HTLV-1 Proviral Load Research Articles

The role of CREB and MAPK signaling pathways in ATLL patients.

HTLV-1 is a worldwide distribution retrovirus with 10-20 million infected individuals. ATLL is an Adult T-cell leukaemia lymphoma caused by aggressive T-cell proliferation that is infected by HTLV-1 and is associated with an inferior prognosis. The exact molecular pathogenesis has yet to be fully understood. CREB, a transcription factor, acts as a molecular switch that controls the expression of numerous genes in response to various extracellular signals. Its activation is primarily mediated through phosphorylation by multiple kinases, including MAPKs. MAPKs, a family of serine/threonine kinases, serve as crucial mediators of intracellular signaling cascades. This study investigated, 38 HTLV-I-infected individuals, including 18 HTLV-1 asymptomatic carriers(ACs) and 20 ATLL subjects. mRNA was extracted and converted to cDNA from Peripheral blood mononuclear cells (PBMCs), and then the expression of TAX, HBZ, CREB, and MAPK was analyzed by TaqMan qPCR. The genomic HTLV-1 Proviral loads were examined among the study group. The data analysis showed a significant difference in the mean of CREB expression amongst study groups (ATLL and carriers,(p = 0.002). There is no statistical difference between the MAPK gene expression (p = 0.35). HBZ, TAX, and HTLV-1 proviral load wereesignificantly higher in ATLL subjectscompared to ACs (p = 0.002, 0.000, and 0.000), respectively. Moreover, our results, demonstrated a direct positive correlation among HBZ, CREB, and TAX gene expression in ATLL patients (p = 0.001), whilst betweenthe ACs, TAX gene expression had a positive significant correlation with HBZ and HTLV-1 proviral load (p = 0.007 and p = 0.004, respectively). The present study demonstrated that CREB gene expression was higher in the ATLL group than ACs, while there was no difference for MAPK. Therefore, this pathway may not strongly involve inthe activation of CREB. The CREB may be a prognostic factor for the development of HTLV-I-associated diseases and can be used as a monitoring marker for the efficiency of the therapeutic regime and prognosis.

B-cell depletion limits HTLV-1-infected T-cell expansion and ameliorate HTLV-1-associated myelopathy.

Human T-cell leukemia virus type 1-associated myelopathy (HAM) is a chronic, progressive, inflammatory disease with unclear pathogenesis and no effective treatments. We aimed to investigate a novel mechanistic theory and treat HAM patients with rituximab, which can deplete CD20+ B lymphocytes in circulation. Single-cell RNA sequencing (scRNA-seq) data was analyzed to identify HTLV-1-associated B cells and their effect on Tcells. An observational analysis of our HAM cohort was conducted to elucidate changes in the immunological microenvironment of these patients. Peripheral blood mononuclear cells (PBMC) from HAM patients were isolated to explore the efficacy of B cell depletion in vitro. To assess the effect of B-cell depletion on HAM patients, eligible participants in our cohort received rituximab therapy (NCT04004819). ScRNA-seq results suggest a significant effect of HTLV-1-associated B cells on Tcells. Additionally, HTLV-1 was found to infect B cells and depletion of B cells inhibited the proliferation of Tcells. Number of B cells in HAM patients had positive correlation with the proviral load and infected cell counts. Depletion of B cells led to a reduction in HTLV-1 proviral load in vitro. Furthermore, in clinical trial, 14 HAM patients were enrolled. Three patients (21.4%) who received rituximab failed to achieve remission, compared to 24 (85.7%) patients received any other therapy that failed to achieve remission. With a low level of circulating B cells, the proportion of Ki67-positive cells in CD4+ Tcells fell. This study provided evidence that depleting B-lymphocytes is an innovative strategy for treating patients with HAM and broadens the understanding of the role of B cells in infectious immunity.

Imbalanced IL10/TGF-β production by regulatory T-lymphocytes in patients with HTLV-1-associated myelopathy/ tropical spastic paraparesis

BackgroundHuman T-cell lymphotropic virus type 1 (HTLV-1), also denominated Human T-cell leukemia virus-1, induces immune activation and secretion of proinflammatory cytokines, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Regulatory T lymphocytes (Tregs) may control of inflammation through the production of regulatory cytokines, including IL10 and TGF-β. In this study we determined the frequencies of CD4 + and CD8 + Tregs in a HAM/TSP population, compared to asymptomatic carriers and uninfected individuals, as well as investigated the profiles of regulatory and inflammatory cytokines.MethodsAsymptomatic HTLV-1 carriers and HAM/TSP patients were matched by sex and age. The frequencies of IL10- and/or TGF-β-producing Tregs were quantified by flow cytometry. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify HTLV-1 proviral load and the mRNA expression of cytokines and cellular receptors in peripheral blood mononuclear cells.ResultsTotal frequencies of CD4 + Tregs, as well as the IL10-producing CD4 + and CD8 + Treg subsets, were statistically higher in patients with HAM/TSP compared to asymptomatic HTLV-1-infected individuals. In addition, a positive correlation was found between the frequency of CD4 + IL10 + Tregs and proviral load in the HAM/TSP patients evaluated. A positive correlation was also observed between gene expression of proinflammatory versus regulatory cytokines only in HAM / TSP group.ConclusionsA higher frequencies of IL10-producing Tregs were identified in patients with HAM/TSP. Imbalanced production of IL10 in relation to TGF-β may contribute to the increased inflammatory response characteristically seen in HAM/TSP patients.

Evaluating tocilizumab safety and immunomodulatory effects under ocular HTLV-1 infection in vitro

There is growing interest in evaluating the safety and therapeutic potential of existing treatments such as tocilizumab (TCZ), an IL-6 receptor antagonist used to treat inflammatory diseases. However, there have been reports of increased inflammation in patients with HTLV-1 uveitis after TCZ treatment, and its ocular safety in the HTLV-1 infected state remains unknown. This study focused on assessing the impact of TCZ on HTLV-1-infected ocular cells using an in vitro model in which retinal pigment epithelial cells were cocultured with irradiated HTLV-1-infected T-cell lines. TCZ did not significantly affect cellular viability, inflammatory markers, or HTLV-1 proviral loads at various concentrations (25/50/100 µg/ml), indicating no increased risk of HTLV-1 viral infection and no exacerbation of the inflammatory aspects of HTLV-1 infection in the ocular cells. These promising results support the potential of TCZ as a safe treatment option for HTLV-1-infected patients, particularly those with eye infections.

Cannabinoid receptors as new targets for HTLV-1 associated myelopathy (HAM/TSP) treatment

Background/AimThe roles of endocannabinoids are described in immune modulation and neuroprotection. HTLV-1-associated myelopathy (HAM/TSP) is an inflammatory neurodegenerative disease. Therefore, in this study, the interactions of HTLV-1 regulatory factors and host cannabinoid receptors (CBRs) were evaluated in HAM/TSP. MethodsNineteen HAM/TSPs, 22 asymptomatic carriers (ACs), and 18 healthy controls (HCs) were enrolled. RNA was extracted from PBMCs and then reverse-transcribed to cDNA. The gene expression of CB1R and CB2R, as well as HTLV-1 proviral load (PVL), Tax and HTLV-1 basic leucine zipper factor (HBZ) were assessed by RT-qPCR. ResultsThe mean expression of CB1R in ACs (8.51 ± 2.76) was significantly higher than HAMTSPs (1.593 ± 0.74, p = 0.05) and also HCs (0.10 ± 0.039, p = 0.001). The CB2R gene expression level in ACs (2.62±0.44) was significantly higher than HAM/TSPs (0.59 ± 0.15, p = 0.001) and HCs (1.00 ± 0.2, p = 0.006). Meanwhile there was a strong correlation between CB1R and CB2R gene expression levels in the HCs and HAM/TSPs (p = 0.001). HTLV-1-Tax expression in HAM/TSPs (386 ± 104) was higher than ACs (75 ± 32) and statistically significant (p = 0.003). While HTLV-1-HBZ was only expressed in three AC subjects and five HAM/TSPs, thus it cannot be analyzed. ConclusionThe up-regulation of CB2R has immunomodulatory effects in inflammatory reactions. While CB1R as a neuroprotective agent may suppress inflammatory reactions in ACs, preventing HAM/TSP. It seems that, like multiple sclerosis (MS), cannabinoid medications are beneficial in HAM/TSP.

Single-Cell RNA Sequencing Revealed the YY1/EZH2/MLH1 Axis As a Possible Therapeutic Target of Intractable Adult T-Cell Leukemia

Background: Due to clinical heterogeneity of adult T-cell leukemia/lymphoma (ATL) and its diverse genetic abnormality, common therapeutic targets of ATL remains unclear. Whereas the YY1/EZH2 axis regulates global epigenetic modification of genes in ATL, its downstream target genes have not been fully elucidated. Meanwhile, DNA mismatch repair proteins may be targeted in ATL. Here we explored underlying molecular axes for ATL progression through comprehensive single-cell RNA-sequencing (sc-RNA-seq). Methods: Peripheral blood of 42 ATL patients and asymptomatic HTLV-1 carriers in addition to two cell lines of ATL43T and ED+ were subjected to the following analyses. Molecular markers associated with their clinical phenotypes were extracted through sc-RNA-seq analysis. Expression levels of DNA mismatch repair proteins (MLH1, MSH2) and target molecules were validated by flow cytometry. Finally, their function was verified through lentiviral shRNA-dependent knockdown and molecular-targeting reagents. Results: While the frequencies of CADM1+ cells among CD4+ T cells were generally correlated with HTLV-1 proviral loads (R = 0.76, p < 0.001), substantial CADM1+ cells were detected but did not increase in some long-remitted cases; thus, CADM1+ cells are biologically heterogeneous. sc-RNA-seq of CADM1+ cells of four ATL cases identified a cluster of CD48-deficient cells. in silico promoter analysis of the cluster extracted a transcription factor YY1 as a candidate for common master regulators. EZH2 and the putative driver genes (CELF2, PTPRC, CBLB, ATXN1, IKZF2) were identified as differentially expressed genes (DEGs) in the cluster. The EZH2-overexpressed cluster was increased in acute-type ATL compared with remitted ATL and smoldering-type ATL. Interestingly, the cluster increased moderately in the other smoldering-type ATL, which required UVB irradiation due to intractable skin lesion. Given that YY1 and EZH2 were up-regulated in acute-type ATL, the cluster may represent tumor aggressiveness. Furthermore, biphasic expression of YY1/EZH2 was observed in 2 acute-type ATL, which indicates that the EZH2 high ATL cells confer malignant signature. In-house analysis on microarray dataset GSE55851 specified the MLH1 associated with YY1/EZH2. MLH1 within CADM1+ fraction was down-regulated in ATL patients with remission (n = 7) but maintained in progressive ATL (n = 6) (p < 0.05). Lentiviral shYY1 knockdown resulted in down-regulation of MLH1 in both ATL43T and ED+ (p < 0.01). The EZH1/2 inhibitor valemetostat down-regulated EZH2 (p = 0.018) and MLH1 (p = 0.014) in primary ATL cells. Finally, knockdown of either YY1 or MLH1 suppressed proliferation of ATL43T and ED+ (p < 0.0001). Conclusions: Our findings indicate that down-regulation of MLH1 through YY1/EZH2 inhibition plays a key role in the treatment of aggressive ATL.

HTLV-1-associated myelopathy in Spain

BackgroundHTLV-1 infection is a neglected disease. Over 10 million people are infected worldwide, with hot spots of high endemicity across all continents. Roughly 5% of HTLV-1 carriers develop HTLV-1-associated myelopathy (HAM), a progressive subacute neurological disabling disease. MethodsWe report the main features of patients diagnosed with HAM up to date in Spain, a non-endemic country with a relatively high migrant flow from Latin America and Equatorial Africa, where HTLV-1 is endemic. ResultsA total of 451 cases of HTLV-1 had been recorded in Spain until the end of year 2022. HAM had been diagnosed in 58 (12.9%). The current incidence is of 2-3 new cases per year. Women represent 76%. Mean age at diagnosis is 49 years-old. Nearly 60% are Latin Americans. Although sexual transmission is the most likely route of HTLV-1 acquisition, up to 6 individuals had been infected following solid organ transplantation. Rapid onset myelopathy developed in all but one of these transplant recipients from three HTLV-1-positive donors. HTLV-1 subtype 1a transcontinental was the only variant recognized in HAM patients. HTLV-1 proviral load was significantly greater in HAM patients than in asymptomatic HTLV-1 carriers (677 vs 104 HTLV-1 DNA copies/104 PBMC; p=0.012). Symptom relief medications and physiotherapy have been the only treatment providing some benefit to HAM patients. Neither significant clinical nor virological efficacy was noticed using antiretrovirals in at least 9 HAM patients. Two thirds of HAM patients ended up in a wheelchair and with urinary/fecal sphincter incontinence. ConclusionHAM is the most frequent clinical manifestation of HTLV-1 infection in Spain, a non-endemic country. Middle aged women migrants from Latin America are the most frequently affected. Two thirds end up in a wheelchair despite using antiretroviral therapy.

HTLV-1 Proviral Load Absolute RT-qPCR Development for Assessing on Clinical Outcomes in HAM/TSP Patients.

The significance of HTLV-1 proviral load as a prognostic biomarker in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) has been a subject of controversy. This study aims to assess the impact of HTLV-1 proviral load (PVL) on the clinical outcome in patients with HAM/TSP. An absolute quantitative HTLV-1 PVL RT-qPCR, TaqMan method was developed with 100% sensitivity and specificity. Then, from 2005-2018, the HTLV-1 PVL of 90 eligible newly diagnosed HAM/TSP patients were assessed for demographic, clinical symptoms and their associations with HTLV-1-PVL. The quality control of the designed RT-qPCR showed a sensitivity and specificity of 100%. Spasticity in lower limbs in 58.9% and urinary symptoms in 17.8% of HAM/TSPs were observed. Using this designed RT-qPCR, the HTLV-1-PVL strongly affected spasticity and sphincter disturbance (p=0.05). The multivariate logistic test showed that only the beginning of lower limb weakness along with tremor was associated with PVL (OR: 2.78. 95% CI (0.99-1.02) and p=0.05). Urinary incontinence was prevalent among these patients; however, no association was identified with the HTLV-1 proviral load (PVL). The absolute RT-qPCR developed for measuring HTLV-1 proviral load (PVL) demonstrated reliable results. Despite a high prevalence of urinary incontinence in these patients, no association was observed with the PVL. Consequently, it appears that HTLV-1 proviral load is specifically associated with developing spasticity in HAM/TSP.

Adult T-cell leukemia/lymphoma in HTLV-1 non-endemic regions

BackgroundHTLV-1 infection is a neglected disease, despite producing neurological and lymphoproliferative severe illnesses and affect over 10 million people worldwide. Roughly 5% of HTLV-1 carriers develop Adult T-cell leukemia/lymphoma (ATLL), one of the most aggressive hematological malignancies. MethodsA national HTLV-1 register exists since 1989 in Spain, a non-endemic country with a large migrant flow from Latin America and Equatorial Africa, where HTLV-1 is endemic. The main features of all patients diagnosed with ATLL in Spain up to date are reported. ResultsA total of 451 cases of HTLV-1 infection had been reported in Spain until the end of year 2022. ATLL had been diagnosed in 35 (7.8%). The current average incidence of ATLL in Spain is of two cases per year. Women represent 57% of ATLL patients. Mean age at diagnosis was 47 years-old. Roughly 57% were Latin Americans and 26% Africans. At diagnosis, the majority presented with acute or lymphoma clinical forms. Survival was shorter than one year in most of them.Mean HTLV-1 proviral load was significantly greater in ATLL patients than in asymptomatic HTLV-1 carriers (2,305 vs 104 copies/104 PBMC). HTLV-1 subtyping in 6 ATLL patients found the 1a transcontinental variant (n = 4) and the Japanese variant (n = 2). All ATLL patients were negative for HIV-1, did not develop HTLV-1-associated myelopathy and were not transplant recipients. ConclusionThe rate of ATLL is very low in Spain and mostly associated to migrants from HTLV-1 endemic regions. Given the poor clinical outcome of ATLL, HTLV-1 testing should be performed at least once in all migrants coming from HTLV-1 endemic countries and in natives who have lived in or had sex partners from such regions.

Mogamulizumab for Treatment of Human T-lymphotropic Virus Type 1-Associated Myelopathy/Tropical Spastic Paraparesis: A Single-Center US-based Series.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic neurological condition characterized by progressive myelopathic symptoms including spasticity, pain, weakness, and urinary symptoms, without proven treatments. Mogamulizumab (MOG) is a monoclonal antibody that binds CCR4 and leads to the clearance of HTLV-1-infected CCR4+ cells. A phase 1-2a study in Japan evaluated MOG for the treatment of HAM/TSP and reported decreases in HTLV-1 proviral load and neuroinflammatory markers, with clinical improvement in some participants. We administered MOG 0.1 mg/kg every 8 weeks to individuals with HAM/TSP as a compassionate and palliative treatment. Patients who received MOG had (1) a positive peripheral HTLV-1 antibody, (2) progressive myelopathic symptoms, and (3) a diagnosis of HAM/TSP. Four female patients, ages 45-68, received MOG (range, 2-6 infusions) between 1 November 2019 and 30 November 2022. Two patients with <3 years of symptoms had milder disease, with Osame scores <4. The other 2, with >7 years of symptoms, had Osame scores >5. One patient, with 6 total treatments, received dose-reduced MOG after she developed a rash at the initial dose. The 2 patients with milder baseline disease reported symptomatic improvement and saw reductions in Osame and/or modified Ashworth scale scores during follow-up. The other 2 patients showed no improvement. All 4 developed rashes after receiving MOG-a treatment-limiting event in some cases. Clinical trials are needed including diverse patient populations to assess the potential role of MOG for HAM/TSP. Our findings may help inform the development of these trials.

HTLV-1 Proviral Load in Vaginal Fluid Correlates with Levels in Peripheral Blood Mononuclear Cells.

The prevalence of human T-lymphotropic virus type-1 (HTLV-1) infection is higher in women, and sexual intercourse has been described as an important route of male-to-female transmission. The present study aimed to quantify HTLV-1 proviral load (PVL) in vaginal fluid, and to investigate correlations with PVL in peripheral blood mononuclear cells (PBMCs). In addition, cytopathological alterations and vaginal microbiota were evaluated. HTLV-1-infected women were consecutively recruited at a multidisciplinary center for HTLV patients in Salvador, Brazil. All women underwent gynecological examinations to obtain cervicovaginal fluid and venipuncture for blood collection. PVL, as measured by real-time quantitative polymerase chain reaction (RT-qPCR), was expressed as the number of copies of HTLV-1/106 cells in blood and vaginal fluid samples. Light microscopy was used to assess cervicovaginal cytopathology and vaginal microbiota. In the 56 included women (43 asymptomatic carriers and 13 diagnosed with HTLV-1-associated myelopathy/tropical spastic paraparesis-HAM/TSP), mean age was 35.9 (SD ± 7.2) years. PVL was higher in PBMCs (median: 23,264 copies/106 cells; IQR: 6776-60,036) than in vaginal fluid (451.9 copies/106 cells; IQR: 0-2490) (p < 0.0001). PVL in PBMCs was observed to correlate directly with PVL in vaginal fluid (R = 0.37, p = 0.006). PVL was detected in the vaginal fluid of 24 of 43 (55.8%) asymptomatic women compared to 12 of 13 (92.3%) HAM/TSP patients, p = 0.02. Cytopathologic analyses revealed no differences between women with detectable or undetectable PVL. HTLV-1 proviral load is detectable in vaginal fluid and correlates directly with proviral load in peripheral blood. This finding suggests that sexual transmission of HTLV-1 from females to males may occur, as well as vertical transmission, particularly in the context of vaginal delivery.

Development and validation of a duplex real-time PCR for the rapid detection and quantitation of HTLV-1

BackgroundThe HTLV-1 prevalence in China varies geographically, while HTLV-2 infection has rarely been found so far. Proviral load is one of the determining factors of pathogenesis and progression of HTLV-1 related diseases. However, neither molecular assays nor commercial kits are available for HTLV-1 diagnosis in China. The objective of the present study was to develop and validate a TaqMan qPCR assay for HTLV-1 proviral load quantification.ResultsA plasmid containing both the HTLV-1 of interest and a fragment of the RNase P (RPPH1) gene was constructed and used to establish the standard curves. The assay has a wide dynamic range (2.5 × 108 copies/reaction ~ 25 copies/reaction) and sensitive to 1 copy for HTLV-1 and RPPH1. The limit of detection for Hut102 cell concentration was 0.0218% (95% confidence interval 0.0179–0.0298%). The assay gave coefficient of variation (CV) for both the HTLV-1 and RPPH1 Ct values. All of the HTLV-1 sero-negative samples and MOT cell line (infected with HTLV-2) amplified only the RPPH1 gene by our method, presenting 100% specificity. 85 Samples confirmed positive or indeterminate by LIA were performed by established qPCR assay and WB. 90.0% (27/30) of LIA-HTLV-1-positive, 33% (2/6) of LIA-untypeable and 2% (1/49) of LIA-indeterminate samples were defined as qPCR-positive. The median PVL of LIA-positive samples (n = 27, 1.780 copies/100 cells) was much higher than that of LIA-untypeable and (n = 2, 0.271 copies/100 cells) indeterminate samples (n = 1, 0.017 copies/ 100 cells). Additionally, compared to WB, the duplex qPCR verified more positive samples, demonstrating a better sensitivity.ConclusionThe duplex qPCR developed here with high sensitivity, good specificity and reproducibility could accurately and quantitatively detect the HTLV-1 PVLs, which can be used to confirm the initial reactive samples for an improved cost/benefit ratio as well as to monitor the clinical progression and efficacy of therapy in patients with HTLV-1 related disease.

XCL1, a serum biomarker in neurological diseases; HTLV-1-associated myelopathy and multiple sclerosis.

The XCL1-XCR1 axis has a potential role in the recruitment of immune cells to the site of inflammation. The present study aimed to examine the relation of XCL1 serum levels with Multiple sclerosis (MS) and HTLV-1-associated myelopathy (HAM), as chronic inflammatory diseases of the central nervous system (CNS). DNA was extracted to evaluate HTLV-1 proviral load (PVL) using real-time PCR. Serum levels of XCL1 was determined by using an ELISA assay. The serum level of XCL1 was significantly higher in patients with HAM than that of asymptomatic carriers (ACs) and healthy controls (HCs) (p<0.001 and p<0.0001, respectively) and was also higher in MS patients compared to HCs (p<0.0001). Moreover, the concentration of XCL1 serum level was significantly different between the ACs and HCs group (p<0.0001). In conclusion, increased expression of XCL1 might contribute to the migration of autoreactive T cells to the central nervous system and play a critical role in the development and pathogenesis of inflammatory neurological diseases including HAM and MS.

Frequency of HTLV-1 seroconversion between pregnancies in Nagasaki, Japan, 2011-2018.

Human T-cell leukemia virus type-1 (HTLV-1) is transmitted vertically from an infected mother to her child via breastfeeding during infancy or horizontally via sexual contact. However, little information is available on the HTLV-1 seroconversion rate in pregnant mothers and the impact of new HTLV-1 infection on mothers and babies during the perinatal period. From the database of a prefecture-wide antenatal adult T-cell leukemia prevention program in Nagasaki, Japan, we extracted data on 57,323 pregnant women who were screened for anti-HTLV-1 antibody during 2011-2018. Data on the 16,863 subjects whose HTLV-1 proviral load (PVL) was measured more than twice were included in our analyses. In total, 133 (0.79%) pregnant women were HTLV-1-positive during their first pregnancy and nine (0.05%) seroconverted before or during subsequent pregnancies (between pregnancies). The median PVL (per 100 peripheral blood mononuclear cells) was significantly lower in the seroconverted mothers (0.10%) than in the initially seropositive mothers (0.15%). A repeated measures correlation analysis for the individual PVLs of the HTLV-1-positive pregnant women showed that PVL increased with parity number (rrm = 0.25) with no perinatal problems. The HTLV-1 seroconversion rate between pregnancies was 0.05%, and their HTLV-1 PVL increased annually but no perinatal problems were noted.

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.

Refining the risk of HTLV-1-associated myelopathy in people living with HTLV-1: identification of a HAM-like phenotype in a proportion of asymptomatic carriers

Up to 3.8% of human T-lymphotropic virus type-1 (HTLV-1)-infected asymptomatic carriers (AC) eventually develop HTLV-1-associated myelopathy (HAM). HAM occurs in patients with high (> 1%) HTLV proviral load (PVL). However, this cut-off includes more than 50% of ACs and therefore the risk needs to be refined. As HAM is additionally characterised by an inflammatory response to HTLV-1, markers of T cell activation (TCA), β2-microglobulin (β2M) and neuronal damage were accessed for the identification of ACs at high risk of HAM. Retrospective analysis of cross-sectional and longitudinal routine clinical data examining differences in TCA (CD4/CD25, CD4/HLA-DR, CD8/CD25 & CD8/HLA-DR), β2M and neurofilament light (NfL) in plasma in ACs with high or low PVL and patients with HAM. Comparison between 74 low PVL ACs, 84 high PVL ACs and 58 patients with HAM revealed a significant, stepwise, increase in TCA and β2M. Construction of receiver operating characteristic (ROC) curves for each of these blood tests generated a profile that correctly identifies 88% of patients with HAM along with 6% of ACs. The 10 ACs with this ‘HAM-like’ profile had increased levels of NfL in plasma and two developed myelopathy during follow-up, compared to none of the 148 without this viral-immune-phenotype. A viral-immuno-phenotype resembling that seen in patients with HAM identifies asymptomatic carriers who are at increased risk of developing HAM and have markers of subclinical neuronal damage.

HTLV-1 encephalomyelitis; a case report of a treatable manifestation of HTLV-1 infection

IntroductionHuman T-cell lymphotropic virus type-1 (HTLV-1) infection remains asymptomatic in &gt;90% of the 20-million people infected worldwide. However in 3%, chronic inflammation within the thoracic spinal cord leads to progressive spastic paraparesis; HTLV-1 associated myelopathy (HAM). This pathological process is not limited to the thoracic cord. We present a case of HTLV-1 encephalomyelitis.CaseA 53-year-old woman with HAM of 9 years duration presented with subacute cerebellar dysfunc- tion, being unable to feed herself, 3-months after cessation of methotrexate. Investigations demonstrated extensive T2-hyperintensity within the brainstem, cortical and subcortical white matter with punctate contrast enhancement; lymphocytic CSF; and high blood (36%) and CSF (72%) HTLV-1 proviral load (DNA copies per 100 lymphocytes). We diagnosed HTLV-1 encephalomyelitis and commenced high dose methylprednisolone with slow steroid taper following which functional independence in the upper limbs was regained.DiscussionRisk of HAM rises exponentially once HTLV-1 proviral load exceeds 1%, while CSF:blood proviral load ratio ≥2:1 indicates CNS infiltration of HTLV-1 infected lymphocytes. This patient’s high HTLV-1 proviral load and widespread MRI changes indicated HTLV-1 associated inflammation of the brain and spine. Prompt immunosuppression resulted in significant recovery and highlights the importance of early rec- ognition and management of extraspinal manifestations of HTLV-1 infection.j.king-robson@nhs.net

The Association of Increase of Human T-Cell Leukemia Virus Type-1 (HTLV-1) Proviral Load (PVL) With Infection in HTLV-1-Positive Patients With Rheumatoid Arthritis: A Longitudinal Analysis of Changes in HTLV-1 PVLs in a Single Center Cohort Study

ObjectiveWe evaluated changes of HTLV-1 proviral loads (PVLs) during treatment for rheumatoid arthritis (RA) and investigated whether these changes affect the clinical course in HTLV-1-positive RA patients.MethodsA total of 41 HTLV-1-positive RA patients were analyzed. Their clinical picture including disease activity [Disease Activity Score in 28 joints-erythrocyte sedimentation rate (DAS28-ESR), DAS28-CRP, simplified disease activity index (SDAI), and clinical disease activity index (CDAI)] and comorbidity were evaluated over a 2-year period. PVLs from peripheral blood mononuclear cells were investigated by real-time polymerase chain reaction (PCR). We investigated whether HTLV-1 PVLs is altered, or which clinical characteristics affect changes of HTLV1-PVLs during 2-year treatment.ResultsClinical disease activity was not changed during the 2-year observational period. The mean HTLV-1 PVL value change from baseline to 2 years was -1.2 copies/1000 PBMCs, which was not statistically significant. No baseline clinical characteristics influenced changes in HTLV-1 PVL. However, a numerical change of HTLV-1 PVLs was increased in 4 patients initiating the new biological/targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) at 2−10 months after starting the new b/ts DMARDs (numerical increase was 24.87 copies/1000 PBMCs). Infection occurred in 4 patients, and 3 of those patients showed an increased HTLV-1 PVL. Univariate analysis revealed an association between increase of HTLV-1 PVL and incidence of infection.ConclusionsOver 2 years, HTLV-1 PVL did not significantly change in our HTLV-1-positive RA patients. Individual changes in HTLV-1 PVL were correlated with incidence of infection but not disease activity which indicate that we may take precaution toward infection at the uptick of HTLV-1 PVL in HTLV-1-positive RA patients.

Monitoring of HTLV-1-associated diseases by proviral load quantification using multiplex real-time PCR.

Proviral load (PVL) is one of the determining factors for the pathogenesis and clinical progression of the human T-lymphotropic virus type I (HTLV-1) infection. In the present study, we optimized a sensitive multiplex real-time PCR for the simultaneous detection and quantification of HTLV-1 proviral load and beta-globin gene as endogenous control. The values obtained for HTLV-1 PVL were used to monitor the clinical evolution in HTLV-1-infected individuals. A vector containing cloned DNA targets of the real-time PCR for the beta-globin gene and the HTLV-1pol region was constructed. For the reaction validation, we compared the amplification efficiency of the constructed vector and MT-2 cell line containing HTLV-1. The analytical sensitivity of the reaction was evaluated by the application of a standard curve with a high order of magnitude. PVL assay was evaluated on DNA samples of HTLV-1 seropositive individuals. The construct showed adequate amplification for the beta-globin and HTLV-1 pol genes when evaluated as multiplex real-time PCR (slope = 3.23/3.26, Y-intercept = 40.18/40.73, correlation coefficient r2 = 0.99/0.99, and efficiency = 103.98/102.78, respectively). The quantification of PVL using the MT-2 cell line was equivalent to the data obtained using the plasmidial curve (2.5 copies per cell). In HTLV-1-associatedmyelopathy/tropical spastic paraparesis patients, PVL was significantly higher (21315 ± 2154 copies/105 PBMC) compared to asymptomatic individuals (1253 ± 691 copies/105 PBMC). The obtained results indicate that the optimized HTLV-1 PVL assay using plasmidial curve can be applied for monitoring and follow-up of the progression of HTLV-1 disease. The use of a unique reference plasmid for both HTLV-1 and endogenous gene allows a robust and effective quantification of HTLV-1 PVL. In addition, the developed multiplex real-time PCR assay was efficient to be used as a tool to monitor HTLV-1-infected individuals.

Following the Clues: Usefulness of Biomarkers of Neuroinflammation and Neurodegeneration in the Investigation of HTLV-1-Associated Myelopathy Progression.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease due to axonal damage of the corticospinal secondary to an inflammatory response against infected T-cells. In the present work, we aimed to evaluate biomarkers of neurodegeneration and neuroinflammation in the definition of HAM/TSP prognosis. Neurofilament light (NfL) and phosphorylated heavy (pNfH) chains, total Tau protein, cellular prion protein (PrPc), inflammatory chemokines, and neopterin were quantified in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n=21), HTLV-1 asymptomatic carriers (AC) (n=13), and HTLV-1 seronegative individuals with non-inflammatory non-degenerative neurological disease (normal-pressure hydrocephalus) (n=9) as a control group. HTLV-1 proviral load in peripheral blood mononuclear cells and the expression of chemokine receptors CCR4, CCR5, and CXCR3 in infected CD4+ T-cells (HTLV-1 Tax+ cells) were also assessed. CSF levels of Tau, NfL, and pNfH were similar between groups, but PrPc and neopterin were elevated in HAM/TSP patients. Most individuals in the control group and all HTLV-1 AC had CSF/serum neopterin ratio < 1.0, and two-thirds of HAM/TSP patients had ratio values > 1.0, which positively correlated with the speed of disease progression and pNfH levels, indicating active neuroinflammation. HAM/TSP patients showed high serum levels of CXCR3-binding chemokines (CXCL9, CXCL10, and CXCL11) and elevated CSF levels of CCL2, CCL3, CCL4, CCL17, CXCL5, CXCL10, and CXCL11. Indeed, CXCL10 concentration in CSF of HAM/TSP patients was 5.8-fold and 8.7-fold higher in than in HTLV-1 AC and controls, respectively, and correlated with CSF cell counts. HAM/TSP patients with typical/rapid disease progression had CSF/serum CXCL10 ratio > 1.0 and a higher frequency of CXCR3+Tax+CD4+ T-cells in blood, which indicated a positive gradient for the migration of infected cells and infiltration into the central nervous system. In conclusion, the slow progression of HAM/TSP abrogates the usefulness of biomarkers of neuronal injury for the disease prognosis. Thus, markers of inflammation provide stronger evidence for HAM/TSP progression, particularly the CSF/serum neopterin ratio, which may contribute to overcome differences between laboratory assays.