Acute-type Adult T-cell Leukemia Research Articles

IPSC-Derived CD4 T Cell Generation and Investigation of CD4/CD8 T Cell Lineage Choice

iPSC-derived functionally rejuvenated antigen-specific cytotoxic T lymphocytes generated from exhausted CTLs show promising antitumor effect towards refractory tumors. Currently, we are preparing for clinical trials using iPSC-derived CTLs for EBV-associated lymphomas. These iPSC-derived T cells (iPSC-Ts) all generated into CD8 T cells, and stable generation of CD4 single positive (SP) T cells from iPSCs has never been accomplished without enforced CD4 gene transduction even though many research groups have challenged to do so. Even when an iPSC is established from a CD4 T cell clone, differentiating this iPSC does not generate into CD4 T cells, but in fact it generates into CD8 T cells. Therefore, we aimed to investigate why iPSC-Ts only differentiate into CD8T cells, and not into CD4T cells. We focused on adult T cell leukemia (ATL), because HTLV-1 infected CD4 T cells clonally expand. We reprogrammed CD4T cells obtained from acute-type ATL patients into iPSCs (ATL-iPSCs), then differentiated them into T cells using our established method. Interestingly, ATL-iPSCs from different donors all successfully differentiated into CD4 SP T cells (ATL-iPSC-Ts). Upon characterization of ATL-iPSC-Ts we confirmed that they showed FOXP3 positive naive regulatory T cell (Treg) phenotype. To investigate the function of these cells, we performed 51Cr-release assay. The cytotoxicity of HTLV-1 Tax-specific CTLs (Tax-CTLs) with or without ATL-iPSC-Ts against primary ATL cells was measured. The percentages of ATL cells lysed by Tax-CTLs was 34 ± 3.2%, whereas by the addition of ATL-iPSC-Ts, the cytotoxicity decreased to 23.4 ± 1.0% revealing the functionally suppressive effect of ATL-iPSC-Ts. In order to further understand the mechanism of CD4/CD8 T cell lineage choice, and discover the key regulators needed for generation of CD4 SP T cells from iPSCs, we performed single cell RNA-sequencing (scRNA-seq) on ATL-iPSC-Ts. As a control we used healthy donor (HD) CD4 SP T cell clone-derived iPSCs (HD-iPSCs), which differentiated into CD8 T cells (HD-iPSC-Ts [CD8 +]). We identified several genes expressed at a significantly higher level on HD-iPSC-Ts (CD8 +) than on ATL-iPSC-Ts (CD4 +). We presumed these genes to be essential for CD8 T cell differentiation. Therefore, we used CRISPR/Cas9 technology to knock out the respective gene in HD-iPSCs and confirmed if these iPSCs could differentiate into CD4 SP T cells, instead of CD8 T cells. This resulted in successful generation of CD4 SP T cells. We confirmed that these CD4 SP T cells were FOXP3 negative and were not Tregs. On the other hand, overexpression of this gene on ATL-iPSCs differentiated into CD8 SP T cells, instead of CD4 T cells. In conclusion, our work represents the first successful generation of natural CD4 SP T cells from iPSCs. The gene we identified appears to be a pivotal regulator of CD4/CD8 T cell lineage choice in our iPSC-T differentiation system. While more investigation is required, our method shows promise towards facilitating stable generation of CD4 T cells for T cell therapy.

Acute type adult T-cell leukemia cells proliferate in the lymph nodes rather than in peripheral blood.

A massive increase in the number of mature CD4+ T-cells in peripheral blood (PB) is a defining characteristic of acute type of adult T-cell leukemia (ATL). To date, the site of proliferation of ATL cells in the body has been unclear. In an attempt to address this question, we examined the expression of the proliferation marker, Ki-67, in freshly isolated ATL cells from PB and lymph nodes (LNs) of patients with various types of ATL. Our findings reveal that LN-ATL cells display higher expression of the Ki-67 antigen than PB-ATL cells in acute type patients. The gene expression of T-cell quiescence regulators such as Krüppel-like factor 2/6 and forkhead box protein 1 was substantially high in acute type PB-ATL cells. The expression of human telomerase reverse transcriptase, which is involved in T-cell expansion, was significantly low in PB-ATL cells from acute type patients, similar to that in normal resting T-cells. These findings suggest that ATL cells proliferate in the LNs rather than in PB.

Overexpression of a cell adhesion molecule, TSLC1, as a possible molecular marker for acute-type adult T-cell leukemia

AbstractAdult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) infection, occurs in 2% to 4% of the HTLV-1 carriers with a long latent period, suggesting that additional alterations participate in the development of ATL. To characterize and identify novel markers of ATL, we examined the expression profiles of more than 12 000 genes in 8 cases of acute-type ATL using microarray. One hundred ninety-two genes containing interleukin 2 (IL-2) receptor α were up-regulated more than 2-fold compared with CD4+ and CD4+CD45RO+ T cells, and tumor suppressor in lung cancer 1 (TSLC1), caveolin 1, and prostaglandin D2 synthase showed increased expression of more than 30-fold. TSLC1 is a cell adhesion molecule originally identified as a tumor suppressor in the lung but lacks its expression in normal or activated T cells. We confirmed ectopic expression of the TSLC1 in all acute-type ATL cells and in 7 of 10 ATL- or HTLV-1–infected T-cell lines. Introduction of TSLC1 into a human ATL cell line ED enhanced both self-aggregation and adhesion ability to vascular endothelial cells. These results suggested that the ectopic expression of TSLC1 could provide a novel marker for acute-type ATL and may participate in tissue invasion, a characteristic feature of the malignant ATL cells.

Complete Sequences of the Human T-Cell Leukemia Virus Type 1 Proviral Genomes from Newly Established Adult T-Cell Leukemia Cell Lines in Oita Prefecture, Japan.

ABSTRACTWe report two complete proviral genome sequences of human T-cell leukemia virus type 1 (HTLV-1) isolated from the peripheral blood specimens of acute type adult T-cell leukemia (ATL) patients in Oita Prefecture, Japan.

Leukemic T cells are specifically enriched in a unique CD3dimCD7low subpopulation of CD4+ T cells in acute‐type adult T‐cell leukemia

The morphological discrimination of leukemic from non-leukemic T cells is often difficult in adult T-cell leukemia (ATL) as ATL cells show morphological diversity, with the exception of typical "flower cells." Because defects in the expression of CD3 as well as CD7 are common in ATL cells, we applied multi-color flow cytometry to detect a putative leukemia-specific cell population in the peripheral blood from ATL patients. CD4(+) CD14(-) cells subjected to two-color analysis based on a CD3 vs CD7 plot clearly demonstrated the presence of a CD3(dim) CD7(low) subpopulation in each of nine patients with acute-type ATL. The majority of sorted cells from this fraction showed a flower cell-like morphology and carried a high proviral load for the human T-cell leukemia virus type 1 (HTLV-I). Genomic integration site analysis (inverse long-range PCR) and analysis of the T cell receptor Vβ repertoire by flow cytometry indicated that the majority of leukemia cells were included in the CD3(dim) CD7(low) subpopulation. These results suggest that leukemic T cells are specifically enriched in a unique CD3(dim) CD7(low) subpopulation of CD4(+) T cells in acute-type ATL.

Leukemic T Cells Are Specifically Enriched In a Unique CD3dimCD7low Subpopulation of CD4+ T Cells In Acute-Type Adult T Cell Leukemia

AbstractAbstract 4144 [Background]Adult T-cell leukemia (ATL) is a malignant disorder caused by human T-cell leukemia virus type I (HTLV-I). Morphological discrimination of leukemic cells from non-leukemic T cells is often difficult in ATL since ATL cells reveal morphological diversity except for typical “flower cells”. Although a study using CD3 gating in flow cytometry reported that ATL cells were distinguishable as a CD3low population from normal lymphocytes, these cells were not well characterised as ATL cells. Considering that defective expression of CD7 as well as CD3 is common in ATL cells, we applied multi-color flow cytometry to detect a putative leukemia-specific cell population in the peripheral blood from ATL patients. [Methods and Results](1) In flow cytometry, after dead-cell and monocyte removal, CD4+ T lymphocytes were gated on the CD3 versus CD4 plot. Based on cell density and fluorescence intensity of CD3 and CD7 in this population, we designated three subpopulations on this plot: CD3highCD7high, CD3dimCD7dim and CD3dimCD7low(Results of a representative ATL and a control sample are shown in Figure). The proportion of the CD3dim/CD7low subpopulation was significantly higher in acute-type ATL CD4+ lymphocytes than in normal controls(Figure). (2) To extensively characterise this subpopulation, we next estimated the HTLV-I proviral load by quantitative real-time PCR after FACS sorting based on this CD3 versus CD7 plot. In all patient samples, HTLV-I proviral integration was detected in all subpopulations. However, the proviral load was significantly higher in the CD3dim/CD7low subpopulation compared to the CD3high/CD7high subpopulation. Almost all of the cells in the CD3dim/CD7low subpopulation were HTLV-I infected. (3) We next examined CCR4 and CD25 expression in each subpopulation. Both CCR4 and CD25 expression levels were maintained at very low and similar levels throughout all subpopulations in normal control cells and in the CD3high/CD7high subpopulation of patients with ATL as well. In contrast, CCR4 expression was significantly up-regulated in CD3dim/CD7low subpopulation of patients with ATL compared to the CD3high/CD7high subpopulation (MFI: 36.5±17.2 vs. 3.8±1.1). The expression of CD25 was also up-regulated in the subpopulation (MFI: 7.8±8.0 vs. 2.7±1.6). (4) Monoclonal expansion of HTLV-I-infected cells in the CD3dim/CD7low subpopulation was indicated by the genomic integration site analysis using a long inverse polymerase chain reaction (PCR) method. (5) We reviewed the glass-slide specimens of FACS-sorted samples to evaluate the morphology of each subpopulation on the CD3 versus CD7 plot. Atypical lymphocytes with morphology such as a notch in the nucleus were observed in all subpopulations. The majority of sorted cells from CD3dim/CD7low subpopulation showed “flower cell”-like morphology. (6) We also detected a small CD3dim/CD7dim subpopulation other than the CD3dim/CD7low and CD3high/CD7high subpopulations in all patients with acute-type ATL who were analysed(Figure). This subpopulation contained the same clone as the CD3dim/CD7low subpopulation, although a phenotypical difference existed between these subpopulations. [Display omitted] [Conclusion](1) Above findings indicate that leukemic T cells are specifically enriched in a unique CD3dim/CD7low subpopulation of CD4+ T cells in acute-type ATL. This multi-color FACS system may be useful for precisely monitoring disease during chemotherapy, detecting minimal residual disease and analysing ATL cells. (2) Previous reports have revealed that HTLV-I-infected cells transform through multi-step oncogenesis. Detailed analysis of these three subpopulations (CD3high/CD7high, CD3dim/CD7dim and CD3dim/CD7low) may give some insight into oncogenesis of HTLV-I-infected cells. Disclosures:No relevant conflicts of interest to declare.

Maintenance and preemptive therapy with ganciclovir for cytomegalovirus colitis with extremely high antigenemia in adult T-cell leukemia

Patients with adult T-cell leukemia (ATL) are susceptible to opportunistic infections. The major cause of death in ATL patients is cytomegalovirus (CMV) pneumonia [1]. However, the onset of CMV infection in a very early phase of ATL is uncommon [2]. In addition, there are no detailed descriptions concerning the effect of oral ganciclovir (GCV) in ATL. Herein, we report a case with ATL who demonstrated CMV colitis on the second day of prednisolone administration. A 48-year-old female was admitted because of fever, erythema, and fatigue. Her white blood cell (WBC) count was 18,400/mm (atypical lymphocytes including flower cells: 48.5%). The left and right cervical, axillary, and right inguinal lymph nodes were swollen. Erythema of the face, limbs, and neck was observed. Histopathologic findings of biopsy specimens from erythema sites demonstrated the possibility of ATL cells having invaded the skin. The lactate dehydrogenase (LDH) score was 505 U/L (more than twice the normal upper limit) and human T lymphotrophic virus type 1 provirus was detected on Southern blot analysis. Therefore, we diagnosed this case as acutetype ATL. The compensated calcium level was normal (9.6 mg/dL), and there were no other sites of involvement such as the liver, spleen, and cerebrospinal fluid. She was started on prednisolone for ATL. On the day following the start of administration, diarrhea and abdominal pain developed. We started chemotherapy primarily according to the LSG 15 protocol [3], because it was possible at that time that her diarrhea was caused by ATL cell invasion of the colon. However, an abdominal computed tomography scan showed ascites and mucosal thickening of the colon, which indicated colitis. Histopathologic findings of biopsy specimens from edematous lesions of the rectum detected by proctoscopy demonstrated intranuclear CMV inclusion bodies in endothelial cells but not the invasion of ATL cells. In addition, there were 3411 and 3258 CMV antigenpositive leukocytes each per 15 9 10 WBCs, and other pathogens which can induce diarrhea were not detected. No other manifestations induced by CMV infection such as CMV pneumonia and retinitis appeared. Therefore, the diagnosis of CMV colitis was confirmed. She received intravenous GCV. Diarrhea improved and the score of CMV antigenemia gradually declined (Fig. 1). After the diarrhea had fully resolved and informed consent was given, we continued maintenance therapy with oral GCV until CMV antigenemia became negative. Thereafter, we chose preemptive therapy involving oral GCV along with the monitoring of CMV antigenemia. This patient received allogeneic bone marrow transplantation (BMT) four months after the diagnosis of CMV colitis. We continued the same preemptive therapy until BMT. CMV infection never recurred before BMT. In addition, chemotherapy was effective. After chemotherapy, atypical lymphocytes in the peripheral blood disappeared, and the score of the soluble interleukin-2 receptor was reduced (Fig. 1). Swollen lymph nodes regressed. In addition, the level of LDH was reduced, but just less than half of the erythematous area S. Nagai M. Toshima K. Sato M. Mori T. Nagai K. Muroi K. Ozawa Division of Hematology, Department of Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 3290498, Japan

Adhesion-dependent growth of primary adult T cell leukemia cells with down-regulation of HTLV-I p40Tax protein: a novel in vitro model of the growth of acute ATL cells

In order to better understand the biology of adult T cell leukemia (ATL), we aimed to establish a novel method, which allows the primary growth of ATL cells using a co-culture system with murine bone marrow-derived stromal cells, MS-5. ATL cells grew in close contact with MS-5 layers and formed so-called "cobblestone areas" (CAs) without the addition of IL-2. In clinical samples, eight of ten (80.0%) cases of acute or lymphoma type ATL cells formed CAs. The frequency of CA forming cells in ATL cells ranged from 0.03 to 1.04%. The morphology, immunophenotyping, and DNA analysis indicated that cells composing CA were compatible with ATL cells, and clonally identical to primary CD4-positive ATL cells. Furthermore, in ATL cells composing CA, the expression of p40Tax was down-regulated in transcriptional and translational level, while that of HTLV-I basic leucine zipper factor (HBZ) gene was comparable to the level of primary ATL cells, resembling expression pattern of proviral genes in in vivo ATL cells. By microarray analysis, several genes which coded products involved in cell-cell interaction, and cellular survival and proliferation, were differentially expressed in ATL cells composing CA compared with primary samples. In conclusion, our co-culture system allows for the first time the growth of primary ATL cells in vitro, and might be useful as an in vitro assay for biological and clinical studies to develop molecular targeting drugs against ATL.

Splenectomy for hypersplenism caused by adult T-cell leukemia: Report of a case

A 45-year-old woman with previously diagnosed chronic type adult T-cell leukemia (ATL) presented with abdominal discomfort and red eruptions on her arms and legs. Anemia, thrombocytopenia, hypercalcemia, and splenomegaly indicated progression to acute-type ATL. Combined chemotherapy resulted in normalization of the serum calcium level and improvement in her symptoms. However, the severe anemia and thrombocytopenia persisted, necessitating transfusions of red blood cells (RBC) and platelets three times a week. We performed splenectomy in an attempt to reduce the total volume of malignant cells and improve the hypersplenism. After the operation, the RBC and platelet counts increased gradually, and the transfusions were stopped on postoperative day (POD) 3. Splenectomy should be considered as an optional treatment for hypersplenism caused by ATL when hypersplenism cannot be controlled by chemotherapy in patients without a high surgical risk.

Highly Enhanced Expression of CD70 on Human T-Lymphotropic Virus Type 1-Carrying T-Cell Lines and Adult T-Cell Leukemia Cells

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). In Japan, the number of HTLV-1 carriers is estimated to be 1.2 million and more than 700 cases of ATL have been diagnosed every year. Considering the poor prognosis and lack of curative therapy of ATL, it seems mandatory to establish an effective strategy for the treatment of ATL. In this study, we attempted to identify the cell surface molecules that will become suitable targets of antibodies for anti-ATL therapy. The expression levels of approximately 40,000 host genes of three human T-cell lines carrying HTLV-1 genomes were analyzed by oligonucleotide microarray and compared with the expression levels of the genes in an HTLV-1-negative T-cell line. The HTLV-1-carrying T-cell lines used for experiments had totally different expression patterns of viral genome. Among the genes evaluated, the expression levels of 108 genes were found to be enhanced more than 10-fold in all of the T-cell lines examined and 11 of the 108 genes were considered to generate the proteins expressed on the cell surface. In particular, the CD70 gene was upregulated more than 1,000-fold and the enhanced expression of the CD70 molecule was confirmed by laser flow cytometry for various HTLV-1-carrying T-cell lines and primary CD4(+) T cells isolated from acute-type ATL patients. Such expression was not observed for primary CD4(+) T cells isolated from healthy donors. Since CD70 expression is strictly restricted in normal tissues, such as highly activated T and B cells, CD70 appears to be a potential target for effective antibody therapy against ATL.

Association of High Proliferation in Adult T-cell Leukemia Cells With Apoptosis, and Expression of p53 Protein in Acute Type ATL

Proliferation, apoptosis and p53 protein expression in adult T-cell leukemia (ATL) cells were investigated. Twenty peripheral blood tissue specimens (PBTS) comprising 7 cases of acute type ATL, 7 cases of chronic type ATL and 6 other leukemias were examined by means of antigen retrieval and the polymer method employing anti-Ki67 antigen (MIB-1), anti-cleaved caspase-3, anti-single stranded DNA and three kinds of anti-p53 protein antibodies including DO7. Most acute and chronic cases of ATL included more than 10% MIB-1-positive proliferating leukemia cells and more than 1% cleaved caspase-3-positive apoptotic cells. Some cells which were positive for both MIB-1 and anti-cleaved caspase-3 antibody were observed in acute type ATL. Nuclear deposition of p53 protein labeled by DO7 was often found in acute type (p < 0.05). Within the medium-sized population of ATL cell nuclei, DO7-positive ATL cells had a smaller nuclear area factor (long axis x short axis) than DO7-negative ATL cells. A few proliferating ATL cells entered apoptosis, and the appearance of a subclone of ATL cells with nuclear deposition of p53 protein labeled by DO7 characterized acute type.

Establishment of Novel Disease Models for Human Acute Type ATL and HTLV-I Carrier Using NOD-scid/IL2rgnull Mouse.

[Purpose] Adult T cell leukemia (ATL) is developed by clonal proliferation of human T cell leukemia virus-type 1 (HTLV-I) infected CD4+ human T cells, and is one of the most intractable leukemia in adults. Excellent animal model has been desired to fully investigate the pathophysiology of HTLV-I infected cells in vivo. However, acute type primary ATL has been previously reported to engraft poorly in NOD-scid recipients and it was difficult to study human T cells physiologically developed from hematopoietic stem cells (HSCs) in vivo. We aimed to develop novel disease models for acute type ATL and HTLV-I carrier using newborn NOD-scid/IL2rgnull mice.[Method and Result] First, to analyze the engraftment levels of acute type ATL cells, 2.5–10 million peripheral blood (PB)-derived CD4+ cells from acute type ATL patients were intravenously transplanted into newborn NOD-scid/IL2rgnull recipients following sublethal irradiation. At 6 weeks after transplantation, the engraftment of hCD45+hCD3dimhCD4+hCD25+ cells were detected in recipients' PB and the engraftment levels increased over time. The phenotypical characterization of hCD3dimhCD4+hCD25+ was similar to that of primary patient sample. Until 16 weeks post-transplantation, recipient exhibited ruffled fur and lethargy and was sacrificed to analyze the engraftment and infiltration of ATL cells. All the engrafted human CD45+ cells expressed CD4 and CD25, and the engraftment levels of human ATL cells were higher than 90% in the PB and the spleen of recipients. Lymphadenopathy and splenomegaly were seen, which were not seen in untransplanted mice or in the mice transplanted with cord blood-derived CD34+CD38− cells. Abnormal multi-lobulated flower-like cells were present in the PB and the spleen of recipients as evidenced by May-Grunwald-Giemsa staining. Infiltration of human CD4+ ATL cells into the spleen, lymph nodes (LNs), and liver of recipients was demonstrated. Nested polymerase chain reaction (PCR) confirmed the presence of HTLV-I genome in the recipient spleen. These results suggest that, after transplantation of primary acute type ATL cells, NOD-scid/IL2rgnull mice properly mimicked the clinical features of ATL, showing efficient infiltration of tumor cells. Next, to understand leukemogenic process of normal CD4+ T cells through transmission of HTLV-I, we infused mitomycin-C (MMC)-treated HTLV-I cell lines (MT-2) into the humanized NOD-scid/IL2rgnull mice which possess human hematopoiesis derived from cord blood HSCs. At 7 to 12 weeks after infusion of MT-2 cells, atypical lymphocytes were found in the PB and LNs cells of recipients. Nested PCR of pX region demonstrated the presence of the HTLV-I genome in the recipient bone marrow cells. These results suggest that the transmission of HTLV-I from MMC-treated MT-2 cells to normal CD4+ T lymphocytes occurred in vivo in the humanized NOD-scid/IL2rgnull recipients. This model may be useful as a model for the carrier state for HTLV-I.[Conclusion] These new disease model mice may recapitulate the pathology of human acute type ATL and HTLV-I carrier state. These models can be useful tool to investigate the pathogenesis of ATL and to develop the curative treatment for ATL.

Clinical efficacy of all-trans retinoic acid for treating adult T cell leukemia

We previously reported that all-trans retinoic acid (ATRA) inhibited growth in human T-cell leukemia virus type I (HTLV-I)-positive T-cell lines and in fresh cells from patients with adult T cell leukemia (ATL). Here, we confirmed the clinical effects of ATRA in 20 patients with ATL. The 20 patients (n = 20) with a median age of 56 (range 35-73) years who were diagnosed with ATL received ATRA orally. The efficacy of treatment was as follows: no complete response (CR), a partial response (PR) in 40% of the patients, no change (NC) in 45% of the patients, and a progressive disease (PD) in 15% of the patients. In seven acute-type ATL patients, a PR was achieved in two (28.5%), NC was observed in two (28.5%), and a PD was observed in three (42.8%). In three lymphoma-type ATL patients, a PR (100%) was achieved. Among four chronic-type ATL patients, a PR was achieved in one (25%) and NC was observed in the remaining three (75%). In six smoldering-type ATL patients, a PR was achieved in two (33.3%) and NC was observed in four (66.6%). The major side effects were headache (n = 5), transient liver dysfunction (n = 2), hyperlipidemia (n = 2), and anorexia (n = 1). These results indicated that ATRA might be a useful agent for the safe treatment of ATL.

Adult T-cell Leukemia Development From a Human T-cell Leukemia Virus Type I Carrier After a Living-Donor Liver Transplantation

Adult T-cell leukemia (ATL) develops in a human T-cell leukemia virus type I (HTLV-I) carrier. The development of malignancy during immunosuppressive treatment following organ transplantation is one of the late fatal complications. We describe the development of three cases of ATL in eight HTLV-I carriers within 164 living-donor liver transplant recipients undergoing immunosuppressive treatment. All three cases were immunosuppressed with tacrolimus. Acute-type ATL was diagnosed at 6, 9, and 25 months after living-donor liver transplantation, based on increased numbers of CD4+25+ lymphocytes exhibiting "flower-like" nuclei, and the elevation of lactate dehydrogenase. Southern blot analysis demonstrated the clonal proliferation of ATL cells in peripheral blood. The ATL cells originated from the recipient, as demonstrated by fluorescence in situ hybridization analysis using sex chromosomal markers. Our observations suggest that immunosuppressive treatment for the prevention of graft rejection after living-donor liver transplantation may induce the development of ATL in an HTLV-I carrier.

Overexpression of survivin in primary ATL cells and sodium arsenite induces apoptosis by down-regulating survivin expression in ATL cell lines

AbstractPatients with acute- or lymphoma-type adult T-cell leukemia (ATL) have a poor outcome because of the intrinsic drug resistance to chemotherapy. Protection from apoptosis is a common feature involved in multidrug-resistance of ATL. IAP (inhibitor of apoptosis) family proteins inhibit apoptosis induced by a variety of stimuli. In this study, we investigated the expression of IAP family members (survivin, cIAP1, cIAP2, and XIAP) in the primary leukemic cells from patients with ATL. We found that survivin was overexpressed in ATL, especially in acute-type ATL. Sodium arsenite was shown to down-regulate the expression of survivin at both the protein and RNA levels in a time- and dose-dependent manner, thus inhibiting cell growth, inducing apoptosis, and enhancing the caspase-3 activity in ATL cells. Nuclear factor-κB (NF-κB) enhances the transcriptional activity of survivin. Sodium arsenite suppressed the constitutive NF-κB activation by preventing the IκB-α degradation and the nuclear translocation of NF-κB. These findings suggest that survivin is an important antiapoptotic molecule that confers drug resistance on ATL cells. Sodium arsenite was shown to down-regulate the expression of survivin through the NF-κB pathway, thus inhibiting cell growth and promoting apoptosis of ATL cells.

Adult T-Cell Leukemia in a Liver Transplant Recipient That Did Not Progress after Onset of Graft Rejection

A liver allograft recipient developed acute-type adult T-cell leukemia (ATL) during tacrolimus treatment, 2 years after undergoing transplantation for subacute fulminant hepatitis. Both donor and recipient were asymptomatic carriers of human T-cell lymphotropic virus type I (HTLV-I), but the ATL cells originated from the recipient. Tacrolimus treatment was discontinued, and combination chemotherapy was administered. The patient achieved complete remission, but the transplanted liver was acutely and chronically rejected. The patient did not respond to rescue therapy with tacrolimus, prednisolone, and mycophenolate mofetil and died of hepatic failure. Liver biopsies showed CD4+ ATL cell infiltration at the onset of ATL but not at the terminal stage. Moreover, Southern blotting revealed clonal integration of HTLV-I into the host genome of lymphoma cells at onset but not at the terminal stage. ATL after liver transplantation has not been previously described. The clinical course of the posttransplantational ATL was atypical, because it did not progress after the onset of rejection.

Infusional Adriamycin Based Chemotherapy Significantly Improves the Survival of Human T-Cell Lymphotropic Virus-I Induced Acute Type Adult T-Cell Leukemia Lymphoma.

Introduction: In this retrospective analysis, we provide a comprehensive account of the pathophysiology, clinical course and outcomes with various therapeutic regimens in HTLV-1 associated Adult T-cell Lymphoma/Leukemia- The Brooklyn Experience.Patients and Methods:We retrospectively reviewed the medical records of all patients identified as having acute type Human T-cell Lymphotropic Virus (HTLV-I) associated Adult T-cell Leukemia/Lymphoma (ATLL), from 1995 to 2005. The diagnosis was made according to REAL and WHO classification and based on clinical presentation, tissue biopsy and positive HTLV-I serology. Since all patients expired during the study, we used analysis of variance to examine the mean survival of these patients and the relationship between types of therapy and the survival.Results:We identified 44 patients with diagnosis of acute type ATLL. The most common disease presentation was lymphomatous in 70% (n=31) and leukemic presentation in 8% (n=3) both leukemia and lymphomatous presentation at diagnosis was seen in 22% (n=10) of patients. The mean survival for the entire group of patients was 31 weeks. The median survival was 16 weeks (range from 1 to 314 weeks). Patients who presented initially without hypercalcemia had a mean survival that was significantly higher than the patients with hypercalcemia 54 weeks versus 20 weeks, p<0.01. Treatment with infusional adriamycin chemotherapy significantly improved survival mean 99 ± 2.2 weeks, compared to CHOP 28 ± 2 weeks and much better than non-adriamycin regimens 21 ± 3 weeks p<0.001. Treatment with infusional adriamycin consistently improved survival in the patients without hypercalcemia 56± 6.6 weeks versus CHOP 21 ± 3.4 versus non-adriamycin containing regimens 10 ± 2.5 weeks, p=0.001. The presence or absence at diagnosis of central nervous system disease, pleural effusion, ascites, extensive liver or skin involvement or blood group were not independently associated with survival after adjusting for either hypercalcemia or infusional adriamycin chemotherapy. In multivariate analysis the presence of hypercalcemia was no longer significant after the therapy with infusional adriamycin.Conclusions: Hypercalcemia portends poor prognosis in all forms of HTLV-1 associated ATLL and should be included in any risk stratification schema for this disease. Our review demonstrates a statistically significant improvement in median overall survival with infusional adriamycin based chemotherapy over traditional CHOP, CVP or the AZT and interferon combination. This superiority in outcomes was evident in patients with or without hypercalcemia at presentation. Although overall survival remains relatively low in the acute form of this disease and most patients remain incurable with current therapies, our findings suggest that the incorporation of infusional adriamycin based regimens in future treatment modalities to include specific and non-specific molecular targeted therapies, would be a prudent strategy in development of more effective therapies for this disease.

HTLV-I viral escape and host genetic changes in the development of adult T cell leukemia

In the pathogenesis of adult T cell leukemia (ATL), an oncogenetic role of the human T cell lymphotropic virus type I (HTLV-I) Tax protein, viral escape from the host immune system, and host genetic changes have been proposed as contributory factors. We examined the premature stop codons in tax gene as one of the mutations that may lead to escape of HTLV-I from the cytotoxic T lymphocyte (CTL) response in HTLV-I carriers, to test whether a putative CTL escape mutant can emerge in the early stage of ATL development and whether HTLV-I infected cells with such a mutation can proliferate subsequently. We also examined deletion of cyclin-dependent kinase inhibitor 4 (INK4) genes and mutation of p53 gene in combination with changes in the HTLV-I genome in acute type ATL to test whether host genetic changes promoted the malignant transformation of ATL cells that carry putative CTL escape mutations. The premature stop codon in tax gene existed in many non-ATL HTLV-I carriers as a minor population but not in the commonest HTLV-I sequence of the individual. This minor population with a premature stop codon did not expand subsequently in 3 asymptomatic carriers tested. There were cases who had a mutation or deletion in HTLV-I who also have either deletion of INK4 genes or mutation in p53 gene. Our findings suggest that CTL escape mutation can occur at an early stage of ATL development, and that certain host genetic changes favor the development of the aggressive form of ATL.

Efficient engraftment of primary adult T-cell leukemia cells in newborn NOD/SCID/β2-microglobulinnull mice

Adult T-cell leukemia (ATL) develops via multiple oncogenic steps in human T-cell leukemia virus type I (HTLV-I) carriers. To better understand pathogenesis of ATL, we developed a novel xenogeneic engraftment model in which primary ATL cells are intravenously transplanted into neonatal nonobese diabetic (NOD)/severe-combined immunodeficiency (SCID)/beta2-microglobulin(null) (NOD/SCID/beta2m(null)) mice. Acute-type ATL cells engrafted in the peripheral blood and in the lymph nodes of recipients at a high efficiency. Engrafted ATL cells were dually positive for human CD4 and CD25, and displayed patterns of HTLV-I integration identical to those of donors by Southern blot analysis. These cells infiltrated into recipients' liver, and formed nodular lesions, recapitulating the clinical feature of each patient. In contrast, in smoldering-type ATL cases, multiple clones of ATL cells engrafted efficiently in NOD/SCID/beta2m(null) mice. When smoldering-type ATL cells were retransplanted into secondary NOD/SCID/beta2m(null) recipients, single HTLV-I-infected clones became predominant, suggesting that clones with dominant proliferative activity can be competitively selected in this xenogeneic system. Taken together, the NOD/SCID/beta2m(null) newborn system is useful to understand kinetics, metastasis, and disease progression of ATL in vivo.

Reduced expression of human mismatch repair genes in adult T-cell leukemia

In this study, we investigated the expression of six human DNA mismatch repair (MMR) genes, human MutS homologues 2 (hMSH2), 3 (hMSH3), and 6 (hMSH6), human MutL homologue 1 (hMLH1), human post-meiotic segregations 1 (hPMS1) and 2 (hPMS2), in primary leukemic cells obtained from 11 patients with acute-type adult T-cell leukemia (ATL) by using reverse transcription-polymerase chain reaction (RT-PCR). In contrast to normal peripheral lymphocytes, all primary ATL samples had reduced or loss of expression of two or more MMR genes, and the expression of several MMR genes was simultaneously suppressed in each ATL patient. Abnormal expression of hMSH2, hMSH3, hMSH6, hMLH1, and hPMS1 was observed more frequently than that of hPMS2. In particular, expression of hMSH2 and hPMS1 was reduced in all cases. Western blot analysis further showed reduced expression of both hMSH2 and hPMS1 proteins in all five cases examined. In three out of the 5 cases, both of the two proteins were undetectable. Interestingly, methylation-specific PCR indicated methylation of hPMS1 promoter in all of four ATL cases examined. hPMS1 expression, but not hMSH2 expression, was restored by treatment with a DNA demethylation agent, 5-aza-2'-deoxycytidine, suggesting that methylation plays a crucial role in inhibition of the hPMS1 gene expression in ATL. Our results demonstrate that defect of both human MutS and human MutL systems in primary ATL cells.