Role In Chronic Lymphocytic Leukemia Research Articles

Written in Blood: Kissing Disease miRNAs Could Predict Outcome of Patients With Chronic Lymphocytic Leukaemia

Written in Blood: Kissing Disease miRNAs Could Predict Outcome of Patients With Chronic Lymphocytic Leukaemia

Role of CD38 Expression in Diagnosis and Pathogenesis of Chronic Lymphocytic Leukemia and Its Potential as Therapeutic Target.

CD38 is widely accepted as a marker for unfavorable prognosis in chronic lymphocytic leukemia (CLL). Nevertheless, its direct contribution to the disease pathogenesis is not very well understood. Recent data indicate that CD38 may promote CLL pathogenesis by enhancing proliferation in synergy with B-cell receptor (BCR) signaling and by supporting migration and homing of CLL cells to secondary lymphoid organs, where the malignant cells receive support from the tumor microenvironment. CD38 may also contribute to a suppressed anticancer immune response through the production of tolerogenic compounds. This review first shortly summarizes the biology, expression, and function of CD38 in general and in CLL cells in more detail. Next, the current literature and open questions regarding a direct contribution of CD38 to CLL pathogenesis are critically reviewed. Finally, I discuss the potential of CD38 as therapeutic target in light of its possible roles in CLL and the promising results of clinical trials with CD38 antibodies in multiple myeloma (MM).

Opposite Prognostic Significance of Cellular and Serum Circulating MicroRNA-150 in Patients with Chronic Lymphocytic Leukemia.

MicroRNAs (or miRs) play a crucial role in chronic lymphocytic leukemia (CLL) physiopathology and prognosis. In addition, circulating microRNAs in body fluids have been proposed as new biomarkers. We investigated the expression of matched cellular and serum circulating microRNA-150 by quantitative real-time PCR (qPCR) from purified CD19(+) cells or from CLL serums obtained at diagnosis in a cohort of 273/252 CLL patients with a median follow-up of 78 months (range 7-380) and correlated it to other biological or clinical parameters. We showed that miR-150 was significantly overexpressed in CLL cells/serums compared with healthy subjects (P < 0.0001). Among CLL patients, a low cellular miR-150 expression level was associated with tumor burden, disease aggressiveness and poor prognostic factors. In contrast, a high level of serum miR-150 was associated with tumor burden markers and some markers of poor prognosis. Similarly, cellular and serum miR-150 also predicted treatment-free survival (TFS) and overall survival (OS) in an opposite manner: patients with low cellular/serum miR-150 levels have median TFS of 40/111 months compared with high-level patients who have a median TFS of 122/60 months (P < 0.0001/P = 0.0066). Similar results were observed for OS. We also found that cellular and serum miR-150 levels vary in an opposite manner during disease progression and that cellular miR-150 could be regulated by its release into the extracellular space. Cellular and serum levels of miR-150 are associated with opposite clinical prognoses and could be used to molecularly monitor disease evolution as a new prognostic factor in CLL.

The Identification of Further Minimal Regions of Overlap in Chronic Lymphocytic Leukemia Using High-Resolution SNP Arrays

Background:Historically, the identification of minimal deleted regions (MDRs) has been a useful approach for pinpointing genes involved in the pathogenesis of human malignancies and constitutional disorders. Microarray technology has offered increased capability for newly identifying or refining existing MDRs and minimal overlapping regions (MORs) in cancer. Despite this, in chronic lymphocytic leukemia (CLL), published MORs that pinpoint only a few candidate genes have been limited and with the advent of NGS, the utility of high resolution array work as a discovery tool has become uncertain. Here, we show that profiling copy number abnormalities (CNAs) and cnLOH using arrays in a large patient series can still be a valuable approach for the identification of genes that are disrupted or mutated in CLL and have a role in CLL development and/or progression.Methods: 250 CLL patient DNAs from individuals enrolled in two UK-based Phase II randomised controlled trials (AdMIRe and ARCTIC trials) were tested using Infinium HumanOmni2.5-8 v1.1 according to manufacturer’s guidelines (Illumina Inc, San Diego, CA). Data were processed using GenomeStudioV2009.2 (Illumina Inc.) and analysed using Nexus Discovery Edition v6.1 (BioDiscovery, Hawthorne, CA). All Nexus plots were inspected visually to verify calls made, identify uncalled events and exclude likely false positives. To exclude common germline CNVs, the Database of Genomic Variants (DGV), a comprehensive catalog of structural variation in control data, was used. Copy number (CN) changes that encompassed fully changes noted in the DGV were excluded from further analysis. Regions of copy neutral loss of heterozygosity (cnLOH) were recorded if >1Mb in size, but were not used to define or refine MORs. Data from 1275 age-appropriate control samples minimised the reporting of common cnLOH events. All genomic coordinates were noted with reference to the GRCh37, hg19 assembly. MORs were investigated using Microsoft Excel filtering functions. A subset of genes (n=91) selected from MORs mainly on the basis of event frequency and/or number of genes within the MOR and/or literature interest were taken forward for targeted sequencing (exons only) of appropriate samples with/without CN Losses or cnLOH (Set 1 n=124; Set 2 n=126). These were tested using custom designed TruSeq Custom Amplicon panels (Illumina Inc) and processed according to manufacturer’s instructions. SAMHD1 was excluded from these panels since it had been studied separately within our laboratory. The data were analysed using an in-house bioinformatics pipeline that uses the sequence aligners MSR and Stampy and the variant callers GATK and Platypus, followed by stringent filtering.Results: Using our datasets we have identified >50 MORs previously unreported in the literature. Six of these showed copy number (CN) losses in >3% of patients studied. Furthermore, we have refined 14 MORs that overlapped with regions described previously and that had also a CN loss frequency of >3%. Thirteen MORs involved only a single reference gene, often a gene implicated previously in cancer (eg. SAMHD1, MTSS1, DCC and RFC1). Of the 91 genes taken forward for targeted sequencing, stringent data filtering led to a subset of 19 genes of interest harbouring exonic mutations. Genes with mutations identified include DCC, BAP1 and FBXW7, also implicated previously in cancer.Conclusion: We have generated high resolution CNA and cnLOH profiles for 250 first-line chemo-immunotherapy treated CLL patients and used this information to document newly identified MORs, to refine MORs reported previously and to identify mutation harbouring genes using targeted NGS. Functional knowledge supports our hypothesis that these genes may have a contributory role in CLL. For two genes, SAMHD1 and FBXW7, relevance in CLL has been established already. Taken together, our data validate the utility of high resolution arrays studies for the identification of candidate genes that may be involved in CLL development or progression when disrupted. Further studies are required to confirm a role for these genes in CLL and to elucidate the nature of the underlying biological mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Chronic lymphocytic leukemia disease progression is accelerated by APRIL-TACI interaction in the TCL1 transgenic mouse model

AbstractAlthough in vitro studies pointed to the tumor necrosis factor family member APRIL (a proliferation-inducing ligand) in mediating survival of chronic lymphocytic leukemia (CLL) cells, clear evidence for a role in leukemogenesis and progression in CLL is lacking. APRIL significantly prolonged in vitro survival of CD5+B220dull leukemic cells derived from the murine Eμ-TCL1-Tg (TCL1-Tg [transgenic]) model for CLL. APRIL-TCL1 double-Tg mice showed a significantly earlier onset of leukemia and disruption of splenic architecture, and survival was significantly reduced. Interestingly, clonal evolution of CD5+B220dull cells (judged by BCR clonality) did not seem to be accelerated by APRIL; both mouse strains were oligoclonal at 4 months. Although APRIL binds different receptors, APRIL-mediated leukemic cell survival depended on tumor necrosis factor receptor superfamily member 13B (TACI) ligation. These findings indicate that APRIL has an important role in CLL and that the APRIL-TACI interaction might be a selective novel therapeutic target for human CLL.

The Bruton's Tyrosine Kinase (BTK) Inhibitor Ibrutinib (PCI-32765) Monotherapy Demonstrates Long-Term Safety and Durability Of Response In Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL) Patients In An Open-Label Extension Study

Abstract Background Bruton’s Tyrosine Kinase (BTK) plays a critical role in chronic lymphocytic leukemia (CLL) cell survival by modulating B-cell receptor signaling. Ibrutinib (PCI-32765), a first-in-class oral inhibitor of BTK, inhibits proliferation, migration and adhesion in CLL cells. A total of 148 patients with CLL/SLL received ibrutinib monotherapy in a Phase 1 multiple ascending dose study (PCYC-04753) or Phase 1b/2 continuous dosing study (PCYC-1102-CA), after which a long-term extension study was available for continued follow-up for safety and efficacy with daily orally-administered ibrutinib monotherapy. The studies included patients with treatment-naïve (TN) and relapsed or refractory (RR) CLL/SLL. The aims of the present analysis were to evaluate safety based on time on ibrutinib therapy (≤ 1 year and &gt; 1 year), summarize safety findings in the TN and RR patient populations, and assess duration of response (DOR). Methods Demographics and baseline characteristics were summarized according to parent study, comprising either TN patients or RR CLL/SLL patients who had received at least one dose of ibrutinib monotherapy. Patient disposition, treatment-emergent adverse events (AEs), best response, overall response rate (ORR), and DOR were determined for the time treated (beginning in the parent studies and extending into the long-term extension study). Results At a median treatment duration of 21.5 months, 109 out of 148 patients continued treatment with ibrutinib for over a year. The percentage of patients who had a grade 3 or higher serious adverse event (SAE) declined over time from 43% within the first year of study treatment to 32% after the first year of treatment. With respect to side effects determined to be related to study drug, the number of grade 3 AEs and SAEs also declined from within the first year of treatment (24% and 8%, respectively) to after the first year of treatment (7% and 0%, respectively). AEs leading to ibrutinib discontinuation occurred in 12 patients within the first year of treatment for all 148 patients and in 6 out of 109 patients after the first year of treatment. Overall, the most frequent AEs grade 3 or higher were pneumonia (16.9%), hypertension (13.5%), neutropenia (11.5%), thrombocytopenia (7.4%), and diarrhea (5.4%), regardless of relationship to study drug. Grade 3 or higher SAEs were reported in RR patients at 62% compared to TN patients at 29%. Pneumonia was reported in TN patients at 6.5% and in RR patients at 19.7%. Within the efficacy population (n = 140), the ORR was 86.2% for TN patients and 88.3% for RR patients who achieved a partial response (PR) or better. The ORR combined with PR with lymphocytosis suggests that 93.1% of TN patients and 93.7% of RR patients achieved an objective response to ibrutinib therapy based on Cheson JCO 2012. After a median follow up of 27.2 months (range 1.9-42 months) for TN and RR responders who achieved PR or better, the median DOR has not been reached. At landmark 30 months, 76.1% of the responders were alive without progression. Conclusions Ibrutinib as a single agent demonstrates long-term safety, tolerability, and durability of response in patients with TN and RR CLL/SLL. Indeed, a decrease in the number of patients experiencing SAEs or AEs grade 3 or higher after 1 year of treatment with ibrutinib resulted in low rates of treatment-related discontinuation after that time point. Grade 3 or higher SAEs were reported at a two-fold higher rate in patients who had received prior therapies, which may be reflective of disease state rather than relationship to ibrutinib. A majority of patients remain on ibrutinib monotherapy with the median DOR not yet reached in the ongoing extension study. Disclosures: O'Brien: Pharmacyclics: Research Funding. Furman:Genentech: Consultancy, Speakers Bureau; GlaxoSmithKline: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy; Gilead: Consultancy. Fowler:Pharmacyclics: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Coutre:Pharmacyclics: Consultancy, Research Funding. Burger:Pharmacyclics: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Jones:Pharmacyclics: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Wierda:Abbott Laboratories: Research Funding; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Research Funding, Speakers Bureau; Genentech/Roche: Consultancy, DSMB, DSMB Other, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Merck: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Sanofi-Aventis: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Tragara: Research Funding. Flinn:Pharmacyclics: Research Funding. Advani:Pharmacyclics: Research Funding; Janssen: Research Funding. Kolibaba:Pharmacyclics: Research Funding. Shaw:Pharmacyclics: Employment, Equity Ownership. Clow:Pharmacyclics: Employment, Equity Ownership. James:Pharmacyclics: Employment, Equity Ownership. Chu:Pharmacyclics: Employment, Equity Ownership. Byrd:Celgene: Consultancy; Johnson and Johnson: Consultancy; Pharmacyclics: Research Funding.

GLI1 Inhibitor GANT61 Deregulates stat3 Phosphorylation In Chronic Lymphocytic Leukemia Cells

BackgroundHedgehog(Hh) family has come to be recognized as key fundamental mediators of many carcinomas, but conflicting results exist about its role in chronic lymphocytic leukemia (CLL). Here we examined the effect of GLI inhibitor GANT61 to investigate the role of the Hh-signaling pathway in CLL. Methods and ResultsWe conduct real-time PCR for Hedgehog family members on isolated mononuclear cells from peripheral blood (n=35) and bone marrow cells(n=6) to evaluate the presence of the Hh-signaling pathway in CLL. There's no significant difference between peripheral blood and bone marrow cells in levels of Hh members. Profiling of cognate Hh pathway members revealed reduced expression of three key Hh signaling effectors, Patched, Smoothened (SMOH) and GLI, in peripheral blood mononuclear cells (PBMC), whereas transcription levels of other investigated members(SHH, IHH, DHH, GLI2, GLI3 etc.) resembled normal B-lymphocyte levels. However, we found a great heterogeneity for the expression levels of the Hh family with a subset of about 25% of CLL PBMC samples showing high transcript levels ( 1.5-fold than the median) for GLI1 and SMO. There is a direct positive correlation between GLI1 expression and SMO expression. We performed western-blot in CLL PBMC samples and found a positive correlation between phosphorylation of stat3 and GLI1 (figure 1). We examined the activity of GANT61 on viability of cell lines and primary CLL cells (N=3) in vitro by CCK8. GANT61 reduced the cell viability to 65% ± 14% after 24 hours of culture at concentration of 20uM (mean +/¨C SD, P < 0.05). We found that the capacity of GANT61 to inhibit CLL cell viability was associated with stat3 phosphorylation, which is time and dose dependent (figure 2). ConclusionThese results suggest that in CLL Hh pathway is closely related to stat3 pathway. Moreover, these studies reveal a potential mechanism for the anti-leukemia activity of GANT61 which might inhibit viability of CLL cells by deregulating stat3 phosphorylation. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia-Derived Exosomes Stimulate Cells From The Microenvironment

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the blood and the primary lymphoid organs of long-lasting, mature, but non-functional B lymphocytes. Although CLL B cells can survive for long time periods in vivo, cells are undergoing apoptosis relatively quickly in vitro. This spontaneous apoptosis and their sensitivity to drugs is strongly reduced in presence of bone marrow mesenchymal stem cells (MSC) and endothelial cells (EC), which provide anti-apoptotic stimuli to CLL cells via direct contact or secretion of soluble factors. We recently reported the first profiling of circulating miRNA obtained from plasma of CLL patients (Moussay et al., PNAS, 2011). Specific miRNAs were found at higher level in the plasma of CLL patients compared to healthy donors. Exosomes, which are small extracellular vesicles of 50-150 nm originating from endosomes, are now known to efficiently transport nucleic acids and transfer mRNA, microRNA and proteins to target cells. Therefore, exosomes constitute a new component of intercellular communication and their role in CLL remains totally unknown.The specific miRNA signature from plasma of CLL patients combined with our observations that primary CLL B cells can transfer vesicles to MSC through 0.4 µm culture inserts in vitro prompted us to investigate whether CLL B cells secrete exosomes that could modify cells of the bone marrow microenvironment to produce tumor growth promoting factors locally in order to favor their own survival.We isolated, purified and characterized exosomes derived from CLL cell lines, primary cells culture supernatants and plasma from CLL patients. Proteins, mRNA and microRNAs contents were evaluated by high-throughput methods (LC-MS, microarrays) revealing in particular the presence of oncogenic molecules. In vitro, purified CLL-exosomes were found to rapidly enter target cells (already after 1h in MSC and endothelial cells) and to transfer proteins and miRNA. Flow cytometry showed that transferred proteins were expressed at cell surface. Luciferase reporter assay confirmed that miRNAs were efficient in targeting cellular mRNA. Exosomes could also be taken up ex vivo and in vivo by mouse bone marrow cells. Functionally, CLL-exosomes activated key signaling pathways (PI3K, AKT, and MAPK) Immunoblotting indicated the rapid phosphorylation of kinases after 5 min of incubation with CLL-exosomes and the subsequent activation of the canonical NF-kB pathway. We also observed that CLL-exosomes modulated gene expression in target cells among which cytokines (BAFF, IL-6, and IL-8), chemokines (CCL2/MCP-1, CCL5/RANTES, and CXCL1), and other factors involved in cell adhesion and migration (ICAM-1 and MMP-1). These factors were also secreted in the supernatants of MSC and EC as detected by antibody arrays. Exosomes were also shown to increase MSC and EC proliferation, to stimulate actin remodeling, cell migration and to enhance EC angiogenic capabilities (tube formation and aortic ring assays).In conclusion, CLL-exosomes contain pro-oncogenic molecules and strongly affect key functions of MSC and EC which are critical component of the bone marrow microenvironment. Activation of these cells by CLL-exosomes led to release of cytokines/chemokines and oncogenic factors that could promote angiogenesis and also favor leukemic cells survival and migration.Our findings may lead to applications in both diagnosis and therapy development. Molecules identified at the surface or inside CLL-exosomes may be further used as cancer biomarkers. Finally, the description of cell-to-cell communication mechanisms will generate opportunities of innovative therapeutic strategies and confirms the crucial role of exosomes in the development of CLL. Disclosures:No relevant conflicts of interest to declare.

Igs Expressed by Chronic Lymphocytic Leukemia B Cells Show Limited Binding-Site Structure Variability

Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated.

The genomic landscape of chronic lymphocytic leukemia: clinical implications

A precise understanding of the genomic and epigenomic features of chronic lymphocytic leukemia (CLL) may benefit the study of the disease’s staging and treatment. While recent reports have shed some light on these aspects, several challenges need to be addressed before translating this research into clinical practice. Thus, even the best candidate driver genes display low mutational rates compared to other tumors. This means that a large percentage of cases do not display clear tumor-driving point mutations, or show candidate driving point mutations with no obvious biochemical relationship to the more frequently mutated genes. This genomic landscape probably reflects either an unknown underlying biochemical mechanism playing a key role in CLL or multiple biochemical pathways independently driving the development of this tumor. The elucidation of either scenario will have important consequences on the clinical management of CLL. Herein, we review the recent advances in the definition of the genomic landscape of CLL and the ongoing research to characterize the underlying biochemical events that drive this disease.

Recurrent ATM and BIRC3 Mutations in Patients with Chronic Lymphocytic Leukemia (CLL) and Deletion 11q22-q23

Abstract 1771 Introduction:Deletion of the long arm of chromosome 11 (11q22-q23) is one of the most common chromosome aberrations (∼12%) in CLL and is associated with rapid disease progression and shorter overall survival (OS). The commonly deleted region contains the genes ATM and BIRC3. ATM (chromosome 11q22.3) plays a central role in activating TP53, DNA recombination processes, and cell cycle control. ATMmut were described to occur in ∼35% of CLL patients with 11q deletions. The apoptosis inhibitor BIRC3 is located on chromosome band 11q22.2, 6.0 Mb proximal of ATM. BIRC3mut have recently been described with a mutation frequency of 4% in CLL (Rossi D, Blood 2012). Aims:1. Determine the frequency of deletions and mutations of ATM and BIRC3 in CLL with 11q deletion. 2. Analyze the association of ATM and BIRC3 mutations with ATM and BIRC3 deletions. 3. Characterization of the landscape and prognostic impact of ATM and BIRC3 mutations. Patient and Methods:The investigated cohort comprised 60 de novo CLL cases harboring del(11q) identified by chromosome banding analysis. The cohort comprised 47 males and 13 females, median age was 68.9 years. Survival data was available in 38 cases. For sensitive mutation detection of the complete coding region of ATM (3056 amino acids) and BIRC3 (604 amino acids) an amplicon deep-sequencing approach (454 Roche Life Sciences, Branford, CT) was developed. Patients were further characterized for mutation status of TP53 (n=60), SF3B1 (n=59), and IGHV (n=60). Additionally, patients were investigated by interphase FISH for ATM (n=60; MetaSystems, Altlussheim, Germany) and BIRC3 (n=39; BAC RP11–177O8, BlueGnome, Cambridge, UK) deletions. Results:Based on FISH results, all 60 cases showed an entire gene deletion of ATM. Of those cases investigated for BIRC3del, 34/39 (87.2%) cases showed a deletion, thus 5/39 (12.8%) carried an ATMdel only. Mutational screening of the ATM and BIRC3 genes identified 24 ATMmut in 19/60 (31.7%) patients and 3 BIRC3mut in 3/60 (5.0%) cases. Additional mutations were detected in TP53 (3/58; 5.2%), and the spliceosome machinery gene SF3B1 (10/59, 16.9%). 49/60 (81.7%) cases were IGHV unmutated, whereas 11/60 (18.3%) cases were IGHV mutated.The majority of ATMmut were found to be missense mutations (n=15) followed by nonsense (n=4), frame-shift (n=4), and in-frame (n=1) variants. In more detail, only one mutation occurred in two distinct patients (p.Ile2888Thr). Most cases showed only one mutation (n=15, 78.9%), whereas 3 cases (15.8%) showed two and one case (5.3%) three mutations. Mutations were spread across the entire coding region (exon 3–59). The median mutational burden as assessed by deep-sequencing read counts was 38.5%, ranging from 4.0–89.5%.For BIRC3 3 deletions (1–4 bp) were observed resulting in 2 frame-shift and 1 splice-site mutation. The median mutation load was 32.5% (range: 20.0%-92.0%). In addition, two cases with BIRC3mut showed also a BIRC3del (n=1 data not available). Interestingly, all of these 3 cases with BIRC3mut were wild-type in the ATM gene.To further address the role of ATMmut association analyses with respect to age, gender, white blood cell count, haemoglobin level, and platelet count were performed, however, no correlations were observed. Further, ATMmut were not correlated with IGHV (3/11 vs 16/49, P=1.00), TP53 (0/3 vs 19/55, P=0.544) or SF3B1 (3/10 vs 15/49, P=1.00) mutation status. However, the presence of ATMmut in CLL with del(11q) was associated with shorter OS (76.2% vs 95.0% at 3 years), although this difference was not significant. However, when comparing OS of ATMmut cases to cases without del(11q) (n=1,245) we detected a trend towards a shorter OS (76.2% vs 94.2% at 3 years, P=0.149) in contrast to cases with del(11q), but without ATMmut in comparison to cases with no del(11q) (95.0% vs 94.2%, P=0.731). Conclusions:1. BIRC3 was mutated in only 5.0%, whereas ATM was mutated in 31.7% of patients with del(11q). 2. No mutation hotspot regions were observed, thus analyses of the whole genes are mandatory. 3. ATMmut were associated with a trend to shorter OS in CLL with del(11q). In comparison to cases without del(11q), cases with ATMmut showed shorter OS, indicating a more relevant role of ATMmut on prognosis versus cases with a del(11q) only. 4. In 38 cases (63.3%) with del(11q) neither ATM nor BIRC3 mutations were detected suggesting that further genomic alterations which are not yet identified play a role in CLL with del(11q). Disclosures:Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weissmann:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Kienast:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

DNA Hypomethylation Leads to Aberrant Expression of PD-1 in Chronic Lymphocytic Leukemia

AbstractAbstract 3504Programmed Death-1 (PD-1) is an inhibitory cell surface receptor of the immunoglobulin superfamily expressed on activated lymphocytes, monocytes and dendritic cells. Although PD-1 function is best characterized in T-cells, it is known that PD-1 also suppresses the immune response of B lymphocytes through protein phosphatase recruitment and dephosphorylation of signaling molecules downstream of the B-cell receptor (BCR). Recent studies have found that PD-1 expression is elevated at the mRNA as well as the protein levels in B cells obtained from chronic lymphocytic leukemia (CLL) patients compared to those from healthy controls. Using genome-wide DNA methylation sequencing, we identified PD-1 as one of the significantly hypomethylated genes in CLL compared to normal B-cell samples. Three differentially methylated regions (DMRs) were discovered in the first intron, proximal promoter and up-stream enhancer regions. We validated these DMRs in 43 CLL and 7 normal control samples using bisulfite pyrosequencing. The pyrosequencing analysis further confirmed that all three regions were significantly hypomethylated in CLL patient samples (p<0.001). These epigenetic changes resulted in the overexpression of PD-1 in primary CLL B cells, which was confirmed by real-time quantitative PCR. In B cells isolated from healthy controls, flow cytometry analysis showed that only approximately 1% expressed PD-1, whereas PD-1 positive B cells in CLL patients ranged from 5% to 64%. No correlation between PD-1 status and IGHV mutations or CD38 expression was observed. To elucidate the mechanisms of epigenetic regulation of PD-1 expression, we studied five non-Hodgkin’s lymphoma cell lines including Mec-1, Granta 519, RL, Raji and DB. Bisulfite pyrosequencing results showed that only the up-stream enhancer is differentially methylated in these cell lines. Treatment of lymphoma cell lines with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors can up-regulate PD-1 expression in RL, Raji and DB cell lines in which the up-stream enhancer region is hypermethylated; however, the same treatments decreased the PD-1 expression in Mec-1 cells, which are demethylated in the PD-1 enhancer region and express PD-1 on the cell surface. Chromatin immunoprecipitation (ChIP) analysis revealed that H3K4me3 and H3K4me1 modifications were significantly enriched in the promoter and enhancer regions in PD-1 positive Mec-1 cells, respectively. However, H3K27me3 modification was enriched in both promoter and enhancer regions in PD-1 negative RL cells, while enrichment of H3K3me3 and H3K4me1 modification was significantly decreased. These results suggest that coordinated regulation of enhancer activity by DNA methylation and histone modification is crucial for PD-1 expression in CLL B cells. Furthermore, we mapped the nucleosome occupancy in the enhancer regions using a high-resolution, single-molecule approach. The nucleosome mapping results revealed nucleosome-depleted regions in Mec-1 cells, but not in RL cells. This novel finding demonstrates the complexity of epigenetic regulation of PD-1 expression. To determine the function of PD-1 in CLL, we co-cultured the Mec-1 cell line and primary CLL B-cells with a hepatocyte cell line Huh7.5 that overexpresses exogenous PD-1 ligand, PD-L1. Surprisingly, unlike PD-1 positive normal B cells, we did not observe increased apoptosis in the co-cultured CLL B cells, suggesting that PD-1 has a different functional role in CLL compared to normal B cells. In summary, we present here the novel finding that DNA hypomethylation in the enhancer region of PD-1 leads to aberrant overexpression of PD-1 on CLL B-cell surfaces and that in CLL PD-1 may have a different function than in normal B cells. Disclosures:No relevant conflicts of interest to declare.

Comparison of HLA Alleles in Follicular Lymphoma and Chronic Lymphocytic Leukemia Patients Referred for Allogeneic Stem Cell Transplantation

AbstractAbstract 4218 Introduction:Data recently published showed that besides the several well-known parameters, long term outcome after allogeneic stem cell transplantation (SCT) in chronic lymphocytic leukemia (CLL) may be influenced by the presence or absence of certain HLA class I alleles (HLA-A1+/A2-/B44-) (Khouri et. Al. Cancer 2011). We have also recently published an 11-year progression free survival (PFS) rate of 72% in relapsed follicular lymphoma (FL) after SCT (Khouri et al. Blood 2012). A higher relapse rate has been observed in CLL patients when compared to FL (50% vs. 4%). Since HLA subtypes played an important role in CLL, our goal in this study was to assess and compare over expressed HLA alleles in FL and CLL patients who received a SCT at our center. Methods:Two cohorts of patients who received SCT were retrospectively studied. Group I consisted of 59 Caucasian patients (23 [39%] F: 36 [61%] M) with FL and Group II consisted of 119 Caucasian patients (27 [23%] F: 112 [77%] M) with CLL. The HLA alleles at HLA-A, -B,-C and -DRB1 loci of both groups were analyzed and the HLA typing was performed by polymerase chain reaction (PCR) amplification and oligonucleotide hybridization using commercial kits from Invitrogen (Carlsbad, Ca) or One Lambda (Canoga Park, Ca) that resulted in intermediate resolution. Patients were also typed for these loci using high-resolution methods with PCR amplification and nucleotide sequencing (Abbott, Abbott Park, Ill).The antigen frequencies of all of the alleles of HLA-A, -B,-C AND DRB1 were calculated. Antigen frequency was defined as the percentage of the population possessing the antigen. Antigen frequency comparisons were only done for North American whites due to sample size and control group constraints. The control group was based on a sample analysis of 643 normal North American Whites. The Pearson x2goodness-of-fit test was used to validate the Hardy-Weinberg genetic equilibrium for phenotypic data. The association of various alleles with the control group was determined by using a chi-square test with Yates correction in a 2 × 2 table with 1 degree of freedom (SAS software, version 6.12, SAS Institute Inc, Cary NC). P values < 0.05 at the 95% confidence interval (95% CI) were considered significant. Results:A male predominance was noted in both patient groups. A total of 17 HLA-A, 29 HLA-B, 13 HLA-C and 13 HLA-DRB1 distinct alleles for FL patients and 16 HLA-A, 24 HLA-B, 13 HLA-C and 11 HLA-DRB1 distinct alleles were identified for the CLL patients. Since the predominant ethnic type in both groups were North American Whites, statistically valid comparisons of HLA antigen frequencies were only possible in this population. The observed heterozygosity for FL/CLL patients was 0.93220./0.831932 for HLA-A, 0.915254/0.949579 for HLA-B, 0.779661/0.882352 for HLA-C and 0.847457/0.941176 for HLA-DRB1. There were no untyped patients and all of the patients underwent hematopoietic stem cell transplantation.Our analysis reveals an over expression of HLA-A*03, HLA-C*04, HLA-DRB1*01, HLA-DRB1*07 and HLA-DRB1*15 with frequencies of 25.4%, 24.1%, 22.9%, 28.8% and 8.5 % in FL patients which was significantly higher than the frequencies of 15.1% for HLA-A*03 (p value 0.005), 1.1% for HLA-C*04 (p value < .00001), 10.3% for HLA-DRB1*01 (p value <.0001), 14.4% for HLA-DRB1*07 (p value < .0001) and 15.7% for HLA-DRB1*15 (p value < .0495) in the normal population showing for the first time an over representation of these alleles in patients with FL.In the CLL group our analysis revealed a 24.8% (p value 0.0014) frequency for HLA-A*01 which was significantly higher than the frequency of 16% (p value 0.0014) in the normal population showing an overrepresentation of this allele. The underrepresented allele was HLA-B*38 with a frequency of 3.4% compared to 12.4% (p value 0.0335) in the normal population.When the two groups FL and CLL patients were analyzed, the significant alleles were HLA-A*01, HLA-A*03, HLA-C*04, HLA-DRB1*01 and HLA-DRB1*07. Conclusion:Our results demonstrate a significant difference in HLA expression in Follicular Lymphoma and Chronic Lymphocytic Leukemia patients with the over representation of HLA-A*03, HLA-C*04, HLA-DRB1*01, HLA-DRB1*07 and HLA-DRB1*15 alleles in FL and HLA-A*01 and HLA-B*38 alleles in CLL. We do not know whether these variances account for a different graft-versus-malignancy susceptibility to donor cells between the two groups and this remains to be studied. Disclosures:No relevant conflicts of interest to declare.

The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia

AbstractB-cell antigen receptor (BCR)–mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium–calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets.

MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia

AbstractMicroRNAs (miRNAs) play a key role in chronic lymphocytic leukemia as well as in normal B cells. Notably, miRNA gene encoding miR-650 and its homologs overlap with several variable (V) subgenes coding for lambda immunoglobulin (IgLλ). Recent studies describe the role of miR-650 in solid tumors, but its role in chronic lymphocytic leukemia (CLL) has not yet been studied. Our experiments demonstrate that miR-650 expression is regulated by coupled expression with its host gene for IgLλ. This coupling provides a unique yet unobserved mechanism for microRNA gene regulation. We determine that higher expression of miR-650 is associated with a favorable CLL prognosis and influences the proliferation capacity of B cells. We also establish that in B cells, miR-650 targets proteins important in cell proliferation and survival: cyclin dependent kinase 1 (CDK1), inhibitor of growth 4 (ING4), and early B-cell factor 3 (EBF3). This study underscores the importance of miR-650 in CLL biology and normal B-cell physiology.

Trisomy 12 and elevated GLI1 and PTCH1 transcript levels are biomarkers for Hedgehog-inhibitor responsiveness in CLL

AbstractHedgehog (HH) signaling is activated in various lymphoid malignancies, but conflicting results exist about its role in chronic lymphocytic leukemia (CLL). Here, we demonstrate that the expression of essential HH pathway components like GLI1, PTCH1, and the HH ligands is highly diverse in CLL. A subset of 36.7% of 60 tested CLL samples responded to all 3 SMOOTHENED (SMO) inhibitors, whereas 40% were completely resistant. Responsiveness correlated with elevated GLI1 and PTCH1 transcript levels and the presence of trisomy 12, whereas no other karyotype correlated with responsiveness. All trisomy 12 CLLs displayed constitutive HH pathway activation driven by autocrine DESERT HH (DHH) ligand secretion, which could be blocked by the HH-blocking Ab 5E1. Cocultures with DHH-expressing BM stromal cells reduced sensitivity of CLLs to SMO-inhibitor treatment by activation of noncanonical ERK phosphorylation directly downstream of the PTCH1 receptor without involvement of SMO and could be overcome by the HH-blocking Ab 5E1 or a combination of SMO and ERK inhibitors. Our results demonstrate that the HH-signaling pathway is an interesting therapeutic target for a subset of patients with CLL, characterized by high GLI1 and PTCH1 transcript levels, and all patients with trisomy 12 and indicate HH-blocking Abs to be favorable over SMO inhibitors in overcoming stroma-mediated protective effects.

SF3B1and Other Novel Cancer Genes in Chronic Lymphocytic Leukemia

The somatic genetic basis of chronic lymphocytic leukemia, a common and clinically heterogeneous leukemia occurring in adults, remains poorly understood. We obtained DNA samples from leukemia cells in 91 patients with chronic lymphocytic leukemia and performed massively parallel sequencing of 88 whole exomes and whole genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in this disease. Nine genes that are mutated at significant frequencies were identified, including four with established roles in chronic lymphocytic leukemia (TP53 in 15% of patients, ATM in 9%, MYD88 in 10%, and NOTCH1 in 4%) and five with unestablished roles (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X). SF3B1, which functions at the catalytic core of the spliceosome, was the second most frequently mutated gene (with mutations occurring in 15% of patients). SF3B1 mutations occurred primarily in tumors with deletions in chromosome 11q, which are associated with a poor prognosis in patients with chronic lymphocytic leukemia. We further discovered that tumor samples with mutations in SF3B1 had alterations in pre-messenger RNA (mRNA) splicing. Our study defines the landscape of somatic mutations in chronic lymphocytic leukemia and highlights pre-mRNA splicing as a critical cellular process contributing to chronic lymphocytic leukemia.

Sensitivity to Wnt Pathway Inhibition in CLL Is Associated with Specific Gene Expression Signatures

Abstract 801We have recently found that the Wnt/b-catenin signaling pathway plays a key role in chronic lymphocytic leukemia (CLL). We were, however, intrigued by the question of whether this aberrant pathway may function differently in independent leukemias, and contribute to disease heterogeneity. To assess differential activity of the Wnt pathway across patients, we tested the effects of blocking Wnt activation on CLL cell survival. We knocked down a key downstream gene, LEF1, which is the most differentially expressed gene in CLL compared to normal B cells (based on gene expression microarrays).Addressing this question requires genetic manipulation of primary normal and malignant human B cells, and yet these cells are notoriously difficult to transfect. We therefore focused on developing a method for introducing siRNAs into normal and malignant B cells. We adapted a novel delivery system consisting of vertical silicon nanowires (SiNWs, Shalek et al PNAS 2010) that penetrate the plasma membrane in a minimally invasive fashion and deliver biomolecular cargo directly into the cytoplasm. We achieved consistent and reliable delivery of fluorescently labeled siRNAs (at 50–200 pmol) into normal and CLL B cells. siRNA was delivered to >90% of cells with >85% cell viability remaining after 48 hours.We used this platform to knockdown LEF1 in 20 CLL-B and 5 normal CD19+ B cell samples, and examined cell survival 48 hours after siRNA delivery using an ATP-based CellTiter-Glo assay. Indeed, our studies revealed a heterogeneous response among CLL-B cells to LEF1 inhibition. As a group, CLL-B cells were significantly more sensitive to LEF1 knockdown with a survival rate of 77% (12% s.e.m) compared to 97% (13% s.e.m) in normal B cells. CLL B cells from different patients showed differential sensitivity to LEF1 knockdown, with 8 non-responders, 8 intermediate responders and 4 strong responders (i.e. significant death). Sensitivity to LEF1 inhibition did not correlate with known CLL cytogenetic prognostic factors.To determine if the differential response to LEF1 knockdown was associated with specific gene signatures, we examined gene expression data generated from CLL-B cells from 12 (4 strong, 3 intermediate, and 5 non-responders) of the 20 CLLs tested (using the Affymetrix U133 Plus 2 Array). To increase statistical power, we used each CLL’s expression profile (using only genes that showed variability across samples) to create clusters of ∼19 CLLs that showed similar expression profiles (using microarray data from our compendium of 177 additional CLLs). We further reduced the number of genes to ∼4000 genes by retaining only those whose expression levels were significantly different in at least one associated cluster relative to normal CD19+ B cell controls (T-test, FDR<10−4; p-values converted using the Benjamini-Hochberg method). These analyses led to the identification of several hundred genes whose expression correlated significantly with LEF1 knockdown’s effect on cell viability. Analysis of these differentially expressed genes identified several potentially important pathways. Ongoing analyses include the identification and validation of a molecular signature for this effect. This signature could enable rapid identification of patients who would be most responsive to therapy with LEF1 inhibitors, which are under development along with other Wnt pathway inhibitors. Disclosures:No relevant conflicts of interest to declare.

Btk Inhibitor, PCI-32765, Delays CLL Progression in a TCL1 Adoptive Transfer Model by Impairing Migration and Cell Proliferation

Abstract 982Bruton's tyrosine kinase (Btk) is critical for B-lymphocyte function, due to its involvement in key functions in B-cells, e.g., maturation, activation, and trafficking. These functions are mediated by several receptors, including the B-cell antigen receptor (BCR) and the chemokine receptor CXCR4. Both BCR and CXCR4 play important roles in chronic lymphocytic leukemia (CLL), providing leukemic cells with survival and proliferation advantages. Initial studies in CLL suggest that inhibiting Btk with PCI-32765 (Pharmacyclics, Inc) is an effective therapeutic, reducing the size of solid lymphoid tissues leading initially to lymphocytosis and eventually to decreased blood absolute lymphocyte counts (ALCs).To understand the effect of blocking Btk-mediated signaling in CLL, we utilized an accelerated adoptive transfer CLL mouse model, injecting 5×106 TCL1 leukemia cells into SCID mice that succumb 5–6 weeks after cell transfer. Total 45 mice were injected, separated into 3 groups, and then treated with PCI-32765, at either 2, 3 or 4 weeks after cell transfer, with 5 mice from each group receiving either vehicle control or PCI-32765 (5 or 25 mg/kg/day) in daily drinking water. Mice were bled to track changes in ALCs.Animals treated at 2-weeks post cell transfer with the suboptimal (5mg/kg/day) and optimal (25mg/kg/day) doses exhibited a transient lymphocytosis at day 4, with a 7- and 10-fold increase in circulating TCL1 leukemia cells, respectively (p=0.002). By day 7, these levels had fallen to those of untreated mice. Until week 6, mice receiving the optimal dose of PCI-32765 at week 2 and 3 but not week 4, appeared healthy and had significantly reduced ALCs (p<0.001). These mice had small or no lymph nodes (LNs) and significantly smaller livers and spleens with markedly reduced leukemic infiltration. In contrast, mice receiving vehicle control or sub-optimal dose of PCI-32765 exhibited lethargy, weight loss, and hunched posture; these animals had massive lymphocytosis, huge hepatosplenomegaly, and lymphadenopathy.Surprisingly, the delayed disease progression by PCI-32765 correlates with blocked CXCR4 surface membrane recycling in CLL cells. In addition, there were significantly repressed levels of phosphorylated phospholipase C-gamma 2 (PLCg2) in optimally treated mice. This is relevant because phosphorylation of PLCg2 by BTK influences chemokine-controlled cell migration, at least partially through levels of CXCL12 or its receptor CXCR4. In vivo studies of CLL patients suggest that peripheral blood cells bearing a CXCR4BRCD5DIM (R4BR) surface phenotype are more likely to re-enter lymphoid tissues, while cells expressing CXCR4DIMCD5BR (R4dim) have more likely recently left solid tissues. Here, we found spleen cells in optimally treated mice had significantly lower percentages of R4BR; while in the blood, there was an increased population of R4DIM cells. These data suggest that the smaller spleen and LN sizes of treated mice are due to promoted migration of CLL cells out of lymphoid tissues and blocked return from blood.In vivo effect of PCI-32765 on TCL1 cell proliferation was also evaluated. BrdU was injected in mice 24 hours prior to sacrifice. As TCL1 cells proliferate in lymphoid organs, significantly repressed BrdU incorporation was demonstrated in spleen and LN cells of optimally treated mice, consistent with PCI-32765 inhibiting proliferation. In addition, in support of enhanced migration of cells out of lymphoid tissues, there was an ∼3-4 fold increase in BrdU-labeled cells in blood after optimal PCI-32765 treatment.Finally, the effects of PCI-32765 on TCL1 cell homing was assessed by injecting SCID mice with 2.5×106 R4DIM blood cells from either controls or optimally treated mice. 24 hours later, mice engrafted with R4DIM cells from PCI-32765 treated animals had ∼30–50 fold fewer leukemic cells in spleen (p=0.018); and ∼1.5 fold increased CLL cells in blood (p=0.021), further supporting blocked homing by PCI-32765.Collectively, our data suggest that targeting Btk, its receptors, and the downstream targets that require its use delays CLL progression. This effect is, at least partially, due to repressed surface CXCR4 expression and blocked cell proliferation, mediated either directly in CLL cells or indirectly, by minimizing the likelihood of receiving trophic stimuli via the BCR or CXCR4 in a solid tissue microenvironment. Disclosures:Buggy:Pharmacyclics, Inc.: Employment. Chang:Pharmacyclics Inc: Employment. Burger:Pharmacyclics, Inc: Research Funding.

Association of an increased frequency of CD14+HLA‐DRlo/neg monocytes with decreased time to progression in chronic lymphocytic leukaemia (CLL)

Association of an increased frequency of CD14⁺HLA‐DR^lo/neg monocytes with decreased time to progression in chronic lymphocytic leukaemia (CLL)