Immune Senescence-Related Gene Expression Profile in CD4+ T-Lymphocytes of HTLV-1 Asymptomatic Carriers and Patients with Adult T-Cell Leukemia/Lymphoma (ATLL): A Brazilian Preliminary Study

Introduction: Adult T-cell leukemia/lymphoma (ATLL) is a rare and aggressive neoplasm caused by the human T-lymphotropic virus type 1 (HTLV-1). It is estimated, worldwide, that at least 5-10 million people carry HTLV-1 and 2-5% out of them will develop ATLL. Previously, our group demonstrated an increase of cells in G0/G1 phase of cell cycle and aneuploidy in CD4+ T-cells in HTLV-1 asymptomatic carriers [1]. These findings may reflect an adverse intracellular environment, caused by genetic stress due to viral particles inserted into host DNA. Intracellular mechanisms aiming to control and prevent replication of cells carrying genetic aberrations in genes involved in cell cycle regulation, DNA repair and apoptosis could be activated to hold cell division while DNA damage is repaired. Based on this hypothesis, delay of cell cycle in G0/G1 may be a step in the process of oncogenesis [1]. Herein, we did a pilot study in order to analyze the pattern of expression of a set of genes involved in cell cycle control and senescence in CD4+ T-lymphocytes of HTLV-1 infected individuals searching for additional genetic abnormalities in this setting. Methods: Peripheral blood samples were tested obtained from five HTLV-1 asymptomatic carriers and four ATLL patients for this pilot study. T-CD4+ cells were isolated in magnetic column followed by RNA extraction. Subsequently, it was converted into cDNA for the assays of the real-time quantitative polymerase chain reaction (qRT-PCR) in microplates of 96 wells previously customized with 44 senescence related genes pursued from ThermoFischer Scientific. A pool of five samples from healthy individuals (control group) was used as a calibrator. RNA transcription was measured using 7500 Fast real time PCR system (Applied Biosystems) and data were collected by the 7500 software v2.0.5 (Applied Biosystems). The expression levels of the target genes were calculated using the Livak and Schmittgen (2001) method [2]. The mRNA expression was normalized using the β-glucuronidase (GUSB) gene (Applied Biosystems, cod. Hs99999908_m1) and those genes with expression ≥ 2x or ≤ 2x in comparison to control group were considered as differentially expressed and were chosen to be validated in a secondary cohort of thirty HTLV-1 infected individuals. Results: In HTLV-1 asymptomatic carriers the median age was 55 years (37-62 years) and 20% (1/5) of them were males, in ATLL patients: 45 years (38-66 years) and 100% (4/4) were females, while the contr