Adult T-cell Leukemia/lymphoma Patients Research Articles

HTLV-1 Hbz protein, but not hbz mRNA secondary structure, is critical for viral persistence and disease development.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic cause of adult T-cell leukemia/lymphoma (ATL) and encodes a viral oncoprotein (Hbz) that is consistently expressed in asymptomatic carriers and ATL patients, suggesting its importance in the development and maintenance of HTLV-1 leukemic cells. Our previous work found Hbz protein is dispensable for virus-mediated T-cell immortalization but enhances viral persistence. We and others have also shown that hbz mRNA promotes T-cell proliferation. In our current studies, we evaluated the role of hbz mRNA on HTLV-1-mediated immortalization in vitro as well as in vivo persistence and disease development. We generated mutant proviral clones to examine the individual contributions of hbz mRNA, hbz mRNA secondary structure (stem-loop), and Hbz protein. Wild-type (WT) and all mutant viruses produced virions and immortalized T-cells in vitro. Viral persistence and disease development were also evaluated in vivo by infection of a rabbit model and humanized immune system (HIS) mice, respectively. Proviral load and sense and antisense viral gene expression were significantly lower in rabbits infected with mutant viruses lacking Hbz protein compared to WT or virus with an altered hbz mRNA stem-loop (M3 mutant). HIS mice infected with Hbz protein-deficient viruses showed significantly increased survival times compared to animals infected with WT or M3 mutant virus. Altered hbz mRNA secondary structure, or loss of hbz mRNA or protein, has no significant effect on T-cell immortalization induced by HTLV-1 in vitro; however, the Hbz protein plays a critical role in establishing viral persistence and leukemogenesis in vivo.

Gene expression and TCR amino acid sequences selected by HLA-A02:01-restricted CTLs specific to HTLV-1 in ATL patients.

Adult T-cell leukaemia/lymphoma (ATL) is an aggressive malignancy of peripheral T cells caused by human T-cell lymphotropic virus type-1 (HTLV-1). Tax is the most important regulatory protein for HTLV-1. We aimed to reveal a unique amino acid sequence (AA) of complementarity-determining region 3 (CDR3) of the T-cell receptor (TCR)β and TCRα chains of HLA-A*02:01-restricted Tax11-19 -specific cytotoxic T cells (Tax-CTLs). The gene expression profiles (GEP) of Tax-CTLs were assessed by the next-generation sequence (NGS) method with SMARTer technology. Tax-CTLs seemed to be oligoclonal, and their gene compositions were skewed. The unique motifs of 'DSWGK' in TCRα and 'LAG' in TCRβ at CDR3 were observed in almost all patients. Tax-CTL clones harbouring the 'LAG' motif with BV28 had a higher binding score than those without either of them, besides a higher binding score associated with longer survival. Tax-CTLs established from a single cell showed killing activities against Tax-peptide-pulsed HLA-A2+ T2 cell lines. GEP of Tax-CTLs revealed that genes associated with immune response activity were well preserved in long-term survivors with stable status. These methods and results can help us better understand immunity against ATL, and should contribute to future studies on the clinical application of adoptive T-cell therapies.

Zidovudine and Interferon Alfa based regimens for the treatment of adult T-cell leukemia/lymphoma (ATLL): a systematic review and meta-analysis

BackgroundATLL (Adult T-Cell Leukemia/Lymphoma) is an aggressive hematological malignancy. This T-cell non-Hodgkin lymphoma, caused by the human T-cell leukemia virus type 1 (HTLV-1), is challenging to treat. There is no known treatment for ATLL as of yet. However, it is recommended to use Zidovudine and Interferon Alfa-based regimens (AZT/IFN), chemotherapy, and stem cell transplant. This study aims to review the outcome of patients with different subtypes of ATLL treated with Zidovudine and Interferon Alfa-based regimens.MethodsA systematic search was carried out for articles evaluating outcomes of ATLL treatment by AZT/IFN agents on human subjects from January 1, 2004, until July 1, 2022. Researchers assessed all studies regarding the topic, followed by extracting the data. A random-effects model was used in the meta-analyses.ResultsWe obtained fifteen articles on the AZT/IFN treatment of 1101 ATLL patients. The response rate of the AZT/IFN regimen yielded an OR of 67% [95% CI: 0.50; 0.80], a CR of 33% [95% CI: 0.24; 0.44], and a PR of 31% [95% CI: 0.24; 0.39] among individuals who received this regimen at any point during their treatment. Our subgroup analyses’ findings demonstrated that patients who received front-line and combined AZT/IFN therapy responded better than those who received AZT/IFN alone. It is significant to note that patients with indolent subtypes of disease had considerably higher response rates than individuals with aggressive disease.ConclusionIFN/AZT combined with chemotherapy regimens is an effective treatment for ATLL patients, and its use in the early stages of the disease may result in a greater response rate.

A contemporary national population-based analysis of demographics and outcomes of Hispanics with adult T-cell leukemia/lymphoma in the US.

e18771 Background: Adult T-cell Leukemia/Lymphoma (ATL) is a malignancy caused by the human T-cell leukemia virus type I with heterogenous clinical course (Blood Adv PMID29545256 J Clin Exp Hematop PMID34937829). There are endemic regions, but emigration has increased its incidence in the United States (US) (Hematol Oncol Clin North AmPMID2834087). There is a need to recognize trends and ethnic patterns to better understand this malignancy (J Oncol Pract PMID28796966 Blood Adv PMID29545256). This is the first national population-based analysis evaluating ethnic differences for HI diagnosed with ATL in the US. Methods: Data were analyzed on ATL patients in the US reported to the Surveillance, Epidemiology, and End Results (SEER) database between 2000-2018. Demographic, clinical and survival patterns were analyzed. Kaplan-Meier and Cox regression analyses were used when appropriate. Multivariate analysis and propensity score matching were performed with adjustment for age, stage and B-symptoms. Results: 598 patients (HI=65, NH=533) were diagnosed with ATL. Male sex predominated in in both. HI were diagnosed at a younger median age, 52 years (y) vs 63y for NH [p<0.001]. Most of HI presented in the age bracket of 40-60y, while NH were identified between 60-80y [p <0.001]. Regarding race, most of HI and NH were whites 82% vs 52% respectively, however, for NH Blacks and Asian/Pacific Islander were noted to be representative in the sample [p <0.001]. The majority of HI were diagnosed between 2010-2014, however for NH, same incidence was reported from 2005-2009 and 2010-2014 [p = 0.107]. For both group the presence of B symptoms was predominantly unknown. The median survival for HI was 1.5y vs 0.8y for NH. The survival probability at 2, 5 and 10y for HI corresponded to 0.474, 0.341 and 0.238, while for NH it was 0.347, 0.244 and 0.153, respectively. The OS probability at 10y had no statistically significant difference but a tendency favoring HI [p = 0.053]. On multivariate analysis, when adjusted for age, those patients who were older than 80y and between 60-80 y, had worse OS compared to those younger than 60y, with Hazard Ratio of 3.9 (95% CI: 2.7 – 5.6) and 1.7 (95% CI: 1.2 – 2.2), respectively. Conclusions: This national population-based study identified similar OS in HI diagnosed with ATL in the US. The median survival time was noted to be higher for HI and the OS had a tendency favoring this group; however, findings are not yet significant. This association should be further analyzed as there may be unique oncologic response in this ethnic minority. HI may have remarkable immunologic reaction to infection due to oncogenic viruses plus the creation of a distinct tumor microenvironment. Recognizing clinical trends and identifying genetic predictors may assist in constructing more accurate prognostic indices to deliver targeted therapy.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy caused by human T-cell leukemia virus type I (HTLV-1), endemic in some areas of the world, such as Brazil. Between 3% and 5% of HTLV-1-infected individuals develop ATL after a long latency. The clinical features are heterogeneous and classified into acute, lymphoma, chronic and smoldering types. Acute and lymphoma types are defined as aggressive whereas the others as indolent ATL. On April 2017, the T-cell Brazil project was launched. Methods: Ambispective observational study design collected 90 ATL patients from 606 registered cases in the T-Cell Brazil Project. Its main goal is to describe demographic and clinical features, analyze the overall and progression-free survival (OS and PFS), and try to identify factors that could influence them. REDcap Platform has been used to collect and store data, and the IBM-SPSS version 24 for statistical analysis. Results: The median age was 50 years (23–88), 59% female; 50% was lymphoma subtype, followed by 33% acute (both aggressive), 12% chronic and 5% smoldering (indolent); 87% had advanced stage disease (III-IV, Ann Arbor), 57% had B symptoms and 11% had Central Nervous System involved. Both indolent and aggressive had elevated serum LDH levels. 25% received chemotherapy plus immunotherapy, whilst 46% took chemotherapy alone with anthracycline-based regimens (46% CHOEP; 33% CHOP; 21% others). The best response (complete plus partial) after 1st line of treatment reached 50%; 31% had no response or progression; 13% was undetermined due early death; 5% were on treatment, and 1% had stable disease (without treatment). Median follow-up was ten months (0–78) and 18 months for 35% of alive cases. OS for 24 months was 34% (95% CI: 24%–44%), whereas 24-month PFS was 20% (95% CI: 12%–28%). Indolent presented better OS and PFS when compared with Aggressive; 86% versus 33%, P = 0.001 for OS and 45% versus 12%, P = 0.002 for PFS. Public medical care had worst OS and PFS than private (P = 0.14 and P = 0.05). The multivariate Cox Regression for OS resulted hypoalbuminemia - albumin <3.5 g/dL (HR 2.13 CI95% 1.10–4.12, P = 0.024) plus LDH twice the normal value (HR 2.49 CI95% 1.17–5.32, P = 0.018) as predictors. And for PFS were male (HR 1.83 CI95% 1.03–3.24, P = 0.037), ECOG > 1 (HR 2.01 CI95% 1.10–3.70, P = 0.023) and hypoalbuminemia (HR 3.35 CI95% 1.82–6.17, P < 0.0001). The research was funded by: Educational Support by Takeda Keyword: aggressive T-cell non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.

Mechanisms of Innate Immune Sensing of HTLV-1 and Viral Immune Evasion.

Human T lymphotropic virus-1 (HTLV-1) was the first identified oncoretrovirus, which infects and establishes a persistent infection in approximately 10-20 million people worldwide. Although only ~5% of infected individuals develop pathologies such as adult T-cell leukemia/lymphoma (ATLL) or a neuroinflammatory disorder termed HTLV-1-asssociated myelopathy/tropical spastic paraparesis (HAM/TSP), asymptomatic carriers are more susceptible to opportunistic infections. Furthermore, ATLL patients are severely immunosuppressed and prone to other malignancies and other infections. The HTLV-1 replication cycle provides ligands, mainly nucleic acids (RNA, RNA/DNA intermediates, ssDNA intermediates, and dsDNA), that are sensed by different pattern recognition receptors (PRRs) to trigger immune responses. However, the mechanisms of innate immune detection and immune responses to HTLV-1 infection are not well understood. In this review, we highlight the functional roles of different immune sensors in recognizing HTLV-1 infection in multiple cell types and the antiviral roles of host restriction factors in limiting persistent infection of HTLV-1. We also provide a comprehensive overview of intricate strategies employed by HTLV-1 to subvert the host innate immune response that may contribute to the development of HTLV-1-associated diseases. A more detailed understanding of HTLV-1-host pathogen interactions may inform novel strategies for HTLV-1 antivirals, vaccines, and treatments for ATLL or HAM/TSP.

Adult T-cell leukemia and lymphoma in French Guiana: a retrospective analysis with real-life data from 2009 to 2019

Adult T-cell leukemia/lymphoma (ATL), one of the most aggressive cancers in the world, occurs in 5% of the 10 million people living with HTLV-1 worldwide. French Guiana, a French overseas territory in South America, is one of the highest endemic areas of HTLV-1 worldwide. Here, we describe the demographic and clinical characteristics and outcome of ATL in this area. We retrospectively collected data from all patients diagnosed between 2009 and 2019. Patients were distributed according to Shimoyama's classification. Prognostic factors were explored through univariate analysis. Over the 10-year study period, 41 patients with a median age of 54 years at diagnosis were identified, among whom 56% were women. Sixteen (39%) patients were Maroons, a cultural group descendant of the runaway enslaved Africans from former Dutch Guiana. Among the study population, 23 (56%) had an acute type, 14 (34%) a lymphoma type, and one and one chronic and primary cutaneous tumour, respectively. First-lines of treatment included either chemotherapy or Zidovudine combined with pegylated interferon alpha. The 4-year overall survival was 11.4% for the entire population with 0% and 11% for lymphoma and acute forms, respectively. The median progression-free survival was 93 and 115 days for the acute and lymphoma groups (p=0.37), respectively. Among the twenty-nine patients who died, 8 (28%) died of toxicity, 7 (24%) died of disease progression and the cause of death remained unknown in 14 (48%) patients. Due to the overall poor prognosis, no significant prognostic factors could be identified. This study provides real-life data from ATL patients in French Guiana, a remote territory in a middle-income region. Patients, mostly Maroons, presented with a younger age and the prognosis was worse than expected compared to Japanese patients. None.

Outcome of Stem Cell Transplantation in HTLV-1-Associated North American Adult T-Cell Leukemia/Lymphoma

Adult T-cell leukemia/lymphoma (ATLL) remains challenging to treat and has dismal outcome. Allogeneic stem-cell transplantation (allo-SCT) has promising results, but data remain scarce. In this single-center retrospective analysis of 100 patients with ATLL from north America (67 acute, 22 lymphomatous), 17 underwent allo-SCT and 5 autologous SCT (ASCT), with a median follow-up of 65 months. Post-transplant 3-years relapse incidence (RI) and non-relapse mortality (NRM) were 51% and 37%, respectively, and 3-year progression-free survival (PFS) and overall survival (OS) were 31% and 35%, respectively. ASCT 1-year RI was 80% compared to 30% in allo-SCT (p = 0.03). After adjusting for immortal-time bias, allo-SCT had significantly improved OS (HR = 0.4, p = 0.01). In exploratory multivariate analysis, patients achieving first complete response and Karnofsky score ≥ 90 had significantly better outcomes, as did Black patients, compared to Hispanics, who had worse outcome. In transplanted patients, 14 died within 2 years, 4 of which ASCT recipients. Our data are the largest ATLL transplant cohort presented to date outside of Japan and Europe. We show that allo-SCT, but not ASCT, is a valid option in select ATLL patients, and can induce long term survival, with 40% of patients alive after more than 5 years.

A decrease in newly diagnosed patients with adult T-cell leukemia/lymphoma in Kagoshima, a highly endemic area of HTLV-1 in southwestern Japan

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy caused by human T-cell leukemia virus type-I (HTLV-1). This study investigated whether the number of newly diagnosed patients with ATL is decreasing in the background of a declining number of individuals infected by HTLV-1 in Kagoshima, Japan, one of the most endemic areas of HTLV-1 in the world. We retrospectively analyzed the number of newly diagnosed patients with ATL between January 2001 and December 2021 in three major hospitals. The number of newly diagnosed patients with B-cell non-Hodgkin lymphoma (B-NHL) in the same period was examined as an internal control. One thousand eighteen and 2,029 patients with ATL and B-NHL were registered, respectively. The age-adjusted incidence of ATL steadily increased between 2001 and 2012, whereas that between 2013 and 2021 decreased. Despite the limitation of its retrospective nature, this is the first report indicating a decrease in ATL patients in Japan.

The PRMT5 inhibitor EPZ015666 is effective against HTLV-1-transformed T-cell lines in vitro and in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) is the infectious cause of adult T-cell leukemia/lymphoma (ATL), an extremely aggressive and fatal malignancy of CD4+ T-cells. Due to the chemotherapy-resistance of ATL and the absence of long-term therapy regimens currently available for ATL patients, there is an urgent need to characterize novel therapeutic targets against this disease. Protein arginine methyltransferase 5 (PRMT5) is a type II PRMT enzyme that is directly involved in the pathogenesis of multiple different lymphomas through the transcriptional regulation of relevant oncogenes. Recently, our group identified that PRMT5 is overexpressed in HTLV-1-transformed T-cell lines, during the HTLV-1-mediated T-cell immortalization process, and in ATL patient samples. The objective of this study was to determine the importance of PRMT5 on HTLV-1 infected cell viability, T-cell transformation, and ultimately disease induction. Inhibition of PRMT5 enzymatic activity with a commercially available small molecule inhibitor (EPZ015666) resulted in selective in vitro toxicity of actively proliferating and transformed T-cells. EPZ015666-treatment resulted in a dose-dependent increase in apoptosis in HTLV-1-transformed and ATL-derived cell lines compared to uninfected Jurkat T-cells. Using a co-culture model of infection and immortalization, we found that EPZ015666 is capable of blocking HTLV-1-mediated T-cell immortalization in vitro, indicating that PRMT5 enzymatic activity is essential for the HTLV-1 T-cell transformation process. Administration of EPZ015666 in both NSG xenograft and HTLV-1-infected humanized immune system (HIS) mice significantly improved survival outcomes. The cumulative findings of this study demonstrate that the epigenetic regulator PRMT5 is critical for the survival, transformation, and pathogenesis of HTLV-1, illustrating the value of this cellular enzyme as a potential therapeutic target for the treatment of ATL.

The oncogenic driving force of CD30 signaling-induced chromosomal instability in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) develops via stepwise accumulation of gene mutations and chromosome aberrations. However, the molecular mechanisms underlying this tumorigenic process are poorly understood. We previously reported the presence of a biological link between the expression of CD30, which serves as a marker for ATL progression, and the actively proliferating fraction of human T-cell leukemia virus type 1 (HTLV-1)-infected cells that display polylobulation. Here, we demonstrated that CD30 signaling induced chromosomal instability with clonal expansion through DNA double-strand breaks (DSBs) via an increase of intracellular reactive oxygen species. CD30+ ATL cells were composed of subclones with additional genomic aberrations compared with CD30- ATL cells in ATL patients. Furthermore, we found an accumulation of copy number loss of DSB repair-related genes as the disease progressed. Taken together, CD30 expression on ATL cells appears to be correlated with genomic instability, suggesting that CD30 signaling is one of the oncogenic factors of ATL progression with clonal evolution. This study provides new insight into the biological roles of CD30 signaling and could improve our understanding of tumorigenic processes of HTLV-1-infected cells.

Tax-targeted dendritic cell vaccine therapy for long-term remission of adult T-cell leukemia-lymphoma

Adult T-cell leukemia-lymphoma (ATL) is a highly aggressive peripheral T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1) infection occurring in approximately 5% of patients after prolonged latent period. ATL relapses within a short period despite its transient response to multiagent chemotherapy and the prognosis is extremely poor due to anticancer drug resistance and immunodeficiency. Although novel agents with different mechanisms, such as molecular targeted agents, have improved the prognosis, the number of cured patients remains limited. Hematopoietic stem cell transplantation resulted in long-term remission, whereas its indication is limited due to treatment-related mortality. As most ATL patients are of advanced age, development of a lesser toxic treatment is necessary. Therefore, we developed a novel therapeutic dendritic cell vaccine targeting the HTLV-1 Tax antigen. The safety profile has been confirmed in a pilot and phase I clinical studies, and a promising long-term clinical efficacy has also been obtained. This novel vaccine is a noninvasive, long-lasting therapy for ATL and can potentially be extended to different applications for low-grade ATL and high-risk HTLV-1 carriers.

The clinical impact of the ratio of C-reactive protein to albumin (CAR) in patients with acute- and lymphoma-type adult T-cell leukemia-lymphoma (ATL).

Recently, the ratio of C-reactive protein to albumin (CAR) is used as an inflammatory marker that has been demonstrated to be a simple and reliable prognostic factor in solid tumors and hematological malignancy. However, no studies of the CAR have been performed in patients with adult T-cell leukemia-lymphoma (ATL). We retrospectively analyzed the clinical features and outcomes in 68 newly diagnosed acute- and lymphoma-type ATL [(acute-(n=42) or lymphoma-type (n=26)] patients in Miyazaki Prefecture from 2013 to 2017. Furthermore, we investigated correlations between pretreatment CAR levels and clinical features. The median age was 67 years (range, 44 - 87). Patients were initially treated by either palliative therapy (n=14) or chemotherapy [n=54; CHOP therapy (n=37)/ VCAP-AMP-VECP therapy (n=17)], and showed median survival durations of 0.5 months and 7.4 months, respectively. The factors affecting OS by multivariate analysis were age, BUN, and CAR. Importantly, we revealed that the high CAR group (optimal cut-off point; 0.553) was a significant indicator of worse OS by multivariate analysis (p< 0.001, HR; 5.46). The median survival of patients with a CAR< 0.553 was 8.37 months, while patients with a CAR>0.553 had a median survival of 3.94 months. The different clinical features between high CAR and low CAR groups were hypoproteinemia and the implementation of chemotherapy. Furthermore, in the chemotherapy group, but not the palliative therapy group, CAR was a significant prognostic marker. Our study indicated that CAR may be a new simple and significant independent prognostic marker in acute- and lymphoma-type ATL patients.

High Expression of Inflammatory Cytokines and Chemokines in Human T-lymphotropic Virus 1-Associated Adult T-cell Leukemia/Lymphoma

Background: Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) without any specific antiviral. Objectives: This study aimed to evaluate the expression level of inflammatory chemokines and pro-inflammatory cytokines in ATLL patients, asymptomatic carriers (ACs), and healthy individuals to assess the role of these inflammatory markers in ATLL pathogenicity. Methods: This study was conducted from May 2021 to August 2022. The ATLL blood samples were collected from the oncology wards of Imam Khomeini, Shariati, and Imam Hossein hospitals, in Tehran, Iran. The blood samples of ACs and normal control subjects were collected from blood donors referred to blood transfusion centers of Tehran and Alborz provinces, Iran. RNA extraction, complementary DNA (cDNA) synthesis, and real-time polymerase chain reaction (PCR) were done in targeted sample groups to investigate the correlation and expression rate of C-C motif chemokine ligand 3 (CCL3), C-C motif chemokine ligand 4 (CCL4), C-X-C motif chemokine ligand 8 (CXCL8), interleukin 23 subunit alpha (IL-23A), and interleukin 17 A (IL-17A). Results: A total of 30 samples were collected from 3 groups. The CCL3, CCL4, CXCL8, and IL-17A messenger RNA (mRNA) expression levels were significantly upregulated in the ATLL groups. There was a significant difference between CCL3 expression between the ACs and ATLL groups. In addition, CCL4 and CXCL8 expression levels were more significant in the ATLL group than in the normal control group. The IL-17A expression level significantly increased between groups. The IL-23A expression levels had no significant differences between the ATLL, ACs, and normal control groups. Conclusions: This study showed significant upregulation of pro-inflammatory cytokines and chemokines mRNAs in HTLV-1–associated ATLL compared to the ACs and normal control groups. Conducting more experiments to investigate the therapeutic effect of chemokines/cytokines in ATLL is essential.

T-Cell Receptor Amino Acid Sequence Selected By Cytotoxic T Cells Specific to HTLV-1 in HLA-A02:01-Positive ATL Patients

Purpose: Adult T cell leukemia/lymphoma (ATL) is an aggressive peripheral malignancy of T cells caused by human T cell lymphotropic virus type-1 (HTLV-1). Its prognosis remains very poor. Tax is the most important regulatory protein for HTLV-1, which is associated with the proliferation of host cells. Tax has been considered as an important target antigen for CD8+cytotoxic T cells (CTLs). We aimed to reveal such a unique sequence of complementarity-determining region 3 (CDR3) of the T cell receptor (TCR)-α and-β chain of HLA-A*02:01-restricted Tax11-19-specific CTLs (Tax-CTL). Methods: We serially analyzed Tax-CTLs from ATL patients with HLA-A*02:01 who had received allogeneic hematopoietic cell transplantation (all-HCT), chemotherapies alone, or had not ever received any treatment. Tax-CTLs were defined as a living CD3+CD8+Tax-tetramer+cells. After sorting Tax-CTLs directly from an actual patient sample into a PCR tube, TCR-α and -β chains of individual CTL clones were determined by the next-generation sequence (NGS) method with SMARTer technology. In addition, we cultured and established individual Tax-CTL clones from a sorted single cell, and finally evaluated the cytotoxicity of Tax-CTLs by calcein-acetoxymethyl (AM)-based cytotoxicity assay. Results: Peripheral blood samples were obtained from 7 patients (Pt-1,-2,-3,-4,-5,-6 and -7). Pt-1 had received allo-HCT and the samples were available 270 days, 1 year, 3 years, 5 years and 7 years after allo-HCT. Samples before treatment were available in the other 5 patient (acute ATL Pt-2,-3, and -6; chronic ATL, Pt-4; HTLV-1 asymptomatic charrier, Pt-5), while the samples after treatment were available in 1 patient (acute ATL Pt-7). Tax-specific CTLs were detected in 0.03-0.58% of total peripheral blood mononuclear cells, and 144-1000 cells per sample (median; 250 cells) were sorted and analyzed for TCR determination. Of them, 2 to 30 Tax-specific CTL clones were identified for each patient. Tax-specific CTLs seemed oligoclonal, and their usage of BV and BJ genes were restricted in TCR-BV28(30.5%), -BV6-5(33.3%), and -BJ2-1(30.5%), while their usage of AV and AJ genes were restricted in TCR-AV12-2(54.5%) and -AJ24(24.2%) gene families. The length of amino acids (AA) in TCR-α and TCR-β CDR3 of all Tax-CTLs clones seemed to be symmetrically distributed, and the most frequently observed AA length was 13 in TCR-α, and 15 in TCR-β. The unique motifs of "DSWGK" in TCR-α and "LAGG" in TCR-β at CDR3 were observed in almost all ATL patients. The 'DSWGK' motif was observed in 0-81.7% (median; 55.5%), and the 'LAGG' motif was observed in 5.2%-80.3% (median; 48%). Pt-2 didn't have any clones with 'DSWGK' motif in TCR-α, whereas regarding 'LAGG' motif in TCR-β, all patients had it in this study. We established Tax11-19-specific CTL clones from a single cell of Pt-4, which showed killing activities against Tax-peptide-pulsed HLA-A2+ T2 cell lines. Conclusion: These methods and results can help us to better understand immunity against ATL, leading to future studies on the clinical application of adoptive T-cell therapies in the future.

Allogeneic but Not Autologous Stem Cell Transplantation Improves Outcome in HTLV-1-Associated North American Adult T-Cell Leukemia/Lymphoma

Background: Adult T-cell leukemia/lymphoma (ATLL) is a rare mature T-cell malignancy driven by the human T-cell lymphotropic virus type I (HTLV-1) that remains challenging to treat and is associated with a dismal prognosis. While some reports have shown encouraging results with allogeneic stem-cell transplantation (allo-SCT), data remains scarce and mostly limited to Japanese patients. Patients and Methods: This is a retrospective single center analysis with 100 HTLV-1-associated North American ATLL patients. Variables collected included age, sex, ethnicity, CMV/HTLV1 serologies, karnofsky performance score (KPS), diagnosis date, disease subtype, number of prior therapy lines, transplant type, status at transplant, transplant date, prior SCT, donor sex, CMV/HTLV1 serologies, stem cell source, donor type, in-vivo T-cell depletion, engraftment, conditioning regimen, GVHD prophylaxis, maximum acute and chronic GVHD grade, relapse date when applicable, and main cause of death. The Kaplan-Meier method was used to calculate overall survival (OS) and progression-free survival (PFS). Cumulative incidence functions were used to estimate relapse incidence (RI) and non-relapse mortality (NRM) in a competing risk setting. Death and relapse were considered as competing events for acute and chronic GVHD. Univariate analyses were done using the Gray's test for cumulative incidence functions and the log rank test for OS and PFS. A Cox proportional hazards model was used for multivariate regression, results were expressed as hazard ratio (HR) with a 95% confidence interval (CI). All tests were two-sided, type-1 error was fixed at 5%. Results: One hundred patients met eligibility criteria, 18-87 years old, 69 of which were Black, 27 Hispanic, and 2 of Asian descent. Sixty-seven patients had acute ATLL and 22 lymphomatous. In total, 22 patients underwent SCT with 17 allo-SCT and 5 autologous SCT (ASCT) between 2005-2020 with a median follow-up of 62 months. Ten patients (45%) were transplanted in first complete remission (CR1), and 15 (68%) had KPS ≥90. Among patients undergoing allo-SCT, 41% had matched sibling donors (MSD), 18% matched unrelated (MUD), and 35% haploidentical. The 1-year and 3-years rates of overall survival from diagnosis of the cohort were 42% and 21%, respectively. Acute ATLL subtype was a strong predictor of worse outcome (Figure 1) with 1-year and 3-years OS of 32% vs 71% (p=0.004) and 11% vs 46% (p=0.002), respectively, compared to lymphomatous disease. To account for immortal time bias, 37 non-transplanted patients with early mortality within 4 months of diagnosis that were not able to make it to transplant were excluded from the Kaplan-Meier survival analysis. Patients undergoing transplant had improved outcomes compared to non-transplanted patients (Figure 1, Table 1), with allo-SCT reaching statistical significance in multivariate analysis compared to the non-transplant group with OS HR of 0.3 (p=0.004) compared to 0.7 (p=0.6) in ASCT vs non-transplant. In transplanted patients, the 3-year RI and NRM were 51% and 37%, respectively, and 3-year PFS and OS were 31% and 39%, respectively. Four of five patients who received ASCT rapidly relapsed with 3 dying from disease progression and one lost to follow-up, with 1-year RI of 80% vs 30% in allo-SCT (p=0.03). In exploratory multivariate analysis, patients achieving CR1 and with good baseline performance scores (KPS >90) had significantly better outcomes compared to their counterparts regardless of transplant type, and surprisingly, race was a strong and significant predictor of outcomes affecting PFS and OS with Hispanic vs Black PFS and OS HRs of 6 (p=0.04) and 10 (p=0.04), respectively. Thirteen patients died within 2 years, eight from relapse/progression including 3 ASCT patients, and 4 from transplant-related toxicities. Conclusion: Our data suggest that allo-SCT, but not ASCT, is a valid option in select ATLL patients. It also appears that patients of Hispanic descent have worse outcome post-transplant but not overall, which could be explained by lack of compatible donors or barriers to early transplant. Although this is the largest NA-ATLL transplant cohort presented to date outside of Japan and Europe, further studies are necessary to fully explain these discrepancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Highly Sensitive Detection Method Reveals Significant Anti-Tumor Activity of Valemetostat in Patients with Relapsed/Refractory Adult T Cell Leukemia

Background: Enhancer of zeste homolog 2 (EZH2) and its close homolog, EZH1, catalyze the attachment of 3 methyl groups to histone H3 at lysine 27 (H3K27me3). H3K27me3 is an epigenetic mark involved in downregulating gene expression associated with tumor suppression and cell differentiation. Adult T-cell leukemia/lymphoma (ATL) is an aggressive non-Hodgkin lymphoma (NHL) subtype that arises from CD4 positive T cells associated with human T-lymphotropic virus type 1 (HTLV-1) infection. During asymptomatic period, genetic and epigenetic abnormalities in the HTLV-1 infected cells accumulates and finally leads to ATL development. Current therapy for aggressive type ATL is still unsatisfactory and prognosis of relapsed and refractory (r/r) ATL after lenalidomide or mogamulizumab treatment is poor. Dysregulation of EZH2/PRC2-mediated epigenome status has been reported to be highly involved in tumorigenesis in ATL as well as other tumors such as B cell lymphoma. Valemetostat is a potent dual inhibitor of EZH1 and 2 which are key catalytic subunits of PRC2. The safety and efficacy of valemetostat in patients with r/r ATL were evaluated in a multicenter, single-arm, open-label phase 2 study (NCT04102150) and the results were presented at ASH 2021. Here we show clinical biomarker data using peripheral blood obtained over time, accompanying clinical outcomes. Also we discuss relevance of the measured data in blood to clinical outcomes among assay formats. Method: In the Ph2 study, valemetostat 200mg was orally administrated once daily in continuous 28-day cycles until disease progression or intolerance. Nine of 25 patients in the Ph2 study had abnormal lymphocytes in their peripheral blood. Peripheral blood from these 9 patients (7 acute and 2 unfavorable chronic) was drawn at pre-dosing and every 4-weeks for the analysis of this study. Abnormal lymphocytes (Ably) and soluble IL-2R (sIL-2R) are indicator of ATL prognosis. For HTLV-1 proviral load (PVL) detection, genomic DNA was extracted from PBMC from each patient. Droplet Digital PCR (ddPCR) was performed using probes specific for HTLV-1 provirus and RNase 5’ pyrophospgohydrolase (RPPH) gene. % PVL was determined from the copy number ratio of HTLV-1 to RPPH1 genes. For HTLV-1 Analysis System (HAS)-flow cytometry, frozen cell suspensions were thawed and stained with anti- CD3/4/7/14/CADM1 Abs. The data from live T cells was analyzed by CD7 versus CADM1 plot. CADM1+CD7- population ratio was denoted as %N. We analyzed changes of each measured parameter in every patient in comparison with clinical outcome. Result: In the 9 patients who met criteria in blood collection for blood biomarker evaluation at screening, 1 CR, 6 PRs and 1 SD were observed as the best response by blood assessment (not evaluable in one patient, who failed to meet criteria for blood assessment at pre-treatment evaluation). Significant decrease in Ably number and blood sIL-2R concentration was observed after 4-week valemetostat treatment in all 9 patients and this decrease was maintained during the treatment until PD. Valemetostat also decreased the %PVL and %N in HAS-flow. The reduction trends were weaker in comparison with Ably and sIL-2R. As for %PVL, most of the patients experienced modest reduction and sustained around 50%. Especially HAS-flow results exhibited two patient groups with either lower (<25%) or higher %N at pre-dosing. Two patients in the lower %N group showed CR or PR, and in overall response the remaining 7 patients with higher %N showed SD or PR. Conclusion: Valemetostat significantly reduced markers of ATL progression. Ably and sIL-2R to very low levels in all patients. In contrast, PVL assay did not exhibit complete %PVL reduction. HAS-flow showed variable %N at pre-dosing and the patients with higher %N exhibited SD or PR throughout the treatment in overall response and blood assessment. These data suggest that PVL assay and HAS-flow can detect ATL cells with a high resolution, which cannot be detected by Ably and sIL-2R, when valemetostat has significant therapeutic efficacy in ATL patients. Further analyses, such as gene mutation, transcription, chromatin state and histone methylation, using the collected blood samples will be performed next.

The Cause and Consequence of Replication Stress in Adult T-Cell Leukemia

EP300 and its homolog CBP are transcriptional co-activators of genes involved in a broad range of cellular processes, including proliferation, differentiation, apoptosis and DNA repair. Inactivating mutations in both EP300 and CBP have been reported in developmental disorders, such as Rubinstein-Taybi Syndrome, and several hematological malignancies. Most recently, somatic hypermutations in EP300 (but not CBP) were identified in Adult T-cell leukemia/lymphoma (ATLL) patients, diagnosed in Northern America. The mechanistic contribution of EP300 dysregulation to NA-ATLL etiology is currently unknown. ATLL is caused by infection by the Human T-cell Lymphotrophic Virus Type-1 (HTLV-1), that is endemic to Japan, the Caribbean basin, South America, southern parts of North America, Eastern Europe and certain areas of Africa. Unlike patients diagnosed in Japan (J-ATLL), NA-ATLL patient display a severe case of chemo-refractory disease with very poor prognosis. While prominent differences in the transcriptional and epigenetic landscape, particularly the p300 mutational status, has been identified between J-ATLL and NA-ATLL patients, the mechanistic contribution of p300 dysregulation to disease severity is yet to be established. Studies have revealed that the independent inhibition of EP300 in human cells results in a unique transcriptional landscape, involving the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Here, we show that EP300-mutated ATLL cells display a protracted S-phase, extensive genomic instability, dysregulations in DNA replication and repair dynamics. Using a powerful locus specific single molecule assay and genome-wide DNA fiber analysis, we show that perturbed DNA replication in EP300-mutated ATLL cells is characterized by elevated origin firing, increased replisome pausing and prominent nucleolytic degradation of nascent DNA at collapsed forks. The elevated origin firing in the absence of EP300 is likely driven by the inherent endogenous increase in c-MYC expression, which has been previously seen in EP300-mutated MCF7 cells. The striking replication pausing, fork collapse and nascent strand degradation observed in EP300-deficient NA-ATLL cells results in the persistence of single stranded DNA (ssDNA) genome-wide, resulting in the hyperphosphorylation of RPA at Ser4/8. Inhibition of Mre11 nuclease partially rescued the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Elevated endogenous genomic instability, observed in ATLL cells during the S-phase is exacerbated by EP300 deficiency, even in the absence of exogenous stress/damage. Analysis of the consequences of S-phase abnormalities and genomic instability revealed elevated micronuclei and cytosolic DNA in ATLL cells exposed to DNA damage. Preliminary studies evaluating the innate immune system activation (cGAS-STING-pIRF3) by immunoblotting, in response to stress, has led to some intriguing results. There is a clear separation in the ability of J-ATLL versus NA-ATLL cells in activating the innate immune system, a response that appears to be EP300 independent. These results raise the possibility of the contribution of a suppressed innate immune response, a phenomenon seen in many fast-progressing cancers, to the etiology of NA-ATLL. In addition to increasing our understanding of the mechanisms contributing to ATLL in general, the results from this study have helped identify new mechanism-based treatment regimens that could target the replicative and innate immune system vulnerabilities observed in North American ATLL patients.

The importance of the clinical classification of adult T-cell leukemia/lymphoma (ATLL) in the prognosis.

BackgroundAdult T-cell leukemia/lymphoma (ATLL), a peripheral T-cell leukemia/lymphoma associated with the human T-cell lymphotropic virus type-1 (HTLV-1), has been classified following the clinical forms defined by Shimoyama in 1991. A suggestion to modify Shimoyama’s classification was proposed in 2007 to differentiate within the smoldering patients those who presented nodules or tumors in the skin without lung involvement, which was named the primary cutaneous tumoral (PCT) form of ATLL. In the present study, according to their clinicopathological characteristics, we estimated the mortality rates of 143 ATLL patients from Bahia, Brazil. We also evaluated the importance of classifying PCT/ATLL separately from the smoldering type on disease prognosis.Methodology/Principal findingsDiagnosis of ATLL was established based on a positive serology for HTLV-1, histopathological and/or cytological diagnosis of peripheral T-cell leukemia/lymphoma. Patients were clinically grouped according to Shimoyama’s classification, considering PCT variants separately from the smoldering cases. Bivariate and multivariable survival analyses were applied to identify factors associated with disease prognosis. Significant differences in the median survival time were observed between the clinical types, with the smoldering type presenting the longest median survival (109 months) compared to the other forms (<50 months); the median survival for PCT/ATLL was 20 months. Multivariable analysis confirmed that ATLL clinical types were associated with survival, with a better prognosis for patients with the smoldering and chronic types. Furthermore, skin involvement was related to a worse outcome in the multivariable analysis, regardless of the clinical form and presence of lymphadenopathy.Conclusions/SignificanceOur results reinforce the importance of considering the PCT/ATLL separately from the smoldering type when classifying ATLL to better define prognosis and treatment, given the significant difference in the survival of patients between the smoldering form and PCT/ATLL. Skin involvement should also be considered an independent prognostic factor in patients with ATLL.

Live attenuated VZV vaccination induces antitumor immunity in ATLL patients

Adult T cell leukemia/lymphoma (ATLL) is a CD4-positive peripheral T cell lymphoma caused by human T cell lymphotropic virus type 1 (HTLV-1). Although ATLL is quite difficult to be cured, up-regulation of cellular immunity such as HTLV-1 Tax-specific cytotoxic T lymphocytes (CTLs) has been proved to be important to obtain long-term survival. At present, no efficacious method to activate ATLL-specific cellular immunity is available. This study aimed to investigate whether live attenuated varicella-zoster virus (VZV) vaccination to ATLL can activate HTLV-1 Tax-specific cellular immune response. A total of 3 indolent- and 3 aggressive-type ATLL patients were enrolled. All aggressive-type patients had the VZV vaccination after completing anti-ATLL treatment including mogamulizumab, which is a monoclonal antibody for C–C chemokine receptor 4 antigen, plus combination chemotherapy, whereas all indolent-type patients had the VZV vaccination without any antitumor treatment. Cellular immune responses including Tax-specific CTLs were analyzed at several time points of pre- and post-VZV vaccination. After the VZV vaccination, a moderate increase in 1 of 3 indolent-type patients and obvious increase in all 3 aggressive-type patients in Tax-specific CTLs percentage were observed. The increase in the cell-mediated immunity against VZV was observed in all indolent- and aggressive-type patients after VZV vaccination. To conclude, VZV vaccination to aggressive-type ATLL patients after mogamulizumab plus chemotherapy led to the up-regulation of HTLV-1 Tax-specific CTLs without any adverse event. Suppression of regulatory T lymphocytes by mogamulizumab may have contributed to increase tumor immunity in aggressive-type ATLL patients. Japan Registry of Clinical Trials number, jRCTs051180107.