Cytokine Receptors in Adult T-cell Leukemia/ Lymphoma?

Soluble cytokine receptors are products of tumor cells infected with viruses such as human T-cell leukemia virus type 1 (HTLV-1). Adult T-cell leukemia/lymphoma (ATLL) is a highly aggressive leukemia/lymphoma caused by HTLV-1. High levels of soluble cytokine receptors are detected in the serum of patients with ATLL. Matrix metalloproteinase (MMP)-9 cleaves the interleukin (IL)-2 receptor α chain (IL-2Rα, CD25), which produces soluble IL-2Rα (sIL-2R) and down-regulates the proliferative capability of T cells that encounter cancer cells. Soluble CD30 (sCD30) impairs the interaction of CD30 ligand+ cells with CD30+ cells, whereas CD30 expression integrates survival signals to NK cells. Accordingly, high level of sIL-2R and sCD30 suggest that not only does impaired tumor surveillance lead to the survival of ATLL cells, but also dysfunction of immune surveillance can cause severe infectious complications. Freshly isolated leukemic cells from acute type ATLL patients express little CD30 on the surface, and predominantly produce sCD30. Proliferating ATLL cells are thought to release sCD30, and excess sCD30 probably blocks CD30L on normal activated T cells and myeloid cells, protecting CD30+ ATLL cells from apoptosis. Regarding CD30, two types of MMPs have been reported, ADAM10 and ADAM17, and Tax expression may be associated with ADAM17 activation in HTLV-1-infected cells. Thus, soluble cytokine receptor, sCD30, is both a serum component and a functional protein. Detection of sCD30 level may be useful for detecting and monitoring Tax-expressing tumor cells in vivo.

IFN-α suppresses HTLV-1 expression via PKR in infected cells and renders them susceptible to AZT through p53 activation in AZT/IFN-α treatment

HTLV-1 expression is maintained at low levels in vivo through unknown mechanisms. Interferon-a (IFN-a) has been used in combination with zidovudin (AZT) to treat adult T-cell leukemia/lymphoma (ATL) patients with favorable effects, although the mechanism is also unclear. We previously reported that HTLV-1 gene expression could be markedly suppressed by stromal cells through a type-I IFN response in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients. Here, we found that treatment with IFN-a alone produced suppressive effects on HTLV-1 gene expression in ILTs and also on the spontaneous HTLV-1 induction in short-term cultured primary ATL cells. Following IFN-a treatment, the levels of intracellular Tax protein decreased earlier than those of HTLV-1 mRNA in ILTs. An RNA-dependent protein kinase (PKR) inhibitor reversed IFN-a-mediated suppression of Tax in ILTs, suggesting the involvement of a post-transcriptional mechanism mediated by PKR in the suppression. IFN-a also induced cell cycle arrest at the G0/G1 phase in ILTs. We further found that AZT combined with IFN-a induced cell apoptosis in ILTs, associated with phosphorylation of p53 and enhanced expression of genes responsive to p53, whereas AZT alone did not affect cell viability or viral expression. Our results suggest that ILTs and ATL cells retain susceptibility to type-I IFNs, which suppress HTLV-1 gene expression primarily at a posttranscriptional level and potentially allow for signaling through the p53 pathway in combination with AZT. These findings would partly explain how HTLV-1 gene expression is regulated in vivo, and how AZT/IFN-a produces therapeutic effects in ATL.

Soluble CD30 and peripheral blood or pulmonary involvement of adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive leukemia/lymphoma which was discovered in Japan [1,2]. The long clinical latency and low incidence of ATL indicate that ATL is an agerelated disease and some genetic changes are involved in malignant transformation and monoclonal expansion of the human T-cell leukemia virus type 1 (HTLV-1)-infected cells [3]. HTLV-1-mediated T-cell transformation presumably arises from a multistep oncogenic process in which the virus induces chronic T-cell proliferation with accumulation of genetic defects followed by dysregulation of cell growth [4]. Monoclonal proliferation of HTLV-1-infected cells is observed in upwards of 5% of patients who ultimately develop ATL, constituting the high risk group for ATL development [5]. We previously reported that HTLV-1-infected cells and ATL cells exhibit CD30 and soluble CD30 (sCD30) is elevated in the sera of patients with ATL [6]. CD30, a 120 kDa type I cell surface glycoprotein, is a member of the tumor necrosis factor receptor superfamily [7]. CD30 expression is dependent on mitogen or viral activation and proliferation of B and T cells. CD30+ cells release sCD30 in vitro and in vivo [8], detected at low levels in the sera of healthy donors [9].The question remains as to where the ATL cells proliferate and produce sCD30? We have focused on the monoclonal proliferation of T cells and chronic inflammation in the microenvironment in vivo.

Differential CD30 expression in adult T-cell leukemia-lymphoma subtypes

Adult T-cell leukemia-lymphoma (ATLL) is caused by HTLV-I, and is a disease with dismal prognosis urging new therapies. Brentuximab vedotin (SGN-35) is an anti-CD30 monoclonal antibody conjugated to a potent microtubule poisoning agent monomethyl auristatin E that is effective in the treatment of CD30-expressing lymphomas. There are conflicting reports on the frequency of ATLL tumors expressing CD30. At our institution we encounter a relatively large number of ATLL cases due to our patient demographics. Thirty-nine out of 156 ATLL cases identified so far have been checked for CD30 status. CD30 expression was evaluated by immunohistochemistry (IHC) performed on either tissue sections as part of the routine pathology workup or cytospins prepared from CD4+ lymphocyte-enriched peripheral blood specimens using 20% expression as a cut-off positive value. The proportion of CD30+ ATLLs was 36% (95% CI 11%-61%), including 47% in lymphomatous-type, 28% in acute-type, and 10% in indeterminate cases. Four of 12 (33%) acute-type ATLL cytospin cases were CD30+, however, a high expression of 80% was observed in only one case. One patient with CD30+ acute-type ATLL with diffuse skin involvement treated with brentuximab had an objective transient response. Our preliminary data show that 36% of ATLLs are CD30+ and therefore may be amenable to anti-CD30 therapy. However, the low percentage of CD30+ cells in acute-type ATLL may limit the response to anti-CD30 target therapies for this subtype. We are currently testing a larger number of tissue and leukemic ATLL specimens available for further immunophenotypic characterization and comprehensive analysis of clinical outcome.

The HTLV-1 encoded bZIP factor promotes cell proliferation and genetic instability through activation of oncogenic microRNAs

Viruses disrupt their host cells microRNAs (miRNAs) network for facilitating their replication. That of HTLV-1 relies on the clonal expansion of its host CD4+ and CD8+ T-cells yet the virus causes adult T-cell leukemia/lymphoma (ATLL) that is regularly of the CD4+ phenotype. Infected cells express Tax and HBZ viral oncoproteins. Tax is expressed in untransformed cells where it promotes cell proliferation, genetic instability and miRNAs deregulation whereas in contrast, HBZ is expressed by untransformed and malignant T-cells where hitherto, it is considered to promote cell proliferation and to silence virus expression. Here we show that an HBZ/miRNAs axis promotes cell proliferation and genetic instability. Infected CD4+ but not CD8+ T-cells were found to overexpress oncogenic miRNAs such as miR-17 and miR-21. HBZ activated these miRNAs via a posttranscriptional mechanism while in addition to promoting cellular growth; HBZ decreased DNA stability. These effects were alleviated by either miR-21/miR-17 knockdown or by the ectopic expression of OBFC2A, a factor that protects genome stability and that we found targeted by miR-17 and miR-21 in HTLV-1 infected CD4+ T-cells. This considerably extends the oncogenic potential of HBZ and suggests that viral expression might be involved in the remarkable genetic instability of ATLL cells.