Poor Prognosis Chronic Lymphocytic Leukemia Research Articles

Laboratory analysis of 124 chronic lymphocytic leukemia cases: Single center study

Abstract BACKGROUND: Chronic lymphocytic leukemia (CLL) is a disease characterized by an uncontrolled proliferation of malfunctioning mature B-lymphocytes. It represents a variable range of clinical presentation and prognoses. Understanding the demographic and cellular characteristics of CLL patients can aid in better disease management and treatment outcomes. OBJECTIVES: To identifying hematological parameters and patterns and potential markers that could assist in better diagnosis and of CLL in the local population. PATIENTS, MATERIALS AND METHODS: A retrospective analysis of 124 cases of newly diagnosed CLL was conducted. Data were collected from reports over 2 years (April 2022-April2024), including demographics, Hb levels, WBC counts, platelet counts and expression of CD markers (5, 19, 20, 23, 200, FMC7, LAIR1 and others) using flow cytometry on peripheral blood specimens. Based on FC findings, patients were categorized into two groups for better assessment; definite diagnosis CLL and possible diagnosis CLL; then further subdivided into poor prognosis CLL and good prognosis CLL. RESULTS: Male patients represented 59.5% of cases and females were 40.5% with a median age of 61 years at diagnosis. The mean Hb level was 109 ± 2 g/L, the mean WBC count was 58.0 ± 25.0 × 109/L, and the mean platelet count was 190 ± 40 × 109/L. CD5 was positive in 97.6% of cases, with CD23 in 96%. CONCLUSION: This study provides an overview of the demographic and cellular marker characteristics of CLL patients in FC unit. The findings underscore the heterogeneity of CLL immunophenotype and the importance of detailed characterization in improving patient management protocols; however, further research is warranted to correlate these findings with other parameters of disease burden and prognosis to optimize the therapeutic approaches for CLL patients.

Specific NOTCH1 antibody targets DLL4-induced proliferation, migration, and angiogenesis in NOTCH1-mutated CLL cells

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

Three Different Jak2/Stat3-Related Pathways Favor the Survival of Chronic Lymphocytic Leukemia Neoplastic Clone

INTRODUCTIONChronic lymphoproliferative disorders are characterized by the expansion of malignant lymphocytes, the most common form being Chronic Lymphocytic Leukemia (CLL). Besides intrinsic abnormalities, the acquisition of the transformed phenotype and the diffusion of disease are related to the favorable cross-talking tumor cell-microenvironment. Furthermore, it has been demonstrated that CLL cells own an amplified mitochondrial respiration leading to an increased Reactive Oxygen Species (ROS) production and intrinsic oxidative stress. Several molecules released by microenvironmental partners signal through JAK-STAT pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. We previously analyzed STAT3 and JAK2 expression, phosphorylation and localization in normal and leukemic B cells, demonstrating an abnormal activation of this pathway in the neoplastic clone with respect to normal B lymphocytes. We focused on STAT3 constitutive phosphorylation at serine (Ser) 727 residue since it has been recently observed that the presence of mt-STAT3 Ser727 promotes mitochondrial respiration and, generally, cell viability. We hypothesized the involvement of JAK2/STAT3 axis in 3 different pathways: (i) canonical “IL-6 related pathway”; (ii) JAK2/STAT3 - BCR/Lyn crosstalk; (iii) STAT3 effects on mitochondrial regulation.METHODSSTAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC). Purified cells (2x106 cells/ml) were cultured, and treated with the JAK2 inhibitor AG490 (10, 50 and 100μM) and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs and with/without Ibrutinib (2.5μM) and Venetoclax (1nM). CLL and normal B cell viability was tested with Annexin V/PI test by FC. P-STAT3 Ser727 expression has been correlated with p44/42, SAPK-JNK, NF-kB p65 and p38 MAPK activation by Reverse Phase Protein Microarrays (RPPA) in 57 therapy-free patients presenting good (mutated IGHV, absence of CD38 or normal karyotype) and poor (unmutated IGHV, CD38 expression or 17p deletion) prognostic factors.RESULTSWe found that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. We demonstrated that STAT3 and JAK2 were similarly overexpressed in both good and poor prognosis CLL patients. However, a significant correlation between STAT3 expression levels and their overall survival was observed. Considering STAT3 over-expression and its correlation with the clinical outcome in CLL and, according to our hypothesis, we moved forward with the analysis of the three different JAK2/STAT3 pathways. We demonstrated that AG490 and Stattic were able to induce a dose-dependent apoptosis in CLL cells, also bypassing environmental protection. AG490, targeting JAK2, inhibited the phosphorylation of SHP-1 at Ser591, activating the phosphatase. In turn, SHP-1 activation led to Lyn Tyr396 dephosphorylation/inactivation. Treatment with Stattic did not affect Lyn and SHP-1 phosphorylation since this inhibitor acts downstream to AG490. In fact, simultaneous administration of Ibrutinib led to an increase of apoptosis only in Stattic, but not in AG490 treated cells. This confirms a possible dual role of JAK2 inhibition. Venetoclax/AG490 and Venetoclax/Stattic co-treatment did not show an increased cell death rate, consistent with a downstream effect of Venetoclax to both AG490 and Stattic. RPPA analysis demonstrated a correlation between STAT3, Ser727 constitutively phosphorylated in CLL, and P-p44/42 Thr202/Tyr204, P-SAPK-JNK Thr-183/Tyr185, NF-kB p65 Ser-536 and p38 MAPK Thr-180/Tyr-182 expression. All these proteins are described as involved in an alternative pathway that allows STAT3 Ser727 to exert a pro-survival effect thorough the regulation of mitochondrial activity.CONCLUSIONSThe analysis of JAK2 and STAT3 expression, activation and inhibition lets us to highlight the importance of JAK2/STAT3 axis in the maintenance of 3 different survival pathways in CLL B cells. Furthermore, the correlation we demonstrated with clinical outcome of patients and the strengthening of Ibrutinib effect when we targeted this axis could represent a starting point for the development of new therapeutic strategies in CLL. DisclosuresTrentin:Abbvie: Honoraria; Gilead: Research Funding; Janssen: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees.

Turning the Heat up a Notch in Biclonal Lymphoproliferative Disorders

Biclonal lymphoid disorders (BLD) are a rare (1.4%) but distinct entity in which Chronic lymphocytic leukaemia (CLL) co-exists with a second lymphoproliferative disorder (LPD) and are poorly understood in terms of pathogenesis and clinical impact. The NOTCH1 pathway is involved in cell proliferation, cell cycle progression and is anti-apoptotic, with mutations of the regulatory PEST domain causing constitutive up-regulation of the pathway. NOTCH1 mutations occur early in CLL leukaemogenesis and are found in 10% of poor prognosis CLL at diagnosis as well as being associated with 50% of Richter's transformation (both Hodgkin's and non-Hodgkin's subtype). We hypothesised that NOTCH1 mutations may be implicated in BLD pathogenesis both because they occur early in leukaemogenesis when clonal divergence is possible and because of its association with Hodgkin's Richter's, which is biologically distinct from CLL.We identified 19 patients with BLD at initial presentation, confirmed by flow cytometry and/or molecular analysis of the Immunoglobulin heavy (IgH) and light (IgL) chain genes between 2008 and 2015. This cohort consisted of 14 males and 5 females with a mean age of 72 (range 47-90) years. BLD was defined as follows: (1) co-existence of cells with different immunophenotypes such as a CD5+/CD19+ and CD5-/CD10- population confirming the co-existence of 2 diagnoses, (2) demonstration of both kappa (κ) and lambda (λ) light chain restriction on a CD5+/CD19+ population and (3) CD5+/CD19+ population without demonstrable surface light chains but with >2 clonal gene rearrangements of their IgH and IgL loci. All 19 cases were tested for a c.7544-7545delCT resulting in truncation of the regulatory C-terminal PEST domain of the NOTCH1 gene by PCR.Eleven of 19 patients had CLL co-existing with a different LPD as follows: Mantle Cell Lymphoma (MCL) (2), Hairy Cell Leukaemia (HCL) (2) and Marginal Zone Lymphoma (7). Of this subgroup 5 of 11 cases (45%) had a NOTCH1 mutation. Four samples in our cohort displayed dual κ and λ light chain expression detected by flow cytometry and confirmed molecularly by identifying 2 clonally distinct populations of LPD and 3 samples (75%) had a NOTCH1 mutation. Four samples were identified with >90% CD5+/CD19+ expression but no monoclonality by flow cytometry, but with 2 distinct populations identified by IgH and IgL rearrangements and of this subgroup 2 (50%) had NOTCH1 mutations. Clinical data was available on 13/19 patients, of whom 11 patients had Binet A and 2 Binet B disease. No patient has required CLL-directed treatment, 2 patients have died of other causes and 11 remain under surveillance. Three of 9 patients with a second LPD have required treatment: both patients with HCL received cladribine for pancytopaenia and a patient with MCL was treated with Rituximab and Bendamustine.Overall, 10 of the 19 (52%) patients with BLD had a NOTCH1 mutation at diagnosis, which is higher than the 10% reported in CLL literature. The absence of any germinal centre cell derived lymphoma in the cohort of patients with additional LPD is intriguing and needs confirmation in other series. The prognostic implications of BLD and NOTCH1 mutations are not possible to assess in this small cohort, but accurate characterization of BLD aids therapeutic decision-making as in the case of concurrent CLL and HCL. Light chain restriction by flow cytometry in a mature B-cell population confirms monoclonality and suggests malignancy, however dual κ and λ expressing CLL may give a polyclonal pattern resulting in failure to make a CLL diagnosis; therefore awareness of this rare subtype of CLL is important.In summary we have added a further association between NOTCH1 mutations and CLL associated disorders which currently includes: poor prognosis CLL at diagnosis, Hodgkin's and non Hodgkin's Richter's transformation and most recently BLD. Increased sensitivity of NOTCH1 detection by next generation sequencing techniques may provide a further insight into the evolutionary origin and pathogenesis of BLD and further elucidate the enigmatic role of NOTCH1. DisclosuresCrotty:BMS, Takeda, Novartis, Janssen, Roche: Honoraria.

Outcome of Patients with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) and/or 17p Deletion/TP53 Mutations Treated with Ibrutinib According to a Named Patient Program (NPP) in Italy: Preliminary Analysis of a Real Life Retrospective Study

Outcome of Patients with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) and/or 17p Deletion/TP53 Mutations Treated with Ibrutinib According to a Named Patient Program (NPP) in Italy: Preliminary Analysis of a Real Life Retrospective Study

Venetoclax responses of pediatric ALL xenografts reveal sensitivity of MLL-rearranged leukemia

AbstractThe clinical success of the BCL-2-selective BH3-mimetic venetoclax in patients with poor prognosis chronic lymphocytic leukemia (CLL) highlights the potential of targeting the BCL-2-regulated apoptotic pathway in previously untreatable lymphoid malignancies. By selectively inhibiting BCL-2, venetoclax circumvents the dose-limiting, BCL-XL-mediated thrombocytopenia of its less selective predecessor navitoclax, while enhancing efficacy in CLL. We have previously reported the potent sensitivity of many high-risk childhood acute lymphoblastic leukemia (ALL) xenografts to navitoclax. Given the superior tolerability of venetoclax, here we have investigated its efficacy in childhood ALL. We demonstrate that in contrast to the clear dependence of CLL on BCL-2 alone, effective antileukemic activity in the majority of ALL xenografts requires concurrent inhibition of both BCL-2 and BCL-XL. We identify BCL-XL expression as a key predictor of poor response to venetoclax and demonstrate that concurrent inhibition of both BCL-2 and BCL-XL results in synergistic killing in the majority of ALL xenografts. A notable exception is mixed lineage leukemia–rearranged infant ALL, where venetoclax largely recapitulates the activity of navitoclax, identifying this subgroup of patients as potential candidates for clinical trials of venetoclax in childhood ALL. Conversely, our findings provide a clear basis for progressing navitoclax into trials ahead of venetoclax in other subgroups.

Mirna-21 Targets Sprouty2 to Activate SYK Mediated BCR and MAPK-Erk Signaling in Chronic Lymphocytic Leukemia

Abstract INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) represents the most common adult leukemia in the western world. Several reports have demonstrated that the constitutively activated MAPK-Erk signaling pathways promote CLL cell proliferation and survival. However, the precise molecular mechanisms that lead to deregulated MAPK signaling in CLL initiation and progression are not fully understood. We have previously reported that Sprouty2 (Spry2) is a negative regulator of BCR and MAPK-Erk signaling in poor prognosis CLL (American Society of Hematology, 56th Annual Meeting, San Francisco, CA, 2014). Here in follow up studies, we set to further determine molecular basis deregulation of Spry2 in patients with poor prognosis CLL. Spry2 is either epigenetically silenced or targeted by Microrna-21 (miR21) in several malignancies. MiR-21 plays a role of oncomir in CLL, which is significantly upregulated in CLL patients with poor prognosis specifically with Del 17p, High 38 and Zap70 expression. Notably, we observed a decrease in Spry2 expression in CLL cells expressing high levels of miR-21. Also, it is already reported that miR21 is regulated by STAT3 transcription factor in CLL patients with poor prognosis in refractory phase. However the precise molecular mechanism by which the miR21 leads to leukemic progression in poor prognosis CLL is not known. Here we demonstrate the molecular mechanism of miR21 to activate Syk mediated BCR and MAPK-Erk signaling by targeting Spry2 in CLL cells from poor prognosis patients. METHODOLOGY: We isolated CLL cells from peripheral blood of newly diagnosed high CD38 poor prognosis patients. For molecular analysis, in addition to primary CLL cells we use Mec-1 CLL cell line. To measure the levels of miR21 we used Taqman assay qPCR from Invitrogen. To decrease miR21 levels we used miR21 inhibitor (Invitrogen) and STAT3 inhibitor (SantaCruz). To overexpress miR21 we used pcDNAmir-21 construct from Addgene. Activation of BCR and MAPK-Erk signaling pathway was measured by levels of p-Erk and p-Syk. Expression of Spry2, Erk and Syk was measured by western blot analysis. RESULTS: To study if miR21 targets Spry2 in CLL cells, we first overexpressed miR21 in Mec-1 cells. We observed a significant decrease in Spry2 expression in Mec-1 cells expressing high levels of miR-21 compared to empty vector control. In our previous studies we observed Spry2 acts as a negative regulator of BCR and MAPK-Erk signaling. Therefore, we next studied the activity of Spry2 targeting signaling pathways in miR21-overexpressing CLL cells. We observed elevated levels of p-Erk and p-Syk in CLL cells overexpressing miR21. For control we used pcDNA-empty vector. Whereas upon miR21 knockdown in CLL cells resulted in stabilization of Spry2 expression and decrease in BCR and MAPK-Erk signaling. Also, targeting miR21 by STAT3 inhibitor leads to induction of spontaneous apoptosis in CLL cells. We next tested the effect of STAT3 inhibitor on primary CLL cells from 13 different patients. We measured the viability of CLL cells using MTT assay, we observed higher efficacy of STAT3 inhibitor over Syk and BRAF inhibitors on CLL cells. CONCLUSION: We conclude that Spry2 is a target of miR21 in CLL cells from poor prognosis patients. MiR21 targets Spry2 to activate Syk mediated BCR and MAPK signaling in CLL patients. Also, STAT3 can be used as therapeutic target for poor prognosis CLL patients with high miR21 expression. Disclosures No relevant conflicts of interest to declare.

Myeloablative Allogeneic Stem Cell Transplantation in CLL: A Single Centre Retrospective Study

Introduction:Chronic lymphocytic leukemia (CLL) is the most common lymphoproliferative disorder in the Western world. Despite recent advances in therapy, CLL remains incurable with conventional chemo-immunotherapy. Allogeneic hematopoietic stem cell transplantation (alloSCT) remains the only curative option in CLL. AlloSCT relies on graft-versus-leukemia effect and can achieve the possibility of long-term disease control even in poor prognosis CLL such as chemotherapy refractory or high-risk cytogenetics. Unfortunately, alloSCT is associated with significant morbidity and mortality risks from regimen related toxicity, graft-versus-host disease (GVHD) and infectious complications. In order to reduce the rates of non-relapse mortality (NRM), reduced intensity conditioning regimens have been routinely adopted in most centers. While these newer regimens have shown lower NRM, they result in higher relapse rates compared to traditional myeloablative (MA) regimens. This retrospective analysis describes the outcomes of using contemporary MA conditioning in a consecutive series of patients transplanted for high-risk CLL including patients with relapsed/refractory disease or 17p deletion.Patients & Methods:We retrospectively reviewed CLL patients who underwent alloSCT between February 2000 to July 2012 at a single center using a uniform myeloablative conditioning regimen comprising Fludarabine 250mg/m2, PK targeted Busulfan 12.8mg/kg IV, and Thymoglobulin 4.5mg/kg (FLUBUPATG) with cyclosporine and methotrexate GVHD prophylaxis. Major eligibility criteria were relapsed CLL or del(17p) CLL in adults (18-70yrs) with an available HLA-compatible donor. Cox regression models were used to evaluate the impact of variables on outcomes including OS, PFS, and NRM. Cumulative incidence function (CIF) was used to estimate relapse. Statistical analysis was conducted using SAS (SAS Institute, Cary, NC) version 9.3 software. A p-value <0.05 was used for all statistical significance. Responses were defined according to the iwCLL criteria.Results:Sixty-one patients (46 males, 15 females) with a median age of 53 years (range, 38-65 years) including 21% ≥ 60 years were analyzed. The median time from diagnosis to alloSCT was 48 months (range, 3-236 months). Fifty-six percent of patients were treated with ≥ 2 regimens prior to alloSCT and 74% were fludarabine-refractory. Forty-three percent were considered chemosensitive prior to alloSCT and 5 (8%) were transplanted due to Richter's transformation. Twenty percent had poor-risk cytogenetics including del17p (n=6) and del11q (n=7). Sixty-nine percent were fully HLA matched including a total of 33 (54%) matched related donors and 9 (15%) matched unrelated donors. A total of 19 (31%) patients had a mismatched donor, including 6 (10%) with 2 mismatches. A complete remission (CR) was achieved in 62%, while 16% had partial remission (PR). Twelve (20%) patients relapsed after achieving CR/PR. The 5-year cumulative incidence of relapse was 26%. Fifty-two percent of patients remain alive at a median follow-up of 83 months. The estimated 5-year overall survival (OS) and progression free survival (PFS) were 61% and 47%, respectively. Twenty-nine patients died, 12 from CLL and 17 NRM. The 2-year NRM was 24%. Although 74% of patients had acute GVHD, only 10% had grade III or IV GVHD. Extensive chronic GVHD developed in 49% of patients, of whom 85% required second-line therapy. Only 6 patients (10%) remain on chronic cGVHD treatment and 6 deaths were caused by GVHD. Based upon multivariate analysis, extensive chronic GVHD was associated with improved OS (HR 0.289, [95% CI, 0.136-0.614, p=0.001]) and PFS (HR 0.136, [95% CI, 0.060-0.305, p=0.001]). A mismatched donor was associated with worse OS (HR 2.347, [95% CI, 1.078-5.109, p=0.03]) and the use of a female donor resulted in worse PFS (HR 2.738, [95% CI, 1.290-5.811, p=0.009]).Conclusions:This study illustrates that myeloablative alloSCT in high-risk CLL patients is both feasible and relatively well tolerated, resulting in excellent survival outcomes with comparable NRM to popular reduced intensity regimens. The positive impact of cGVHD confirms a graft versus leukemia effect with this MA regimen. These favorable PFS and OS rates support the value of allogeneic transplantation in the treatment of CLL. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Real-World Results on Ibrutinib in Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL): Data from 97 Swedish Patients Treated in a Compassionate Use Program

Background: Patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) and those with del 17p CLL have a poor prognosis. Ibrutinib (ImbruvicaTM) is a first-in-class Bruton´s tyrosine kinase (BTK) inhibitor that has recently been approved for previously treated patients with CLL or those with del 17p after having demonstrated convincing effects in pivotal clinical studies. However, there is often a discrepancy between data from patients strictly included in clinical trials and those later obtained in routine health care. We report here the first Real-World results on ibrutinib in 97 consecutive Swedish patients with poor-prognosis CLL who received their treatment in a compassionate use program (CUP).Methods: Patients with CLL were eligible for the CUP if they a) had a confirmed diagnosis of CLL or small lymphocytic lymphoma (SLL) b) had relapsed after or were refractory to conventional CLL therapy or had del 17p / TP53 disruption in any disease phase c) had a treatment indication for CLL d) had no contraindication for ibrutinib such as receiving a strong CYP3A inhibitor or warfarin treatment e) had a neutrophil count of at least 0.5x109/L and a platelet count above 30x109/L.Patients received 420 mg oral ibrutinib once daily until progression. Endpoints were overall response rate (ORR) with or without persistent lymphocytosis, complete remission (CR) rate with or without (CRi) complete blood count recovery, duration of response (DOR), progression-free survival (PFS), overall survival and safety of ibrutinib. The IWCLL response criteria were applied.Results: Fourenrolled patients were excluded as they died or were diagnosed with Richter transformation (RT) and never received ibrutinib. Ninety-seven patients (95 with CLL, 2 with SLL) were included between May 15, 2014 and May 31, 2015. The median age was 69 years (24% were 75 years or older). FISH or TP53 mutation test was performed in 82 patients and 61% had del 17p / TP53 disruption. Del 11q was present in 17%. Baseline characteristics included 65% of patients with Rai Stage III or IV, 48% with at least one lymph node >5 cm and 25% had performance status grade 2 or 3. A median of 3 prior therapies (range 0-10) were reported. At the time of writing, 81 patients (86%) had been enrolled for least 2 months and were included in the preliminary efficacy analysis. All patients who had received at least one dose of ibrutinib (n=92) at the time of writing were included in the safety analysis. The median treatment duration at analysis was 6.8+ months (range 0-12.7+ months) and 78 patients (85%) remain on continuous ibrutinib therapy. Investigator-assessed ORR was 80% including 25% PR with lymphocytosis (PR-L). No CR or CRi were reported. At a median follow-up time of 6.8+ months (range 0.5-12.7+ months) the median PFS and DOR had not been reached.Ninety- two patients were evaluable for toxicity. Twenty-five patients (27%) were hospitalized due to grade 3 or worse infection. Neutropenia grade 4 occurred in 16% of the patients. Diarrhea of any grade was reported in 17% (4% were grade 3, none was grade 4). Arthralgia or muscle pain was reported in 15% (all but one were grade 1-2) and any hemorrhage occurred in 30 patients (33%). Atrial fibrillation occurred in 3%. Three patients had a secondary tumor during ibrutinib treatment. Ibrutinib was permanently stopped in 13 patients for the following reasons: CLL progression (n=1), RT (n=4, after a median time of 7.6 months, range 3.7-10.3), cutaneous adverse event (n=2), congestive heart failure (n=1), infections (n=3) or other malignancy (n=2). Twelve patients have died. The reasons were progression (n=3), RT (n=2), congestive heart failure (n=1), infection (n=3), secondary tumor (n=2) or unknown (n=1).Conclusions: This is the first Real-World analysis on ibrutinib treatment of poor prognosis CLL patients from a well-defined geographical region. The efficacy of ibrutinib was similar to that of a previous pivotal clinical trial (Byrd et al NEJM 2013) and with a similar adverse event profile as previously reported (Byrd et al, NEJM 2014) despite that our patients were older, had a lower performance status and with a majority having del 17p / TP53 disruption. The median PFS has not yet been reached and most patients remain on continuous ibrutinib therapy. Updated results will be presented at the meeting. DisclosuresHansson:Jansse Cilag: Research Funding. Winqvist:Janssen Cilag: Research Funding. Asklid:Janssen Cilag: Research Funding. Lundin:Janssen: Research Funding; Novartis: Research Funding. Mattsson:Janssen: Consultancy. Österborg:Janssen, Pharmacyclics, Gilead: Consultancy, Research Funding; Novartis: Research Funding.

Abstract 3624: A novel, selective DNA-PK inhibitor sensitizes poor prognosis chronic lymphocytic leukemia (CLL) cells to mitoxantrone

Abstract Approximately 20% of Chronic Lymphocytic Leukaemia (CLL) cases carry 11q deletions and 10% carry 17p deletions (associated with loss of p53 function), both of which are poor prognostic markers. Ataxia telangiectasia mutated kinase (ATM) is located on chromosome 11q23 and 30% of del11q patients lack ATM function. Previous work has shown that the DNA-PK inhibitor NU7441 chemosensitises CLL cells to fludarabine ex vivo (Willmore E et al 2008), and monotherapy with KU60648, a dual inhibitor of PI3kδ and DNA-PK has been shown to be effective in ATM deficient cells (Riabinska A et al 2013). Furthermore, a dual DNA-PK/mTOR inhibitor, CC-115, is now in clinical trials (NCT01353625). Here, we aimed to validate the selective inhibition of DNA-PK over related kinases as a therapeutic approach in CLL, using a novel, more selective compound (NDD0004, cell-free IC50 10nM). Both primary CLL cells and an isogenic pair of DNA-PK-deficient and proficient HCT116 cells were used as models. Primary CLL cells were cultured ex vivo and treated with the topoisomerase II poison mitoxantrone in the presence and absence of NU7441 and NDD0004. Cell viability was determined after 48h by XTT assay. Apoptosis and DNA damage (yH2AX foci) were measured by caspase 3/7GloAssay and immunofluorescence, respectively. ATM function was determined by the cells ability to autophosphorylate ATM on serine 1981 after ionising radiation. Chemosensitisation to mitoxantrone in primary CLL cells was substantial and it was higher with NDD0004 than with NU7441 (mean 202- vs. 69-fold, ranges 1-1000 vs. 1-543-fold). Intriguingly, cells lacking ATM or p53 function were also sensitised by DNA-PK inhibition (mean 15- and 21-fold, respectively). HCT116 cells lacking DNA-PK were not chemosensitised by NDD0004 but DNA-PK proficient cells were sensitised 5-fold. Mitoxantrone-induced DSBs (as measured by yH2AX foci) were repaired by 24h, but persisted in the presence of DNA-PK inhibitors. In keeping with this observation, apoptosis as measured by caspase 3/7 activity was potentiated by DNA-PK inhibitors. NDD0004 and KU60648 were also evaluated in a panel of ATM deficient (HT144, H1395) and ATM proficient (A549, A375) cell lines. ATM status had no impact on (a) the level of chemosensitisation to mitoxantrone by both inhibitors and (b) single agent activity for NDD0004 and KU60648. In conclusion, our data show that selective inhibition of DNA-PK chemosensitises primary CLL cells even when p53 is mutated or ATM function is lost. Current work is focused on identifying biomarkers which predict good response to DNA-PK inhibitors. Citation Format: Gesa Junge. A novel, selective DNA-PK inhibitor sensitizes poor prognosis chronic lymphocytic leukemia (CLL) cells to mitoxantrone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3624. doi:10.1158/1538-7445.AM2015-3624

Role of Sprouty2 in the Pathogenesis of Chronic Lymphocytic Leukemia

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults representing about 35% of all adult leukemias in the United States. CLL is a B-cell neoplasm which is characterized by accumulation of long lived mature B-cells in peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). We and others have reported that CLL cells in tissue microenvironments have constitutively activated MAPK-Erk and AKT signaling pathways, which promotes their proliferation and survival. However, the molecular mechanisms and the gene(s) responsible for the activation of these pathways in CLL are not clearly understood. We performed gene expression profiling of CLL cells from PB, BM and LN and transcriptome analyses of CLL cells from PB from good and poor prognosis patients. Our studies identified Sprouty 2 (Spry2), a negative feedback regulator as one of the key genes involved in the regulation of MAPK-Erk and Akt pathways in CLL. We observed that Spry2 levels were down-regulated in poor prognosis CLL cells. Interestingly, Spry2 is epigenetically silenced in most human and mouse lymphomas and ectopic expression of Spry2 induces apoptosis in mouse B cells. We categorized CLL patients into good and poor prognosis to further evaluate Spry2 in CLL biology. Poor prognosis patients were defined by unmutated immunoglobulin variable heavy chain (IgVH), 11q22 deletion, 17p deletion, trisomy 12 or high CD38 and Zap70 expression. Good prognosis patients were defined as having mutated IgVH, 13q14 deletions or low CD38 and Zap70 expression.RESULTS: We found that Spry2 expression was significantly decreased by 3.2 log2-fold, (p=0.0001) at transcript and protein levels, respectively, in CLL cells from patients with poor prognosis compared to patients with good prognosis. Spry2 is a negative feedback regulator which attenuates cell proliferation, migration and survival. To determine the effect of Spry2 on CLL cells we transfected Mec-1 cells (stable human CLL cell line) and primary human CLL cells with a Spry2 cDNA containing vector. Ectopic expression of Spry2 induced spontaneous apoptosis in Mec-1 cells and primary CLL cells from poor prognosis patients. These results indicate that Spry2 negatively regulates the survival of human CLL cells. CLL cells have been shown to have active B-cell receptor (BCR) signaling promoting proliferation in vivo. To study the effect of BCR stimulation on Spry2 we stimulated BCR on Mec-1 and primary CLL cells by BCR cross-linking. Unexpectedly, the rate of proliferation of Mec-1 cells decreased upon BCR stimulation. We were able to demonstrate a transient increase in Spry2 levels that peaked at 12 hours post BCR stimulation, which we suspected was mediating the decrease in proliferation observed. We therefore repeated the BCR stimulation experiment using siRNA to inhibit the transient increase in Spry2 in primary CLL cells from seven good prognosis patients and in the Mec-1 cells. As predicted, we observed a significant increase in proliferation in all primary CLL samples and in the Mec-1 cells upon Spry2 knock down following BCR stimulation. Spry2 attenuates ligand induced MAPK-Erk and AKT signaling leading to suppression of proliferation and survival. We also examined the effect of Spry2 on MAPK-Erk and Akt signaling in CLL cells. Upon induction of exogenous Spry2 expression in Mec-1 cells there was a significant decrease in p-Akt and p-Erk levels. Conversely, Spry2 knock down resulted in an increase in p-Erk and p-Akt and enhanced cell proliferation.CONCLUSIONS: Spry2 is significantly down-regulated in the B-cells from patients with poor prognosis CLL. Down-regulation of Spry2 in CLL cells leads to hyperactivation of MAPK-Erk and Akt signaling and exogenous and endogenous expression of Spry2 leads to spontaneous apoptosis in human CLL cells. These results identify Spry2 as a negative regulator of CLL cell survival and proliferation and provide a potential molecular target for therapeutic intervention. DisclosuresBociek:Seattle Genetics, Inc.: Research Funding.

Proteomics-based strategies to identify proteins relevant to chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), a malignant B-cell disorder, is characterized by a heterogeneous clinical course. Two-dimensional nano liquid chromatography (2D-nano-LC) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) (LC-MALDI) was used to perform qualitative and quantitative analysis on cellular extracts from 12 primary CLL samples. We identified 728 proteins and quantified 655 proteins using isobaric tag-labeled extracts. Four strategies were used to identify disease-related proteins. First, we integrated our CLL proteome with published gene expression data of normal B-cells and CLL cells to highlight proteins with preferential expression in the transcriptome of CLL. Second, as CLL's outcome is heterogeneous, our quantitative proteomic data were used to indicate heterogeneously expressed proteins. Third, we used the quantitative data to identify proteins with differential abundance in poor prognosis CLL samples. Fourth, hierarchical cluster analysis was applied to identify hidden patterns of protein expression. These strategies identified 63 proteins, and 4 were investigated in a CLL cohort (39 patients). Thyroid hormone receptor-associated protein 3, T-cell leukemia/lymphoma protein 1A, and S100A8 were associated with high-risk CLL. Myosin-9 exhibited reduced expression in CLL samples from high-risk patients. This study shows the usefulness of proteomic approaches, combined with transcriptomics, to identify disease-related proteins.

Role Of Allogeneic Transplant In Poor Risk CLL Patients: A Long-Term Monocentric Experience In 39 Patients

BackgroundAllogeneic stem cell transplantation (SCT) remains the only curative option in poor risk chronic lymphocytic leukemia (CLL). Reduced intensity conditioning regimens (RIC) have improved outcome and enlarged number of patients eligible to SCT. However it is still controversial whether patient characteristics, CLL treatment, conditioning regimen or GVH prophylaxis influence the outcome. Here we present the analysis of our cohort of CLL patients who underwent RIC SCT. Patients and methodsThis is a retrospective single center study Primary endpoint was overall survival; secondary endpoints were cumulated incidence of relapse and non-relapse mortality. ResultsFrom 2000 to 2012, Thirty-nine patients with a sex ratio of 2 were transplanted. At time of transplantation, median age was 59 years (27-68), 82% patients displayed performans status (PS) of 0 and 20% had a comorbidity score ≥3. More than half patients (54%) were Fludarabine refractory and FISH 17p deletion was found in 10 out of 23 tested patients (43%). Median delay between diagnosis and transplant was 92 months (4-182). Patients received a median of 3 (1-9) prior therapies. Last treatment before transplant was fludarabine-based in 12 patients, alemtuzumab in 13, and other (RCHOP, R-Bendamustine, RDHAP) in 14. Median delay between the last treatment and transplantation was 2 months (1-33). At time of transplant, overall response rate was 82% including 33% complete response (CR). Four-color phenotypic MRD was negative (<10-4) in 9 patients (23%). Donor type was HLA matched relative for 22 patients, unrelated for 13 and cord blood for 4. Conditioning regimen varied according to protocols and donor. It consisted of fludarabine, busulfan and antithymocyte globulin in 21 patients, fludarabine, TBI and rituximab in 14 patients and fludarabine, cyclophosphamide and total body irradiation (TBI) in 4 and. Graft versus host disease (GVHD) prophylaxis was provided by cyclosporine alone in 54% patients, and cyclosporine and mycophenolate mofetil in 46%.After transplant, overall response rate was 90% with 17 patients evolving from partial response or stable disease to complete response and MRD was negative in 28/33 (85%) patients. Donor chimerism was performed for 27 patients, chimerism >90% was obtained in all patients except one, after a median of 90d (19-180) post transplant. Acute and chronic extensive GVHD occurred in 44% and 38% patients respectively.With a median follow-up of 59 months, median overall survival (OS) was 97 months, 2- and 5-years OS were 72% and 59% respectively. Fifteen patients died, 7 from GVHD, 4 from sepsis, 1 from CLL and 3 from other causes. 2- and 5-years cumulated incidences (CI) of non-relapse mortality (NRM) were 25.6% and 38.3% respectively. Three patients relapsed after transplant, 2 are still alive 82 and 98 months post-transplant. 2- and 5-years cumulated incidences of relapse were 2.8% and 6.8% respectively. In univariate analysis, disease status at transplant and number of prior therapies were significantly associated with better OS (p=0.0009 and p=0.03 respectively), whereas a trend to correlation between PS and OS was observed (p=0.063). In multivariate analysis, PS and achievement of CR or PR before transplant correlated significantly to OS (p=0.015 and p=0.05 respectively). Clearance of MRD before transplant perhaps suggested a better survival (p=0.158). ConclusionThis retrospective study highlights the ability of allogeneic transplant to improve OS in otherwise very poor prognosis CLL patients and deserve further investigations. Disclosures:Aurran-Schleinitz:Roche: Honoraria.

Progressive Chronic Lymphocytic Leukemia After Allogeneic Hematopoietic Cell Transplantation

This case report presents a patient with poor-prognosis chronic lymphocytic leukemia (CLL) who was treated with chemotherapy and underwent allogeneic hematopoietic cell transplant (alloHCT) but ultimately progressed. The application of the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for CLL and the impact of alloHCT on secondary therapy for progressive CLL are discussed.

Allogeneic Stem Cell Transplantation for Relapsed or Refractory Lymphoma after Conditioning with BEAM/Fludarabine/TBI

Allogeneic stem cell transplant (SCT) after high-dose conditioning with BEAM/fludarabine/total body irradiation (TBI) in patients with relapsed or refractory lymphoma has shown promising results in a pilot study. In this trial, we treated 50 consecutive patients with refractory or relapsed lymphoma or chronic lymphocytic leukemia (CLL). The patients included were considered to have poor-prognosis disease (eg, one-third was chemo-refractory at transplantation and more than one-half had failed previous autologous or allogeneic SCT). All patients engrafted and achieved full donor chimerism. Grade II-IV acute graft-versus-host disease (aGVHD) occurred in 64% of patients (95% confidence interval [CI], 52% to 79%), and chronic GVHD (cGVHD) in 51% (95% CI, 36% to 66%). At 3 years, overall survival was 61% (95% CI, 46% to 75%). Progression-free survival was 55% (95% CI, 40% to 70%), with 30% (95% CI, 19% to 47%) transplantation-related mortality and a relapse incidence of 15% (95% CI, 7% to 32%). Disease classification and stage as well as remission status at transplantation and type of previous treatment (including previous SCT) had no significant impact on transplantation outcome. In conclusion, allogeneic SCT after BEAM/fludarabine/TBI provides excellent tumor control with complete and durable remissions in patients with poor-prognosis lymphoma and CLL. High rates of GVHD and GVHD-related mortality associated with this regimen are a major concern and warrant modification of the regimen in the future.

Low dose alemtuzumab in patients with fludarabine-refractory chronic lymphocytic leukemia

Alemtuzumab has been shown to be effective in poor-prognosis chronic lymphocytic leukemia (CLL); treatment, however, has been associated with significant toxicity. With the aim of seeking better tolerability, we treated 39 patients with fludarabine-refractory CLL subcutaneous alemtuzumab 10 mg three times a week, for 18 weeks. In 18 randomly selected patients, after obtaining lymphocyte count reduction by 1 Log, the antibody was administered once weekly at the dose of 30 mg. Overall response rate was 44%, including 8% complete remissions. Median overall survival and progression free survival were 29.1 and 10.3 months, respectively. Treatment was well tolerated, severe non-CMV infection occurred in 7% of the patients. CMV reactivation was detected in 27% of the patients, with only one case of CMV disease. No deaths occurred during therapy. In conclusion, low-dose alemtuzumab shows a promising safety profile coupled with satisfactory effectiveness in this category of poor prognosis CLL patients.

Single-agent rituximab in treatment-refractory or poor prognosis patients with chronic lymphocytic leukemia

Objective:Rituximab in combination with fludarabine and cyclophosphamide has significantly improved outcomes for patients with chronic lymphocytic leukemia (CLL) and an improvement in overall survival has recently been shown for the first time in the history of CLL treatment. However, the chemotherapy portion of this regimen may be unsuitable for elderly patients or those with significant comorbidities. We investigated the safety and tolerability of single-agent rituximab in 23 consecutive patients presenting with CLL at a single institution.Research design and methods:Patients received eight cycles of weekly rituximab (375 mg/m2 initially). Patients progressing on rituximab treatment could receive up to five further courses of single-agent rituximab in escalating doses up to 3 gm/m2 per dose. Previously untreated and previously treated patients, including those refractory to fludarabine, were eligible for the study.Results:Single-agent rituximab was efficacious, even in patients with treatment-refractory or poor-prognosis CLL. The overall response rate was 90.9% with a CR rate of 63.6%. The median PFS was 28.5 months, and median duration of response was 26 months. Responses were seen regardless of prior treatment including in patients refractory to fludarabine. Especially encouraging results were seen in patients receiving rituximab maintenance therapy who had a median duration of response substantially longer than those who did not receive maintenance (35 months vs. 14 months, respectively).Conclusions:Rituximab was well tolerated with no unexpected adverse events even at the highest dose. These results indicate that single-agent rituximab is effective and has a place in the treatment of CLL.

Novel X‐linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL‐mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis

Given that aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukaemia (CLL), alternative therapeutic approaches are needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumour-specific apoptosis. However, apoptosis might be inhibited by elevated levels of X-linked inhibitor of apoptosis (XIAP). Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-mediated apoptosis. A novel small molecule, compound A (CA), an inhibitor of XIAP, was used in combination with TRAIL to induce apoptosis in primary CLL cells (n = 48). XIAP was significantly more highly expressed in primary CLL cells (n = 28) compared to healthy B cells (n = 16) (P = 0·02). Our data obtained by specific knock-down of XIAP by siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Combined treatment with CA/TRAIL significantly increased apoptosis compared to untreated (P = 8·5 × 10⁻¹⁰), solely CA (P = 4·1 × 10⁻¹²) or TRAIL treated (P = 4·8 × 10⁻¹⁰) CLL cells. CA rendered 40 of 48 (83·3%) primary CLL samples susceptible to TRAIL-mediated apoptosis. In particular, cells derived from patients with poor prognosis CLL (ZAP-70(+) , IGHV unmutated, 17p-) were highly responsive to this drug combination. Our highly-effective XIAP inhibitor CA, in concert with TRAIL, shows potential for the treatment of CLL cases with poor prognosis and therefore warrants further clinical investigation.

ATM Mutant Chronic Lymphocytic Leukaemia Cells are Chemosensitized by Inhibition of DNA-Dependent Protein Kinase

AbstractAbstract 433Defects in ataxia telangiectasia mutated kinase (ATM) confer poor survival in B-cell chronic lymphocytic leukaemia (CLL). ATM gene deletion (11q23) occurs in approximately 20% of cases, and of these, 38% harbor an ATM mutation on the second allele, resulting in complete loss of ATM function. This prevents signaling of DNA double strand break (DSB) damage and compromises homologous recombinational (HR) repair. Importantly, del(11q) cases without corresponding ATM mutation display wild type ATM activity, with an intact DNA damage response and better outcome than ATM mutated cases1. Thus, in terms of chemotherapeutic resistance, it has been established that not all del(11q) cases should be placed in one group, since those with wild type ATM function have a distinct response. DNA-dependent protein kinase (DNA-PK) is also important for DSB repair and mediates the alternative non homologous end-joining (NHEJ) pathway. We previously showed that increased DNA-PK activity and expression is frequent in poor prognosis CLL and is associated with shorter survival2. We hypothesized that targeting the NHEJ pathway (via DNA-PK inhibition), in ATM deficient CLL cells that already have a defective DNA damage response, would completely compromise the ability of these cells to perform DSB repair, resulting in selective killing of ATM mutant CLL cells.The ATM status of CLL cases was determined using denaturing HPLC followed by confirmatory direct sequencing. Only those cases with pathogenic mutations (e.g. predicted to cause truncations or change in protein structure, or previously reported in A-T patients) were classed as ‘ATM mutant’. The resulting defects in ATM activity in these cases was confirmed by quantifying phosphorylation of downstream ATM targets (e.g autophosphorylation of ATM (ser1981) or phosphorylation of SMC-1) in lysates of ionizing-radiation-treated cells. Ex vivo cytotoxicity using freshly isolated CLL cells was possible in 77 cases of known ATM status. Cells were exposed to increasing concentrations of Fludarabine in combination with the DNA-PK inhibitor, NU7441 (1μ M), for 72 hr, and viability was assessed using an XTT kit. NU7441 potentiated the cytotoxicity of Fludarabine in 31 cases, including 8 cases with a known ATM mutation. As expected, ATM mutant CLL cells were more drug-resistant than those with wild type ATM, but even highly resistant (>20μ M) ATM mutant cases were resensitized up to 20-fold by NU7441. Pilot data using a similar approach showed ATM mutant cases to be 5-fold more resistant to mitoxantrone, but NU7441 potentiated the cytotoxicity of mitoxantrone in ATM mutant cases (n = 3) and ATM wild type cases (n = 5) by up to 6-fold or 3-fold respectively.Preliminary data (n =18) using an ex vivo assay dot blot assay to measure DNA-PK activity correlated DNA-PK activity to expression of the DNA-PK catalytic subunit as determined by Western blotting. Furthermore, DNA-PK activity was significantly higher in ATM mutant, compared to wild type CLL cells (p = 0.008). Current studies are exploring the formation and longevity of γH2AX foci in ATM mutant CLL cells treated with DNA damaging agents, and the role of DNA-PK in repair of DSB in these cells. Also, the link between DNA-PK activity and ATM mutation will be further examined as it provides a possible mechanism and proof of concept of increased sensitization by DNA-PK inhibitors. These results suggest that DNA-PK inhibition can sensitize poor prognosis, ATM mutant CLL cells to chemotherapeutics. Our data are consistent with the concept of synthetic lethality, where tumor cells harboring a DNA repair defect can be killed by targeting the compensatory DNA repair pathway, and suggest a group of patients that may benefit from this combination.1 Austen et al., Journal of Clin. Oncol. 20082 Willmore et al., Clin. Can. Res. 2008 Disclosures:No relevant conflicts of interest to declare.

Novel X-Linked Inhibitor of Apoptosis (XIAP) Inhibiting Compound as Sensitizer for TRAIL-Mediated Apoptosis In Chronic Lymphocytic Leukemia with Poor Prognosis

AbstractAbstract 1375 Background:Since aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukemia (CLL), alternative therapeutic approaches are needed. Moreover, there is an essential need to improve specific therapeutic regimes for “non-fit” patients, which cannot receive myeloablative therapies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumor-specific apoptosis. However, apoptosis might be inhibited by elevated X-linked inhibitor of apoptosis (XIAP) level, the only cellular protein capable to bind to and effectively inhibit caspases. Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-induced lysis. Experimental design:We compared XIAP protein levels between freshly purified CD5+CD19+ primary CLL cells (n=28) and CD19+ B cells from healthy donors (n=16) by western blotting. In a knockdown approach, specific siRNAs against XIAP were nucleofected to check whether XIAP expression prevents TRAIL-mediated apoptosis in CLL. After proof of concept, we applied the novel small molecule IAP antagonizing compound (IAC), an inhibitor of XIAP, in combination with TRAIL to induce apoptosis in primary CLL cells (n=48). Compound A (CA) was developed based on the crystal structure of four amino acids of SMAC, which enabling SMAC to efficiently bind the BIR3 domain of XIAP. In contrast to the active compound CA, which consists of an amino terminal methyl alanine, the inactive compound CB used in our studies as a negative control has an amino terminal methyl glycine. This specific substitution results in a significant reduction of IAP binding capability of CB as CA has binding affinity to XIAP in the picomolar range and CB is a weak binder with micromolar binding affinity to XIAP. Results:XIAP is significantly higher expressed in primary CLL cells (n=28) compared to healthy B cells (n=16) (P=0.02). Our data obtained by specific knockdown of XIAP via siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Based on these results we used IAC in combination with TRAIL. Combined treatment with both drugs significantly increased apoptosis compared to untreated (P=8.5×10-10), solely IAC (P=4.1×10-12) or TRAIL treated (P=4.8×10-10) CLL cells. As a potent cellular caspase inhibitor, we also examined the involvement of caspases in CA/TRAIL-mediated apoptosis. Not surprisingly, co-application of pan-caspase inhibitor zVAD.fmk inhibited cell death induced by CA/TRAIL underscoring the apoptotic caspase-dependent cytotoxicity of CA/TRAIL treatment in CLL cells. IAC rendered 40 of 48 (83.3%) primary CLL samples susceptible towards TRAIL-mediated apoptosis. Especially cells derived from patients with poor prognosis (ZAP-70+, IGHV unmutated, 17p-) were highly responsive to this drug combination. Furthermore, this study reveals that TRAIL application alone induces apoptosis in poor-prognosis CLL samples (13,8% in ZAP-70+ (n=10) vs. 2,3 in ZAP-70- (n=9); P=0.0008), which correlates with the elevated expression levels of TRAIL-R1 and –R2 on ZAP-70+ CLL cells. To assess whether TRAIL treatment is CLL cell specific, healthy B cells (n=4) were exposed to TRAIL alone or CA(CB)/TRAIL and showed significantly lower susceptibility towards CA/TRAIL administration than CLL cells. Conclusion:XIAP is over-expressed in CLL and displays a suitable target to induce TRAIL-mediated apoptosis. The novel XIAP inhibitor used in our study was able to inhibit XIAP function at a concentration of 0,1μM. CA/TRAIL administration was also shown not to induce apoptosis in healthy donor B cells and might therefore also display an attractive option for “non-fit” CLL patients. Our highly effective XIAP inhibitor CA, in concert with TRAIL, shows potential for treatment of CLL of those cases with poor prognosis and therefore warrants further clinical investigation. Disclosures:No relevant conflicts of interest to declare.