Aggressive Chronic Lymphocytic Leukemia Research Articles

Untargeted metabolomics identifies metabolic dysregulation of sphingolipids associated with aggressive chronic lymphocytic leukaemia and poor survival.

Metabolic dependencies of chronic lymphocytic leukaemia (CLL) cells may represent new personalized treatment approaches in patients harbouring unfavourable features. Here, we used untargeted metabolomics and lipidomics analyses to isolate metabolomic features associated with aggressive CLL and poor survival outcomes. We initially focused on profiles associated with overexpression of the adverse metabolic marker glycosyltransferase (UGT2B17) associated with poor survival and drug resistance. Leukaemic B-cell metabolomes indicated a significant perturbation in lipids, predominantly bio-active sphingolipids. Expression of numerous enzyme-encoding genes of sphingolipid biosynthesis pathways was significantly associated with shorter patient survival. Targeted metabolomics further exposed higher circulating levels of glucosylceramides (C16:0 GluCer) in CLL patients relative to healthy donors and an aggressive cancer biology. In multivariate analyses, C16:0 GluCer and sphinganine were independent prognostic markers and were inversely linked to treatment-free survival. These two sphingolipid species function as antagonistic mediators, with sphinganine being pro-apoptotic and GluCer being pro-proliferative, tested in leukemic B-CLL cell models. Blocking GluCer synthesis using ceramide glucosyltransferase inhibitors induced cell death and reduced the proliferative phenotype, which further sensitized a leukaemic B-cell model to the anti-leukaemics fludarabine and ibrutinib in vitro. Specific sphingolipids may serve as prognostic markers in CLL, and inhibiting enzymatic pathways involved in their biosynthesis has potential as a therapaeutic approach.

CD49d Shapes B Cell Receptor Responsiveness and Tissue Distribution in an Aggressive Chronic Lymphocytic Leukemia Murine Model

Background: High expression of the very late antigen-4 (VLA-4) integrin subunit CD49d predicts disease progression in chronic lymphocytic leukemia (CLL). We previously demonstrated that B cell receptor (BCR) signals induce activation of VLA-4 (Tissino et al., J Exp Med. 2018) and characterized Eµ-TCL1-transgenic (TCL1-tg) mice as an adequate model for the human high-risk CD49d-high CLL patient subgroup, with VLA-4 inhibition hampering the disease (Szenes et al., Leukemia 2020). Here, we address the consequences of genetic B cell-specific ablation of CD49d on disease development, engraftment capacity and treatment susceptibility in a novel CLL mouse model. Methods: We crossed ITGA4fl/fl mice, kindly provided by T. Papayannopoulou, University of Washington, with TCL1-tg mice and CD19 Cre mice, subsequently named CD49d∆B TCL1-tg. Tumor development was continuously monitored by regular blood checks and mice were sacrificed at 10 months. Tumor infiltration was quantified in peritoneal cavity (PC), spleen and bone marrow by flow cytometry using anti-CD5/CD19 antibodies. Integrin and chemokine receptor expression was profiled. In vitro responsiveness to BCR stimulation of CD49d-deficient and proficient leukemic cells was assessed using phospho-specific flow cytometry. Furthermore, purified leukemic cells derived from PC or spleen were subjected to RNA-seq analyses. CD49d-deficient or proficient leukemic cells were transplanted into wild-type C57BL/6J recipients in short and long term experiments. Ongoing: CD49d-deficient or proficient leukemic cells were transplanted competitively into C57BL/6J recipients and are receiving continuous Bruton's tyrosine kinase (BTK)-inhibitor (acalabrutinib) treatment upon tumor onset. Results: CD49d∆B TCL1-tg mice showed a B cell-specific integrin loss in all organs. Blood tumor burden was measured monthly. At months seven and eight, leukemic cells of CD49d∆B TCL1-tg mice were reduced by approx. 25% compared to TCL1-tg littermates (Fig. 1A). This trend was reversed at month nine, with approx. 45% increased lymphocytosis in CD49d∆B TCL1-tg mice. CD49d expression in leukemic cells of TCL1-tg mice was unaffected by disease progression. At sacrifice, CD49d∆B TCL1-tg mice showed an 80% ( p=<0.0001) reduction of tumor burden in the bone marrow (Fig. 1B), but comparable leukemic cell numbers in PC and spleen. Leukemic splenocytes derived from CD49d∆B TCL1-tg mice displayed 38% ( p=0.032) higher IgM and 33% higher CD44 expression but 43% ( p=0.015) reduced expression of CXCR4. BCR signaling was modulated by the integrin loss and BCR stimulation with anti-IgM induced 43% ( p=0.022) higher PLCγ2 and 45% ( p=0.022) higher SYK phosphorylation in leukemic cells derived from CD49d∆B TCL1-tg mice compared to littermates, but comparable BTK phosphorylation. RNA-seq analysis revealed strong differences in leukemic cell transcriptomes between PC and spleen, indicating tissue dependent effects on leukemic cells. Particularly, TNF-α signaling via NF-κB, MTORC1, MAPK signaling and JAK-STAT signaling pathways were upregulated in leukemic cells derived from spleen compared to PC irrespective of CD49d expression, while chemokine signaling pathways were downregulated in PC. When comparing CD49d deficient and proficient leukemic cells, the gene set MYC targets v1 was enriched in deficient cells. When injecting CD49d-deficient leukemic splenocytes intravenously, more cells were recovered after 5 days from blood ( p=0.048) and spleen compared to CD49d-proficient cells, but did not engraft in bone marrow or peritoneum. When injected intraperitonally, CD49d-deficient leukemic cells were found in peritoneum in contrast to TCL1-tg littermates ( p=<0.0001). Conclusion: The TCL1 mouse model resembles a human high-risk CLL (e.g. IGHV unmutated, CD49d-expressing), and thus allows to investigate the impact of CD49d expression in this CLL subset. Loss of CD49d expression resulted in an altered redistribution of leukemic cells in tissue sites with a significant reduction of leukemic cells in the bone marrow. Lack of integrin signals resulted in increased antigen-induced BCR signaling and IgM expression, as well as a B cell receptor activation phenotype associated with MYC gene set enrichment, indicating a feedback loop from integrins to the BCR. Currently, the susceptibility of CD49d∆B TCL1-tg mice to BTK inhibitors is under investigation.

Characterization of Three Somatic Mutations in the 3'UTR of RRAS2 and Their Inverse Correlation with Lymphocytosis in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by progressive accumulation of a rare population of CD5+ B-lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. CLL exhibits remarkable clinical heterogeneity, with some patients presenting with indolent disease and others progressing rapidly to aggressive CLL. The significant heterogeneity of CLL underscores the importance of identifying novel prognostic markers. Recently, the RAS-related gene RRAS2 has emerged as both a driver oncogene and a potential marker for CLL progression, with higher RRAS2 expression associated with poorer disease prognosis. Although missense somatic mutations in the coding sequence of RRAS2 have not been described in CLL, this study reports the frequent detection of three somatic mutations in the 3' untranslated region (3'UTR) affecting positions +26, +53, and +180 downstream of the stop codon in the mRNA. An inverse relationship was observed between these three somatic mutations and RRAS2 mRNA expression, which correlated with lower blood lymphocytosis. These findings highlight the importance of RRAS2 overexpression in CLL development and prognosis and point to somatic mutations in its 3'UTR as novel mechanistic clues. Our results may contribute to the development of targeted therapeutic strategies and improved risk stratification for CLL patients.

CLL Cells within the Proliferation Centers of the Patient Lymph Nodes Are Enriched for Notch Signaling, Thus NOTCH a Viable Target for CLL Therapy

CLL cells within the Proliferation Centers of the Patient Lymph Nodes are enriched for Notch signaling, thus a Target for CLL Therapy. Shantaram S. Joshi, Michael Pitner, Matt Kling, Furqan Mehdi, Mamta Shukla, Ashima Shukla, Vipul Shukla, Nagendra Chaturvedi, Hamid Band , Javeed Iqbal The tissue microenvironment, particularly the lymph node (LN), plays a major role in regulating the leukemic progression and clinical heterogeneity of chronic lymphocytic leukemia (CLL). CLL cells proliferate in the patient lymph nodes, resulting in the formation of proliferation centers (PCs). The molecular basis of the CLL cell proliferation within PCs is not well known. Several reports have implicated the activated Notch signaling pathway in the pathogenesis of CLL. Using the IRF4 -/-Vh11 mouse model of CLL, we have previously shown that Notch signaling is indispensable for CLL development (Vipul Shukla et al, Oncotarget, 2016). Based on these studies, we hypothesized that CLL cell proliferation in the lymph node PCs involves activated NOTCH signaling. To this end, we have examined the expression levels of Notch signaling associated molecules namely IRF4, Notch 1, Notch 2, Nedd4, Fbxw7 and Itch1 on CLL cells within the patient lymph node proliferation centers (n=12) using immunohistochemistry and western blotting. Expression levels were scored based on the percentage of cells with positive staining: 5+ (>80%), 4+ (60-80%), 3+ (40-60%), 2+ (20-40%), 1+ (<20%) and 0 (No staining). IHC analyses showed that the expression levels of IRF4 to be 2+, Notch 1 was 4+, Notch 2 was -3 to 4+, Nedd4 was 1+, Fbxw7 was 3+ and ITCH1 was 1+. Western blotting of CLL cells from the lymph node proliferation centers (n=6) confirmed a similar pattern of expression of the Notch-associated genes. Together, these results validate the association of Notch signaling in CLL cells of the patient lymph node PCs similar to CLL cells from the IRF4 -/-Vh11 mouse model. Therefore, as a next logical step we treated the MEC-1 and OSU-CLL cells in culture and primary CLL cell from patients with NOTCH inhibitor RO4929097 (RO), a γ-secretase inhibitor for 24, 48 and 72 hours in vitro. There was a significant decrease in the expression NOTCH 1, NOTCH 2 in MEC-1, OSU-CLL and primary CLL cells determined using western blot technique. Also, we observed a significant decrease in proliferation of CLL cells from MEC-1 and OSU-CLL cell lines, following treatment with NOTCH inhibitor RO. In addition, NOTCH inhibitor combined with Bruton's Tyrosine Kinase (BTK) a member of the B-Cell Receptor signaling, showed a further significant decrease in the proliferation of CLL cells in culture. Together these experiments demonstrated that NOTCH targeting strategy is a viable therapy for aggressive CLL warranting further preclinical and clinical studies. This work was partially supported by bridge funding from the American Association of Hematology awarded to Dr. Runqing Lu, who passed away during the pursuit of this study.

Progressive Gut Microbiome Dysbiosis Is Associated with Chronic Lymphocytic Leukemia Pathogenesis and Development in the Eµ-TCL1 Mouse Model

Introduction: The gut microbiome is home to a community of microorganisms such as bacteria, fungi, viruses, archaea, and protozoa. Maintaining a balance between “favorable” and “unfavorable” bacteria within the gut microbiome is essential for homeostatic physiological processes including proper immune system development, regulation of metabolic pathways, protection against pathogens, and gut epithelium integrity. Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by the accumulation of CD5 + B-cells in the blood, bone marrow, and secondary lymphoid tissues. The pathogenesis of CLL is related to the activation of critical survival pathways such as B-cell and toll-like receptor signaling pathways, where microbial antigens are known to be key stimulators driving CLL B-cell proliferation. In this study, we investigated how the gut microbiota composition contributes and correlates to CLL disease progression. Methods: Using a well-established murine model representative of CLL development in humans, we profiled the gut microbiota of transgenic Eµ-TCL1 mice and age-matched wild-type B6 (WT B6) mice over the course of 12 months (n = 7-10/group). Fecal pellets were collected aseptically from study mice at four time points (4, 7, 10, and 12 months) for DNA extraction using the QIAamp® PowerFecal® Pro DNA Kit. To characterize the gut microflora, we utilized 16S rRNA sequencing using the Illumina 16S metagenomics protocol. Bacterial taxonomy was assigned and analyzed using the QIIME2™ platform. Linear Discriminant Analysis (LDA) Effect Size (LEfSe) analysis was employed to identify differentially expressed bacterial taxa between cohorts. Beta diversity indices were used to evaluate intersample differences in the fecal microbiota. Mice were monitored for peripheral expansion of leukemia cells (%CD45 +/CD19 +/CD5 + cells) via flow cytometry. Next, we evaluated the microbiome in an aggressive model of CLL. Utilizing the Eµ-TCL1 adoptive transfer murine model, similar methods were adopted to profile the gut microbiome over the course of 7 weeks. Lastly, to evaluate the contribution of the gut microbiota to CLL development, we employed an antibiotic-mediated gut microflora ablation to the adoptive transfer Eµ-TCL1 model, with leukemia expansion monitored post-engraftment on a weekly basis. Results: A unique microbiome signature was identified in both the indolent and aggressive model of CLL. Evaluation of beta diversity, using Bray-Curtis distance metrics, confirmed distinct differences in the gut microbial communities between Eµ-TCL1 and WT B6 cohorts. At the phylum level, the gut microbiota of the Eµ-TCL1 and WT B6 cohorts were similarly dominated by Firmicutes and Bacteroidota. However, Proteobacteria Deferribacterota and Desulfobacterota were more abundant in the Eµ-TCL1 mice over 12 months as compared to age-matched WT B6. Abundance of Actinobacteriota increased specifically at 12 months in the Eµ-TCL1 mice. Additionally, LEfSe analysis showed enrichment of certain phyla in the Eµ-TCL1 mice at 4 months, 10 months, and 12 months. Presence of these bacteria paralleled with expansion of disease. In the aggressive model of CLL, beta diversity confirmed distinct differences in gut microbial communities as CLL disease progressed. Moreover, certain phyla were enriched in leukemic mice at 7 weeks post-engraftment including Proteobacteria and Actinobacteriota. Lastly, in the antibiotic-mediated gut flora ablation model, a delay of disease onset was reflected in mice with ablated gut microbiomes compared to mice with intact gut microbiomes, resulting in prolonged overall survival. Conclusion: We provide novel results associating the gut microbiome with the initiation and progression of CLL disease. In both the indolent and aggressive CLL disease models, we showed that there is a distinct gut microbiome as evident by beta diversity. A dysbiotic signature represented by increasing Proteobacteria abundance was similarly identified in both models. Proteobacteria are known to be evident in diseases characterized by inflammation. These findings support future studies to determine a potential causal mechanism between gut microbiota changes and CLL pathogenesis.

Nucleosome repositioning in chronic lymphocytic leukemia.

The location of nucleosomes in the human genome determines the primary chromatin structure and regulates access to regulatory regions. However, genome-wide information on deregulated nucleosome occupancy and its implications in primary cancer cells is scarce. Here, we conducted a genome-wide comparison of high-resolution nucleosome maps in peripheral blood B cells from patients with chronic lymphocytic leukemia (CLL) and healthy individuals at single-base-pair resolution. Our investigation uncovered significant changes of nucleosome positioning in CLL. Globally, the spacing between nucleosomes-the nucleosome repeat length (NRL)-is shortened in CLL. This effect is stronger in the more aggressive IGHV-unmutated CLL subtype than in the IGHV-mutated CLL subtype. Changes in nucleosome occupancy at specific sites are linked to active chromatin remodeling and reduced DNA methylation. Nucleosomes lost or gained in CLL marks differential binding of 3D chromatin organizers such as CTCF as well as immune response-related transcription factors and delineated mechanisms of epigenetic deregulation. The principal component analysis of nucleosome occupancy in cancer-specific regions allowed the classification of samples between cancer subtypes and normal controls. Furthermore, patients could be better assigned to CLL subtypes according to differential nucleosome occupancy than based on DNA methylation or gene expression. Thus, nucleosome positioning constitutes a novel readout to dissect molecular mechanisms of disease progression and to stratify patients. Furthermore, we anticipate that the global nucleosome repositioning detected in our study, such as changes in the NRL, can be exploited for liquid biopsy applications based on cell-free DNA to stratify patients and monitor disease progression.

Identifying CLL Patients at High Risk of Infection on Treatment Using Machine Learning

Background. Targeted therapies, including BTK inhibitors like ibrutinib, have transformed the treatment of patients with chronic lymphocytic leukemia (CLL), improving overall survival and progression-free survival, in particular for patients with the most aggressive CLL disease. However, due to relapse after stopping treatment and the rarity of reaching undetectable minimum residual disease, treatment with ibrutinib requires long-term maintenance therapy. Thus, adverse events (AEs) may have a great impact on the patient's quality of life and overall survival. Several studies have reported higher risk of AEs including severe infections, bleeding, and cardiac events like atrial fibrillation (AF) and hypertension in CLL patients treated with ibrutinib. Identifying patients that might be at a higher risk of developing AEs at the time of treatment initiation could inform individualized treatment and may reduce morbidity and mortality. In this study, we used Machine learning (ML) to predict the risk of developing severe infection (≥ grade 3) after treatment with ibrutinib and/or other standard treatment regimens. Methods. The dataset consisted of 647 patients with CLL that were treated prior to December 2020. Using the Danish CLL registry, the Persimune data warehouse, and Electronic Health Record (EHR) data sources, we extracted baseline features such as age (at the diagnosis and treatment), sex, body mass index (BMI), smoking status, and a range of medical features (with cut-off date set as the date of treatment initiation) including routine laboratory tests, microbiology cultures, known CLL prognostics and historical data on previous infections and type of treatments. The treatment data and AEs were collected by manual review of EHRs as well as through direct data extraction. Features with more than 80% missing values were discarded. For continuous features, missing values were imputed by the mean value of each feature estimated from the training set. Overall, 167 features were used in the modeling. Since some patients received multiple lines of treatment, the dataset consisted of 1400 events of patients receiving treatment. Using an ML algorithm (XGBoost), Cox regression analyses were performed to predict severe infections (≥ Grade 3). Stratified group cross-validation was used to preserve the proportion of samples with severe infection across splits. The contribution of each feature across the study was measured using SHapley Additive exPlanations (SHAP). Results. We identified a high-risk and a low-risk group with 57% and 28% estimated risk of a severe infection within one year of treatment initiation, respectively (Figure 1). The median survival times for the high-risk and low-risk group were 7 (CI%95: 5.0-12.4) and 44 (CI%95: 39.0-56.2) months, respectively. The model could also discriminate the risk of infection within the group of ibrutinib treated and non-ibrutinib treated patients. A c-index of 0.687 (CI%95: 0.649-0.725) for all treatments, 0.672 (CI%95: 0.647-0.696) for ibrutinib, and 0.659 (CI%95: 0.597-0.721) for other treatments (including all treatments except ibrutinib) were achieved using 4-fold cross-validation. To assess impact of different features on the risk of severe treatment related infection, we performed SHAP analysis of feature contribution (Figure 2). The results showed that the targeted therapy (Targeted treatment) and ibrutinib treatment correlated with increased risk of infection. This may in part reflect that targeted therapy was primarily used for patients with aggressive CLL (TP53 aberration and/or relapsed/refractory CLL). In addition, the number of blood cultures drawn prior to treatment correlated with the risk of severe infection. Furthermore, several routine laboratory tests measuring immunoglobulin G, c-reactive protein, and high-density lipoprotein levels were predictive of severe infection. Conclusion. This study confirms the advantage of an ML based approach to stratify patients with CLL prior to initiation of treatment for risk of severe infections within and across treatment regimens. As infections are the main cause of death in CLL, this represents the first steps towards individualized therapy based on risk of AEs. Future work will focus on predicting cardiac events including AF. Validation of the findings in clinical trial populations and other real world data cohorts are awaited before clinical implementation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MER Signaling Axis: A Novel Therapeutic Target in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+/CD23+ monoclonal B-cells in peripheral blood, bone marrow and lymphoid tissues with a highly variable disease course. B-cell receptor (BCR)-signal inhibitors, specifically Ibrutinib which targets Bruton's tyrosine kinase (BTK) and Idelalisib which targets PI3Kδ, are effective in relapsed/refractory CLL patients. Despite BCR-signal-inhibitors are changing the management of CLL, these agents are not curative and resistance can develop, often leading to the transformation of CLL into more aggressive B-cell lymphomas limiting their therapeutics options. Thus, improved understanding of the nature and extent of other active receptor tyrosine kinase (RTK) signals in CLL cells is critical for development of additional therapies to treat these high-risk patients. Our recent discovery of MER, a RTK of the TAM (Tyro3, AXL, MER) family, in CLL cells may offer such an option. In order to analyze MER expression status in CLL cells from previously untreated CLL patients, we employed western blot, flow cytometry and qRT-PCR analyses. We detected low to high level expression of MER in CLL cells from ~50% of CLL patients as compared to normal, purified B cells. We also found that MER in CLL cells remained constitutively phosphorylated (active). Furthermore, our preliminary analysis suggested that MER expression in CLL cells may be positively associated with high-risk, unmutated IGHV CLL patients. In vitro ligation of Gas6 (growth arrest-specific gene 6), a specific ligand for all the TAM receptors, robustly activated MER as compared to AXL or Tyro3. Interestingly, activation of MER preferentially activated Erk1/2, NF-κB, and BTK of the BCR pathway in CLL cells. Partial depletion of MER in CLL cells using a specific-siRNA resulted in inhibition of P-Erk1/2, P-NF-κB and P-BTK, but not P-AKT, suggesting that MER may crosstalk with the BCR signaling pathway in CLL cells likely via interaction with CD79a, LYN or BTK. To address this hypothesis, we immunoprecipitated MER from CLL cell lysates and found a direct complex formation between MER and LYN. We also analyzed leukemic B cells from the Eµ-TCL1 transgenic mice which recapitulate highly aggressive human CLL for the expression of MER by western blot/flow cytometry. Results showed robust expression of MER in TCL1 leukemic B cells purified from mice with advanced CLL. Finally, treatment of CLL mice with a high-affinity MER inhibitor (UNC-2025) via oral gavage significantly reduced leukemic tumor burden. In total, our findings suggest that expression/activation of MER may potentiate CLL cell survival via a crosstalk with the BCR signal thus, could be an attractive therapeutic target for the progressive CLL patients.

Enhanced IFN Sensing by Aggressive Chronic Lymphocytic Leukemia Cells.

Type I IFN is made by cells in response to stress. Cancer cells exist in a state of stress, but their IFN response is complex and not completely understood. This study investigated the role of autocrine IFN in human chronic lymphocytic leukemia (CLL) cells. CLL cells were found to make low amounts of IFN via TANK-binding kinase 1 pathways, but p-STAT1 and -STAT2 proteins along with IFN-stimulated genes that reflect IFN activation were variably downregulated in cultured CLL cells by the neutralizing IFNAR1 Ab anifrolumab. Patients with CLL were segregated into two groups based on the response of their leukemia cells to anifrolumab. Samples associated with more aggressive clinical behavior indicated by unmutated IGHV genes along with high CD38 and p-Bruton's tyrosine kinase expression exhibited responses to low amounts of IFN that were blocked by anifrolumab. Samples with more indolent behavior were unaffected by anifrolumab. Hypersensitivity to IFN was associated with higher expression of IFNAR1, MX1, STAT1, and STAT2 proteins and lower activity of negative regulatory tyrosine phosphatases. Autocrine IFN protected responsive CLL cells from stressful tissue culture environments and therapeutic drugs such as ibrutinib and venetoclax in vitro, in part by upregulating Mcl-1 expression. These findings suggest hypersensitivity to IFN may promote aggressive clinical behavior. Specific blockade of IFN signaling may improve outcomes for patients with CLL with higher-risk disease.

Pharmacological modulation of Kv1.3 potassium channel selectively triggers pathological B lymphocyte apoptosis in vivo in a genetic CLL model

BackgroundIon channels are emerging as promising oncological targets. The potassium channels Kv1.3 and IKCa are highly expressed in the plasma membrane and mitochondria of human chronic lymphocytic leukemia (CLL) cells, compared to healthy lymphocytes. In vitro, inhibition of mitoKv1.3 by PAPTP was shown to kill ex vivo primary human CLL cells, while targeting IKCa with TRAM-34 decreased CLL cell proliferation.MethodsHere we evaluated the effect of the above drugs in CLL cells from ibrutinib-resistant patients and in combination with Venetoclax, two drugs used in the clinical practice. The effects of the drugs were tested also in the Eμ-TCL1 genetic CLL murine model, characterized by a lympho-proliferative disease reminiscent of aggressive human CLL. Eμ-TCL1 mice showing overt disease state were treated with intraperitoneal injections of non-toxic 5 nmol/g PAPTP or 10 nmol/g TRAM-34 once a day and the number and percentage of pathological B cells (CD19+CD5+) in different, pathologically relevant body districts were determined.ResultsWe show that Kv1.3 expression correlates with sensitivity of the human and mouse neoplastic cells to PAPTP. Primary CLL cells from ibrutinib-resistant patients could be killed with PAPTP and this drug enhanced the effect of Venetoclax, by acting on mitoKv1.3 of the inner mitochondrial membrane and triggering rapid mitochondrial changes and cytochrome c release. In vivo, after 2 week- therapy of Eμ-TCL1 mice harboring distinct CLL clones, leukemia burden was reduced by more than 85%: the number and percentage of CLL B cells fall in the spleen and peritoneal cavity and in the peripheral blood, without signs of toxicity. Notably, CLL infiltration into liver and spleen and splenomegaly were also drastically reduced upon PAPTP treatment. In contrast, TRAM-34 did not exert any beneficial effect when administered in vivo to Eμ-TCL1 mice at non-toxic concentration.ConclusionAltogether, by comparing vehicle versus compound effect in different Eμ-TCL1 animals bearing unique clones similarly to CLL patients, we conclude that PAPTP significantly reduced leukemia burden in CLL-relevant districts, even in animals with advanced stage of the disease. Our results thus identify PAPTP as a very promising drug for CLL treatment, even for the chemoresistant forms of the disease.

Efficacy of Ibrutinib Monotherapy in Pre-Clinical Mouse Models of Richter Transformation: Ibrutinib Effectively Reduces the Incidence of Richter Transformation but Fails in Treating Transformed Lymphoma, Especially in Primary Lymphoid Tissue

Richter's transformation (RT) is a complication of chronic lymphocytic leukemia (CLL) characterized by the development of an aggressive lymphoma, typically diffuse large B cell lymphoma (DLBCL). RT is associated with high expression of activation-induced cytidine deaminase (AID) and aberrant somatic hypermutation of non-immunoglobulin genes. Current frontline therapy of CLL, ibrutinib, eliminates AID + CLL cells and alters tissue microenvironment to Th1-biased niches; however approximately 10-20% of CLL patients develop RT upon ibrutinib treatment. In addition, ibrutinib leads to a clinical response in only ~40% of RT patients. Thus, RT remains an important unmet challenge for CLL patients and the mechanism of RT development during ibrutinib treatment is poorly understood.To determine the contributions of AID and T cells in RT development, and to understand the efficacy of ibrutinib in the treatment of RT, we first created patient-derived xenografts (PDXs) with samples from three RT patients who were refractory to (immuno)chemotherapy but had not previously received ibrutinib. In each case, the B-lymphocytes were larger in size and expressed lower levels of CD5 but higher levels of activation markers compared with the CLL cell counterpart. In PDXs, ibrutinib treatment led to significantly less RT cells in spleen with percentage of inhibition ranging from 28-60% for all three cases. Notably, AID levels were similar in RT cells residing in spleen of ibrutinib-treated and control mice. The numbers of T cells were also similar, although Th1:Th2 ratios were significantly increased by ibrutinib (control vs ibrutinib group: 33% vs 42%, P=0.01). Interestingly, the growth and survival of RT cells located in bone marrow (BM) were not affected by ibrutinib.We then investigated the efficacy of ibrutinib in the prevention and treatment of RT in a novel strain of TCL-1 mice, TCL1/Igκ-AID mice, that have AID overexpressed solely in B lymphocytes. These mice develop accelerated CLL disease, have shorter overall survival (OS), and bear increased numbers of AID signature mutations in genes that are drivers in CLL and DLBCL.First we injected primary splenocytes from a TCL1/Igκ-AID mouse (A-31) into wild-type C57BL/6 mice. A week later, mice were treated with vehicle or ibrutinib continuously for 3 weeks (N=5 per group, duplicated experiments). At the end of treatment, RT cells were found in 25% of recipients in the control group and only in 10% of ibrutinib-treated recipients. Ibrutinib-treated animals had overall smaller spleens and lymph nodes (LNs) filled with significantly less CLL or RT cells. Importantly, the balance of Th1/Th2 cytokines was also significantly changed by ibrutinib (Th1/Th2 in control vs ibrutinib: 25% vs 47%, P=0.01). However, again RT cells in BMs of treated and control animals were similar.We then repeated the experiment to determine overall survival by giving the mice vehicle or ibrutinib continuously. Compared to the TCL1/Igκ-AID (A-31) donors, recipients treated with control vehicle developed more aggressive CLL with shorter OS. However, Ibrutinib did not change the OS; both control and ibrutinib treated recipients had similar OS with all the mice developed and died from RT eventually.Finally, to determine the efficacy of ibrutinib in the treatment of RT in TCL1/Igκ-AID mice, we sorted A-31-derived RT cells collected from LNs, and injected these into C57BL/6 mice. Mice then received treatment for 3 weeks. Notably, ibrutinib did not change the numbers or sizes of LNs or spleens. The numbers of RT cells at all sites, including BM, remained the same. In both groups, the number of T cells and the Th1:Th2 were not altered by ibrutinib.In summary, our data suggest that ibrutinib is somewhat effective against primary RT cells in the spleen in PDX models but not at all in the aggressive TCL1/IgK-AID mouse model. This might be because ibrutinib does not reduce AID levels in tissue-resident RT cells. Notably, although RT cells in spleens and LNs respond to ibrutinib, those residing in BM are resistant in both PDX and TCL1/IgK-AID models. This suggests different clones or different ibrutinib-responsiveness of RT cells in primary versus secondary lymphoid tissues. Overall, these results suggest that ibrutinib does not prevent or cure RT. Additionally, TCL1/Igκ-AID mice might provide a model to study the development, prevention, and treatment of RT without the influence of prior chemo- or other therapies. DisclosuresWirtz: Abbvie: Current Employment. Szafer-Glusman: AbbVie: Current Employment, Other: Stock or other ownership.

Comparison with Private and Public Clonotypes Reveals That, While CLL Type Stereotyped BCR Are Produced in 90% of Healthy Subjects from 18 to 78 Years, They Are Not Accumulated and Are Mostly Found in Immature and Naïve B-Cells

Introduction:One third of Chronic Lymphocytic Leukemia (CLL) patients harbor stereotyped B-Cell receptor (BCR). This is in contrast with the 1012 potential different BCRs that can be produced by the immune system. Some stereotyped BCR are also highly associated with prognosis. For example, the stereotyped subset #2 identifies aggressive CLLs with frequent SF3B1 and ATM alterations. This underlies an important role of BCR selection for emergence of CLL clones.Aim and methods:We raised the question of the existence and quantification of B-cells with stereotyped BCR of the CLL-type in a series of 69 healthy subjects (HS) from 18 to 78 years old, without any past of hematological history nor any detectable monotypic B-cells by flow cytometry.HS were divided in age three groups, adults (18 - 55 years, n= 31), senior-1 (55 - 65 years, n = 14) and senior-2 (> 65 years, n = 24).The variable region of the immunoglobulin heavy chain gene (IGHV) was amplified from genomic DNA of purified B-cells sequenced on an Illumina MiSeq and annotated with the online IMGT HighV-Quest tool.Clonotypes were defined as IGHV genes with the same rearranged V and J segment and with the same CDR3 amino acid sequence with one mismatch tolerance, and if a minimal threshold read number was reached (ranging 5 to 15 according to the DNA input and sequencing depth).Public clonotypes were those shared in between at least 4 (5%) HS.Detection of the 19 major stereotyped CLL's BCRs was performed with an in house R script. And was confirmed with the ARResT/AssignSubsets online tool (V Bistry et al, Bioinformatics, 2015).Exploration of the IGHV repertoire of the different B-cell subpopulations was done from public data (Mitsunaga, Mol Cell Proteomics, 2020).Results:To be independent of the number of reads and to compare HS to each other, clonotypic frequencies were ranked in 150 increasing intervals. In each case, the mean rank of clonotypic frequency ranged 3 to 7 with an SD in between 2 and 6 and was independent of the age category. A Z-score was calculated for each clonotype. Less than 1% of private clonotypes had a Z-score>1.96, while 10% to 30% of public clonotypes had a significant Z-score>1.96 (figure 1). This indicates that public clonotypes are often accumulated when compared to their private counterpart.A stereotyped CLL's BCR was found in 90% HS all stereotypes together. Subsets #2 and #5 were found in more than 50% of cases (figure 2). Frequencies of subsets #3, #59, #7C, #202, #14, #64B, decreased from 23% to 6% cases. Subsets #1, #4, #6, #8, #12, #16, #99 and #201 were never found.Frequencies of stereotyped BCRs ranged 5.2x10 -5 to 1.2x10 -4, being similar in the different HS age categories. Ranks of stereotyped BCR frequencies were almost always below the mean rank of private clonotypes, that indicates a very low abundance (figure 3). Junction analyses suggest that B-cells with stereotyped BCRs were polyclonal since they were very likely to derive from different B-cell precursors. Exploring the longitudinal repertoires of Mitsunaga and Snyder(Mol Cell Proteomics 2020), showed that stereotyped BCRs were almost restricted to immature and naïve B-cells and were fugace while public clonotypes were recurrently and frequently found in memory B-cells and could be stable over time for many of them.Conclusion:B-cells with stereotyped CLL BCRs were easily detected in healthy people whatever their age category. Remarkably, subsets #2 and #5 that are associated with aggressive CLL were found in more than 50% healthy donors at any age. Comparison with private and public clonotypic repertoire reveals that abundance of stereotyped BCR was almost always very low and was restricted to immature and naïve B-cells.Therefore, B-cells with stereotyped BCRs are produced throughout life in almost everybody. But these B-cells do not accumulate and do not seem to undergo to B-cell maturation.This suggests that stereotyped BCRs are not intrinsically prone to transformation and that clonal selection of CLL cells would involve additional events such as antigenic encounter or oncogenic alterations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BET Inhibition As a Targeted Epigenetic Approach to Reverse T Cell Dysfunction in Chronic Lymphocytic Leukemia

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by the clonal expansion of mature CD19+/CD5+ lymphocytes in the peripheral blood and secondary lymphoid organs. The accumulation of B-CLL cells yields profound immune defects in the CLL tumor microenvironment (TME), promoting evasion of immune surveillance that contributes to tumor persistence and thus relapsed/refractory disease. The bromodomain and extra-terminal domain (BET) family of proteins are epigenetic readers that bind acetylated histone residues to regulate transcription of numerous genes involved in critical CLL protumor pathways. Of the BET family proteins, BRD4 is overexpressed in CLL and highly enriched at super-enhancers of genes that regulate CLL-TME interactions such as B cell receptor pathway components, chemokine/cytokine receptors, and immune checkpoint molecules. Pan BET inhibitors (BET-i), such as PLX51107 (Plexxikon Inc.) significantly improve survival in aggressive CLL murine models. Here we demonstrate that blocking BRD4 function with PLX51107 (PLX5) can alleviate the inherent immune defects observed in CLL, hence reducing B-CLL induced T cell dysfunction and allowing for robust B-CLL cell elimination. This therapeutic strategy may be vital in overcoming frequent drug resistance and/or bolstering the anti-tumor effect of current CLL therapies.Methods: Primary leukemic B cells were isolated from the peripheral blood of CLL patients and co-cultured with healthy donor T cells to evaluate the effect of PLX5 (0.1-0.5μM) on CLL-induced T cell immunosuppression ex vivo via an array of flow cytometry assays. T cell proliferation was assessed using CFSE after 96 h co-culture with α-CD3/α-CD28 stimulation. Effector cytokine production was evaluated after 48 h co-culture in the presence of PMA/ionomycin (final 6 h) and brefeldin A (final 5 h). Immune inhibitory molecule surface expression was measured following 48 h co-culture with α-CD3/α-CD28 stimulation. To further validate our ex vivo findings, the E μ-TCL1 adoptive transfer model was used. Once disease onset was confirmed in recipient WT B6 mice (>10% CD45+/CD19+/CD5+ peripheral blood lymphocytes), mice were randomized to receive either PLX5 (20 mg/kg) or vehicle (VEH) equivalent daily by oral gavage for 4 weeks. Following treatment, mouse spleens were processed to evaluate exhaustion marker expression, T cell proliferation (CellTrace™ Violet, 72 h a-CD3/α-CD28 stimulation ex-vivo), and T-cell effector function (ex-vivo mitogenic stimulation, 6 h).Results: T cell proliferation indices were reduced following ex vivo co-culture with primary B-CLL cells (mean ± SEM for T cells vs. co-culture, 2.0 ± 0.13 vs. 1.57 ± 0.05; P<0.01). This suppression was significantly alleviated in 0.5μM PLX5-treated co-cultures (1.84 ± 0.08; P<0.01). In a similar fashion, the percentage of polyfunctional TNF-α+/IFN-γ+ CD4+ T cells markedly increased in PLX5-treated co-cultures (VEH vs. 0.5μM PLX5, 10.0% ± 0.76% vs. 15.2% ± 0.92%; P<0.01). Notably, BET inhibition with PLX5 also bolstered T cell inflammatory function (%TNF-α+/IFN-γ+) in the absence of B-CLL cells (VEH vs. PLX5, 12.9% ± 1.0% vs. 15.3% ± 0.69%; P<0.05). Remarkably, the expression of numerous immune inhibitory molecules (e.g., PDL1, PD1, CTLA4, LAG3) was consistently reduced between 1.8- and 3-fold in PLX5-treated co-cultures (0.1μM). In the adoptive transfer E μ-TCL1 model, mice receiving PLX5 displayed reduced expansion of B-CLL cells and increased T cell infiltration in the spleen (Fig. 1A). Splenic CD4+ T cells from PLX5-treated mice had significantly greater proliferative capacity (Fig. 1B) and pro-inflammatory functionality (Fig. 1C). Finally, PLX5 treatment markedly reduced the surface expression of immune inhibitory molecules (e.g., PDL1, LAG3, VISTA) on CD4+ and CD8+ T cells in the spleen (Fig. 1D). Studies to evaluate the effects of PLX5 on malignant B-CLL and T cells within the bone marrow niche and soluble factors in the plasma are ongoing. Collectively, our data indicate that the novel BET-i, PLX5, exerts beneficial immunomodulatory effects on T cells within the CLL TME.Conclusion: Epigenetic-targeted therapies such as BET-i have the potential to alleviate CLL-induced T cell dysfunction while eliminating B-CLL cells and preventing tumor expansion. Future profiling studies are pending to further illuminate how BET proteins regulate immune function in CLL. [Display omitted] DisclosuresLunning: AstraZeneca: Consultancy; Legend: Consultancy; Acrotech: Consultancy; ADC Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Myeloid Therapeutics: Consultancy; Beigene: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Morphosys: Consultancy; TG Therapeutics: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Spectrum: Consultancy; Kite, a Gilead Company: Consultancy. Vose: Kite, a Gilead Company: Honoraria, Research Funding. Powell: Plexxikon Inc.: Current Employment.

Distinctive Signaling Profiles With Distinct Biological and Clinical Implications in Aggressive CLL Subsets With Stereotyped B-Cell Receptor Immunoglobulin.

The ontogeny and evolution of chronic lymphocytic leukemia (CLL) are critically dependent on interactions between leukemic cells and their microenvironment, including antigens, the latter recognized through the clonotypic B-cell receptor immunoglobulin (BcR IG). Antigen selection is key to the pathogenesis of CLL, as evidenced by the remarkable skewing of the BcR IG gene repertoire, culminating in BcR IG stereotypy, referring to the existence of subsets of patients with (quasi)identical BcR IG. Notably, certain of these subsets have been found to display distinct, subset-biased biological background, clinical presentation, and outcome, including the response to treatment. This points to BcR IG centrality while also emphasizing the need to dissect the signaling pathways triggered by the distinctive BcR IG expressed by different subsets, particularly those with aggressive clinical behavior. In this mini-review, we discuss the current knowledge on the implicated signaling pathways as well as the recurrent gene mutations in these pathways that characterize major aggressive stereotyped subsets. Special emphasis is given on the intertwining of BcR IG and Toll-like receptor (TLR) signaling and the molecular characterization of signaling activation, which has revealed novel players implicated in shaping clinical aggressiveness in CLL, e.g., the histone methyltransferase EZH2 and the transcription factor p63.

Richter transformation of chronic lymphocytic leukaemia: a British Society for Haematology Good Practice Paper.

Richter transformation of chronic lymphocytic leukaemia: a British Society for Haematology Good Practice Paper.

Combined ibrutinib and venetoclax treatment vs single agents in the TCL1 mouse model of chronic lymphocytic leukemia

AbstractThe covalent inhibitor of Bruton's tyrosine kinase ibrutinib and the specific Bcl-2 inhibitor venetoclax are both highly efficacious single-agent drugs in the treatment of chronic lymphocytic leukemia (CLL). Based on their complementary modes of action, ibrutinib and venetoclax are hypothesized to act in a synergistic fashion. Currently, it is unclear whether combined treatment is indeed superior to continuous single-agent treatment and what mechanisms underlie the resistance to combination treatment. In addition, the effects of such treatment on the skewed T-cell compartment characteristic of CLL are as yet unknown. In the murine Eµ-TCL1 adoptive transfer model resembling aggressive CLL, we found that combined treatment resulted in the deepest responses, with the longest duration related to a combination of decreased proliferation and increased induction of apoptosis. In addition, alterations in T-cell subsets were most prominent after combination treatment, with increased naive cells and reduced effector memory cells. Remarkably, effects of single agents but also combination treatment were eventually interrupted by relapse, and we found downregulation of BIM expression as a plausible cause of acquired drug resistance. Nevertheless, in this murine model, the combination of venetoclax and ibrutinib has increased efficacy over single agents, accompanied by a restoration of the T-cell compartment.

Development and characterization of prototypes for in vitro and in vivo mouse models of ibrutinib-resistant CLL

AbstractAlthough ibrutinib improves the overall survival of patients with chronic lymphocytic leukemia (CLL), some patients still develop resistance, most commonly through point mutations affecting cysteine residue 481 (C481) in Bruton's tyrosine kinase (BTKC481S and BTKC481R). To enhance our understanding of the biological impact of these mutations, we established cell lines that overexpress wild-type or mutant BTK in in vitro and in vivo models that mimic ibrutinib-sensitive and -resistant CLL. MEC-1 cell lines stably overexpressing wild-type or mutant BTK were generated. All cell lines coexpressed GFP, were CD19+ and CD23+, and overexpressed BTK. Overexpression of wild-type or mutant BTK resulted in increased signaling, as evidenced by the induction of p-BTK, p-PLCγ2, and p-extracellular signal–related kinase (ERK) levels, the latter further augmented upon IgM stimulation. In all cell lines, cell cycle profiles and levels of BTK expression were similar, but the RNA sequencing and reverse-phase protein array results revealed that the molecular transcript and protein profiles were distinct. To mimic aggressive CLL, we created xenograft mouse models by transplanting the generated cell lines into Rag2−/−γc−/− mice. Spleens, livers, bone marrow, and peripheral blood were collected. All mice developed CLL-like disease with systemic involvement (engraftment efficiency, 100%). We observed splenomegaly, accumulation of leukemic cells in the spleen and liver, and macroscopically evident necrosis. CD19+ cells accumulated in the spleen, bone marrow, and peripheral blood. The overall survival duration was slightly lower in mice expressing mutant BTK. Our cell lines and murine models mimicking ibrutinib-resistant CLL will serve as powerful tools to test reversible BTK inhibitors and novel, non–BTK-targeted therapeutics.

ENDOG Impacts on Tumor Cell Proliferation and Tumor Prognosis in the Context of PI3K/PTEN Pathway Status.

Simple SummaryThe PI3K/AKT pathway is involved in cell survival and proliferation. Molecular aberrations and/or hyperactivation of the PI3K-PTEN-AKT axis are frequent in distinct cancer types such as endometrial carcinoma, the most common type of cancer of the female genital tract, and chronic lymphocytic leukemia (CLL), a mature B-cell neoplasm depending on B-cell receptor (BCR) activity, which induces chronical activation of this pathway. The mitochondrial nuclease ENDOG was found to influence PI3K/AKT activity in somatic cells. Our aim was to assess the value of ENDOG gene silencing to block cancer cell proliferation and to evaluate the relevance of ENDOG as prognostic marker. In vivo, in vitro and in silico experiments show that Endog/ENDOG silencing blunts proliferation of tumor cells dependent on high p-AKT/low PTEN activity and that ENDOG has prognostic value in specific cancer types.EndoG influences mitochondrial DNA replication and is involved in somatic cell proliferation. Here, we investigated the effect of ENDOG/Endog expression on proliferation in different tumor models. Noteworthy, ENDOG deficiency reduced proliferation of endometrial tumor cells expressing low PTEN/high p-AKT levels, and Endog deletion blunted the growth of PTEN-deficient 3D endometrial cultures. Furthermore, ENDOG silencing reduced proliferation of follicular thyroid carcinoma and glioblastoma cell lines with high p-AKT expression. High ENDOG expression was associated with a short time to treatment in a cohort of patients with chronic lymphocytic leukemia (CLL), a B-cell lymphoid neoplasm with activation of PI3K/AKT. This clinical impact was observed in the less aggressive CLL subtype with mutated IGHV in which high ENDOG and low PTEN levels were associated with worse outcome. In summary, our results show that reducing ENDOG expression hinders growth of some tumors characterized by low PTEN activity and high p-AKT expression and that ENDOG has prognostic value for some cancer types.

CD4+ T cells sustain aggressive chronic lymphocytic leukemia in Eμ-TCL1 mice through a CD40L-independent mechanism

Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40−/−), or CD8+ T cells (TCL1+/+TAP−/−), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP−/− mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L−/− mice, and TCL1+/+CD40−/− mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms.

High CD52 mRNA Expression Is An Adverse Prognostic Factor In Fludarabine-Refractory CLL Treated With Alemtuzumab On The Gcllsg CLL2H Trial

Alemtuzumab is a humanized mAb that targets CD52 and has been widely used for the treatment of chronic lymphocytic leukemia (CLL) patients with refractory disease or loss / mutation of TP53. The cell-surface marker CD52 is variably expressed on peripheral blood mononuclear cells and shows relatively high expression levels on the surface of both healthy B lymphocytes and on CLL cells, but the biologic function of CD52 has not been sufficiently clarified yet.To elucidate the prognostic value of CD52 expression, we analyzed a large gene expression dataset. Gene expression profiling was performed on Affymetrix Exon 1.0 ST Arrays. Data sets were generated for 75 fludarabine-refractory CLL patients treated within the multicenter phase II CLL2H trial of the GCLLSG (3x30 mg Alemtuzumab s.c. per week, max. 12 weeks). Normalization of the arrays was conducted by using the RMA algorithm. Statistical analysis was performed with the R software version 2.15.1.Patients were grouped into quartiles (Q1-Q4) according to CD52 expression levels (CD52 low: Q1, n=19; CD52 intermediate: Q2-Q3, n=37; CD52 high: Q4, n=19).Progression free survival (PFS) and overall survival (OS) were markedly different for patients from Q1 and Q4 (median PFS: Q1=413 days vs. Q4=145 days (p=0.0102); median OS: Q1= 879 days vs. Q4=413 days (p=0.0164)). Patients with progressive disease were overrepresented in Q4 (PD 7/18 (38.8%); ORR 5/18 (27.7%)) while rare in Q1 (PD 2/19 (10.5%); ORR 9/19 (47.3%)). Patients with low expression of CD52 had less frequently genetic/biologic and clinical characteristics of aggressive disease (Q1 vs. Q4; TP53 mutation/17p deletion: 47.36% vs. 36.84%; IGHV unmutated: 61.11% vs. 73.68%; leukocyte counts (median): 45.8 G/L vs. 55.4 G/L; thymidine kinase: 22.2 U/L vs. 30.4 U/L; β2-microglobulin: 3.3 mg/l vs. 5.2 mg/l; no palpable lymph nodes: 47.36 % vs. 10.52 %).Mutations of SF3B1 and NOTCH1 were present in 4/17 (23.5%) and in 5/17 (29.4%) cases in Q1. In contrast, cases from Q4 harbored mutations of SF3B1 and NOTCH1 in 2/18 (11.1%) and 1/18 (5.5%).Cases with high CD52 levels showed overexpression (FDR<1e-07;FC>2) of genes predominantly involved in signaling of the B-cell-, toll-like- and interleukin-receptor (BTK, SYK, CD79B; TLR7, TLR10, IL27RA, IL2RG, TRAF3IP3, TRAF5).The corresponding gene set overrepresentation analysis on KEGG pathways was significantly enriched for gene sets related to cell-extracellular matrix interaction, focal adhesion, regulation of actin cytoskeleton and cell adhesion molecules as well as associated signaling pathways like WNT- and MAPK-signaling.In conclusion, high CD52 expression is an adverse prognostic factor in refractory CLL treated with Alemtuzumab. Although suggestive, the level of CD52 expression and subsequent antibody binding capacity appear not to determine the effectivity of treatment. The association with genetic and biological characteristics indicates an independent role in the biology of aggressive CLL and highlights a potential function in cell-microenvironmental interactions for CLL cells. [Display omitted] Disclosures:Stilgenbauer:Genzyme: Honoraria, Research Funding.