Chronic Adult T-cell Leukemia Research Articles

Therapeutic advances for the management of adult T cell leukemia: Where do we stand?

Adult T cell leukemia (ATL) is an aggressive blood malignancy secondary to chronic infection with the human T cell leukemia virus type I (HTLV-1) retrovirus. ATL encompasses four subtypes (acute, lymphoma, chronic, and smoldering), which exhibit different clinical characteristics and respond differently to various treatment strategies. Yet, all four subtypes are characterized by a dismal long-term prognosis and a low survival rate. While antiretroviral therapy improves overall survival outcomes in smoldering and chronic subtypes, survival remains poor in lymphoma subtypes despite their good response to intensive chemotherapy. Nonetheless, acute ATL remains the most aggressive form associated with profound immunosuppression, chemo-resistance and dismal prognosis. Targeted therapies such as monoclonal antibodies, epigenetic therapies, and arsenic/IFN, emerged as promising therapeutic approaches in ATL. Allogeneic hematopoietic cell transplantation is the only potentially curative modality, alas applicable to only a small percentage of patients. The recent findings demonstrating the expression of the viral oncoprotein Tax in primary ATL cells from patients with acute or chronic ATL, albeit at low levels, and their dependence on continuous Tax expression for their survival, position ATL as a virus-addicted leukemia and validates the rationale of anti-viral treatment strategies. This review provides a comprehensive overview on conventional, anti-viral and targeted therapies of ATL, with emphasis on Tax-targeted therapied in the pre-clinical and clinical settings.

A protective role of HTLV-1 gp46-specific neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies in progression to adult T-cell leukemia (ATL).

Human T-cell leukemia virus type-1 (HTLV-1) establishes a long-term persistent infection in humans and causes malignant T-cell leukemia, adult T-cell leukemia (ATL). HTLV-1-specific cytotoxic T lymphocytes have been suggested to play a major role in the immunosurveillance of HTLV-1-infected T cells. However, it remains unclear whether HTLV-1-specific functional antibodies are also involved in the host defense. To explore the role of antibodies in the course of HTLV-1 infection, we quantitated HTLV-1-specific neutralizing and antibody-dependent cellular cytotoxicity (ADCC)-inducing antibody levels in plasma from asymptomatic carriers (ACs) and ATL patients. The levels of neutralizing antibodies, as determined by a syncytium inhibition assay, were significantly lower in acute and chronic ATL patients than in ACs. The levels of ADCC-inducing activity were tested using an autologous pair of HTLV-1-producing cells and cultured natural killer (NK) cells, which showed that the ADCC-inducing activity of IgG at a concentration of 100 µg/ml was comparable between ACs and acute ATL patients. The anti-gp46 antibody IgG levels, determined by ELISA, correlated with those of the neutralizing and ADCC-inducing antibodies. In contrast, the proviral loads did not correlate with any of these antibody levels. NK cells and a monoclonal anti-gp46 antibody reduced the number of HTLV-1 Tax-expressing cells in cultured peripheral blood mononuclear cells from patients with aggressive ATL. These results suggest a protective role for HTLV-1 neutralizing and ADCC-inducing antibodies during the course of HTLV-1 infection.

Arsenic Trioxide and Interferon-Alpha Eradicate Adult T Cell Leukemia Initiating Cells through PML/p53 Senescence and Activation of Innate Immunity

Adult T cell leukemia (ATL) is an aggressive chemo-resistant T cell malignancy secondary to chronic HTLV-I infection. The viral oncoprotein Tax initiates ATL in transgenic mice. We previously showed that arsenic trioxide (As) and interferon-α (IFN) combination, known to trigger proteasome mediated Tax degradation and apoptosis in ATL cells, cures murine ATL through clearance of leukemia initiating cells (LIC). We also showed that the triple combination of As, IFN and zidovudine is highly effective in the treatment of chronic ATL patients, inducing a shift from an immunosuppressive Treg/Th2 profile at diagnosis (high IL-10; low IL2; low IFN-γ) towards a Th-1 immunocompetent profile (high IFN-γ; high IL2; low IL-10).To dissect the molecular basis of As/IFN-induced ATL LIC eradication, primary ATL SCID mice were treated with As/IFN for 3 days. Spleen derived ATL cells were injected into secondary recipient SCID mice left untreated and sacrificed on a weekly basis. Whereas, secondary mice derived from untreated primaries succumbed from ATL between 3 and 4 weeks after transplantation, we observed a biphasic growth intersected by a spontaneous regression phase in untreated secondary mice transplanted from As/IFN treated primary mice. This process was associated with telomere shortening, DNA damage, activation of the PML/P53 axis, and senescence in tumor cells. Interestingly, injection of spleen derived ATL cells from secondary SCID sacrificed before ATL senescence, into untreated tertiary and quaternary SCID, resulted in normal ATL development, indicating that senescence of ATL cells in secondary SCID mice resulted in a change of the microenvironment that culminated in ATL cell apoptosis and loss of ATL LIC activity. Furthermore, injection of spleen derived ATL cells from untreated secondary SCID sacrificed before or after ATL senescence, into untreated tertiary NSG mice, resulted in normal ATL development. These NSG mice, contrary to SCID mice, lack natural killer (NK) cells and have an impaired macrophage activity. In agreement with these results, injection of spleen derived ATL cells from treated primary SCID into untreated secondary NSG mice, resulted in normal ATL development. RT-PCR and ELISA analysis of sorted CD25 positive (ATL) or negative (microenvironment) cells revealed treatment-induced upregulation of MIP1, RANTES, and IFN-γ in the CD25 negative fraction in untreated secondary SCID, following ATL senescence, and concomitant with apoptosis and loss of LIC activity. Conversely, and as previously seen in treated ATL patients, IL-10 expression was rapidly and dramatically downregulated in the CD25 positive fraction in untreated secondary SCID mice. Addition of the proteasome inhibitor PS-341 to AS/IFN treatment of primary mice reverted all above results, presumably indicating the role of As/IFN-induced Tax degradation in the observed phenotypes.Collectively, these results demonstrate that As/IFN-induced, PML/p53 dependent, senescence of ATL cells resulted in activation of the innate immunity that culminated in apoptosis and loss of ATL LIC activity. The striking similarity of these results with patients' data suggests a critical role for innate immunity in ATL cure. DisclosuresHermine:Novartis: Research Funding; Hybrigenics: Research Funding; Celgene: Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; INatherys: Equity Ownership, Research Funding.

Germinal epimutation of Fragile Histidine Triad (FHIT) gene is associated with progression to acute and chronic adult T-cell leukemia diseases

BackgroundHuman T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation.MethodsHere, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples.ResultsWe found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients’ samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation.ConclusionsThese results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current “wait and see approach” in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.

Short‐term cultured autologous peripheral blood mononuclear cells as a potential immunogen to activate Tax‐specific CTL response in adult T‐cell leukemia patients

Activation of CD8+ Tax‐specific CTL is a new therapeutic concept for adult T‐cell leukemia (ATL) caused by HTLV‐1. A recent clinical study of the dendritic cell vaccine pulsed with Tax peptides corresponding to CTL epitopes showed promising outcomes in ATL patients possessing limited human leukocyte antigen (HLA) alleles. In this study, we aimed to develop another immunotherapy to activate Tax‐specific CTL without HLA limitation by using patients’ own HTLV‐1‐infected cells as a vaccine. To examine the potential of HTLV‐1‐infected T‐cells to activate CTL via antigen presenting cells, we established a unique co–culture system. We demonstrated that mitomycin C‐treated HLA‐A2‐negative HTLV‐1‐infected T‐cell lines or short‐term cultured peripheral blood mononuclear cells (PBMC) derived from ATL patients induced cross–presentation of Tax antigen in co–cultured HLA‐A2‐positive antigen presenting cells, resulting in activation of HLA‐A2‐restricted CD8+ Tax‐specific CTL. This effect was not inhibited by a reverse transcriptase inhibitor. IL‐12 production and CD86 expression were also induced in antigen presenting cells co–cultured with HTLV‐1‐infected cells at various levels, which were improved by pre–treatment of the infected cells with histone deacetylase inhibitors. Furthermore, monocyte‐derived dendritic cells induced from PBMC of a chronic ATL patient produced IL‐12 and expressed enhanced levels of CD86 when co–cultured with autologous lymphocytes that had been isolated from the same PBMC and cultured for several days. These findings suggest that short‐term cultured autologous PBMC from ATL patients could potentially serve as a vaccine to evoke Tax‐specific CTL responses.

FHIT as a biomarker for early screening of adult T-cell leukemia.

Adult T-cell leukemia (ATL) is an incurable leukemia deriving from human T-cell leukemia virus (HTLV-I) infected cells. In our most recent study, we discovered that methylation of the tumor suppressor, fragile histidine triad gene (FHIT), exists in the majority of acute and chronic ATL patients. Methylation was seen in non-tumorigenic cells, in cells with low levels of HTLV-I integrated DNA, in longitudinal samples from HTLV-I carriers, in a percentage of HTLV-I carriers, and in direct descendants of ATL patients. Overall, this suggests that FHIT methylation is likely present in patients, prior to HTLV-I infection, and predisposes HTLV-I carriers to ATL development. In this commentary we discuss the importance of developing diagnostic tools for the early detection of FHIT methylation and the possibility that prior FHIT methylation may predispose any individual to the development of cancer.

A-109 A novel multi-cytokine inhibitory strategy in treating HTLV-1 diseases

Human T-cell leukemia virus-I (HTLV-1) is the most oncogenic human virus and arguably the most carcinogenic agent to humans (Gallo, Willems and Tagaya, 2017, Martin et al, 2018). Nevertheless, HTLV-1 is not receiving the deserving attention. We have been advocating for HTLV-1 to enhance the visibility of its research and to make the public aware of its biological and social importance for the past few years as a part of HTLV-1 task force of Global Virus Network (GVN), and we believe we are seeing some promising changes. Despite nearly 40 years of extensive research on the mechanisms by which HTLV-1 causes multiple human diseases including adult T-cell leukemia (ATL), HAM/TSP (HTLV-1 associate myelopathy/Tropical Spastic Paraparesis), and immunosuppressive condition leading to bacterial infections, no reliable cures are available for these diseases. HTLV-1 infection to CD4 T-cells enhances the production of multiple cytokines by CD4 T-cells through the transactivating nature of virus genes such as Tax. This causes a by-stander activation of CD8 T-cells and turn them into inflammatory cells, which contributes to worsening the disease burden in HTLV-1 diseases. For example, the damages to spinal cord cells in HAM/TSP is caused by the inflammatory CD8 T-cells. We have shown that such activation of CD8 T-cells is mainly caused by the cooperation of IL-2 and IL-15, 2 sibling cytokines belonging to the common gamma (γc)-family (Nata et al 2015, Massoud et al 2015) and that only the co-inhibition of these 2 cytokines, but not the inhibition of either of them, effectively suppresses the activation of CD8 T-cells isolated from HAM/TSP patients in an ex vivo assay system. The co-inhibition of 2 cytokines is a clinical challenge because currently available modalities do not allow to accomplish the goal without adverse effects. For example, monoclonal antibodies need to be combined, but is infeasible in the clinic from the viewpoint of cost. Bispecific antibodies do not exist to co-inhibit these 2 cytokines. Signal inhibitors such as Jak kinase inhibitors will have profound side effects by broadly blocking non-γc cytokines such as Erythropoietin and Thrombopoietin and causing severe anemia and bleeding tendency. Even specific JAk3 inhibitors, if available, will cause immunodeficiency by blocking IL-7 and significantly reducing the number of T-cells when used chronically. We thus developed a novel technology to develop inhibitors which block only desired members of the γc-family cytokines. We noted that all γc-tyokines (eg, IL-2, -4, -7, -9-, 15, and −21) use their D-helices to interact with the shared γc-receptor subunit. Accordingly, we found that the D-helices from 6 γc-cytokine show modest conservation in their primary structure. We postulated that a short peptide mimicking these D-helices might inhibit multiple γc-cytokines by competing with them in the binding to the γc, and that leveraging the conserved and non-conserved positions in this region across all γc-cytokines might allow us to synthesize a specific inhibitor to a given choice of γc-cytokines. We in fact successfully synthesized several peptides showing inhibition to only selected γc-cytokines, but not all γc-cytokines with different target spectrums. Among them is the BNZ-1 peptide which blocks IL-2, IL-15 and IL-9. In an ex vivo assay using T-cells derived from HAM/TSP patients, BNZ-1 effectively blocked the proliferation and activation of HAM/TSP T-cells whereas mAb for IL-2 or IL-15 only showed marginal inhibition, suggesting that this compound represents a novel therapeutic candidate for HAM/TSP. It is likely that BNZ-1 could treat chronic ATL as well since chronic ATL has been shown to pathogenically involve IL-2 and IL-15 produced by HTLV-1 infected CD4 T-cells. We have tested the safety, pharmacokinetics and pharmacodynamics of the PEGylated form of the BNZ-1 peptide (BNZ-1/PEG) in animal models (mouse and non-human primate) and in healthy human volunteers (Phase I study) and observed that it blocks IL-2 and IL-15 activity effectively in humans, and that a weekly or biweekly administration maintains the drug level above the minimum effective dose. BNZ-1/PEG has been approved as an IND for treating HAM/TSP and T-cell malignancies (CD8 type LGL-leuekmia/cutaneous T-cell Lymphoma (CTCL)). Currently we are conducting a phase II clinical trial involving LGL-leukemia/CTCL (see a poster presented by J Geh et al) and are aiming at starting a trial involving HAM/TSP in the near future.

Chronic adult T-cell Leukemia in a young male after blood transfusion as a newborn

Human T-cell Lymphotropic virus type 1 (HTLV-1) is the etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HTM/TSP). Areas of extremely high HTLV-1 prevalence are surrounded by areas of middle or very low prevalence. ATLL is an aggressive lymphoproliferative malignancy of peripheral T cells, with an incidence of less than 5% in HTLV-1-infected individuals. ATLL developed in the majority of cases in individuals who were infected with HTLV-1 by their mothers due to prolonged breastfeeding. In non-endemic areas, ATLL is usually limited to immigrants, their sexual partners and descendants from endemic regions. Very few cases of ATLL have been diagnosed in recipient patients few years after an organ transplantation or blood transfusion worldwide. Achieving an accurate and fast diagnosis of ATLL can be challenging due to the lack of professional experience, delayed consultation and difficulty in its sub-classification. We present a case of a delayed onset of a chronic ATLL in an 18-years-old male who was transfused with blood components as a premature newborn in Buenos Aires, a non-endemic city of South America.

Selective targeting of JAK/STAT signaling is potentiated by Bcl-xL blockade in IL-2-dependent adult T-cell leukemia

Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. The HTLV-1-encoded protein Tax up-regulates Bcl-xL expression and constitutively activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems resulting in amplified JAK/STAT signaling. Consequently, inhibition of JAK signaling reduces ex vivo proliferation of PBMCs from ATL patients in smoldering and chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in IL-2-dependent and IL-2-independent ATL cell lines. The highly selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen and the Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. An examination of the mechanistic underpinnings of this combination highlighted a stimulation of Bim and PUMA expression and reduced phosphorylation of BAD upon cellular exposure to ruxolitinib. The combination was noted to strongly activate BAX, effect mitochondrial depolarization and increase caspase 3/7 activity that leads to PARP and Mcl-1 cleavage. Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy while the combination dramatically lowered tumor burden and prolonged survival in an aggressive ATL murine model. Critically, this combination strongly blocked ex vivo proliferation of five ATL patients’ PBMCs. These studies provide support for a therapeutic trial in patients with smoldering and chronic ATL using a drug combination that inhibits JAK signaling and anti-apoptotic protein Bcl-xL.

Achievement of Complete Hematological Remission and Reversal of High HTLV-1 Viral Loads with Anti CCR-4 Monoclonal Antibody Administration in Afro Caribbean Patients with Relapsed Chronic Adult T Cell Leukaemia (ATLL)

IntroductionThe management of heavily pre-treated patients with relapsed ATLL is challenging. These patients display chemo-refractory disease with limited therapeutic options. The Kyowa Hakko Kirin protocol 0761-009 is a randomized controlled trial of anti-CCR4 monoclonal antibody therapy (mogamulizumab) compared with physician choice of salvage chemotherapy in relapsed ATLL. ATLL arises in HTLV-1 infected patients with high pre-morbid HTLV-1 proviral loads (>4–10 HTLV-1 DNA copies/ml)1.MethodsThree patients, female, age 38 - 72 years and of Afro Caribbean origin, with relapsed chronic ATLL, have been recruited to 0761-009 in London. All patients had received two or more lines of therapy: #1 zidovudine (ZDV)/Interferon-a (IFN), anti-CD25/bortezomib; #2 ZDV/INF, anti-CD25/bortezomib, etoposide, Gemcitabine/Oxaloplatin; #3 sodium valproate, ZDV/IFN, etoposide. Lymphocyte counts at study entry were 7.8 - 33.5 x 109/L with high HTLV-1 proviral loads (range 25.4 - 122.4 HTLV-1 DNA copies/100 PBMC). Anti-CCR4 mAb was administered at a dose of 1mg/kg over 1 hour, weekly for 4 weeks and every 2 weeks thereafter. Data were censored 17/06/2014.ResultsAll three patients achieved complete hematological remission (CR) after 1 – 2 infusions. Patient #1 remains on treatment and in CR day 397. Patient #2 received 3 treatment cycles with normalization of lymphocyte count and complete resolution of extensive cutaneous lesions (baseline SWAT= 93.6) but treatment was discontinued after 3 cycles due to, now resolved, drug toxicity. Patient #3 remains on treatment and in CR at day 139. In each case HTLV proviral load fell to 0.02% < 0.7% and have remained <1% for the duration of therapy.ConclusionsAlthough a small case series these data illustrate the efficacy of anti-CCR4 mAb in ATLL, particularly in the chronic form, despite prior treatment failure and support the data observed in the Japanese cohort where CR was achieved in patients with blood compartmental disease, particularly chronic ATLL2. However, the extent of reduction in HTLV-1 proviral load with anti-CCR4 was not observed previously in these patients (or others who achieved CR with zidovudine/interferon the first line treatment of chronic ATLL). The reduction in HTLV-1 proviral burden to <1% suggests not only clearance of circulating HTLV-1 infected malignant clones but additional depletion of HTLV-1 infected non-malignant clones. This will be confirmed by unique integration site analysis.Reference List(1) Hodson A, Laydon D, Bain BJ, Fields P, Taylor GP. Pre-morbid HTLV-1 proviral load, rather than percentage of abnormal lymphocytes, is associated with an increased risk of aggressive ATLL. Haematologica2013; 98(3):385-388.(2) Ishida T, Joh T, Uike N, Yamamoto K, Utsunomiya A, Yoshida S et al. Defucosylated anti-CCR4 monoclonal antibody (KW-0761) for relapsed adult T-cell leukemia-lymphoma: a multicenter phase II study. J Clin Oncol 2012; 30(8):837-842. DisclosuresNo relevant conflicts of interest to declare.

Impact of miR-155 and miR-126 as novel biomarkers on the assessment of disease progression and prognosis in adult T-cell leukemia

Objective: Micro RNAs (miRNAs) provide new insight in the development of cancer, but little is known about their clinical relevance as biomarkers in the assessment of diagnosis, classification, progression and prognosis of various cancers. To explore a potential novel biomarker, we examined the cellular and plasma miRNA profiles in adult T-cell leukemia (ATL) characterized by diverse clinical features. Methods and results: Using CD4-positive cells isolated from 2 non-infected healthy individuals, 3 chronic ATL patients and 3 acute ATL patients, cellular miRNAs were profiled by microarray. The microarray screened 5 miRNAs namely miR-155, let-7g, miR-126, miR-130a and let-7b because of the large difference in their expression in diseased vs. that of healthy controls. The expression levels of before 5 miRNAs re-quantified by reverse transcription quantifiable polymerase chain reaction (RT-qPCR) were not always accordant in cells and plasma. The high and low plasma levels of miR-155 and miR-126 changed with ATL stage. Conclusion: The present study revealed that there is a quantitative discrepancy between cellular and plasma miRNAs. The elevation of plasma miR-155 and the reduction in miR-126 correlated with poor prognosis, indicating their usefulness as a novel biomarker for the assessment of disease stage.

Functional impairment of Tax-specific but not cytomegalovirus-specific CD8+ T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8+ T-cells in various clinical status of HTLV-1 infection.ResultsBy using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8+ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8+ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8+ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8+ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8+ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8+ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8+ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8+ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8+ T-cells.ConclusionsThese findings indicated that Tax-specific CD8+ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8+ T-cells in early stages.

Functional impairment of Tax-specific but not CMV-specific CTLs in a minor population of asymptomatic HTLV-1-carriers

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T cell response. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) also showed impaired T cell responses against the major target antigen Tax. However, it is unclear whether the impaired HTLV-1-specific T cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, we investigated the function of Tax-specific cytotoxic T lymphocytes (CTLs) using tetramers consisting of major CTL epitopes and HLA-A0201, A2402, or A1101 in various HTLV-1-infected individuals possessing these HLAs. Tax-specific CTLs were detected in 33.3% (n=7/21), 100% (n=18/18), and 86.4% (n=19/22) of chronic ATL (cATL) patients, HAM/TSP patients, and AC, respectively. Tax-specific CTLs of all HAM/TSP patients tested proliferated well in culture and produced IFN-γ when stimulated with Tax peptides, while Tax-specific CTLs detected in cATL patients did not. In ACs, the Tax-specific CTL responses were retained in most cases. However, Tax-specific CTLs in one AC hardly produced IFN-γ and failed to proliferate or express a degranulation (CD107a) marker upon Tax peptide stimulation. In contrast, cytomegalovirus (CMV) pp65-specific CTLs in the same donor could be fully activated by pp65 peptide stimulation. These findings indicated that HTLV-1-specific T cell function is impaired in a limited population in an HTLV-1-specific manner during an asymptomatic stage.

Donor-derived adult T-cell leukaemia

In July, 2008, a 55-year-old woman was referred to us with a 2-month history of progressive aphasia after a febrile episode. She had been diagnosed with chronic adult T-cell leukaemia in June, 1999, and had had a nonmyeloablative peripheral-blood stem-cell transplant with conditioning of pentostatin and 2 Gy total body irradiation in March, 2002. The stem cells had been harvested from her brother, an asymptomatic carrier of human T-cell lymphotropic virus type 1 (HTLV-1). After treatment, she had complete remission. Ciclosporin was discontinued 6 months after the transplant ation because of graft-versus-host disease. Our patient had had no bacterial or viral infections. She had no skin lesions, lymphadenopathy, or splenomegaly. Her leucocyte count was 8·74×109/L with 2·5% adult T-cell leukaemia-like cells. The leukaemia was therefore con sidered to be in remission. CD3-positive cells in the peripheral blood were confi rmed as donor derived by XY-fl uorescence insitu hybridisation (XY-FISH). Serum IgG was 13·7 g/L, IgA was 2·82 g/L, and IgM was 1·16 g/L. Our patient was immunocompetent; absolute number of circulating cells positive for CD4 was 1·74×109/L, CD8 0·475×109/L, CD19 0·522×109/L, and CD56 0·326×109/L. Brain MRI showed hyperintense, non-enhancing lesions in the left temporoparietal lobe and right frontal lobe (fi gure A), which suggested encephalitis, progressive multiple leukoencephalopathy, demyelinating disorder, or lymphoma. Serum concentrations of calcium, lactate dehydro genase, soluble interleukin-2 receptor, and β-2 microglobulin were not raised. CSF examination, culture, and PCR, abdominal ultrasonography, endoscopy, and 18F-fl urodeoxyglucose (18F-FDG) PET were normal. Craniotomy and biopsy of the left temporal lobe was done in July, 2008. Histopathology showed perivascular accumulation and cerebral infi ltration by mononuclear tumour cells with large, indented nuclei (fi gure B). Tumour cells were positive for CD3, CD4, CD8, and CD45RO, but negative for B-cell markers (CD20 and CD79a), suggesting cerebral invasion by adult T-cell leukaemia cells (webappendix). XY-FISH showed an XY genotype of the tumour cells (fi gure C), suggesting that they had originated from the donated leucocytes. After whole brain irradiation and localised irradiation of the left temporoparietal lobe, our patient’s aphasia improved remarkably, and repeat MRI showed shrinkage of the brain lesions. In October, 2010, our patient was fully recovered. For patients with adult T-cell leukaemia, asymptomatic carriers of HTLV-1 have been widely considered acceptable as donors in allogeneic stem-cell transplantation. The immune system is thought to have an important role in leukaemia occurrence in HTLV-1-seropositive carriers. The only reported case of donor-derived adult T-cell leukaemia was in 2006, in an immunosuppressed recipient of a peripheral-blood stem-cell transplant. No cases of donorderived adult T-cell leukaemia have been reported in immunocompetent patients. The prognosis tends to be better for these patients than for those with CNS involvement subsequent to systemic adult T-cell leukaemia. Only two cases of isolated CNS involvement have been reported so far. The function of CD4-positive cells was not fully analysed, so there is a possibility of some functional defi cit in immune reconstitution. Our patient’s case suggests that the consent practices in patients who receive HTLV-1-positive cells should be re-evaluated.

Autocrine/paracrine cytokine stimulation of leukemic cell proliferation in smoldering and chronic adult T-cell leukemia

AbstractAdult T-cell leukemia (ATL), a heterogeneous disease, can be divided into smoldering, chronic, lymphoma, and acute types clinically. In addition to different clinical manifestations, different stages of ATL have different molecular signatures. Here, we demonstrated that smoldering/chronic ATL peripheral blood mononuclear cells spontaneously proliferated ex vivo in a cytokine (interleukin -12 [IL-12]/IL-9/IL-15)–dependent manner, while acute-type ATL peripheral blood mononuclear cells did not proliferate or proliferated independent of cytokines. Smoldering/chronic ATL cells produced IL-2 and IL-9 in 6-day ex vivo cultures. Interestingly, the addition of an anti–IL-2R-α monoclonal antibody profoundly inhibited IL-9 expression, suggesting optimal expression of IL-9 was dependent on IL-2 signaling in these patients. To determine whether there would be autonomous proliferation of ATL leukemic cells, we purified leukemic cells from patients with smoldering/chronic ATL. Purified leukemic cells cultured alone produced IL-2/IL-9, and the downstream Janus kinase/signal transducer and activator of transcription pathway was activated. However, the leukemic cells did not proliferate independently, but required coculture with autologous monocytes to induce proliferation. Moreover, interaction between leukemic cells and monocytes was contact dependent, and major histocompatibility complex class II expression may have contributed to this interaction. In conclusion, our data provide evidence that there is autocrine/paracrine cytokine stimulation of leukemic cell proliferation in patients with smoldering/chronic ATL that could be targeted for treatment.

Adult T-Cell Leukemia/Lymphoma with CLL-Like Morphology—A Case Report

Adult T-cell Leukemia/Lymphoma (ATL) is rarely seen in the U.S. and Europe, usually limited to African Americans from the southeastern U.S. and immigrants from HTLV-1 endemic areas. Reaching an accurate and timely diagnosis of ATL in such nonendemic areas can be challenging, owing to limited exposure, diverse manifestations, and varying cell morphology. We present a case of chronic adult T-cell leukemia (ATL) with Chronic Lymphocytic Leukemia- (CLL-) like morphology that remained untreated for ten years and then developed treatment refractory acute ATL crisis.

Multiple osteolytic bone lesions with high serum levels of interleukin‐6 and CCL chemokines in a patient with adult T cell leukemia

A 37-year-old woman was diagnosed as having chronic adult T-cell leukemia (ATL) of the skin by a skin biopsy and human T-cell leukemia virus type-1 serology at our hospital in August 1992. The skin lesions of ATL were improved by treatment with psoralen ultraviolet ray A. She complained of severe pain in her bilateral forearms, hands and ankles, and X-ray examination in July 1999 revealed multiple punched-out lesions of the extremities. Serum levels of parathyroid hormone-related peptide, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha and total serum receptor activator of nuclear factor kappaB ligand were not elevated. However, serum levels of IL-6, CCL2 monocyte chemoattractant protein-1 (MCP-1), CCL3 [macrophage inflammatory protein-1alpha (MIP-1alpha)] and CCL4 (MIP-1beta) were markedly elevated. Here, we have discussed the possible mechanism underlying the onset of the osteolytic lesions.

Zidovudine Blocks NF-κB activity in Vivo in Adult T-Cell Leukemia

Adult T-cell leukemia (ATL) is a lymphoid malignancy caused by the human T-cell leukemia virus type I (HTLV-I), which carries a poor prognosis. A hallmark of ATL is the high constitutive expression of NF-κB, which predominantly exerts an anti-apoptotic effect contributing to chemotherapy resistance. Many of the elegant studies about the pathogenesis of ATL have focused on Tax, a viral transactivator of NF-κB, using HTLV-I expressing cell lines and mouse models, however in primary tumors the virus remains latent and Tax is not detected. We and other investigators have demonstrated the clinical efficacy of Zidovudine (AZT) and interferon-alpha (IFNα) combination therapy in both chronic and acute ATL subtypes with some patients achieving clinical remission or stable disease for many years while on maintenance therapy. The exact mechanisms of these antiviral drugs in ATL remain unclear. In a recent analysis of primary ATL tumors, we implicated the expression of both c-Rel and the NF-κB target gene product IRF-4/MUM-1 in AZT/IFN resistant disease. We have recently opened to accrual a Phase II clinical trial titled Prospective Study of the Molecular Characteristics of Sensitive and Resistant Disease in Patients with HTLV-I Associated Adult T Cell Leukemia Treated with Zidovudine Plus Interferon alpha-2b, which includes the novel use of pegylated interferon-alpha and valproic acid (as HDAC inhibitor) in the maintenance phase as an attempt to eradicate residual ATL clones, which usually occurs after AZT and IFNα therapy even after longterm remission. Our goals are also to study the anti-tumor mechanisms of these drugs in ATL, and define molecular criteria for response. As part of the correlative studies in our Phase II trial, we have analyzed leukemic ATL cells collected from patients during the first 48 hours of treatment (AZT given alone prior to IFN) and found in vivo stabilization of IκB (the repressor protein of NF-κB) by Western Blot in patients responding to the treatment, suggesting a role for this antiviral drug in blocking NF-κB activity as previously hypothesized in our laboratory. We also examined the expression of NF-κB related genes using a custom designed gene expression array by a novel technology (NanoString Inc.) of selected NF-κB target genes and found downregulation of most these genes in vivo by AZT alone. So far, all ATL tumors analyzed exhibited high expression of many NF-κB target genes, and over forty of these are differentially overexpressed in ATL specimens as compared to normal CD4+ T-cells. Some the differentially expressed genes include those encoding NF-κB/Rel, interferon regulatory factor (IRF), and bcl-2 related proteins. A comprehensive analysis of over forty ATL tumors, including specimens collected in both Miami and Brazil, is ongoing and expected to be completed soon. Baseline tumor characteristics and prognostic variables of previously collected tumors, as well interim results of our clinical and molecular studies will be reported.

Promoter methylation of the bone morphogenetic protein‐6 gene in association with adult T‐cell leukemia

Bone morphogenetic proteins (BMP), belonging to the transforming growth factor-beta superfamily, are multifunctional regulators of cell proliferation, differentiation and apoptosis in various types of malignant cells. In this study, we investigated BMP-6 promoter methylation in patients with various types of leukemias. The BMP-6 methylation was found preferentially in adult T-cell leukemia (ATL) (49 of 60, 82%) compared with other types of leukemias studied including acute myeloid leukemia (3 of 67, 5%), acute lymphoblastic leukemia (6 of 38, 16%) and chronic lymphocytic leukemia (1 of 21, 5%). Among subtypes of ATL, the BMP-6 gene was more frequently methylated in aggressive ATL forms of acute (96%) and lymphoma (94%) types than less malignant chronic ATL (44%) and smoldering ATL (20%). We also analyzed the methylation status of peripheral blood mononuclear cells from healthy donors and nonmalignant lymph nodes with reactive lymphadenopathy, none of which showed detectable BMP-6 methylation in this study. The BMP-6 methyaltion was correlated with decreased mRNA transcript and protein expression. Expression of BMP-6 was restored by the demethylating agent 5-aza-2'-deoxycytidine, suggesting that methylation was associated with the transcriptional silencing. Serial analysis demonstrated an increasing methylation of CpG sites in the BMP-6 promoter and the resultant suppression of BMP-6 expression as ATL progressed. These findings suggested that BMP-6 promoter methylation is likely to be a common epigenetic event at later stages of ATL and that the methylation profiles may be useful for the staging of ATL as well as for evaluation of the individual risk of developing the disease.

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.