A Comprehensive Study of the Immunophenotype and its Clinicopathologic Significance in Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell tumor caused by human T-lymphotropic virus type 1 (HTLV-1). The typical ATLL immunophenotypes are described in the 2017 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues (positive: CD2, CD3, CD5, CD4, and CD25; negative: CD7, CD8, and cytotoxic markers; and partially positive: CD30, CCR4, and FOXP3). However, limited studies are available on the expression of these markers, and their mutual relationship remains unknown. Furthermore, the expression status of novel markers associated with T-cell lymphomas, including Th1 markers (T-bet and CXCR3), Th2 markers (GATA3 and CCR4), T follicular helper markers (BCL6, PD1, and ICOS), and T-cell receptor (TCR) markers, and their clinicopathologic significance is unclear. In this study, we performed >20 immunohistochemical stains in 117 ATLL cases to determine the comprehensive immunophenotypic profile of ATLL, which were compared on the basis of clinicopathologic factors, including morphologic variants (pleomorphic vs anaplastic), biopsy locations, treatments, Shimoyama classification-based clinical subtype, and overall survival. CD3+/CD4+/CD25+/CCR4+ was considered a typical immunophenotype of ATLL, but approximately 20% of cases did not conform to this pattern. Simultaneously, the following new findings were obtained: (1) most cases were negative for TCR-ß and TCR-δ (104 cases, 88.9%), indicating the usefulness of negative conversion of TCR expression to provide differentiation from other T-cell tumors; (2) the positivity of CD30 and CD15 and the negativity of FOXP3 and CD3 were significantly associated with anaplastic morphology; and (3) atypical cases, such as T follicular helper marker-positive (12 cases, 10.3%) and cytotoxic molecule-positive cases (3 cases, 2.6%), were identified. No single markers could predict the overall survival among patients with acute/lymphoma subtypes of ATLL. The results of this study illustrate the diversity of ATLL phenotypes. In T-cell tumors occurring in HTLV-1 carriers, the possibility of ATLL should not be eliminated even when the tumor exhibits an atypical phenotype, and the confirmation of HTLV-1 in the tissue is recommended.

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.

Elevation of the Plasma Levels of TNF Receptor 2 in Association with Those of CD25, OX40, and IL-10 and HTLV-1 Proviral Load in Acute Adult T-Cell Leukemia.

Adult T-cell leukemia/lymphoma (ATL) cells express TNF receptor type-2 (TNFR2) on their surface and shed its soluble form (sTNFR2). We previously reported that sTNFR2 levels were highly elevated in the plasma of patients with acute ATL. To investigate whether its quantitation would be helpful for the diagnosis or prediction of the onset of acute ATL, we examined the plasma levels of sTNFR2 in a large number of specimens obtained from a cohort of ATL patients and asymptomatic human T-cell leukemia virus type 1 (HTLV-1) carriers (ACs) and compared them to those of other candidate ATL biomarkers (sCD25, sOX40, and IL-10) by enzyme-linked immunosorbent assays (ELISA) and HTLV-1 proviral loads. We observed that sTNFR2 levels were significantly elevated in acute ATL patients compared to ACs and patients with other types of ATL (chronic, smoldering, and lymphoma). Importantly, sTNFR2 levels were significantly correlated with those of sCD25, sOX40, and IL-10, as well as proviral loads. Thus, the present study confirmed that an increase in plasma sTNFR2 levels is a biomarker for the diagnosis of acute ATL. Examination of plasma sTNFR2 alone or in combination with other ATL biomarkers may be helpful for the diagnosis of acute ATL.

Clinicopathological features of adult T-cell leukemia/lymphoma with HTLV-1-infected Hodgkin and Reed-Sternberg-like cells.

Hodgkin and Reed-Sternberg (HRS) cells, a hallmark of classic Hodgkin lymphoma (CHL), are occasionally detected in non-Hodgkin lymphomas, including adult T-cell leukemia/lymphoma (ATLL), a lymphoid neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). HRS-like cells associated with ATLL have been described to be of B-cell lineage and infected with Epstein-Barr virus (EBV), not HTLV-1. We herein describe clinicopathological findings in 8 cases (4 males and 4 females; median age, 73 years [range, 55-81 years]) of ATLL with HTLV-1-infected HRS-like cells identified by ultrasensitive RNA in situ hybridization for HTLV-1 basic leucine zipper factor (HBZ-ISH), a specific viral transcript of HTLV-1. All patients showed nodal or mediastinal lesions, and 5 of the 8 patients were at an advanced disease stage. HRS-like cells were positive for CD30, CD15, MUM1, CD25, and HBZ-ISH and negative for B-cell markers, including PAX5, pan-T-cell antigens, and EBV in all cases. Five cases were positive for CD4, and 6 cases were positive for fascin. HBZ was identified in both HRS-like cells and surrounding lymphoid cells in 1 case with an aggressive clinical course and only HRS-like cells in 7 cases, most of whom showed a clinical response regardless of the chemotherapeutic regimen. Even though the definitive lineage typing of the HTLV-1-infected HRS cells is one of the limitations of this study in the absence of single-cell microdissection for polymerase chain reaction analysis, the combination of diffuse HBZ-ISH positivity and negativity for PAX5 and EBV deemed these cases distinct from CHL arising in HTLV-1 carriers.

Genetic profile of adult T-cell leukemia/lymphoma in Okinawa: Association with prognosis, ethnicity, and HTLV-1 strains.

Genetic alterations in adult T-cell leukemia/lymphoma (ATLL), a T-cell malignancy associated with HTLV-1, and their clinical impacts, especially from the perspective of viral strains, are not fully elucidated. We employed targeted next-generation sequencing and single nucleotide polymorphism array for 89 patients with ATLL in Okinawa, the southernmost islands in Japan, where the frequency of HTLV-1 tax subgroup-A (HTLV-1-taxA) is notably higher than that in mainland Japan, where most ATLL cases have HTLV-1-taxB, and compared the results with previously reported genomic landscapes of ATLL in mainland Japan and the USA. Okinawan patients exhibited similar mutation profiles to mainland Japanese patients, with frequent alterations in TCR/NF-ĸB (eg, PRKCB, PLCG1, and CARD11) and T-cell trafficking pathways (CCR4 and CCR7), in contrast with North American patients who exhibited a predominance of epigenome-associated gene mutations. Some mutations, especially GATA3 and RHOA, were detected more frequently in Okinawan patients than in mainland Japanese patients. Compared to HTLV-1-taxB, HTLV-1-taxA was significantly dominant in Okinawan patients with these mutations (GATA3, 34.1% vs 14.6%, P = .044; RHOA, 24.4% vs 6.3%, P = .032), suggesting the contribution of viral strains to these mutation frequencies. From a clinical viewpoint, we identified a significant negative impact of biallelic inactivation of PRDM1 (P = .027) in addition to the previously reported PRKCB mutations, indicating the importance of integrated genetic analysis. This study suggests that heterogeneous genetic abnormalities in ATLL depend on the viral strain as well as on the ethnic background. This warrants the need to develop therapeutic interventions considering regional characteristics.

A new diagnostic algorithm using biopsy specimens in adult T-cell leukemia/lymphoma: combination of RNA in situ hybridization and quantitative PCR for HTLV-1.

Histopathological distinction between adult T-cell leukemia/lymphoma (ATLL) and other T-cell neoplasms is often challenging. The current gold standard for the accurate diagnosis of ATLL is the Southern blot hybridization (SBH) assay, which detects clonal integration of human T-cell leukemia virus type I (HTLV-1) provirus. However, SBH cannot be performed with small biopsy or formalin-fixed paraffin-embedded (FFPE) tissue samples because this assay requires a large amount of DNA without degradation. Here we developed a new diagnostic algorithm for the accurate diagnosis of ATLL using FFPE samples. This method combines two HTLV-1 detection assays, namely, ultrasensitive RNA in situ hybridization using RNAscope for HTLV-1 bZIP factor (HBZ-RNAscope), and quantitative PCR targeting the tax gene (tax-qPCR). We analyzed 119 FFPE tissue specimens (62 ATLL, and 57 non-ATLL, including 41 HTLV-1 carriers) and compared them with the SBH results using the corresponding fresh-frozen samples. As a result, tax-qPCR had a higher ATLL identification rate than HBZ-RNAscope (88% [52/59], and 63% [39/62], respectively). However, HBZ-RNAscope clearly visualized the localization of HTLV-1-infected tumor cells and its identification rate increased to 94% (17/18) when the analysis was limited to samples up to 2 years old, indicating its usefulness in the daily diagnosis. The diagnostic algorithm combining these two assays successfully evaluated 94% (112/119) of samples and distinguished ATLL from non-ATLL cases including HTLV-1 carriers with 100% sensitivity and specificity. This method is expected to replace SBH and increase the accuracy of the diagnosis of ATLL.

Co-occurrence of EBV-positive classic Hodgkin lymphoma and B-cell lymphomas of different clonal origins: A case report and literature review.

Although cases with metachronous or synchronous co-occurrence of classic Hodgkin lymphoma (CHL) and B-cell non-Hodgkin lymphoma (B-NHL) have been reported, few reports have analyzed the clonal relationship between both lesions in detail, especially in Epstein-Barr virus (EBV)-positive settings. Here, we report a case of a 38-year-old male with CHL, followed by the recurrence of EBV-positive mucocutaneous ulcers of the large intestine and EBV-positive diffuse large B-cell lymphoma in the liver. Surprisingly, polymerase chain reaction analysis for immunoglobulin heavy chain gene rearrangement revealed that all lesions were clonally distinct. We further reviewed the literature on synchronous and metachronous co-occurrence of CHL and B-NHL in EBV-positive settings. In contrast to EBV-negative settings, all evaluable cases showed clonally distinct multiple lesions. These findings suggest that histologically and clonally distinct B-cells could simultaneously proliferate in EBV-associated settings, providing a new insight into the pathogenesis of EBV-associated lymphoproliferative disorders.

HLA loci predisposing to immune TTP in Japanese: potential role of the shared ADAMTS13 peptide bound to different HLA-DR.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. In white individuals, HLA allele DRB1*11 is a predisposing factor for iTTP, whereas DRB1*04 is a protective factor. However, the role of HLA in Asians is unclear. In this study, we analyzed 10 HLA loci using next-generation sequencing in 52 Japanese patients with iTTP, and the allele frequency in the iTTP group was compared with that in a Japanese control group. We identified the following HLA alleles as predisposing factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR], 3.06; corrected P [Pc] = .005), DRB3/4/5*blank (OR, 2.3; Pc = .007), DQA1*01:03 (OR, 2.25; Pc = .006), and DQB1*06:01 (OR,: 2.41; Pc = .003). The estimated haplotype consisting of these 4 alleles was significantly more frequent in the iTTP group than in the control group (30.8% vs 6.0%; Pc < .001). DRB1*15:01 and DRB5*01:01 were weak protective factors for iTTP (OR, 0.23; Pc = .076; and OR, 0.23, Pc = .034, respectively). On the other hand, DRB1*11 and DRB1*04 were not associated with iTTP in the Japanese. These findings indicated that predisposing and protective factors for iTTP differ between Japanese and white individuals. HLA-DR molecules encoded by DRB1*08:03 and DRB1*11:01 have different peptide-binding motifs, but interestingly, bound to the shared ADAMTS13 peptide in an in silico prediction model.

Proteomic profiling of HTLV-1 carriers and ATL patients reveals sTNFR2 as a novel diagnostic biomarker for acute ATL.

Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type 1 (HTLV-1)-associated T-cell malignancy with generally poor prognosis. Although only ∼5% of HTLV-1 carriers progress to ATL, early diagnosis is challenging because of the lack of ATL biomarkers. In this study, we analyzed blood plasma profiles of asymptomatic HTLV-1 carriers (ACs); untreated ATL patients, including acute, lymphoma, smoldering, and chronic types; and ATL patients in remission. Through SOMAscan, expression levels of 1305 plasma proteins were analyzed in 85 samples (AC, n = 40; ATL, n = 40; remission, n = 5). Using gene set enrichment analysis and gene ontology, overrepresented pathways in ATL vs AC included angiogenesis, inflammation by cytokines and chemokines, interleukin-6 (IL-6)/JAK/STAT3, and notch signaling. In selecting candidate biomarkers, we focused on soluble tumor necrosis factor 2 (sTNFR2) because of its active role in enriched pathways, extreme significance (Welch's t test P < .00001), high discrimination capacity (area under the curve >0.90), and novelty in ATL research. Quantification of sTNFR2 in 102 plasma samples (AC, n = 30; ATL, n = 68; remission, n = 4) using enzyme-linked immunosorbent assay showed remarkable elevations in acute ATL, at least 10 times those of AC samples, and return of sTNFR2 to AC state levels after achieving remission. Flow cytometry and immunostaining validated the expression of TNFR2 in ATL cells. No correlation between sIL-2 and sTNFR2 levels in acute ATL was found, suggesting the possibility of sTNFR2 as an independent biomarker. Our findings represent the first extensive blood-based proteomic analysis of ATL, suggesting the potential clinical utility of sTNFR2 in diagnosing acute ATL.

Phosphorylated STAT3 expression predicts better prognosis in smoldering type of adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm, and is divided into 2 indolent (smoldering and chronic) and 2 aggressive (acute and lymphoma) clinical subtypes. Based on previous integrated molecular analyses suggesting the importance of the JAK-STAT pathway in ATLL, we attempted to clarify the clinicopathological significance of this pathway. Clinical and morphological findings were reviewed in 116 cases with ATLL. The nuclear localizations of phosphorylated STAT3 (pSTAT3), pSTAT5, and pSTAT6 were analyzed by immunohistochemistry. Targeted sequencing was undertaken on the portion of STAT3 encoding the Src homology 2 domain. Expression of pSTAT3 was observed in 43% (50/116) of ATLL cases, whereas pSTAT5 and pSTAT6 were largely undetected. Cases with the lymphoma type showed significantly less frequent pSTAT3 expression (8/45, 18%) than those with the other subtypes (41/66, 62%; P < .001). STAT3 mutations were detected in 36% (10/28) and 19% (12/64) of cases with the smoldering and aggressive types of ATLL, respectively. The correlation between STAT3 mutation and pSTAT3 expression was not significant (P = .07). Both univariate and multivariate analysis revealed that pSTAT3 expression was significantly associated with better overall survival and progression-free survival in the smoldering type of ATLL, whereas STAT3 mutation was not related to a line of clinical outcome. Collectively, our data show that only the lymphoma type showed a low prevalence of tumor cells positive for pSTAT3 expression, and raises the possibility that pSTAT3 expression is a novel biomarker to predict better prognosis in the smoldering type of ATLL.

Evaluation of two prognostic indices for adult T-cell leukemia/lymphoma in the subtropical endemic area, Okinawa, Japan.

Aggressive adult T-cell leukemia/lymphoma (ATL) has an extremely poor prognosis and is hyperendemic in Okinawa, Japan. This study evaluated two prognostic indices (PIs) for aggressive ATL, the ATL-PI and Japan Clinical Oncology Group (JCOG)-PI, in a cohort from Okinawa. The PIs were originally developed using two different Japanese cohorts that included few patients from Okinawa. The endpoint was overall survival (OS). Multivariable Cox regression analyses in the cohort of 433 patients revealed that all seven factors for calculating each PI were statistically significant prognostic predictors. Three-year OS rates for ATL-PI were 35.9% (low-risk, n = 66), 10.4% (intermediate-risk, n = 256), and 1.6% (high-risk, n = 111), and those for JCOG-PI were 22.4% (moderate-risk, n = 176) and 5.3% (high-risk, n = 257). The JCOG-PI moderate-risk group included both the ATL-PI low- and intermediate-risk groups. ATL-PI more clearly identified the low-risk patient subgroup than JCOG-PI. To evaluate the external validity of the two PIs, we also assessed prognostic discriminability among 159 patients who loosely met the eligibility criteria of a previous clinical trial. Three-year OS rates for ATL-PI were 34.5% (low-risk, n = 42), 9.2% (intermediate-risk, n = 109), and 12.5% (high-risk, n = 8). Those for JCOG-PI were 22.4% (moderate-risk, n = 95) and 7.6% (high-risk, n = 64). The low-risk ATL-PI group had a better prognosis than the JCOG-PI moderate-risk group, suggesting that ATL-PI would be more useful than JCOG-PI for establishing and examining novel treatment strategies for ATL patients with a better prognosis. In addition, strongyloidiasis, previously suggested to be associated with ATL-related deaths in Okinawa, was not a prognostic factor in this study.

Characterization of patients with aggressive adult T-cell leukemia-lymphoma in Okinawa, Japan: a retrospective analysis of a large cohort.

Okinawa Prefecture, located in the subtropics, is an area of endemic adult T-cell leukemia-lymphoma (ATL) in Japan. We retrospectively analyzed 659 patients with aggressive ATL in seven institutions in Okinawa between 2002 and 2011. The median patient age was 68 years. More patients were aged ≥90 years (2.6 %), in this study, than in a nationwide survey (<1 %). The median survival time (MST) of the entire cohort was 6.5 months. Of the 217 patients who had a clinical status similar to that stated in the eligibility criteria of JCOG9801 (a randomized phase III study comparing VCAP-AMP-VECP with CHOP-14), 147 who received the CHOP regimen had a poorer MST than those in the CHOP-14 arm of JCOG9801 (8 vs 11 months). The prevalence of strongyloidiasis in the ATL patients was much higher (12.4 %) than in the historical cohort who visited the University of the Ryukyus Hospital (3.4 %). Furthermore, strongyloidiasis may be associated with ATL-related deaths. These findings suggest that, compared with other areas in Japan, in Okinawa, the proportion of patients aged ≥90 years with clinical features of aggressive ATL is higher, outcomes are poorer, and the disease is associated with a higher prevalence of strongyloidiasis.

Overexpression of caveolin-1 in adult T-cell leukemia.

Caveolin-1 is implicated in the regulation of signal pathways. Adult T-cell leukemia (ATL) is a T-cell malignancy causatively associated with human T-cell leukemia virus type 1 (HTLV-1). To determine the role of caveolin-1 in leukemogenesis, we examined caveolin-1 expression levels in HTLV-1-infected T-cell lines and ATL cells. These cells expressed high levels of caveolin-1 compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Caveolin-1-positive ATL cells were detected in ATL lymph nodes and skin lesions, and caveolin-1 was also detected in the plasma of patients with ATL. Infection of a human T-cell line, an epithelial cell line, and normal PBMCs with HTLV-1 induced caveolin-1 expression. The viral protein Tax transcriptionally activated caveolin-1 gene through nuclear factor-kappaB and cAMP response element binding protein signal pathways. HTLV-1-infected T-cell lines, and ATL cells are known to be resistant to transforming growth factor beta (TGF-beta)-induced growth inhibition. Caveolin-1 was colocalized with TGF-beta type I receptor in HTLV-1-infected T-cell lines and suppressed TGF-beta signaling. Caveolin-1 knockdown in an HTLV-1-infected T-cell line exhibited susceptibility to TGF-beta. Thus, we describe a new function for Tax, repression of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.

Aberrant expression of the transcription factor Twist in adult T-cell leukemia.

Adult T-cell leukemia (ATL) is a T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Twist, a highly conserved basic helix-loop-helix transcription factor, is a newly identified oncogene. However, there are no reports on Twist expression in ATL. To define the role of Twist in leukemogenesis of ATL, we examined its expression in T-cell lines and PBMC. HTLV-1-infected T-cell lines and ATL cells expressed high levels of Twist compared with uninfected T-cell lines and normal PBMC. Immunohistochemistry showed immunostaining for Twist in ATL cells in ATL lymph nodes and skin lesions. Infection of normal PBMC with HTLV-1 induced Twist expression. Induction of the viral protein Tax in a human T-cell line led to upregulation of Twist. Tax-induced Twist expression involved the NF-kappaB and CREB signaling pathways. Twist augmented Tax-mediated HTLV-1 LTR and NF-kappaB activation. Short interfering RNA against Twist inhibited cell growth of HTLV-1-infected T-cell lines and downregulation of Twist expression in an HTLV-1-infected T-cell line inhibited the expression of Akt1, interleukin-2 receptor alpha chain, and Tax as well as the known target genes of Twist, YB-1 and Akt2. In conclusion, the results suggest that Tax-induced induction of Twist contributes to leukemogenesis of ATL.

Overexpression of Aurora A by loss of CHFR gene expression increases the growth and survival of HTLV-1-infected T cells through enhanced NF-kappaB activity.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia (ATL). Aurora A, a mitotic checkpoint protein, is overexpressed in human cancer cells. The cell cycle-dependent turnover of Aurora A is regulated by E3 ubiquitin ligases such as checkpoint with fork head-associated and ring finger (CHFR). Here, we found overexpression of Aurora A protein in HTLV-1-infected T-cell lines and primary ATL cells. The expression of CHFR mRNA was reduced in these cells by abnormal methylation of CHFR promoter region. Knockdown of Aurora A using small interfering RNA suppressed the growth of HTLV-1-infected T-cell line. Transfection of Aurora A expression plasmid enhanced Tax-induced nuclear factor-kappaB (NF-kappaB) reporter activity. Transfection of CHFR expression plasmid into an HTLV-1-infected T-cell line reduced cell growth, Aurora A protein level and constitutive NF-kappaB reporter activity. Aurora kinase inhibitor suppressed the growth and survival of HTLV-1-infected T-cell lines and primary ATL cells. It also reduced constitutive NF-kappaB activity in an HTLV-1-infected T-cell line by reducing IkappaB kinase beta phosphorylation and the expression of antiapoptotic protein survivin. Our results suggested that loss of CHFR expression resulted to accumulation of Aurora A, which increased NF-kappaB activity. These findings highlight the critical role of Aurora A in HTLV-1-infected T cells, making this molecule a potentially suitable target for future therapies for ATL.

Anti-adult T-cell leukemia effects of a novel synthetic retinoid, Am80 (Tamibarotene).

Clinical trials for treatment of adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) using all-trans-retinoic acid (ATRA) have shown satisfactory therapeutic responses, although efficacies were limited. Recently, many synthetic retinoids have been developed and among them, a novel synthetic retinoid, Am80 (Tamibarotene) is an RARalpha- and RARbeta-specific retinoid expected to overcome ATRA resistance. The present study examined the inhibitory effects of Am80 on HTLV-I-infected T-cell lines and ATL cells. Am80 had negligible growth inhibition of peripheral blood mononuclear cells but marked growth inhibition of both HTLV-I-infected T-cell lines and ATL cells. Am80 arrested cells in the G1 phase of the cell cycle and induced apoptosis in HTLV-I-infected T-cell lines. It inhibited also the phosphorylation of IkappaBalpha and NF-kappaB-DNA binding, in conjunction with reduction of expression of proteins involved in the G1/S cell cycle transition and apoptosis. Am80 also inhibited the expression of JunD, resulting in suppression of AP-1-DNA binding. Furthermore, severe combined immunodeficient mice with tumors induced by subcutaneous inoculation of HTLV-I-infected T cells, responded to Am80 treatment with partial regression of tumors and no side-effects. These findings demonstrate that Am80 is a potential inhibitor of NF-kappaB and AP-1, and is a potentially useful therapeutic agent against ATL.

Anti-adult T-cell leukemia effects of brown algae fucoxanthin and its deacetylated product, fucoxanthinol.

Adult T-cell leukemia (ATL) is a fatal malignancy of T lymphocytes caused by human T-cell leukemia virus type 1 (HTLV-1) infection and remains incurable. Carotenoids are a family of natural pigments and have several biological functions. Among carotenoids, fucoxanthin is known to have antitumorigenic activity, but the precise mechanism of action is not elucidated. We evaluated the anti-ATL effects of fucoxanthin and its metabolite, fucoxanthinol. Both carotenoids inhibited cell viability of HTLV-1-infected T-cell lines and ATL cells, and fucoxanthinol was approximately twice more potent than fucoxanthin. In contrast, other carotenoids, beta-carotene and astaxanthin, had mild inhibitory effects on HTLV-1-infected T-cell lines. Importantly, uninfected cell lines and normal peripheral blood mononuclear cells were resistant to fucoxanthin and fucoxanthinol. Both carotenoids induced cell cycle arrest during G(1) phase by reducing the expression of cyclin D1, cyclin D2, CDK4 and CDK6, and inducing the expression of GADD45alpha, and induced apoptosis by reducing the expression of Bcl-2, XIAP, cIAP2 and survivin. The induced apoptosis was associated with activation of caspase-3, -8 and -9. Fucoxanthin and fucoxanthinol also suppressed IkappaBalpha phosphorylation and JunD expression, resulting in inactivation of nuclear factor-kappaB and activator protein-1. Mice with severe combined immunodeficiency harboring tumors induced by inoculation of HTLV-1-infected T cells responded to treatment with fucoxanthinol with suppression of tumor growth, showed extensive tissue distribution of fucoxanthinol, and the presence of therapeutically effective serum concentrations of fucoxanthinol. Our preclinical data suggest that fucoxanthin and fucoxanthinol could be potentially useful therapeutic agents for patients with ATL.

Activation of hypoxia-inducible factor 1 in human T-cell leukaemia virus type 1-infected cell lines and primary adult T-cell leukaemia cells.

HTLV-1 (human T-cell leukaemia virus type 1) is the causative agent for ATL (adult T-cell leukaemia). HTLV-1 Tax can activate the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, which is responsible for survival of HTLV-1-infected T-cells. HIFs (hypoxia-inducible factors) are transcriptional regulators that play a central role in the response to hypoxia. Overexpression of HIF-1alpha in many cancers is associated with a poor response to treatment and increased patient mortality. Our objectives in the present study were to investigate whether HIF-1 was activated in HTLV-1-infected T-cells and to elucidate the molecular mechanisms of HIF-1 activation by focusing on the PI3K/Akt signalling pathway. We detected a potent pathway that activated HIF-1 in the HTLV-1-infected T-cells under a normal oxygen concentration. Enhanced HIF-1alpha protein expression and HIF-1 DNA-binding activity were exhibited in HTLV-1-infected T-cell lines. Knockdown of HIF-1alpha by siRNA (small interfering RNA) suppressed the growth and VEGF (vascular endothelial growth factor) expression of the HTLV-1-infected T-cell line. HIF-1 protein accumulation and transcriptional activity were enhanced by Tax, which was inhibited by dominant-negative Akt. Importantly, mutant forms of Tax that are defective in activation of the PI3K/Akt pathway failed to induce HIF-1 transcriptional activity. The PI3K inhibitor LY294002 suppressed HIF-1alpha protein expression, HIF-1 DNA-binding and HIF-1 transcriptional activity in HTLV-1-infected T-cell lines. In primary ATL cells, HIF-1alpha protein levels were strongly correlated with levels of phosphorylated Akt. The results of the present study suggest that PI3K/Akt activation induced by Tax leads to activation of HIF-1. As HIF-1 plays a major role in tumour progression, it may represent a molecular target for the development of novel ATL therapeutics.

A modified version of galectin-9 suppresses cell growth and induces apoptosis of human T-cell leukemia virus type I-infected T-cell lines.

ATL is a fatal malignancy of T lymphocytes caused by HTLV-I infection and remains incurable. Galectins are a family of animal lectins that function both extracellularly (by interacting with cell surface and extracellular matrix glycoproteins and glycolipids) and intracellularly (by interacting with cytoplasmic and nuclear proteins) to modulate signaling pathways. We found that protease-resistant galectin-9 by modification of its linker peptide, hG9NC(null), prevented cell growth of HTLV-I-infected T-cell lines and primary ATL cells. The suppression of cell growth was inhibited by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for hG9NC(null)-induced cell growth suppression. hG9NC(null) induced cell cycle arrest by reducing the expression of cyclin D1, cyclin D2, cyclin B1, Cdk1, Cdk4, Cdk6, Cdc25C and c-Myc, and apoptosis by reducing the expression of XIAP, c-IAP2 and survivin. Most of these genes are regulated by NF-kappaB, which plays a critical role in oncogenesis by HTLV-I. hG9NC(null) suppressed IkappaBalpha phosphorylation, resulting in suppression of NF-kappaB. Most importantly, treatment with hG9NC(null) (6.7 mg/kg injected intraperitoneally every day) reduced tumor formation from an HTLV-I-infected T-cell line when these cells were inoculated subcutaneously into SCID mice. Our results suggest that hG9NC(null) could be a suitable agent for the management of ATL.