La leucemie/lymphome T de l’adulte (ATL) est la premiere maladie humaine maligne associee a une infection chronique par un retrovirus, HTLV-1. Elle se manifeste chez environ 5 % des 10 a 20 millions de personnes infectees, apres une longue periode de latence. L’ATL a un pronostic effroyable avec une mediane de survie de moins d’un an, du fait d’une resistance a la chimiotherapie et d’un deficit immunitaire profond. L’oncoproteine virale, Tax, est un acteur important a l’origine de l’ATL et qui agit en interferant avec la proliferation cellulaire, le cycle cellulaire, l’apoptose ainsi que la reparation de l’ADN. La classification de Shimoyama decrit quatre formes cliniques (aigue, lymphome, chronique, et smoldering). Dans les formes agressives (aigue et lymphome), les essais cliniques, principalement effectues au Japon, ont demontre que des associations de chimiotherapies peuvent induire des taux de reponse acceptables dans l’ATL lymphome mais pas dans la forme aigue. Cependant, le pronostic a long terme reste mediocre dans les deux cas, en raison d’un taux de rechute eleve. De meme, les formes dites indolentes (smoldering et chronique) gardent un mauvais pronostic qu’elles soient prises en charge sous la forme d’une observation sans traitement watch and wait ou traitees par chimiotherapie. Nous avons recemment realise une meta-analyse internationale qui a montre que l’association de deux agents antiviraux, la zidovudine et l’interferon-alpha, est tres efficace dans les formes leucemiques d’ATL et doit etre consideree comme le traitement standard de premiere ligne, dans ce cadre. Cette association a change l’histoire naturelle de la maladie et a induit une amelioration significative de la survie a long terme des patients atteints de l’ATL chronique ou smoldering ainsi que d’un sous-ensemble de patients atteints d’ATL aigue. Les patients atteints de la forme lymphomateuse beneficient toujours d’une chimiotherapie d’induction, en association simultanee ou sequentielle, avec une therapie antivirale a base de zidovudine et d’interferon-alpha. L’allogreffe de cellules souches hematopoietiques reste une voie prometteuse et potentiellement curative mais pour un faible nombre de malades. De nouveaux medicaments tels que le trioxyde d’arsenic associe a l’interferon-alpha ou les anticorps monoclonaux tels que l’anti-CXCR4 ont montre des resultats prometteurs.
Adult T-cell leukemia-lymphoma (ATL), is a highly malignant T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1), characterized by a poor prognosis. Two viral proteins, Tax-1 and HBZ play important roles in the pathogenesis of ATL. While Tax-1 can be found in both cytoplasm and nucleus of HTLV-1 infected patients, HBZ is exclusively localized in the cytoplasm of HTLV-1 asymptomatic carriers and patients with chronic neurologic disease HAM/TSP, and only in the nucleus of ATL cell lines, suggesting that the nuclear localization of HBZ can be a hallmark of neoplastic transformation. To clarify this crucial point, here we investigated in detail the pattern of HBZ expression in ATL patients. We made use of our monoclonal antibody 4D4-F3, that at present is a uniquely reported reagent, among the few described, able to detect endogenous HBZ by immunofluorescence and confocal microscopy in cells from asymptomatic carriers, HAM/TSP and ATL patients.
We found that HBZ localizes both in the cytoplasm and in the nucleus of cells of ATL patients irrespective of their clinical status, with a strong preference for the cytoplasmic localization. Also Tax-1 localized in both compartments. As HBZ is exclusively localized in the cytoplasm in asymptomatic carriers and in non-neoplastic pathologies, this finding shows that neoplastic transformation consequent to HTLV-1 infection is accompanied and associated with the capacity of HBZ to translocate to the nucleus, which suggests a role of cytoplasmic-to-nuclear translocation in HTLV-1-mediated oncogenesis
The combined application of linear amplification-mediated PCR (LAM-PCR) protocols with next-generation sequencing (NGS) has had a large impact on our understanding of retroviral pathogenesis. Previously, considerable effort has been expended to optimize NGS methods to explore the genome-wide distribution of proviral integration sites and the clonal architecture of clinically important retroviruses like human T-cell leukemia virus type-1 (HTLV-1). Once sequencing data are generated, the application of rigorous bioinformatics analysis is central to the biological interpretation of the data. To better exploit the potential information available through these methods, we developed an optimized bioinformatics pipeline to analyze NGS clonality datasets. We found that short-read aligners, specifically designed to manage NGS datasets, provide increased speed, significantly reducing processing time and decreasing the computational burden. This is achieved while also accounting for sequencing base quality. We demonstrate the utility of an additional trimming step in the workflow, which adjusts for the number of reads supporting each insertion site. In addition, we developed a recall procedure to reduce bias associated with proviral integration within low complexity regions of the genome, providing a more accurate estimation of clone abundance. Finally, we recommend the application of a "clean-and-recover" step to clonality datasets generated from large cohorts and longitudinal studies. In summary, we report an optimized bioinformatics workflow for NGS clonality analysis and describe a new set of steps to guide the computational process. We demonstrate that the application of this protocol to the analysis of HTLV-1 and bovine leukemia virus (BLV) clonality datasets improves the quality of data processing and provides a more accurate definition of the clonal landscape in infected individuals. The optimized workflow and analysis recommendations can be implemented in the majority of bioinformatics pipelines developed to analyze LAM-PCR-based NGS clonality datasets.
Mature T-cell neoplasms (MTCN) are heterogeneous diseases with dismal prognosis. Differentiating between the many entities requires specialized pathology expertise, and studies show up to 30% of minor or major diagnostic reclassifications following expert review of cases. Assay for transposase-accessible chromatin sequencing (ATAC-seq) is a simple technique to profile open chromatin regions, which has been shown to be highly discriminative for clustering solid tumors and acute myeloid leukemias. We applied ATAC-seq to MTCN to explore the epigenetic landscape of these different entities, and built a predictive model to aid in diagnosis. FACS-sorted tumor cells from primary MTCN samples and 50µm sections of frozen tumor tissue from the French TENOMIC T-cell lymphoma consortium were processed according to previously published FAST- and OMNI-ATAC protocols, respectively. In parallel, we applied FAST-ATAC to several normal T and NK cell subsets sorted from PBMC or lymph node suspensions of healthy donors. Sequencing data were processed by an adapted version of the ENCODE ATAC-seq pipeline using a custom Hg38 genome version including HTLV1 sequence. A total of 678 normal and tumor samples were sequenced to provide a comprehensive landscape of chromatin accessibility in MTCN. Unsupervised clustering of normal NK and T cell subtypes (N = 49) and sorted tumoral lymphoma cells (N = 104) confirmed that AITL are derived from TFH cells, HSTL and LGL are closely segregated with NK and gd-T cells. We also found that T-PLL likely derive from the transformation of naïve T cells. Epigenetic profiling by ATAC-seq of FACS-sorted tumor samples resulted in a complete segregation of the known MTCN entities (TFH, ALK+ and ALK- ALCL, HSTL, CTCL, ATLL, LGL and T-PLL). Most PTCL-NOS (13/17) clustered with a predefined MTCN subtype (mainly AITL/TFH-phenotype PTCL, CTCL and lymphomas exhibiting cytotoxic features), showing that this waste basket subgroup is merely virtual, at least from an epigenetic point of view. Using unsupervised deconvolution approaches, we were able to discriminate different MTCN subtypes from 223 processed frozen bulk samples. All known MTCN subtypes were identified by ATAC-seq but a novel subset of PTCL-NOS representing ∼5% of cases was pinpointed, showing high GATA3 transcription factor activity. Subsequent exome sequencing revealed numerous copy number alterations and TP53 (8/12) and NCOR1 mutations (7/12). A support vector machine model was trained to predict diagnosis and showed accurate classification performance by cross-validation and on external validation cohort collected from 5 tertiary care centers. ATAC-seq is a rapid and cost-effective technique with high classification accuracy for PTCL subtypes. Among GATA3-expressing PTCL that spread across multiple epigenetic subgroups, we identified a specific entity with recurrent NCOR1 mutations. Keywords: aggressive T-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, pathology and classification of lymphomas No conflicts of interests pertinent to the abstract.
