Role In Pathogenesis Of Chronic Lymphocytic Leukemia Research Articles

B cell receptor signaling and associated pathways in the pathogenesis of chronic lymphocytic leukemia.

B cell antigen receptor (BCR) signaling is a key driver of growth and survival in both normal and malignant B cells. Several lines of evidence support an important pathogenic role of the BCR in chronic lymphocytic leukemia (CLL). The significant improvement of CLL patients' survival with the use of various BCR pathway targeting inhibitors, supports a crucial involvement of BCR signaling in the pathogenesis of CLL. Although the treatment landscape of CLL has significantly evolved in recent years, no agent has clearly demonstrated efficacy in patients with treatment-refractory CLL in the long run. To identify new drug targets and mechanisms of drug action in neoplastic B cells, a detailed understanding of the molecular mechanisms of leukemic transformation as well as CLL cell survival is required. In the last decades, studies of genetically modified CLL mouse models in line with CLL patient studies provided a variety of exciting data about BCR and BCR-associated kinases in their role in CLL pathogenesis as well as disease progression. BCR surface expression was identified as a particularly important factor regulating CLL cell survival. Also, BCR-associated kinases were shown to provide a crosstalk of the CLL cells with their tumor microenvironment, which highlights the significance of the cells' milieu in the assessment of disease progression and treatment. In this review, we summarize the major findings of recent CLL mouse as well as patient studies in regard to the BCR signalosome and discuss its relevance in the clinics.

TRAF3 Alterations Identify a Subgroup of Patients with Adverse Clinical Outcome and Induce Metabolic Reprogramming in Chronic Lymphocytic Leukemia

The TNF receptor-associated factor3 (TRAF3) gene located in 14q32 is frequently altered by deletion and/or mutation in multiple myeloma, lymphoma and other B-cell malignancies such as chronic lymphocytic leukemia (CLL). TRAF3 is a negative regulator of the non-canonical NF-KB signaling, and recent data uncovered additional functions of this gene in inflammation, antiviral immunity or cellular metabolism. Nevertheless, the biological and clinical significance of TRAF3 alterations in CLL is unknown. For that, the aim of this study was to assess the clinical impact of TRAF3 alterations in a CLL cohort as well as its biological role in CLL pathogenesis through CRISPR/Cas9-generated models. TRAF3 alterations (mutations and copy number variations-CNVs) were assessed by NGS in our previously published cohort of 340 CLLs (Pérez Carretero, AJH 2022). A total of 14 patients exhibited a TRAF3 CNV loss, and 10 of them showed additional TRAF3 loss-of-function mutations, indicating a biallelic inactivation of this gene in CLL. Interestingly, the presence of TRAF3 alterations was significantly associated with shorter time to first treatment (6 vs 75 months, p<0.001), being an independent risk factor in the multivariate analysis (p=0.029). To further investigate the biological role of TRAF3, we used the CRISPR/Cas9 system to model TRAF3 loss-of-function mutations in the CLL-derived cell line PGA1, and characterized these cells by transcriptomics, metabolomics and functional studies. RNA-seq analyses identified 54 dysregulated genes enriched in pathways such as NIK/NF-kB signaling (FDR=0.029). Concretely, NIK (a direct target of TRAF3-mediated degradation) was significantly upregulated (Q<0.001) in TRAF3mut clones, which correlated with a higher nuclear activity of the non-canonical NF-kB transcription factors p52 (p=0.05) and RelB (p=0.02). Moreover, we validated these results in primary CLL cells (n=15; 3 TRAF3mut cells). Besides, PGA1-TRAF3mut cells showed increased levels of BAX (p=0.05) and more sensitivity to venetoclax (IC50 3.1 vs 13.8μM, p=0.003). In the metabolomic study, we found an enrichment of metabolites involved in Warburg effect, citric acid cycle (TCA) and glutamate metabolism (FDR<0.001, 0.003, 0.1 resp.), with significant higher levels of α-ketoglutarate (TCA intermediate synthesized from glutamate), pyruvate and lactate (products of glycolysis) (p<0.001, p<0.001, p=0.002) in PGA1-TRAF3mut cells. To better analyze their metabolic specificities, we evaluated the mitochondrial oxygen consumption rate (OCR) extracellular acidification (ECAR) and cell viability at basal state and in response to metabolic inhibitors. Of note, PGA1-TRAF3mut cells exhibited distinct mitochondrial characteristics: higher basal and maximal respiration levels and a more important spare capacity and generation of ATP-linked to mitochondrial respiration. Glycolysis and glycolytic reserve were also enhanced in the TRAF3mut cells. In this way, they were more sensitive to 2-deoxy-D-glucose (glucose uptake inhibitor), and more resistant to oxamate (lactate-dehydrogenase inhibitor, marker of anaerobic glycolysis). Overall, these results indicate, unlike WT, TRAF3mut cells instead utilize a mitochondrial glycolytic metabolism. Surprisingly, by using UK5099 (inhibitor of the mitochondrial pyruvate transporter that regulates mitochondrial glycolysis) mutant cells showed higher maximal respiration levels than WT, suggesting a metabolic reprogramming towards an alternative metabolic way fueling mitochondrial metabolism. We therefore tested the effect of C968 (glutaminolysis inhibitor) and we found that TRAF3mut cells were also more resistant to this drug, what reveals a specific metabolic plasticity (mitochondrial glycolysis/glutaminolysis) in the PGA1-TRAF3mut cells that does not exist in the WT. Interestingly, the combined treatment (UK5099 and C968) induced more cell death in the TRAF3mut than in the WT cells (p=0.03), suggesting a new potential opportunity to treat patients with TRAF3 alterations. In conclusion, TRAF3 deficiency appeared to be an independent prognostic factor of time to first treatment in CLL. This alteration results in a non-canonical NF-KB activation and in a metabolic reprogramming towards an enhanced mitochondrial glycolysis and glutaminolysis, which could be potentially targetable in CLL patients. Fund.: PI21/00983 FI19/00191 FEHH

Exhausted PD-1hi T-Cells Are Enriched in Secondary Lymphoid Organs of CLL Patients

Chronic lymphocytic leukemia (CLL) is associated with increased numbers of T-cells that exhibit substantial alteration in composition and function. But their role in CLL pathogenesis remains largely elusive. On the one side, T-cells provide proliferation and pro-survival signals to CLL cells, but on the other side, they show features of activation and exhaustion, including overexpression of inhibitory molecules, like PD-1, suggesting their involvement in anti-tumoral activity. Since CLL T-cells retain the ability to produce cytokines and cytotoxic molecules, they were termed pseudo-exhausted. Results of previous studies were mostly based on blood samples of CLL patients, allowing only limited conclusions as interactions of immune cells and CLL cells mainly occur in lymphoid organs.In the present study, we aimed at deciphering differences in phenotype, differentiation and functional status of T-cells in paired peripheral blood (PB) and lymph node (LN) samples of CLL patients, particularly focusing on T-cell exhaustion. We applied 12-color flow cytometry and ex vivo stimulation of primary CLL T-cells followed by intracellular cytokine and cytotoxic molecule detection. In addition, we quantified transcription factors that are known to regulate T-cell differentiation by intracellular flow cytometry.In line with published data, we detected an increased proportion of CD45RO- CCR7- CD8+ effector T-cells as well as increased numbers of PD-1+ CD8+ T-cells in PB of CLL patients (n=22), compared to age-matched healthy individuals (n=19), and a positive correlation of these numbers with disease load. Despite of their enhanced expression of PD-1 and other inhibitory receptors, PB CD8+ T-cells from CLL patients exhibited increased production of granzyme B (GzmB), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in comparison to cells from age-matched healthy donors, indicating disease-associated activation of PB CD8+ T-cells with intact functional capacity.Subsequently, we compared CD8+ T-cells from paired LN and PB samples of CLL patients (n=7). This analysis revealed that LNs were enriched in CD45RO+ CCR7- effector memory T-cells (TEM cells) and contained significantly smaller numbers of terminally differentiated effector memory cells re-expressing CD45RA (TEMRA cells). Moreover, LN CD8+ T-cells strongly expressed the activation marker CD69, yet presented with higher levels of PD-1 compared to PB CD8+ T-cells. In line with high PD-1 expression, LN CD8+ T-cells exhibited a significantly lower production of IFNγ and TNFα, and a substantially reduced cytotoxic capability as indicated by lower GzmB production compared to PB T-cells. TEM cells in CLL patients' LNs exhibited different levels of PD-1 expression, and we observed a significantly higher percentage of cells with high PD-1 expression (PD-1hi) in LNs compared to PB, which was mainly composed of cells with intermediate PD-1 expression (PD-1int), (Figure 1). Comparison of PD-1hi and PD-1int subsets in LNs showed that PD-1hi cells expressed higher levels of CD69 compared to PD-1int cells, but were substantially inferior in GzmB production. Furthermore, PD-1hi cells expressed significantly higher levels of EOMES, a transcription factor that is known to drive terminal differentiation and exhaustion of T-cells in chronic infections. In contrast, PD-1int cells showed higher expression of the transcription factors T-BET and TCF7, which mediate long-term endurance of T-cells in chronic situations.In summary, this study shows that exhausted T-cells are enriched in LNs rather than PB of CLL patients. Our data suggests that the increased leukemia-associated activation of CD8+ T-cells in patients' LNs drives the accumulation of PD-1hi exhausted T-cells. A better understanding of these processes will be essential for the development of novel immunotherapeutic approaches for CLL.Figure 1:(A) CD8+ T-cells from paired peripheral blood (PB) and lymph node (LN) samples of CLL patients (n=7) were analyzed by flow cytometry for the expression of PD-1 (representative of seven samples). (B) The relative abundance of PD-1low, PD-1int and PD-1hi subsets within the CD45RO+ CCR7- CD8+ effector memory (EM) population in PB and LN was quantified. [Display omitted] DisclosuresStilgenbauer:AbbVie: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding.

T Cells in Chronic Lymphocytic Leukemia: A Two-Edged Sword.

Chronic lymphocytic leukemia (CLL) is a malignancy of mature, antigen-experienced B lymphocytes. Despite great progress recently achieved in the management of CLL, the disease remains incurable, underscoring the need for further investigation into the underlying pathophysiology. Microenvironmental crosstalk has an established role in CLL pathogenesis and progression. Indeed, the malignant CLL cells are strongly dependent on interactions with other immune and non-immune cell populations that shape a highly orchestrated network, the tumor microenvironment (TME). The composition of the TME, as well as the bidirectional interactions between the malignant clone and the microenvironmental elements have been linked to disease heterogeneity. Mounting evidence implicates T cells present in the TME in the natural history of the CLL as well as in the establishment of certain CLL hallmarks e.g. tumor evasion and immune suppression. CLL is characterized by restrictions in the T cell receptor gene repertoire, T cell oligoclonal expansions, as well as shared T cell receptor clonotypes amongst patients, strongly alluding to selection by restricted antigenic elements of as yet undisclosed identity. Further, the T cells in CLL exhibit a distinctive phenotype with features of “exhaustion” likely as a result of chronic antigenic stimulation. This might be relevant to the fact that, despite increased numbers of oligoclonal T cells in the periphery, these cells are incapable of mounting effective anti-tumor immune responses, a feature perhaps also linked with the elevated numbers of T regulatory subpopulations. Alterations of T cell gene expression profile are associated with defects in both the cytoskeleton and immune synapse formation, and are generally induced by direct contact with the malignant clone. That said, these abnormalities appear to be reversible, which is why therapies targeting the T cell compartment represent a reasonable therapeutic option in CLL. Indeed, novel strategies, including CAR T cell immunotherapy, immune checkpoint blockade and immunomodulation, have come to the spotlight in an attempt to restore the functionality of T cells and enhance targeted cytotoxic activity against the malignant clone.

Telomere Dysfunction in Chronic Lymphocytic Leukemia

Telomeres are nucleprotein structures that cap the chromosomal ends, conferring genomic stability. Alterations in telomere maintenance and function are associated with tumorigenesis. In chronic lymphocytic leukemia (CLL), telomere length is an independent prognostic factor and short telomeres are associated with adverse outcome. Though telomere length associations have been suggested to be only a passive reflection of the cell’s replication history, here, based on published findings, we suggest a more dynamic role of telomere dysfunction in shaping the disease course. Different members of the shelterin complex, which form the telomere structure have deregulated expression and POT1 is recurrently mutated in about 3.5% of CLL. In addition, cases with short telomeres have higher telomerase (TERT) expression and activity. TERT activation and shelterin deregulation thus may be pivotal in maintaining the minimal telomere length necessary to sustain survival and proliferation of CLL cells. On the other hand, activation of DNA damage response and repair signaling at dysfunctional telomeres coupled with checkpoint deregulation, leads to terminal fusions and genomic complexity. In summary, multiple components of the telomere system are affected and they play an important role in CLL pathogenesis, progression, and clonal evolution. However, processes leading to shelterin deregulation as well as cell intrinsic and microenvironmental factors underlying TERT activation are poorly understood. The present review comprehensively summarizes the complex interplay of telomere dysfunction in CLL and underline the mechanisms that are yet to be deciphered.

PF371 DNA METHYLTRANSFERASES GENE EXPRESSION LEVELS IN CHRONIC LYMPHOCYTIC LEUKEMIA ‐ IMPACT IN DEVELOPMENT, DIAGNOSIS AND PROGNOSIS

Background:Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the western world and is characterized by the accumulation in blood, bone marrow and secondary lymphoid organs of long‐lived resting clonal B lymphocytes with a very low proliferation index and/or that proliferate abnormally. The disease has a notable clinical course diversity, with different prognosis and distinct treatment needs. However, the exact etiology is still unknown. Since cancer cells have a global DNA hypomethylation and local hypermethylation, there are evidences that epigenetic modifications have an important role in CLL pathogenesis. DNA methyltransferases (DNMT), including DNMT1, DNMT3A and DNMT3B, are responsible for DNA methylation and changes in the expression of these enzymes were observed in other hematological malignancies.Aims:The aim of this study was to evaluate the expression levels of DNMT1, DNMT3A and DNMT3A genes in chronic lymphocytic leukemia patients and analyze its correlation with clinical and laboratory data, in order to identify potential biomarkers of diagnosis and/or prognosis.Methods:We evaluated the DNMTs in peripheral blood of 68 CLL patients and 23 controls (CTL). Informed consent was obtained in accordance with the Helsinki Declarations. qPCR was used to evaluate the gene expression levels of DNMT1, DNMT3A, DNMT3B and GUSB (endogenous control). Mann–Whitney U and Kruskal–Wallis tests were used to assess the statistical significance between groups. Receiver operating characteristic (ROC) curves analysis was performed to analyze variables accuracy as diagnostic biomarker. Survival analysis was performed by Kaplan Meier method. Patients were dichotomized according to the cut off points obtained from the ROC curves constructed to predict death. All statistical analysis were two sided and a p &lt; 0.05 was considered statistically significant.Results:This study enrolled 68 CLL patients, 37 males and 31 females with a median age of 65 years (48–101), and 23 CTL, 8 males and 15 females with a median age of 72 years (58–88). According with Binet staging system 54 patients (89%) were low risk (A), 2 (3%) intermediate (B) and 5 (8%) high risk (C). Cytogenetic abnormalities usually associated with good prognosis were found in 28 patients (62%) and with bad prognosis in 17 patients (38%), including 12 with del(17p) (27%). We observe that CLL patients had a higher DNMT1 expression [median: 0691; interquartile range (IR): 0.990] and a significant lower DNMT3B gene expression levels [median: 0.023, IR: 0.040; p = 0.020] compared with controls [DNMT1 median: 0.603, IR: 0.995; DNMT3B median: 0.038; IR: 0.267] and in 12 patients (18%) no expression of DNMT3B was detected. On the other hand, DNMT3A expression levels are similar in patients and controls (CLL median: 0.281, IR: 0.283; CTL median: 0.283, IR: 0.268). Using ROC analysis, DNMT3B expression levels shown to be a possible diagnosis biomarker (AUC: 0.720; IC95%: 0.596–0,843; cut‐off&lt;0.026; sensitivity: 63%; specificity: 78%; p = 0.002). The expression levels of DNMT1, DNMT3A and DNMT3B show no correlation with clinical and laboratory data, including Binet staging system or cytogenetic abnormalities. According to ROC analysis and Kaplan Meier curves, CLL patients with DNMT3B expression levels under 0.031 present a tendency for worse survival.Summary/Conclusion:This study suggests that DNMT3B expression levels may contribute to CLL development and may be potential new diagnostic and prognostic biomarkers.The work was supported by CIMAGO (Project 02/18) and FCT (SFRH/BD/51994/2012).

THE ROLE OF THE GENETIC ABNORMALITIES, EPIGENETIC AND microRNA IN THE PROGNOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) isincreased proliferation ofB-cells with peripheral blood and bone marrow involvement, which is usually observed in older people. Genetic mutations, epigenetic changes and miRs play a role in CLL pathogenesis. Del 11q, dell17q, del6q,trisomy 12, p53 and IgVH mutations are the most important genetic changes in CLL. Deletion of miR-15a and miR-16a can increase bcl2 gene expression, miR-29 and miR-181 deletions decrease the expression of TCL1, and miR-146a deletion prevents tumor metastasis. Epigenetic changes such as hypo- and hypermethylation, ubiquitination, hypo- and hyperacetylation ofgene promoters involved in CLL pathogenesis can also play a role in CLL. Expression of CD38 and ZAP70, presence or absence of mutation in IgVH and P53mutation are among the factors involved in CLL prognosis. Use of monoclonal antibodies against surface markers of B-cells like anti-CD20as well as tyrosine kinase inhibitors are the most important therapeutic approaches for CLL.

Proteomics Profiling of CLL Versus Healthy B-cells Identifies Putative Therapeutic Targets and a Subtype-independent Signature of Spliceosome Dysregulation

Chronic lymphocytic leukemia (CLL) is a heterogeneous B-cell cancer exhibiting a wide spectrum of disease courses and treatment responses. Molecular characterization of RNA and DNA from CLL cases has led to the identification of important driver mutations and disease subtypes, but the precise mechanisms of disease progression remain elusive. To further our understanding of CLL biology we performed isobaric labeling and mass spectrometry proteomics on 14 CLL samples, comparing them with B-cells from healthy donors (HDB). Of 8694 identified proteins, ∼6000 were relatively quantitated between all samples (q<0.01). A clear CLL signature, independent of subtype, of 544 significantly overexpressed proteins relative to HDB was identified, highlighting established hallmarks of CLL (e.g. CD5, BCL2, ROR1 and CD23 overexpression). Previously unrecognized surface markers demonstrated overexpression (e.g. CKAP4, PIGR, TMCC3 and CD75) and three of these (LAX1, CLEC17A and ATP2B4) were implicated in B-cell receptor signaling, which plays an important role in CLL pathogenesis. Several other proteins (e.g. Wee1, HMOX1/2, HDAC7 and INPP5F) were identified with significant overexpression that also represent potential targets. Western blotting confirmed overexpression of a selection of these proteins in an independent cohort. mRNA processing machinery were broadly upregulated across the CLL samples. Spliceosome components demonstrated consistent overexpression (p = 1.3 × 10-21) suggesting dysregulation in CLL, independent of SF3B1 mutations. This study highlights the potential of proteomics in the identification of putative CLL therapeutic targets and reveals a subtype-independent protein expression signature in CLL.

CD73, a potential diagnostic marker in Egyptian patients with chronic lymphocytic leukemia

Objective This work aimed to study CD73 expression in chronic lymphocytic leukemia (CLL) patients and its correlation with other disease characteristics. Background CLL is a common B-lymphoproliferative disorder, which can be diagnosed in most cases by morphology and flowcytometry. It displays heterogeneity in clinical presentation, course, and response to treatment, which is likely due to the diversity of the biological nature of the disease. Diagnostic difficulty might arise in some patients due to atypical morphology and/or immunophenotype. CD73 is an extracellular enzyme that hydrolyses adenosine monophosphate to adenosine and is expressed on a subset of T and B lymphocytes. A number of studies have shown CD73 as a contributing factor in the multistep process of carcinogenesis by inducing favorable environment for tumor growth. However, it was shown in some studies to induce apoptosis. Patients and methods CD73 expression on both B and T lymphocytes was measured in 25 cases of newly diagnosed CLL and 15 healthy controls. The levels of expression were correlated to other disease characteristics. Results CD73 expression on CD19 + B lymphocytes was significantly lower in CLL patients compared with controls (mean 3.74 vs. 29.35%, respectively, P Conclusion CD73 expression is a potential diagnostic marker in CLL. This can be particularly relevant in cases with atypical immunophenotype using the current CLL scoring system. CD73 may have a role in CLL pathogenesis as low expression can lead to failure of termination of immune response with subsequent proliferation and clonal expansion of the involved clone.

Circulating microRNA-192 as a diagnostic biomarker in human chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is known as the most common lymphoid malignancy in the Western world. MicroRNAs (miRNAs) are a class of small noncoding RNAs with pivotal roles in cellular and molecular processes related to different malignancies including CLL. Recently, some studies have shown that miR-192 plays a key role in CLL pathogenesis through increasing CDKN1A/p21 levels, suppression of Bcl-2 and enhancement of wild-type P53 and cell cycle arrest. Forty samples, including 20 patients with CLL, diagnosed in Omid hospital (Isfahan, Iran) and 20 healthy controls were sampled during a period of 4 months. Using real-time PCR method, expression of miR-192 was analyzed in peripheral blood mononuclear cells (PBMCs) of CLL patients in comparison with healthy subjects. In silico molecular signaling pathway enrichment analysis was also performed on validated and predicted targets (targetome) of miR-192 in DAVID database to explore possible role of miR-192 in some pathways. The expression of miR-192 was found to be significantly reduced (~2.5-folds) in CLL patients compared with healthy subjects (P=0.002). In silico molecular signaling pathway enrichment analysis detected cell indicated signaling pathway as one of the most statistically relevant pathway with miR-192 targetome. Our findings showed that miR-192 could be a biomarker for early diagnosis of CLL.

Ibrutinib downregulates a subset of miRNA leading to upregulation of tumor suppressors and inhibition of cell proliferation in chronic lymphocytic leukemia.

The lymph node (LN) is the site of chronic lymphocytic leukemia (CLL) cell activation and proliferation. Aberrant microRNA (miRNA) expression has been shown to have a role in CLL pathogenesis; however, a comparison of miRNA expression between CLL cells in the LN and the peripheral blood (PB) has previously not been reported. On the basis of the analysis of 17 paired LN and PB samples from CLL patients, we identify a panel of miRNAs that are increased in LN CLL cells correlating with an activation phenotype. When evaluated in CLL cells from 38 patients pre and post treatment with ibrutinib, a subset of these miRNAs (miR-22, miR-34a, miR-146b and miR-181b) was significantly decreased in response to ibrutinib. A concomitant increase in putative miRNA target transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) was also observed. Functional studies confirmed targets of ibrutinib-responsive miRNAs to include messenger RNA transcripts of multiple tumor suppressors. Knockdown of endogenous miR-34a and miR146b resulted in increased transcription of tumor suppressors and inhibition of cell proliferation. These findings demonstrate that ibrutinib downregulates the expression of a subset of miRNAs related to B-cell activation leading to increased expression of miRNA targets including tumor suppressors and a reduction in cell proliferation.

Ibrutinib Responsive Micro-RNAs and Upregulation of Tumor Suppressor Targets in Chronic Lymphocytic Leukemia

Background: MicroRNAs (miRNAs, miRs) are small (~22nt) noncoding RNAs that are involved in post-transcriptional regulation of specific mRNA targets. Aberrant miRNA expression has been shown to play a role in chronic lymphocytic leukemia (CLL) pathogenesis and to have prognostic implications. The lymph node (LN) has been demonstrated to be the site of CLL cell proliferation and activation. In particular, CLL cells in the LN display significantly increased BCR and NF-kB signaling in comparison to CLL cells in the peripheral blood (PB). Ibrutinib is a clinically approved BTK inhibitor that disrupts BCR and NF-kB signaling; and has been shown to be effective in relapsed or refractory CLL. We hypothesized that 1) miRNAs would be differentially expressed between activated CLL cells in the LN and CLL cells in the PB; and 2) ibrutinib would alter miRNA expression in CLL, which may affect therapeutic response by regulating the expression of miR target genes.Methods: RNA was isolated from CD19 positive selected cells from two separate cohorts of CLL patients. The first group included 17 treatment naïve patients donating paired LN and PB CD19 positive cells (10 IGHV un-mutated [59%], 4 IGHV mutated [23.5%] and 3 IGHV unknown [17.5%]); the second group contained 38 pairs of PB CD19 positive cells (22 IGHV un-mutated [58%] and 16 IGHV mutated [42%]) before and after ibrutinib treatment on day 28. A panel of 22 miRs previously reported to play a role in CLL pathogenesis, prognosis, or BCR signaling was profiled by qPCR. The expression level of a custom panel of genes encoding putative miR targets was quantified using the nanoString nCounter assay and verified by qPCR. Functional studies were performed in MEC-1 and SP-53 cell lines, which were treated separately with 1 μM ibrutinib or 1 μM fludarabine for 48 hours. Live cells were identified by trypan blue staining; miR and mRNA expression was quantified by qPCR.Results: Nineteen of 22 (86.4%) miRs were significantly increased (p < 0.05) in CLL cells from the LN in comparison to paired CLL cells from the PB, correlating with an activated B cell phenotype. Examination of mRNA transcripts of putative miR targets in 13 paired LN and PB samples showed that multiple tumor suppressor transcripts including CYLD, ARID1B and FOXP1 were significantly decreased in LN-derived CLL cells. Assay of 22 miRs in PB CLL cells from 38 patients before and after ibrutinib treatment indicated that a subset of miRs (miR-22, miR-34a, miR-146b and miR-181b) were significantly decreased (p < 0.05) in response to ibrutinib. Quantification of mRNA transcripts of putative targets of the ibrutinib responsive miRs by the nanoString nCounter assay showed that several tumor suppressor transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) were significantly increased. Functional studies in MEC-1 and SP-53 cell lines confirmed that miR-22, miR-34a, miR-146b and miR-181b were down-regulated and the tumor suppressor targets of these miRs (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, PTEN and SMAD4) were significantly increased after ibrutinib treatment. The alteration of miR and gene expression was specific to the effects of ibrutinib as similar changes were not observed post treatment with the chemotherapeutic agent fludarabine.Conclusions: miRNA expression patterns are significantly altered between CLL cells in the LN and those in the PB. An overall increase in the expression of miRs and down-regulation of mRNAs of tumor suppressor genes (ARID1B, CYLD and FOXP1) was observed in CLL cells in the LN, correlating with an activated B-cell phenotype. Ibrutinib treatment and inhibition of BCR signaling resulted in down-regulation of miR-22, miR-34a, miR-146b and miR-181b and concomitant up-regulation of multiple tumor suppressor targets including ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4 in PB CLL cells. Similar findings were also observed in ibrutinib treated MEC-1 and SP-53 cell lines. Additional studies are needed to address how much the down-regulation of miRs and consequent up-regulation of tumor suppressors in response to ibrutinib contributes to the overall disease response and whether this effect could sensitize CLL cells to treatment with cytotoxic agents. DisclosuresWiestner:Pharmacyclics: Research Funding.

Genetic abnormalities in chronic lymphocytic leukemia: where we are and where we go.

Chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are detected in up to 80% of patients. Among them, deletions of 11q, 13q, 17p, and trisomy 12 have a known prognostic value and play an important role in CLL pathogenesis and evolution, determining patients outcome and therapeutic strategies. Standard methods used to identify these genomic aberrations include both conventional G-banding cytogenetics (CGC) and fluorescence in situ hybridization (FISH). Although FISH analyses have been implemented as the gold standard, CGC allows the identification of chromosomal translocations and complex karyotypes, the latest associated with poor outcome. Genomic arrays have a higher resolution that allows the detection of cryptic abnormalities, although these have not been fully implemented in routine laboratories. In the last years, next generation sequencing (NGS) methods have identified a wide range of gene mutations (e.g., TP53, NOTCH1, SF3B1, and BIRC3) which have improved our knowledge about CLL development, allowing us to refine both the prognostic subgroups and better therapeutic strategies. Clonal evolution has also recently arisen as a key point in CLL, integrating cytogenetic alterations and mutations in a dynamic model that improve our understanding about its clinical course and relapse.

Involvement Of Autoreactive T Lymphocytes In Pathogenesis Of Chronic Lymphocytic Leukemia (CLL): Specific T-Cell Immune Responses Against Autoantigens Recognized By CLL Cells

The etiology of chronic lymphocytic leukemia (CLL) has remained unknown but recently it has been speculated that antigens stimulation could be involved in the pathogenesis of CLL. Cytoskeletal proteins exposed on the surface of apoptotic cells could represent autoantigens involved in the development of CLL since monoclonal antibodies derived from CLL-specific B-cell receptors (BCR) recognized MYHIIA (non-muscle myosin heavy chain IIA), vimentin, and cofilin-1. Since the humoral immune response against these autoantigens could play a crucial role in pathogenesis of CLL we suspect that it might be accompanied by T-cell immune responses. We defined HLA-A2-restricted epitopes in silico using two independent computer prediction algorithms. Next 36 most immunogenic peptides derived from MYHIIA, cofilin-1 and vimentin were selected and synthesized. To determine whether peptides could bind to HLA-A2 also in vivo we performed cellular-peptide binding affinity assay utilizing T2 cell line. The expression of HLA-A2 was measured using flow cytometry with FITC-conjugated anti-HLA-A2 monoclonal antibodies. Fluorescence index (FI) for particular peptides was calculated as mean fluorescence intensity of HLA-A2 on T2 cells with peptide versus without peptide. We selected peptides with the highest FI M6 (KLKDVLLQV, FI=0.43), M1 (GLLRVISGV, FI=0.20), M2 (YLLEKSRAI, FI=0.16), M3 (VLMIKALEL, FI=0.15) derived from MYHIIA, V1 (SLQEEIAFL, FI=4.93) and V4 (ILLAELEQL, FI=1.49) derived from vimentin and C1 (ALYDATYET, FI=0.60) C7 (LQANCYEEV, FI=0.68), C11 (ILEEGKEIL FI=0.88) derived from cofilin-1. Mixed lymphocyte/peptide cultures (MLPC) were performed to define immunogenicity of synthesized peptides in vitro. CD8+ cells from HLA-A2 positive 15 CLL patients and 8 healthy volunteers were selected by magnetic-activated cell sorting. Afterwards, CD8- antigen presenting cells were irradiated and pulsed for 2 hours with respective peptides derived from MYHIIA, vimentin and cofilin-1 or control peptide at a concentration of 10 μg/ml and without peptide for negative control. Cells were supplemented with IL-2 and IL-7 on day +1 and restimulated every week. At day +21 of MLPC, cells were harvested and evaluated for their specific cytotoxicity in ELISpot assays with peptides, with the unspecific positive control Pokeweed Mitogen (PWM), or without peptide, as negative control. The ELISpot assay was performed under the conditions recommended by the Cancer Immunotherapy Monitoring Panel. We detected specific cytotoxic immune responses for peptides derived from MYHIIA in 71%, vimentin in 83% and cofilin-1 in 50% CLL patients. Additionally, we found positive responses in 37.5% healthy volunteers. Finally, we assessed the frequency of the specific T cells in 14 CLL patients along with detailed phenotypical characteristics using dextrameric peptide-specific complexes in 7-colour panel using FACSCantoII Flow Cytometer ex vivo. To characterize naïve/effector/effector-memory and memory cells were stained for CD8, CD3, CCR7, CD45RA with exclusion of CD19+, CD56+ and CD4+ cells and 7AAD+ dead cells. We detected low frequency of peptide-specific T cells, with the highest level of 3.6% CD8+ cells specific for M3 peptide. Most cells (83%) represented effector-memory phenotype. Subsequently, we isolated RNA from 100 CLL patients to assess MYHIIA, VIM, and CFL-1 expression using qRT-PCR. Quantity mean values for gene expression were calculated according to the Standard Curve method. GAPDH was used as a constitutive gene. All three genes were overexpressed with no difference between males and females, Binet stages A, B and C nor different cytogenetic and molecular groups of CLL patients. These data suggest that MYHIIA, VIM and CFL-1 might be considered as a novel constitutive genes, highly expressed in CLL. In summary, our findings suggest presence of cytotoxic immune responses against three autoantigens recognized by BCR of CLL cells. The existence of autoreactive CD8+ T cells against MYHIIA, vimentin, cofilin-1 in CLL patients points to the involvement of antigen-specific autoreactive T cells in pathogenesis of CLL. Disclosures:No relevant conflicts of interest to declare.

The Role of the BCR Class Expressed by Eμ-TCL1tg Mice and Human CLL

AbstractAbstract 182The B-cell antigen receptor (BCR) plays a critical role in the development and progression of B-cell lymphomas. In chronic lymphocytic leukemia (CLL), the existence of stereotyped heavy-chain complementarity determining regions (HCDR3) suggested that binding of external antigens might play a role in CLL pathogenesis. In contrast, we recently reported that BCRs derived from both human CLL patients and from Eμ-TCL1tg mice have the unique function to induce antigen-independent signaling. This capacity is mediated by the HCDR3 through binding to a BCR internal motif in adjacent BCRs on the same cell (Dühren-von Minden et al., Nature 2012). Mature B cells as well as CLL B cells co-express IgM and IgD with the same HCDR3. In this study, we address the respective roles of these expressed BCR classes in lymphoma pathogenesis in Eμ-TCL1tg mice and in human CLL.By mating Eμ-TCL1tg-mice with IgM−/− mice, which lacks the μ constant heavy domain (μCH) and instead expresses IgD in all developmental stages, we demonstrate a significantly lower frequency of CD19+CD5+IgM+IgDlow lymphoma cells in heterozygous IgM+/−TCL1 mice compared to conventional IgM+/+TCL1 mice (p=0.007). Furthermore, IgM+/−TCL1 mice show a delayed or slowed disease progression compared to TCL1tg mice carrying both IgM alleles. In both TCL1tg mice strains, lymphoma development was exclusively linked to expression of surface IgM, since no IgD single positive lymphoma was detected in IgM+/−TCL1 mice (n=12). TCL1tg mice that lack both μCH alleles showed an accumulation of CD19+CD5+ cells in the spleen at the age of 6 months. According to the genotype of these mice, this population was indeed IgD single positive. However, no further progression could be observed during follow-up to an age of 8 months, indicating a benign form of lymphoproliferation. In contrast to BCRs derived from Eμ-TCL1 mice, analysis of the signaling properties of BCRs derived from IgM−/−TCL1 mice failed to show any autonomous signaling capacity, even when they were expressed as IgM. To address whether IgD in general is able to mediate autonomous signaling reported for TCL1tg- and CLL-derived BCRs, we tested these receptors for autonomous signaling capacity when expressed as IgD. However, expression as IgD led to a complete loss of autonomous signaling capacity in all cases (n=10).In conclusion, whereas autonomous signaling is a characteristic feature of TCL1tg- and CLL-derived BCRs, the pathogenesis of CLL is dependent on the expression of their BCR as the IgM isotype. Expression of IgD-BCRs leads to loss of autonomous signaling capacity, and mice that lack μCH fail to develop malignant lymphoproliferation.To address the question if differential expression of IgD and IgM also had an impact on the clinical behavior of human CLL, we measured the relative expression levels of surface IgD and IgM on circulating lymphoma cells from 67 CLL patients by flow cytometry with simultaneous staining. According to previous reports (Mockridge et al., Blood 2007), unmutated (UM-CLL) cases (n=22) had a higher level of total surface Ig compared to mutated CLL (M-CLL) cases (n=45). Based on our results that IgM is more potent to drive lymphoproliferation, we calculated the ratio of mean fluorescence intensities for IgD over IgM, further called DvM-Score, for every case. A significant difference in the expression pattern as represented by the DvM-Score was observed for UM-CLL and M-CLL (p=0.003) as well as for ZAP70+ and ZAP70- cases (p=0.0002). Both UM-CLL cases as well as ZAP70+ cases show a higher amount of IgM compared to IgD represented by a DvM-Score of <1, whereas the majority of M-CLL and ZAP70− cases express less IgM than IgD and show a DvM-Score of >1. Based on receiver operating characteristics, an DvM cut-off value of 1.15 was identified to optimally discriminate mutational status (AUC: 0.72) and ZAP70 expression (AUC: 0.78). Preliminary data show that CLL samples with a DvM-Score <1.15 had a more aggressive disease course as indicated by a median time to first treatment (TTFT) of 39 months, whereas cases with a DvM-Score of >1.15 show a median TTFT of 154 months (log rank test, p=0.0014).In summary, our results demonstrate an important role of the BCR class, especially with respect to the pathogenetic role of autonomously active IgM BCRs expressed by CLL B cells, for the outcome of the disease. In addition, the DvM score may represent a convenient and novel prognostic marker for CLL. Disclosures:No relevant conflicts of interest to declare.

Epigenetic Profiling of Primary CLL Reveals Novel DNA Methylation-Based Clusters and Novel Mechanisms of Leukemogenesis

AbstractAbstract 3877 Introduction:Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world with nearly 15,000 new cases diagnosed every year in the USA. The characterization of CLL has resulted in the identification of important disease biomarkers: these include the recurrent genomic deletions del17p and del11q, genomic complexity, TP53 mutations, the expression level of ZAP70 and the mutational status of IgVH. While genomic and transcriptional profiling of CLL identified clinically and biologically relevant markers, there is still significant uncertainty about the pathobiology and the origin of CLL. It is increasingly clear that epigenetic deregulation plays an important role in the biology of all lymphomas/leukemias including CLL. Methods:We hypothesized that DNA methylation profiling would allow us to identify new, biologically significant CLL subtypes and yield greater insight into the biology of this disease. We therefore examined the DNA methylation of over 240 patients with CLL using the HELP assay and hybridization to high density custom microarray that reports on the methylation status of more than 250,000 CpGs corresponding to 20,401 genes. Gene expression profiling and SNP array-based copy number assessments and targeted gene resequencing were available on most of these cases. We performed unsupervised analysis on the most variable probesets (standard deviation > 1.3) using K-means consensus clustering. Results:The experimental approach reproducibly identified three robust CLL subtypes based on epigenetic profiles. To identify the genes that define these three subtypes we next performed unequal variance t-test of the CLL subtypes comparing them to Peripheral Blood CD19+ B cells as a normal control, and identified that clusters are defined by differential methylation of 3719, 6145 and 3349 genes (selected probes displayed changes in methylation of at least 30% and FDR corrected p-value < 0.05), The three clusters featured respectively i) aberrant methylation of MYC and WNT target genes, ii) aberrant methylation of NOTCH1 targets and iii) aberrant methylation of bcl6 and inflammatory cytokines. There was inverse correlation between gene expression and cytosine methylation, suggesting that DNA methylation had an impact on the transcriptional programming of these CLL cases. Strikingly the CLL MYC/WNT cluster displayed poorer prognosis as opposed to the CLL BCL6 cluster (HR=0.14 95% CI: 0.07–0.30). The CLL NOTCH1 cluster had an intermediate prognosis. It was also notable that all CLL patients exhibited deregulation of the B-cell receptor pathway as compared to normal CD19+ B-cells, consistent with the notion that this pathway plays a critical role in CLL pathogenesis. Finally, we divided the cohort into training and testing cohorts and used a machine learning BDVAL algorithm to identify DNA methylation outcome classifiers. This procedure identified a 40-probeset classifier that accurately predicted outcome (Area Under the ROC Curve of 0.77; performance was assessed with 10 fold cross-validation in a training set with 76 patients; validation on an independent set of 105 samples). Conclusion:This large epigenetic profiling study in CLL identifies aberrant epigenetic regulation as a core part of the pathobiology of CLL and identifies novel CLL clusters with distinct effects on survival. MYC-WNT pathway inhibitors are warranted for use in clinical trials for patients belonging to this aggressive epigenetically defined subtype. Disclosures:No relevant conflicts of interest to declare.

Downregulated Dicer expression predicts poor prognosis in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Alterations in microRNAs (miRNAs) expression have been proposed to play a role in CLL pathogenesis. Dicer and Drosha are the main regulators of miRNA biogenesis, and deregulation of their expression has been indicated as a possible cause of miRNA alterations observed in various cancers. To investigate the role of Dicer and Drosha in CLL, we assessed the expression of Dicer and Drosha and their correlation with other prognostic factors, including Binet stages, immunoglobulin heavy chain variable gene (IGHV) mutation status, TP53 mutation status, ZAP-70 protein and CD38 expression level in 165 CLL patients by using real-time polymerase chain reaction methods. Patients with unmutated IGHV genes had significantly lower expression of Dicer than patients with IGHV mutations. The lower expression level of Dicer was also significantly associated with higher level of CD38 and ZAP-70, and more aggressive Binet stage. We also analyzed Dicer expression in different cytogenetic subgroups. Lower Dicer level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22.3) in contrast to higher level in good risk cytogenetics (deletion in 13q14 as the sole abnormality). Furthermore, the lower expression of Dicer in CLL shows a strong association with shorter overall survival (OS) (P = 0.0046) as well as with reduced treatment free survival (TFS) (P = 0.0006). By contrast, no differences in the expression of Drosha among these groups of patients were observed. Our data suggest that Dicer expression may play an important role in the progression and prognosis of CLL.

Aberrant Microrna Expression In Chinese Patients with Chronic Lymphocytic Leukemia.

AbstractAbstract 4605MicroRNAs (miRNAs) are a class of small endogenous RNAs that play important regulatory roles by targeting mRNAs for cleavage or translational repression. Many reports have indicated that microRNAs (miRNAs) play a critical role in malignancies, and regulations in the progression of leukemia. However, the miRNAs expression level in Chinese patients with chronic lymphocytic leukemia (CLL), and its prognostic value remain elusive. We identified various degrees of down-regulation of miR-15a, miR-16-1, miR-29b, miR-181a and miR-181b in CLL mononuclear cells of 138 patients. Moreover, we have identified that miR-29b, miR-181a and miR-181b expression was significantly correlated with IGHV mutatioal status (the P value was 0.057, 0.034 and 0.004, respectively). Down-expression of miR-181b was significantly associated (P=0.001) with overall survival in patients while expression for miR-181a was of borderline significant (P=0.09). To further determine the consequence of miR-181-down regulation, we sought to discover targets of miR-181 family. Transcript levels of predicted target genes BCL-2 and TCL-1 were determined, and the expression levels were significantly upregulated in CLL patients compared with normal controls (P<0.001). Higher expression of TCL-1 was significantly correlated with aggressive disease features. In addition, an inverse correlation was observed in the CLL samples between miRNAs (miR-16-1, miR-181a, miR-181b) and BCL-2 level (r=-0.216, P=0.012; r=-0.274, P=0.002; r=-0.184, P=0.036, respectively); furthermore, an inverse correlation was also observed between miRNAs (miR-16-1, miR-181a, miR-181b) and TCL-1 (r=-0.272, P=0.002; r =-0.224, P=0.009; r=-0.152, P=0.054, respectively), which suggest that these miRNAs may implicated in negatively regulating target mRNA at a transcriptional level. For further study, our study showed that miR-181a and miR-181b functioned as tumor suppressors that induced apoptosis in CLL cells by regulates BCL-2/MCL-1 protein expression. The luciferase activity of a BCL-2-3‘-untranslated region-based reporter construct in CLL cells suggests that a new target site in the 3'UTR of BCL-2 of the mature miR-181a/b. MCL-1 is a BCL-2 family member and pro-survival protein associated with drug resistance in leukemia. Using a luciferase construct containing the MCL-1 3‘ UTR we observed a decrease in luciferase expression upon miR-181b over-expression. It was suggested that miR-181b directly targets MCL-1 mRNA. In addition, enforced miR-181a/b expression in primary CLL cells (n= 52) reduced BCL-2/MCL-1 protein level and sensitized primary CLL B cells to fludarabine-induced apoptosis and also had effect in B-CLL cells from 7 p53 attenuated patients. Taken together, our results suggest that these deregulated miRNAs may play an important role in pathogenesis of CLL and might be applied for the assessment of prognosis in patients with CLL. Among which, miR-181a/b could function as tumor suppressors in human CLL cells, at least in part, by modulation of apoptosis via targeting BCL-2 and MCL-1. Disclosures:No relevant conflicts of interest to declare.

High Expression of LAG3, LPL and ZAP-70 Genes in B-CLL Strongly Correlates with Unmutated IgVH and Early Therapy Requirement.

Mutational status of immunoglobulin genes coding for heavy chain variable region (IgVH) stratifies patients with B-cell chronic lymphocytic leukemia (CLL) into two distinct prognostic groups. Expression level of specific genes such as LPL, ZAP-70, ADAM29 or CLLU1 has previously been shown to distinguish these two subgroups, although their exact role in CLL pathogenesis remains unclear. In our study we performed microarray and Real-time PCR analyses of gene expression in B-CLL cells with different IgVH mutational status in an attempt to further identify potential discriminating genes. Heparinised peripheral blood samples were collected from 92 previously untreated B-CLL patients (40 IgVH unmutated vs. 52 IgVH mutated) with a median age of 62 years (ranging from 40 to 81 years). B-cells were separated using cocktails of selected bispecific antibodies directed against cell surface antigens of hematopoietic cells and glycophorin A (RosetteSep® B Cell Enrichment Kit, StemCell Technologies). Great attention was paid to T-cell depletion using Human CD3 Depletion Cocktail. Samples with lower percentage of B-CLL cells were further processed by immunomagnetic separation (EasySep® FITC Selection Cocktail, anti-CD5FITC) and only samples with B-CLL cell content higher than 95 % (assessed using flow cytometry) were used for the gene expression analysis. Microarray gene expression analysis of 20 B-CLL patients (10 with mutated and 10 with unmutated IgVH) was performed using Agilent Human 1A Arrays and obtained data were statistically analysed using SAM (Significance Analysis of Microarrays). Among approx. 50 diferentially expressed genes, high expression of LPL and LAG3 (Lymphocyte- Activation Gene 3) was the most prominently associated with unmutated IgVH subtype. The mRNA levels of selected genes (LAG3, LPL, ZAP-70, AICDA, BCL2, CLLU1, TCF7, SEPT10, ADAM29) were further tested on a cohort of 92 B-CLL patients using quantitative RT-PCR (TaqMan® Gene Expression Assay, Applied Biosystems). Among them, LAG3, LPL and ZAP-70 reached the best statistical significance (p-value < 0.001; Wilcoxon's rank-sum test). LAG3 expression level can predict unmutated IgVH with 100% sensitivity and could be used as a novel potential CLL prognostic marker. LAG3 plays role in T-cell dependent B-cell activation during affinity maturation and its over-expression in cells with unmutated IgVH may be a consequence of improper signalling in this process. Combination of expression data of three genes LAG3, LPL and ZAP-70 reliably assigned 95% of CLL samples into a correct IgVH subtype and seems to be better predictor of IgVH status than either of these genes itself. Moreover, we have analysed the correlation between expression of those three IgVH surrogate markers and necessity of therapeutical intervention. The patients manifesting high expression of LAG3, LPL and ZAP-70 required therapy significantly earlier than patients with low expression of those genes as calculated by Kaplan-Meier estimator (median time to the first therapy was 797 days in patients with high expression of surrogate markers, median was not reached in patients with low expression of surrogate markers). Differences of time to the first therapy between patients with differential expression of three IgVH surrogate markers (p=4.4*10−8, log-rank test) and differences between patients with different IgVH status (p=7.5*10−8, log-rank test) showed high significance. These results suggest that IgVH status could be precisely assessed using expression analysis of three genes LAG3, LPL and ZAP-70. The expression data of those surrogate markers provide even better statistical concordance with time to requirement of the first therapy than IgVH mutational status itself and possibly could be used in the diagnostics.

Regulation of Chronic Lymphocytic Leukemia Cell Proliferation by CTLA4.

B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia with heterogenous clinical course. Prognostic markers such as CD38 expression has proven to be reliable in predicting clinical outcome where high CD38 expression correlates with poor prognosis and low CD38 expression is associated with good prognosis. Recently, our lab has shown that expression of CTLA4 in CLL cells is inversely correlated with CD38 expression using microarray profiling and real time PCR. Microarray analysis showed significant differential expression of CTLA4 between high and low CD38 subgroups (n=49) (Joshi et al. Clinical Cancer Research, 2007, in press). However, whether CTLA4 plays a direct role in CLL pathogenesis or not is unknown. Therefore to study the potential role of CTLA4 in CLL, CTLA4 expression was down regulated in primary CLL cells from six different patients using antisense oligonucleotide. The down regulation of CTLA4 expression was confirmed by RT-PCR analysis. The proliferative capability of the CTLA4-down regulated CLL cells was determined using MTT assay as well as radiolabeled thymidine uptake assay at 24, 48 and 72 hours in culture. There was a significant increase in the proliferation of CTLA4-down regulated CLL cells compared to untreated control CLL cells or CLL cells treated with an irrelevant oligonucoeotide. To further understand the CTLA4 mediated regulation of CLL cell proliferation, expression levels of downstream target molecules of CTLA4 such as NFATC2 and c-FOS was studied using RT-PCR. There was a significantly increased expression of target genes NFATC2 and c-FOS, molecules in the CTLA4-downregulated CLL cells compared to control CLL cells. In addition, further analyses of the microarray data revealed that CD38 high expressing CLL cells (n=22) has significant (p<0.05) higher NFATC2 and c-FOS expression compared to CD38 low expressing CLL cells (n=29). This was further confirmed by real time PCR analyses (figure1). Thus, these results clearly demonstrate that CTLA4 is involved in the regulation of proliferation of CLL cells via regulating the expression of NFATC2 and c-FOS, the downstream signaling molecules involved in the cell proliferation. (This work was supported by the CLL Foundation, Houston, TX). [Display omitted]