ALBERT

Description: CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor. Macrophage migration inhibitory factor binding to CD74 induces its intramembrane cleavage and the release of its cytosolic intracellular domain (CD74–ICD), which regulates cell survival. In the present study, we characterized the transcriptional activity of CD74–ICD in chronic lymphocytic B cells. We show that following CD74 activation, CD74–ICD interacts with the transcription factors RUNX (Runt related transcription factor) and NF-κB and binds to proximal and distal regulatory sites enriched for genes involved in apoptosis, immune response, and cell migration. This process leads to regulation of expression of these genes. Our results suggest that identifying targets of CD74 will help in understanding of essential pathways regulating B-cell survival in health and disease.

Description: Introduction: BDNF is a neuronal growth factor that previously showed to exert survival effect in B cells. The role of BDNF in CLL is unknown. Objectives: To study the circulating levels of BDNF in CLL patients and their association with disease prognosis. We also looked for CXCR-4 as a possible mechanisms underlying BDNF effect in CLL. Design and methods: The total BDNF levels and the levels of the BDNF precursor; proBDNF were quantified in the serum of 36 CLL patients and 5 healthy controls by commercial ELISA kits (DY248 and DY3175 DuoSet, respectively, from R&D Systems, Minneapolis, MN, USA). The patients' BDNF levels were correlated to the disease characteristics and clinical course. mRNA and protein expression of the high affinity receptor for BDNF; TrkB were evaluated by real time PCR and flow cytometry respectively. The effect of BDNF on CXCR4 surface expression and migration of CLL cells towards SDF-1 were studied in-vitro. Results: The total serum levels of BDNF in CLL patients was not statistically different compared to healthy controls (mean±SD; 19.9±15.1ng/ml vs. 10.5±9.5ng/ml respectively, p=0.19). Serum proBDNF levels in both CLL patients and healthy controls were under the detection limit of the kit. Within the CLL group we found higher total BDNF levels in patients with mutated immunoglobulin variable heavy-chain (IGHV) status than in patients with unmutated IGHV (25.2±14.6ng/ml vs. 14.1±13.0 in mutated and unmutated status, respectively, p=0.028). We also found higher serum levels of total BDNF in CLL patients in Binet stage A compared to those with a more advance clinical stage (mean±SD ; 24.5±14.6ng/ml vs. 17.0±16.9 and 10.2±9.6 in A,B and C, respectively, p=0.013). Accordantly, higher BDNF levels were detected in patients who had a clinically stable disease compared to patients who had disease progression and required treatment (23.1±14.7ng/ml vs. 13.4±14.5, respectively, p=0.013). The mRNA levels of the BDNF receptor; TrkB, were twofold lower in CLL cells compared to normal B cells. However, the overall TrkB mRNA transcripts were very low in CLL cells and normal B cells compared to the normalized housekeeping genes of Beta-2-microglobulin and CD19. Likewise, surface expression of TrkB in CLL was undetectable using several commercial monoclonal Abs by flow cytometry. In vitro culture of CLL cells in serum free media for 24h resulted in an increased CXCR4 surface expression and greater apoptosis. Culture of CLL cells in the presence of recombinant BDNF (50ng/ml) resulted in downregulation CXCR-4 surface levels and protection from spontaneous apoptosis, irrespectively of the IGHV mutational status. The effect of BDNF on CXCR4 downregulation was also confirmed at 1h culture as it shows to induce similar effect to SDF-1 (50ng/ml) and additive effect when combined with SDF-1. Finally, possible competition between BDNF and SDF-1 was indicated as pretreatment of CLL cells with BDNF inhibit their migration toward SDF-1. Discussion: Our findings show association between high BDNF levels and favorable disease prognosis in CLL. Undetected TrkB expression in CLL cells is compatible with previous reports showing sequestration of TrkB in normal and malignant B cells and suggests TrkB independent effect of BDNF in CLL. The effect of BDNF on the survival and migration of CLL cells via CXCR-4 needs to be further investigated. Disclosures No relevant conflicts of interest to declare.

Description: The combination of fludarabine, cyclophosphamide and rituximab (FCR) is still currently regarded as the standard regimen for treatment of physically fit patients with chronic lymphocytic leukemia (CLL). This therapy can be associated with significant toxicity, and patient adherence to the protocol may often be difficult outside of clinical trials. This retrospective study aimed to evaluate the efficacy and safety of FCR therapy in the real life setting, with particular focus on the influence of dose reduction on treatment outcome. A total of 132 CLL patients (≤70 years of age) treated with FCR as frontline therapy from 10 medical centers, were reviewed. The majority of patients were males (73.5%, n=97) and younger than 60 years (78%, n=103). Eleven patients had Binet stage A (8.3%), 72 (54.5%) were stage B and 49 (37.1%) had Binet stage C. Results of FISH analysis were available for 99 patients, with high risk cytogenetics of del(11q) in 21 patients (21.2%) and del(17p) in 9 cases (9.1%). The majority (56.5%, n=74) received rituximab at a dose of 500mg/m2 and the rest 375mg/m2. Almost half of the patients (49.2%, n=65) were given a reduced dose of chemotherapy (

Description: Introduction: Autologous Stem Cell Transplantation (ASCT) has become the cornerstone of managing relapsed lymphomas. However, its use in elderly patients or those with comorbidities is limited because of high regimen related toxicities (RRT). Several chemotherapy regimens have been suggested to minimize RRT while maintaining the antitumor effect. TECAM (Thiotepa 160 mg/m2 over 4 days , Etoposide and Cytarabine 800mg/m2 over 4 days, Cytoxan 60 mg/kg, and Melphalan 120 mg/m2 over 2 days) is a reduced toxicity modification for the commonly used BEAM protocol. Aim: This study aimed to compare the safety and efficacy or the TECAM regimen with that of its more intense BEAM counterpart. Methods: A retrospective cohort study of all patients who underwent ASCT for treatment of lymphoma with either BEAM or TECAM conditioning regimens at the Tel Aviv Medical Center between October 1999 and January 2014. We compared early (≤100 days) mortality, RRT rate, time to engraftment and length of hospitalization as well as overall and progression free survival. Results: A total of 146 patients (76 BEAM, 70 TECAM) were included in the analysis. Median follow up was 47 months. Patient treated with TECAM were older (54.5 vs. 45.5, p=0.001), and nearly 20% (n=13) of them had dose reductions (average 17±7%), as opposed to a single patient in the BEAM arm. Conversely, TECAM patients had a longer time interval from first diagnosis to transplant (22 vs. 13 months, p

Description: Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B lymphocytes, involves blood, bone marrow, and secondary lymphoid organs such as the lymph nodes (LN). A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations, but its contribution in vivo remains ill-defined. To elucidate the effects of tumor-host interactions in vivo, we purified tumor cells from 24 treatment-naive patients. Samples were obtained concurrently from blood, bone marrow, and/or LN and analyzed by gene expression profiling. We identified the LN as a key site in CLL pathogenesis. CLL cells in the LN showed up-regulation of gene signatures, indicating B-cell receptor (BCR) and nuclear factor-κB activation. Consistent with antigen-dependent BCR signaling and canonical nuclear factor-κB activation, we detected phosphorylation of SYK and IκBα, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL, indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation, quantified by the expression of the E2F and c-MYC target genes and verified with Ki67 staining by flow cytometry, was highest in the LN and was correlated with clinical disease progression. These data identify the disruption of tumor microenvironment interactions and the inhibition of BCR signaling as promising therapeutic strategies in CLL. This study is registered at http://clinicaltrials.gov as NCT00019370.

Description: Chronic lymphocytic leukemia (CLL) is an incurable malignancy of mature B-cells. CLL cell proliferation and survival depends on host factors that are present in the tissue microenvironment. In–vitro these cells rapidly undergo apoptosis but can be supported by culture on stroma cells. The extracellular matrix is an important component of the microenvironment. Interactions between tumor cells and the extracellular matrix are, in part, mediated by CD44, whose principle ligand is hyaluronic acid. To explore a possible role of this adhesion receptor in CLL, we evaluated the effect of CD44 engagement on the survival of CLL cells and the induction of drug resistance. Dimerization of CD44 activated the PI3K/AKT and MAPK/ERK pathways and resulted in increased levels of MCL-1. There was no increase in MCL mRNA in CD44 stimulated cells, consistent with an effect on protein stabilization. Consistent with the induction of these anti-apoptotic mechanisms, CD44 protected CLL cells from spontaneous apoptosis: CD44 stimulated CLL cells had a 46% increase (range 7% – 181%) in viability over the corresponding unstimulated control cells (n=20, p

Description: Proposed mechanisms on how the monoclonal anti-CD20 antibody rituximab (R) depletes B-cells include antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. In vitro studies have suggested that R induced pro-apoptotic signals contribute to clinical efficacy and may sensitize cells to chemotherapy. To investigate the effect of R on tumor biology in vivo, we analyzed the molecular changes in leukemic cells of 12 previously untreated CLL patients during the first R (375mg/m2) infusion. The median reduction of circulating tumor cells within 24h was 50% (range 0–67%). We first determined whether R affects gene expression in CLL cells obtained before and at 6h and 24h after the start of R. Cells were purified by CD19+ selection and gene expression was measured on Affymetrix HU133A 2.0 arrays. A one-way ANOVA test with a stringent cutoff (false discovery rate of 50% at 6h compared to pre treatment, and 31 genes whose expression decreased by 〉30%. Most of the up-regulated genes are known to be regulated by interferon (IFN) and include the pro-apoptotic genes IRF1, STAT1, FAS and OAS2. Of 12 cytokines assayed in the serum, we found that only IFNy, IL–6, IL–8, IL–10, and TNFa were induced by R with a peak at 2h. Consistent with a dominant role of IFNy on gene expression in the CLL cells, STAT1, a direct and essential mediator of IFNy signaling, was activated in circulating leukemic cells in vivo. In addition, when comparing the response between patients, IFNy serum protein levels correlated strongly with the intensity of the gene expression changes in the tumor cells (r=0.83, p=0.008). We could not detect any IFNy mRNA in CLL cells and conclude that the IFNy is most likely released by NK cells activated through FcyRIII signaling. Considering the long half-life of R, we were surprised to see that both cytokine serum levels and gene expression changes almost completely subsided by 24h. Intriguingly, among the few genes that were down-regulated by treatment, the gene encoding CD20 was the most strongly and consistently affected showing a 50% decrease in expression at 24h. We also assessed CD20 protein levels by Western blotting. Total CD20 levels were markedly decreased already at 6h and by 24h almost all CD20 had been lost. The more rapid and more pronounced decrease of CD20 protein as opposed to mRNA levels is consistent with a process previously described as shaving, during which R bound CD20 is pulled of the cell surface (Kennedy AD, J Immunol. 2004). Despite the absence of a clinical cytokine release syndrome, we observed basically identical changes in serum cytokines and gene expression with subsequent infusions in 2 patients analyzed. In summary, R induced a characteristic gene expression signature in CLL cells that is dominated by IFN response genes, many of which have well characterized pro-apoptotic functions. Thus, our data suggest that signaling for apoptosis is not so much a direct effect of R, but due to a complex immune response to the R coated CLL cells. Modified treatment schedules capable of delivering sustained pro-apoptotic signals hold promise for improved efficacy of R and should be explored.

Description: The host microenvironment is important for proliferation and survival of leukemic cells in chronic lymphocytic leukemia (CLL). Numerous molecules, signaling pathways and cell types have been reported to enhance CLL cell survival. To date, most reports on such interactions are derived from in-vitro studies, where each study focused on a specific ligand/receptor interaction or candidate pathway. Here, we adopted a more global approach to evaluate in-vivo effects of the microenvironment on leukemic cell biology. CLL cells from 15 patients were obtained on the same day from 3 different compartments: peripheral blood (PB), bone marrow (BM) and lymph node (LN), from which a single cell suspension was prepared. Tumor cells from all three compartments were purified by CD19 selection to purity 〉98%. Patients were assigned to prognostic subtypes based on immunoglobulin sequencing (Ig) and ZAP70 expression: 10 patients had the more progressive subtype (Ig-unmutated, ZAP70+) and 5 patients belonged to the more indolent subtype. Cells were analyzed for surface markers by flow cytometry and by gene expression profiling on Affymetrix HG U133 Plus 2.0 arrays. By flow cytometry, CLL cells in LN expressed higher levels of activation markers including CD69 and CD38 compared to CLL cells in PB (% CD19+/69+; 71 ±27 vs. 35 ±28, p

Description: Background: The Israel National Cancer Registry (INCR) is a population based, national passive tumor registry established in 1960. Reporting by hospitals, laboratories, and other providers has been mandatory in Israel since 1982 and most cancer cases are registered on the basis of pathology reports and hospital records. In some hematopoietic malignancies where the diagnosis was not based on tissue pathology and patients initially received no inpatient treatment, cases may not have been reported to the registry, or reporting to the INCR may have been delayed, resulting in an underestimation of the true burden of disease. One diagnosis for which there is particular concern in this regard is CLL/SLL and here we used active surveillance to estimate the true incidence of CLL/SLL in Israel. Here we present the interim results Methods: We attempted to estimate the incidence of CLL in Israel more accurately,recognizing the fact that the exact incidence may never be known. The Israel Chronic Lymphocytic Leukemia Study Group, working with the Israel Center for Disease Control of the Ministry of Health, actively documented new cases of CLL/SLL in Israel for calendar years 2011 and 2012. All flow cytometry laboratories in Israel provided lists of patients with B cell clones. Israeli hematologists diagnosing CLL were asked to verify which of the B cell clones indicated a diagnosis of CLL, SLL, PLL or MBL and to fill out an internet-based reporting form. Diagnoses based on flow cytometry were verified by medical record review. Cases identified through active surveillance were pooled with cases known to INCR in order to estimate the true annual incidence of CLL/SLL and assess the completeness of the INCR data. Results: We identified 432 and 396 CLL/SLL cases for 2011 and 2012, respectively of whom 57.4% were males. The average age was 68.8. The corresponding age-adjusted[1]incidence rates per 100,000 (ASR) were 4.26 for 2011 and 3.79 for 2012. In comparison, the INCR registered 295 new CLL cases in 2011 (ASR=2.78) and 232 in 2012 (ASR=2.19), 54.5% of them males. The average age at diagnosis was 69.9. These data indicate a gap between true and reported incidence (1.48 and 1.60/100,000 in 2011 and 2012, respectively). However, it should be noted that the INCR will be fully updated for 2012 only by the beginning of 2015. Of active surveillance cases, 157 (2011) and 152 (2012) were registered in the INCR. Most cases missing in the INCR were diagnosed based on flow cytometry, peripheral blood samples and FISH (85.9% in 2011, 89.8% in 2012) without histo-pathological confirmation. Of the CLL/SLL cases existing in the INCR dataset for 2011-12 but not detected by active surveillance (138 in 2011; 80 in 2012), most (76.8% in 2011, 63.8% in 2012) had been diagnosed earlier and the remainder were coded as diagnoses other than CLL/SLL. Omitting these cases from the INCR dataset substantially increased the observed gaps in the true and the registered annual incidence of CLL/SLL (from 1.48 to 2.80 per 100,000 in 2011 and from 1.60 to 2.37 per 100,000 in 2012). Conclusions: The true incidence of CLL is unknown, but it is clear that there is under reporting to cancer registries. Completeness of CLL/SLL data requires accurate reporting of cases by hematologists and other care providers in the community. In Israel, this issue has been addressed by publishing updated guidelines for mandatory reporting stressing the requirement for reporting of hematologic malignancies. [1] Age-adjustment made on the basis of the world standard population Disclosures Ruchlemer: Roche: Research Funding.

Description: Introduction: Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase, is an established therapeutic agent in a variety of B-cell lymphoproliferative disorders. Ibrutinib induces platelet dysfunction and concurrent treatment with ibrutinib and warfarin was shown to significantly increase the risk of bleeding. The current study was designed to investigate the safety of direct oral anticoagulants (DOACs) in patients receiving ibrutinib, considering their expanding employment together with the lack of data regarding their safety in patients receiving ibrutinib. Methods: We conducted a retrospective cohort study to evaluate risks of major bleeding in patients with B-cell lymphoproliferative disorders (CLL, MCL, DLBCL, MZL or WM) that were treated with ibrutinib and DOACs but without concurrent antiplatelet therapy, between January 2010 and October 2018 in 5 participating centers. Patient medical charts were reviewed for demographic parameters, comorbidities, ibrutinib dosage, DOACs dosage (including the adjustment for renal function), blood count and chemistry tests, bleeding site and grade. Results: The study included 30 patients, median age at starting concurrent administration of ibrutinib and DOACs was 71.58 years (range 50.9-88.2). Most patients were treated for CLL (n=18, 60%) and MCL (n=8, 26%). The most common daily doses of ibrutinib were 420 mg and 560 mg in 63.3% and 30% of patients respectively. None of the patients received an additional antiplatelet agent. Twenty-three patients were treated with apixaban (76.7%), 4 with rivaroxaban (13.3%) and 3 (10%) with dabigatran. The main indications for DOACs were atrial fibrillation and VTE (venous thromboembolism). The median follow-up after initiation of the ibrutinib-DOAC combination was 13.4 months (range 1.8-47.9 months). Bleeding was reported in 22 patients (73.3%), mostly mucocutaneous (n=12, 40%) and gastrointestinal tract (n=7, 23.3%), followed by CNS bleeding (n=4, 13.3%). Mucocutaneous bleedings were all grade 1-2 and gastrointestinal tract and CNS bleeding events were grade 1-4. Major bleeding events, defined as grade 3 or 4, occurred in 5 patients (16.6%) and did not result in death of any of the patients. The median time for bleeding following ibrutinib-DOAC initiation was 5.6 months. Over a follow-up period of 21 months of combined treatment, the incidence of bleeding events (of all grades) increased to 75% (Figure 1). Incidence of bleeding events (including all grades) was quite similar between all DOAC subtypes (73.9% with apixaban, 75% with rivaroxaban and 66.7% with dabigatran). No statistically significant predictors for increased risk of bleeding in patients receiving ibrutinib combined with DOACs were detected. Ibrutinib was stopped in 8 patients (26.7%) due to grade 1 to 4 bleeding events and was re-initiated in 6 patients, resulting in recurrent grade 3 and 4 bleeding events in 2 patients. Conclusions: Concurrent administration of DOACs and ibrutinib appears to be feasible. However, risk of bleeding is not neglectable, and treatment resumption in patients that experienced a significant bleeding event should be considered with caution. Disclosures Arnason: Celgene/Juno: Consultancy; Regeneron Pharmaceuticals, Inc.: Consultancy. Herishanu:Roche: Honoraria; AbbVie: Honoraria; Janssen: Honoraria.