Maria Antonietta Demontis

HTLV-1 viral RNA is detected rarely in plasma of HTLV-1 infected subjects.

Plasma of patients infected with HTLV‐1 is considered non‐infectious but detection of HTLV‐1 genomic RNA in plasma has been recently reported. The aim of this project was to detect and quantify HTLV‐1 RNA in plasma and assess its potential value in diagnosis and prognosis. RNA from 1 ml of plasma from 65 subjects infected with HTLV‐1 (27 asymptomatic carriers [AC]), 17 patients with HTLV‐1‐associated myelopathy (HAM/TSP), 14 with adult T‐cell leukemia/lymphoma (ATLL), two co‐infected with HIV, and five with other HTLV‐1‐associated disease, was extracted and reverse transcribed. HTLV‐1 specific nested PCR was performed using primers to amplify the conserved Tax region. All samples were run in quadruplicate, nested PCR products were detected by gel electrophoresis. HTLV‐1 RNA was detected in plasma from 18 (28%) patients, always at a very low copy number (3–13 copies viral cDNA per milliliter of plasma). Mean values of HTLV‐1 proviral load did not differ between patients in whom HTLV‐1 RNA was detected and patients in whom it was not possible to detect HTLV‐1 RNA in plasma. HTLV‐1 genomic RNA can be detected in the plasma of a minority of patients but not at a level or frequency to be useful clinically or diagnostically. Lack of transmission of HTLV‐1 by plasma is due to the rare presence of HTLV‐1 virions, regardless of any other factor. J. Med. Virol. 87:2130–2134, 2015.

Ciclosporin A Proof of Concept Study in Patients with Active, Progressive HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis

Introduction Patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) become progressively impaired, with chronic pain, immobility and bladder, bowel and sexual dysfunction. Tested antiretroviral therapies have not been effective and most patients are offered a short course of corticosteroids or interferon-α, physiotherapy and symptomatic management. Pathogenesis studies implicate activated T-lymphocytes and cytokines in tissue damage. We therefore tested the hypothesis that inhibition of T-cell activation with ciclosporin A would be safe and clinically beneficial in patients with early and/or clinically progressing HAM/TSP. Materials and Methods Open label, proof of concept, pilot study of 48 weeks therapy with the calcineurin antagonist, ciclosporin A (CsA), in seven patients with ‘early’ (50% deterioration in timed walk during the preceding three months) HAM/TSP. Primary outcomes were incidence of clinical failure at 48 weeks and time to clinical failure. Results All patients completed 72 weeks study participation and five showed objective evidence of clinical improvement after 3 months treatment with CsA. Two patients exhibited clinical failure over 6.4 person-years of follow-up to week 48. One patient had a >2 point deterioration in IPEC (Insituto de Pesquisa Clinica Evandro Chagas) disability score at weeks 8 and 12, and then stopped treatment. The other stopped treatment at week 4 because of headache and tremor and deterioration in timed walk, which occurred at week 45. Overall pain, mobility, spasticity and bladder function improved by 48 weeks. Two patients recommenced CsA during follow-up due to relapse. Conclusions These data provide initial evidence that treatment with CsA is safe and may partially reverse the clinical deterioration seen in patients with early/progressive HAM/TSP. This trial supports further investigation of this agents safety and effectiveness in larger, randomised controlled studies in carefully selected patients with disease progression.

Risk stratification of adult T-cell leukemia/lymphoma using immunophenotyping

Adult T‐cell leukemia/lymphoma (ATL), a human T‐lymphotropic virus type 1 (HTLV‐1)‐associated disease, has a highly variable clinical course and four subtypes with therapeutic and prognostic implications. However, there are overlapping features between ATL subtypes and between ATL and nonmalignant (non‐ATL) HTLV‐1 infection complicating diagnosis and prognostication. To further refine the diagnosis and prognosis of ATL, we characterized the immunophenotype of HTLV‐1‐infected cells in ATL and non‐ATL. A retrospective study of peripheral blood samples from 10 HTLV‐1‐uninfected subjects (UI), 54 HTLV‐1‐infected patients with non‐ATL, and 22 with ATL was performed using flow cytometry. All patients with ATL had CD4+ CCR4+ CD26− immunophenotype and the frequency of CD4+ CCR4+ CD26− T cells correlated highly significantly with the proviral load in non‐ATL suggesting CD4+ CCR4+ CD26− as a marker of HTLV‐1‐infected cells. Further immunophenotyping of CD4+ CCR4+ CD26− cells revealed that 95% patients with ATL had a CD7− (≤30% CD7+ cells), whereas 95% HTLV+ non‐ATL had CD7+ (>30% CD7+ cells) immunophenotype. All patients with aggressive ATL had a CCR7+ (≥30%), whereas 92% with indolent ATL and 100% non‐ATL had a CCR7− (<30%) immunophenotype. Patients with nonprogressing indolent ATL were CD127+ but those with progressive lymphocytosis requiring systemic therapy had a CD127− (≤30%) immunophenotype. In summary, HTLV‐1‐infected cells have a CD4+ CCR4+ CD26− immunophenotype. Within this population, CD7− phenotype suggests a diagnosis of ATL, CCR7+ phenotype identifies aggressive ATL, while CCR7− CD127− phenotype identifies progressive indolent ATL.

The clinical utility of HTLV-1 viral load measurement

High levels of HTLV-1 proviral load in peripheral blood mononuclear cells (PBMCs) have been observed in patients with Adult T-cell Leukaemia Lymphoma (ATLL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). The proviral load has been reported to fluctuate in both asymptomatic carriers and in symptomatic patients. We aimed to quantify intra-patient variability, to identify “outliers”, and to characterise the range of viral loads in asymptomatic carriers and in patients with HTLV-1-associated diseases.

Circularised 1 and 2 LTR DNA circles are present in freshly- and chronically-infected cell lines and patient PBMCs, indicating ongoing reverse transcriptase usage

Background After cell entry, HTLV-1 RNA is reverse transcribed and integrated into the host genome using its own reverse transcriptase (RT) and integrase (IN) enzymes. However, some unintegrated DNA circularises into 1 or 2 LTR DNA. Little is known about these unintegrated HTLV-1 DNA circles. Similar to HIV an inhibition of RT should decrease, and an inhibition of IN increase the 1/2 LTR DNA levels.

Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo

Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the “mitotic” spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset.

Effect of Pulsed Methylprednisolone on Pain, in Patients with HTLV-1-Associated Myelopathy

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an immune mediated myelopathy caused by the human T-lymphotropic virus type 1 (HTLV-1). The efficacy of treatments used for patients with HAM/TSP is uncertain. The aim of this study is to document the efficacy of pulsed methylprednisolone in patients with HAM/TSP. Data from an open cohort of 26 patients with HAM/TSP was retrospectively analysed. 1g IV methylprednisolone was infused on three consecutive days. The outcomes were pain, gait, urinary frequency and nocturia, a range of inflammatory markers and HTLV-1 proviral load. Treatment was well tolerated in all but one patient. Significant improvements in pain were: observed immediately, unrelated to duration of disease and maintained for three months. Improvement in gait was only seen on Day 3 of treatment. Baseline cytokine concentrations did not correlate to baseline pain or gait impairment but a decrease in tumour necrosis factor-alpha (TNF-α) concentration after pulsed methylprednisolone was associated with improvements in both. Until compared with placebo, treatment with pulsed methylprednisolone should be offered to patients with HAM/TSP for the treatment of pain present despite regular analgesia.

CD25+CCR4+ cells as a marker of HTLV-1-infected cells in peripheral blood

The diagnosis of Human T cell Lymphotropic Virus type 1 (HTLV-1) infection is based on serology and PCR. The burden of HTLV-1 viral infection is monitored by measuring the proviral load [PVL] in peripheral blood. This assay is not useful for isolation of HTLV-1-infected cells. HTLV-1 Tax upregulates CD25 expression on infected CD4+ T cells. However, CD4+CD25+ is also the phenotype of activated T cells. CCR4 is a chemokine receptor expressed on subsets of activated T cells. HTLV-1-infected cells secrete the CCR4 ligand CCL22, and preferentially infect CCR4+ Cells. We studied the expression of CD25 and CCR4 in lymphocytes and its relation to PVL. We performed 11-colour immunophenotyping and HTLV-1 PVL quantification on 53 samples obtained from 36 HTLV-1 infected patients, [10 asymptomatic carriers (AC); 11 patients with HTLV-1-associated myelopathy (HAM); 4 co-infected with HIV; 11 with chronic/smouldering adult T-cell leukaemia/lymphoma (ATL)] and 3 uninfected individuals. Increased frequency of CD25+CCR4+ T cells [median: 14.7%, range: 1.95-91.3%] was observed in all HTLV-1 infected patients; the frequency correlated with PVL [Spearman r=0.89, P <0.001, Linear R2 = 0.61]. CD4+ and CD8+ cells from 12 patients [6ACs and 6 HAMs] were separated for PVL estimation. CD25+ CCR4+ cell counts correlated closely with PVL in CD4+ cells [Spearman r=0.816, P <0.001, linear R2=0.886] but not in CD8+ cells. Frequency of CD25+CCR4+ T cells correlated with PVL change in treated ATL patients. We conclude that CD25+CCR4+ cells can be used as a specific marker for monitoring of HTLV-1 infection and isolation of HTLV-1 infected cells.

A proof of concept study of Infliximab for the treatment of HTLV-1-associated myelopathy

Background Disease modifying treatment options for patients with HAM are limited. Most studies have included all patients regardless of duration, disability or disease activity. The Medical Research Council UK funded a series of proof of concept studies for patients with early (50% deterioration during preceding 3 months). The results of the first study of ciclosporin have been published. The second study, of the anti-TNF monoclonal antibody Infliximab, is presented.

The potential of CD127 as a prognostic and residual disease marker in chronic adult T cell leukaemia/lymphoma

Adult T cell Leukaemia Lymphoma [ATL], a mature T-cell neoplasm has been classified into 4 subtypes: smouldering; chronic leukaemia; lymphoma and acute leukaemia. The diagnosis depends on clinical features, the immunophenotype, and demonstration of HTLV-1 infection & ideally of monoclonal proviral integration. The typical immunophenotype of ATL is not specific. The methods used to detect monoclonality are labour-intensive and/or expensive and are not widely available. We developed a flow cytometry assay for diagnosis and monitoring of ATL. We performed 11-colour immunophenotyping, HTLV-1 proviral quantification and proviral integration site [IS] analysis on 53 samples from 36 patients [25 non ATL HTLV-1-infected, 11 chronic/smouldering ATL], 3 uninfected individuals and 2 HTLV-1-immortalized cell lines. The non-ATL patients had CD127+ & CCR7-lo expression in CD4+ CD25+CCR4+ cells, and a polyclonal distribution on IS analysis. FourATL patients had CD127+ & CCR7-lo expression in CD4+CD25+CCR4+ cells and polyclonal distribution on IS analysis. These patients had an excellent outcome achieving remission with either PUVA or no therapy. Eight ATL patients had CD127-lo expression on CD4+ CD25+ CCR4+ cells with mono/oligoclonal distribution on IS analysis. One of nine patients with chronic ATLL had high CCR7 expression. Foxp3 expression was variable. All 8 patients required systemic ATL treatment and longitudinal study of 5 patients found the change in frequency of CD4+CD25+CCR4+ to correlate with PVL whilst CD127 expression correlated with IS analysis (p<0.005) and disease remission status. CD127 expression appears to be useful to identify patients needing treatment and for monitoring the treatment of chronic ATL.