High-content multimodal analysis supports the IL-7/IL-7 receptor axis as a relevant therapeutic target in primary Sjögren's syndrome.

OBJECTIVE: While the involvement of IL-7/IL-7R axis in pSS has been described in relation to T cells, little is known about the contribution of this pathway in relationship with other immune cells, and its implication in autoimmunity. Using high-content multiomics data, we aimed at characterizing IL-7R expressing cells and the involvement of IL-7/IL-7R pathway in pSS pathophysiology. METHODS: An IL-7 signature established using RNA-sequencing of human PBMCs incubated with IL-7 was applied to 304 pSS patients, and on RNA-Seq datasets from tissue biopsies. High-content immunophenotyping using flow and imaging mass cytometry was developed to characterize peripheral and in situ IL-7R expression. RESULTS: We identified a blood 4-gene IL-7 module (IKZF4, KIAA0040, PGAP1 and SOS1) associated with anti-SSA/Ro positiveness in patients as well as disease activity, and a tissue 5-gene IL-7 module (IL7R, PCED1B, TNFSF8, ADAM19, MYBL1) associated with infiltration severity. We confirmed expression of IL-7R on T cells subsets, and further observed upregulation of IL-7R on double-negative (DN) B cells, and especially DN2 B cells. IL-7R expression was increased in pSS compared to sicca patients with variations seen according to the degree of infiltration. When expressed, IL-7R was mainly found on epithelial cells, CD4+ and CD8+ T cells, switched memory B cells, DN B cells and M1 macrophages. CONCLUSION: This exhaustive characterization of the IL-7/IL-7R pathway in pSS pathophysiology established that two IL-7 gene modules discriminate pSS patients with a high IL-7 axis involvement. Their use could guide the implementation of an anti-IL-7R targeted therapy in a precision medicine approach.

Deciphering the maturation of tertiary lymphoid structures in cancer and inflammatory diseases of the digestive tract using imaging mass cytometry.

Persistent inflammation can promote the development of tertiary lymphoid structures (TLS) within tissues resembling secondary lymphoid organs (SLO) such as lymph nodes (LN). The composition of TLS across different organs and diseases could be of pathophysiological and medical interest. In this work, we compared TLS to SLO in cancers of the digestive tract and in inflammatory bowel diseases. Colorectal and gastric tissues with different inflammatory diseases and cancers from the department of pathology of CHU Brest were analyzed based on 39 markers using imaging mass cytometry (IMC). Unsupervised and supervised clustering analyses of IMC images were used to compare SLO and TLS. Unsupervised analyses tended to group TLS per patient but not per disease. Supervised analyses of IMC images revealed that LN had a more organized structure than TLS and non-encapsulated SLO Peyer's patches. TLS followed a maturation spectrum with close correlations between germinal center (GC) markers' evolution. The correlations between organizational and functional markers made relevant the previously proposed TLS division into three stages: lymphoid-aggregates (LA) (CD20+CD21-CD23-) had neither organization nor GC functionality, non-GC TLS (CD20+CD21+CD23-) were organized but lacked GC's functionality and GC-like TLS (CD20+CD21+CD23+) had GC's organization and functionality. This architectural and functional maturation grading of TLS pointed to differences across diseases. TLS architectural and functional maturation grading is accessible with few markers allowing future diagnostic, prognostic, and predictive studies on the value of TLS grading, quantification and location within pathological tissues in cancers and inflammatory diseases.

Machine Learning for the Identification of a Common Signature for Anti-SSA/Ro 60 Antibody Expression Across Autoimmune Diseases.

OBJECTIVE: Anti-Ro autoantibodies are among the most frequently detected extractable nuclear antigen autoantibodies, mainly associated with primary Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), and undifferentiated connective tissue disease (UCTD). This study was undertaken to determine if there is a common signature for all patients expressing anti-Ro 60 autoantibodies regardless of their disease phenotype. METHODS: Using high-throughput multiomics data collected from the cross-sectional cohort in the PRECISE Systemic Autoimmune Diseases (PRECISESADS) study Innovative Medicines Initiative (IMI) project (genetic, epigenomic, and transcriptomic data, combined with flow cytometry data, multiplexed cytokines, classic serology, and clinical data), we used machine learning to assess the integrated molecular profiling of 520 anti-Ro 60+ patients compared to 511 anti-Ro 60- patients with primary SS, patients with SLE, and patients with UCTD, and 279 healthy controls. RESULTS: The selected clinical features for RNA-Seq, DNA methylation, and genome-wide association study data allowed for a clear distinction between anti-Ro 60+ and anti-Ro 60- patients. The different features selected using machine learning from the anti-Ro 60+ patients constituted specific signatures when compared to anti-Ro 60- patients and healthy controls. Remarkably, the transcript Z score of 3 genes (ATP10A, MX1, and PARP14), presenting with overexpression associated with hypomethylation and genetic variation and independently identified using the Boruta algorithm, was clearly higher in anti-Ro 60+ patients compared to anti-Ro 60- patients regardless of disease type. Our findings demonstrated that these signatures, enriched in interferon-stimulated genes, were also found in anti-Ro 60+ patients with rheumatoid arthritis and those with systemic sclerosis and remained stable over time and were not affected by treatment. CONCLUSION: Anti-Ro 60+ patients present with a specific inflammatory signature regardless of their disease type, suggesting that a dual therapeutic approach targeting both Ro-associated RNAs and anti-Ro 60 autoantibodies should be considered.

Identification of altered cell signaling pathways using proteomic profiling in stable and progressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by significant biologic and clinical heterogeneity. This study was designed to explore CLL B-cells' proteomic profile in order to identify biologic processes affected at an early stage and during disease evolution as stable or progressive. Purified B cells from 11 untreated CLL patients were tested at two time points by liquid chromatography-tandem mass spectrometry. Patients included in the study evolved to either progressive (n = 6) or stable disease (n = 5). First, at an early stage of the disease (Binet stage A), based on the relative abundance levels of 389 differentially expressed proteins (DEPs), samples were separated into stable and progressive clusters with the main differentiating factor being the RNA splicing pathway. Next, in order to test how the DEPs affect RNA splicing, a RNA-Seq study was conducted showing 4217 differentially spliced genes between the two clusters. Distinct longitudinal evolutions were observed with predominantly proteomic modifications in the stable CLL group and spliced genes in the progressive CLL group. Splicing events were shown to be six times more frequent in the progressive CLL group. The main aberrant biologic processes controlled by DEPs and spliced genes in the progressive group were cytoskeletal organization, Wnt/ß-catenin signaling, and mitochondrial and inositol phosphate metabolism with a downstream impact on CLL B-cell survival and migration. This study suggests that proteomic profiles at the early stage of CLL can discriminate progressive from stable disease and that RNA splicing dysregulation underlies CLL evolution, which opens new perspectives in terms of biomarkers and therapy.

A new molecular classification to drive precision treatment strategies in primary Sjögren's syndrome.

There is currently no approved treatment for primary Sjögren's syndrome, a disease that primarily affects adult women. The difficulty in developing effective therapies is -in part- because of the heterogeneity in the clinical manifestation and pathophysiology of the disease. Finding common molecular signatures among patient subgroups could improve our understanding of disease etiology, and facilitate the development of targeted therapeutics. Here, we report, in a cross-sectional cohort, a molecular classification scheme for Sjögren's syndrome patients based on the multi-omic profiling of whole blood samples from a European cohort of over 300 patients, and a similar number of age and gender-matched healthy volunteers. Using transcriptomic, genomic, epigenetic, cytokine expression and flow cytometry data, combined with clinical parameters, we identify four groups of patients with distinct patterns of immune dysregulation. The biomarkers we identify can be used by machine learning classifiers to sort future patients into subgroups, allowing the re-evaluation of response to treatments in clinical trials.

A genome database for a Japanese population of the larvacean Oikopleura dioica.

The larvacean Oikopleura dioica is a planktonic chordate and is a tunicate that belongs to the closest relatives to vertebrates. Its simple and transparent body, invariant embryonic cell lineages, and short life cycle of 5 days make it a promising model organism for the study of developmental biology. The genome browser OikoBase was established in 2013 using Norwegian O. dioica. However, genome information for other populations is not available, even though many researchers have studied local populations. In the present study, we sequenced using Illumina and PacBio RSII technologies the genome of O. dioica from a southwestern Japanese population that was cultured in our laboratory for 3 years. The genome of Japanese O. dioica was assembled into 576 scaffold sequences with a total length and N50 length of 56.6 and 1.5 Mb, respectively. A total of 18,743 gene models (transcript models) were predicted in the genome assembly, named OSKA2016. In addition, 19,277 non-redundant transcripts were assembled using RNA-seq data. The OSKA2016 has global sequence similarity of only 86.5% when compared with the OikoBase, highlighting the sequence difference between the two far distant O. dioica populations on the globe. The genome assembly, transcript assembly, and transcript models were incorporated into ANISEED (https://www.aniseed.cnrs.fr/) for genome browsing and BLAST searches. Mapping of reads obtained from male- or female-specific genome libraries yielded male-specific scaffolds in the OSKA2016 and revealed that over 2.6 Mb of sequence were included in the male-specific Y-region. The genome and transcriptome resources from two distinct populations will be useful datasets for developmental biology, evolutionary biology, and molecular ecology using this model organism.

Epigenetics in Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (SjS) is a chronic and systemic autoimmune epithelitis with predominant female incidence, which is characterized by exocrine gland dysfunction. Incompletely understood, the etiology of SjS is multi-factorial and evidence is growing to consider that epigenetic factors are playing a crucial role in its development. Independent from DNA sequence mutations, epigenetics is described as inheritable and reversible processes that modify gene expression. Epigenetic modifications reported in minor salivary gland and lymphocytes from SjS patients are related to (i) an abnormal DNA methylation process inducing in turn defective control of normally repressed genes involving such matters as autoantigens, retrotransposons, and the X chromosome in women; (ii) altered nucleosome positioning associated with autoantibody production; and (iii) altered control of microRNA. Results from epigenome-wide association studies have further revealed the importance of the interferon pathway in disease progression, the calcium signaling pathway for controlling fluid secretions, and a cell-specific cross talk with risk factors associated with SjS. Importantly, epigenetic modifications are reversible thus opening opportunities for therapeutic procedures in this currently incurable disease.

JAK Inhibitors Suppress Innate Epigenetic Reprogramming: a Promise for Patients with Sjögren's Syndrome.

Pathogenesis of primary Sjögren's syndrome (SjS) remains obscure. However, recent data demonstrate the implication of epigenetic alterations in the DNA methylation/hydroxymethylation process in SjS mostly affecting genes regulated by two innate cytokines, interferon α (IFNα) and IFNγ as well as the oxidative stress pathways. The Janus kinase (JAK) signal transducer and activator of transcription (STAT) pathway is known to be activated by IFN and reactive oxygen species (ROS). This prompts us to test the potential implication of JAK/STAT signaling on DNA methylation/hydroxymethylation alterations in SjS. For this purpose, the human salivary gland (HSG) cell line was used and cells were treated with both types of IFNs and H2O2 to mimic activated salivary gland epithelial cells (SGEC) as observed in SjS patients. Afterwards, the global DNA level of methylcytosine and hydroxymethylcytosine, the expression of the DNA methylating enzymes (DNMTs) and ten-eleven translocation (TETs) methyl cytosine dioxygenase that controls DNA hydroxymethylation, both at transcriptional and at protein level, as well as STAT phosphorylation and ROS status were determined. Our results showed that expression of TET3 and in turn global DNA hydroxymethylation is controlled through the induction of STAT3 mediated by IFNα, IFNγ, and H2O2. On the other hand, treatment with JAK inhibitors (AG490 and ruxolitinib) reverses this process, suggesting a novel treatment pathway for patients with autoimmune diseases and Sjögren's syndrome.

ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates.

ANISEED (https://www.aniseed.cnrs.fr) is the main model organism database for the worldwide community of scientists working on tunicates, the vertebrate sister-group. Information provided for each species includes functionally-annotated gene and transcript models with orthology relationships within tunicates, and with echinoderms, cephalochordates and vertebrates. Beyond genes the system describes other genetic elements, including repeated elements and cis-regulatory modules. Gene expression profiles for several thousand genes are formalized in both wild-type and experimentally-manipulated conditions, using formal anatomical ontologies. These data can be explored through three complementary types of browsers, each offering a different view-point. A developmental browser summarizes the information in a gene- or territory-centric manner. Advanced genomic browsers integrate the genetic features surrounding genes or gene sets within a species. A Genomicus synteny browser explores the conservation of local gene order across deuterostome. This new release covers an extended taxonomic range of 14 species, including for the first time a non-ascidian species, the appendicularian Oikopleura dioica. Functional annotations, provided for each species, were enhanced through a combination of manual curation of gene models and the development of an improved orthology detection pipeline. Finally, gene expression profiles and anatomical territories can be explored in 4D online through the newly developed Morphonet morphogenetic browser.

Linking genetic variation with epigenetic profiles in Sjögren's syndrome.

DNA methylation represents an important regulatory event governing gene expression that is dysregulated in Sjögren's syndrome (SjS) and a number of autoimmune/inflammatory diseases. As disease-associated single-nucleotide polymorphisms (SNPs) have relevance in controlling DNA methylation, 94 non-HLA SjS-SNPs were investigated, among them 57 (60.6%) with widespread effects on 197 individual DNA methylation quantitative trait loci (meQTL) were selected. Typically, these SNPs are intronic, possess an active promoter histone mark, and control cis-meQTLs located around transcription start sites. Interplay is independent of the physical distance between SNPs and meQTLs. Using epigenome-wide association study datasets, SjS-meQTLs were characterized (41 genes and 13 DNA methylation CpG motifs) and for the most part map to a pro-inflammatory cytokine pathway, which is important for the control of DNA methylation in autoimmune diseases. In conclusion, exploring meQTLs represents a valuable tool to predict and investigate downstream effects of genetic factors in complex diseases such as SjS.

JAK Inhibitors and Oxidative Stress Control.

Primary Sjögren's syndrome (SjS) is a complex autoimmune epithelitis, with few treatment options, but the use of Janus kinase (JAK) inhibitors is promising because suppression of the JAK/signal transducer and activator of transcription (STAT) pathway improves sicca manifestations. Playing a primary and pathogenic role in disease development, the oxidative stress response is upregulated in activated salivary gland epithelial cells (SGECs) from patients with SjS. Therefore, the aim of this study was to investigate whether JAK inhibitors would suppress SGEC activation in response to an oxidative stress. For this purpose, the human salivary gland (HSG) cell line was used, and cells were treated with the reactive oxygen species (ROS) inducer hydrogen peroxide (H2O2) or with interferons (IFN Type I and Type II), used as positive controls, to mimic activated SGECs as observed in SjS patients. Afterward, the levels of the intracellular adhesion molecule-1 (ICAM-1) and the regulatory programmed-death ligand-1 (PD-L1) were measured by real-time PCR and flow cytometry, and the STAT1/3 phosphorylation status was assessed by Western blotting. Using the HSG cell line, our results showed that both ICAM-1 and PD-L1 are induced by ROS through pSTAT3, and that this activation pathway is reversed by the use of JAK inhibitors, AG490 and ruxolitinib, as well as by N-acetylcysteine, which is a direct inhibitor of ROS. These findings open new perspectives regarding the pathogenesis and therapeutic possibilities for SjS.

The regulatory capacity of B cells directs the aggressiveness of CLL.

Chronic lymphocytic leukemia (CLL) is associated with abnormal T-cell responses responsible for defective anti-tumor activities. Intriguingly, CLL B cells share phenotypical characteristics with regulatory B (Breg) cells suggesting that they might negatively control the T-cell activation and immune responses. We elaborated an in vitro co-culture system with T cells to evaluate the Breg capacities of CLL B cells following innate Toll-like receptor 9 (TLR9) engagement. We demonstrated that B cells from half of the patients exhibited regulatory capacities, whilst B cells from the remaining patients were unable to develop a Breg function. The T cell sensitivities of all patients were normal suggesting that defective Breg activities were due to intrinsic CLL B cell deficiencies. Thus, TLR-dedicated gene assays highlighted differential signature of the TLR9 negative regulation pathway between the two groups of patients. Furthermore, correlations of the doubling time of lymphocytosis, the time to first treatment, the mutational status of IgVH and the Breg functions indicate that patients with efficient Breg activities have more aggressive CLL than patients with defective Breg cells. Our in vitro observations may open new approaches for adjusting therapeutic strategies targeting the Breg along with the evolution of the disease.

Evolution of embryonic cis-regulatory landscapes between divergent Phallusia and Ciona ascidians.

Ascidian species of the Phallusia and Ciona genera are distantly related, their last common ancestor dating several hundred million years ago. Although their genome sequences have extensively diverged since this radiation, Phallusia and Ciona species share almost identical early morphogenesis and stereotyped cell lineages. Here, we explored the evolution of transcriptional control between P. mammillata and C. robusta. We combined genome-wide mapping of open chromatin regions in both species with a comparative analysis of the regulatory sequences of a test set of 10 pairs of orthologous early regulatory genes with conserved expression patterns. We find that ascidian chromatin accessibility landscapes obey similar rules as in other metazoa. Open-chromatin regions are short, highly conserved within each genus and cluster around regulatory genes. The dynamics of chromatin accessibility and closest-gene expression are strongly correlated during early embryogenesis. Open-chromatin regions are highly enriched in cis-regulatory elements: 73% of 49 open chromatin regions around our test genes behaved as either distal enhancers or proximal enhancer/promoters following electroporation in Phallusia eggs. Analysis of this datasets suggests a pervasive use in ascidians of "shadow" enhancers with partially overlapping activities. Cross-species electroporations point to a deep conservation of both the trans-regulatory logic between these distantly-related ascidians and the cis-regulatory activities of individual enhancers. Finally, we found that the relative order and approximate distance to the transcription start site of open chromatin regions can be conserved between Ciona and Phallusia species despite extensive sequence divergence, a property that can be used to identify orthologous enhancers, whose regulatory activity can partially diverge.

Convergent Acquisition of Nonembryonic Development in Styelid Ascidians.

Asexual propagation and whole body regeneration are forms of nonembryonic development (NED) widespread across animal phyla and central in life history and evolutionary diversification of metazoans. Whereas it is challenging to reconstruct the gains or losses of NED at large phylogenetic scale, comparative studies could benefit from being conducted at more restricted taxonomic scale, in groups for which phylogenetic relationships are well established. The ascidian family of Styelidae encompasses strictly sexually reproducing solitary forms as well as colonial species that combine sexual reproduction with different forms of NED. To date, the phylogenetic relationships between colonial and solitary styelids remain controversial and so is the pattern of NED evolution. In this study, we built an original pipeline to combine eight genomes with 18 de novo assembled transcriptomes and constructed data sets of unambiguously orthologous genes. Using a phylogenomic super-matrix of 4,908 genes from these 26 tunicates we provided a robust phylogeny of this family of chordates, which supports two convergent acquisitions of NED. This result prompted us to further describe the budding process in the species Polyandrocarpa zorritensis, leading to the discovery of a novel mechanism of asexual development. Whereas the pipeline and the data sets produced can be used for further phylogenetic reconstructions in tunicates, the phylogeny provided here sets an evolutionary framework for future experimental studies on the emergence and disappearance of complex characters such as asexual propagation and whole body regeneration.

ANISEED 2017: extending the integrated ascidian database to the exploration and evolutionary comparison of genome-scale datasets.

ANISEED (www.aniseed.cnrs.fr) is the main model organism database for tunicates, the sister-group of vertebrates. This release gives access to annotated genomes, gene expression patterns, and anatomical descriptions for nine ascidian species. It provides increased integration with external molecular and taxonomy databases, better support for epigenomics datasets, in particular RNA-seq, ChIP-seq and SELEX-seq, and features novel interactive interfaces for existing and novel datatypes. In particular, the cross-species navigation and comparison is enhanced through a novel taxonomy section describing each represented species and through the implementation of interactive phylogenetic gene trees for 60% of tunicate genes. The gene expression section displays the results of RNA-seq experiments for the three major model species of solitary ascidians. Gene expression is controlled by the binding of transcription factors to cis-regulatory sequences. A high-resolution description of the DNA-binding specificity for 131 Ciona robusta (formerly C. intestinalis type A) transcription factors by SELEX-seq is provided and used to map candidate binding sites across the Ciona robusta and Phallusia mammillata genomes. Finally, use of a WashU Epigenome browser enhances genome navigation, while a Genomicus server was set up to explore microsynteny relationships within tunicates and with vertebrates, Amphioxus, echinoderms and hemichordates.

Combining cytogenetic and epigenetic approaches in chronic lymphocytic leukemia improves prognosis prediction for patients with isolated 13q deletion.

Background: Both defective DNA methylation and active DNA demethylation processes are emerging as important risk factors in chronic lymphocytic leukemia (CLL). However, associations between 5-cytosine epigenetic markers and the most frequent chromosomal abnormalities detected in CLL remain to be established. Methods: CLL patients were retrospectively classified into a cytogenetic low-risk group (isolated 13q deletion), an intermediate-risk group (normal karyotype or trisomy 12), and a high-risk group (11q deletion, 17p deletion, or complex karyotype [≥ 3 breakpoints]). The two 5-cytosine derivatives, 5-methylcytosine (5-mCyt) and 5-hydroxymethylcytosine (5-hmCyt), were tested by ELISA (n = 60), while real-time quantitative PCR was used for determining transcriptional expression levels of DNMT and TET (n = 24). Results: By using global DNA methylation/demethylation levels, in the low-risk disease group, two subgroups with significantly different clinical outcomes have been identified (median treatment-free survival [TFS] 45 versus > 120 months for 5-mCyt, p = 0.0008, and 63 versus > 120 months for 5-hmCyt, p = 0.04). A defective 5-mCyt status was further associated with a higher percentage of 13q deleted nuclei (> 80%), thus suggesting an acquired process. When considering the cytogenetic intermediate/high-risk disease groups, an association of 5-mCyt status with lymphocytosis (p = 0.0008) and the lymphocyte doubling time (p = 0.04) but not with TFS was observed, as well as a reduction of DNMT3A, TET1, and TET2 transcripts. Conclusions: Combining cytogenetic studies with 5-mCyt assessment adds accuracy to CLL patients' prognoses and particularly for those with 13q deletion as a sole cytogenetic abnormality.

Control of nerve cord formation by Engrailed and Gooseberry-Neuro: A multi-step, coordinated process.

One way to better understand the molecular mechanisms involved in the construction of a nervous system is to identify the downstream effectors of major regulatory proteins. We previously showed that Engrailed (EN) and Gooseberry-Neuro (GsbN) transcription factors act in partnership to drive the formation of posterior commissures in the central nervous system of Drosophila. In this report, we identified genes regulated by both EN and GsbN through chromatin immunoprecipitation ("ChIP on chip") and transcriptome experiments, combined to a genetic screen relied to the gene dose titration method. The genomic-scale approaches allowed us to define 175 potential targets of EN-GsbN regulation. We chose a subset of these genes to examine ventral nerve cord (VNC) defects and found that half of the mutated targets show clear VNC phenotypes when doubly heterozygous with en or gsbn mutations, or when homozygous. This strategy revealed new groups of genes never described for their implication in the construction of the nerve cord. Their identification suggests that, to construct the nerve cord, EN-GsbN may act at three levels, in: (i) sequential control of the attractive-repulsive signaling that ensures contralateral projection of the commissural axons, (ii) temporal control of the translation of some mRNAs, (iii) regulation of the capability of glial cells to act as commissural guideposts for developing axons. These results illustrate how an early, coordinated transcriptional control may orchestrate the various mechanisms involved in the formation of stereotyped neuronal networks. They also validate the overall strategy to identify genes that play crucial role in axonal pathfinding.

Alterations in DNA methylation/demethylation intermediates predict clinical outcome in chronic lymphocytic leukemia.

Cytosine derivative dysregulations represent important epigenetic modifications whose impact on the clinical outcome in chronic lymphocytic leukemia (CLL) is incompletely understood. Hence, global levels of 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) and 5-hydroxymethyluracil were tested in purified B cells from CLL patients (n = 55) and controls (n = 17). The DNA methylation 'writers' (DNA methyltransferases [DNMT1/3A/3B]), 'readers' (methyl-CpG-binding domain [MBD2/4]), 'editors' (ten-eleven translocation [TET1/2/3]) and 'modulators' (SAT1) were also evaluated. Accordingly, patients were stratified into three subgroups. First, a subgroup with a global deficit in cytosine derivatives characterized by hyperlymphocytosis, reduced median progression free survival (PFS = 52 months) and shorter treatment free survival (TFS = 112 months) was identified. In this subgroup, major epigenetic modifications were highlighted including a reduction of 5-mCyt, 5-hmCyt, 5-CaCyt associated with DNMT3A, MBD2/4 and TET1/2 downregulation. Second, the cytosine derivative analysis revealed a subgroup with a partial deficit (PFS = 84, TFS = 120 months), mainly affecting DNA demethylation (5-hmCyt reduction, SAT1 induction). Third, a subgroup epigenetically similar to controls was identified (PFS and TFS > 120 months). The prognostic impact of stratifying CLL patients within three epigenetic subgroups was confirmed in a validation cohort. In conclusion, our results suggest that dysregulations of cytosine derivative regulators represent major events acquired during CLL progression and are independent from IGHV mutational status.