ALBERT

Description: BACKGROUND: In paroxysmal nocturnal hemoglobinuria (PNH), the lack of the GPI-anchored terminal complement inhibitor CD59 on erythrocytes renders these cells susceptible to continuous complement mediated hemolysis. Hemolysis leads to anemia, fatigue, red blood cell transfusions, renal impairment and increases the risk of thromboembolic events. Urinary iron loss is also common in patients with PNH due to intravascular hemolysis. Eculizumab is a terminal complement inhibitor that has been evaluated in two phase III studies. In previous studies eculizumab significantly reduced intravascular hemolysis (characterized by significant reduction of LDH levels) and significantly reduced transfusion requirements as well as thromboembolic events. In this study we evaluated the change in hemolytic parameters and levels of ferritin parameters in PNH patients treated with eculizumab in two centers (Essen and Homburg/Saar). METHODS: Nineteen PNH patients were treated with eculizumab as follows: 4 x 600mg IV every 7±2 days; 900 mg 7±2 days later; and then 900 mg every 14±2 days. The median therapy duration was 31 months (range 1–40 months). Hemolysis, transfusion requirements and serum iron parameters were analyzed over time of treatment. RESULTS: Eculizumab effectively inhibited intravascular hemolysis in all PNH patients as evidenced by an 79% decrease in LDH levels (mean±SD: 2,110±802 to 349±182 U/l (normal range: 100–247); p=0.0001) and reduced transfusion requirements. Persistent elevation of reticulocytes as well as the reduction of haptoglobin and hemopexin were observed in most patients, which suggest continuing extravascular hemolysis. Interestingly, we observed an 709% increase in ferritin levels in patients treated with eculizumab (median from 73 to 965 μg/l (normal range: 20–290 μg/L); p=0.001). This was more pronounced in patients still requiring some transfusions. Three patients were started on oral iron depletion therapy. No thromboembolic or serious adverse events were observed in eculizumab-treated patients. Two PNH patients were diagnosed with PNH-associated hematological disease (MDS, myelofibrosis). SUMMARY/CONCLUSIONS: Eculizumab is safe and well tolerated in the analyzed cohort of PNH patients. Iron parameters in PNH patients treated with eculizumab should be monitored to determine if iron supplementation should be altered or iron depletion therapy should be considered. While some extravascular hemolysis may persist, intravascular hemolysis is effectively controlled with eculizumab and is associated with a concomitant improvement in anemia and quality of life.

Description: Abstract 2607 Introduction: Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. Current chemotherapeutic regimens are associated with short- and long-term toxicities. Therefore, novel, less toxic therapies are needed. Mer receptor tyrosine kinase (RTK) is ectopically expressed in ALL cell lines and patient samples. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity, and delays development of leukemia in vivo. Mer tyrosine kinase inhibitors (TKIs) are excellent candidates for targeted therapies. We report here the first small molecule inhibitor for Mer RTK (UNC569) and demonstrate efficacy as a novel strategy in the treatment of ALL. Methods: UNC569 is a substituted pyrazolopyrimidine. Inhibition of Mer kinase activity by UNC569 was determined by a microfluidic capillary electrophoresis assay. Western blot analysis was used to determine inhibition of phospho-Mer and downstream signaling by UNC569 in 697 (B-ALL) and Jurkat (T-ALL) cells. UNC569-mediated anti-leukemia activity was determined in short- (MTT) and long-term (colony-formation) assays. Diagnostic bone marrow or peripheral blood samples were obtained at the Children's Hospital Colorado. We established a luciferase expressing ALL xenograft model in NOD scid gamma (NSG) mice to evaluate the effects of UNC569 on leukemia progression. NSG mice were transplanted with luciferase-tagged 697 cells and treated for three weeks with an orally bioavailable UNC569 formulation (15 mg/kg body weight, qd). Response was monitored twice weekly using the IVIS200 Imaging System. To examine effects of UNC569 on leukemia progression in a second model, we used transgenic zebrafish ectopically expressing human MYC. These fish develop T-cell malignancy at high penetrance. Cancers in these fish are labeled by T-lymphocyte-specific enhanced green fluorescent protein (EGFP), allowing measurement of treatment responses. Leukemic adult zebrafish were treated over three weeks with 3 μM or over 2 weeks with 4 μM UNC569. Tumor responses were monitored weekly using an Olympus MVX10 Imaging System. Results: UNC569 is a novel Mer TKI with potent activity against Mer RTK (IC50 = 2.9 nM). In cell-based assays, UNC569 inhibited accumulation of phospho-Mer in ALL cell lines (697 IC50 = 141±15 nM, Jurkat IC50 = 193±56 nM). Treatment with UNC569 resulted in inhibition of phosphorylation of Erk1/2 and Akt. Reduced proliferation/survival was observed in ALL cells treated with UNC569 (697 IC50 = 0.5±0.1 μM, Jurkat IC50 = 1.2±0.2 μM). Treatment with UNC569 also resulted in a statistically significant, dose-dependent decrease in colony-formation compared to control cultures in Jurkat (100.1±23.4 vs 25.6±6.4 colonies, p=0.04, n=3) and 697 cells (95.9±16.8 vs 14.8±12.8 colonies, p=0.02, n=3). Similarly, treatment with UNC569 reduced colony formation in methylcellulose compared to control cultures in one of three Mer RTK positive primary ALL patient samples (270.1±18.9 vs 134.0±6.4 colonies). No significant reduction of colony formation was observed in five Mer negative primary ALL patient samples. NSG mice transplanted with luciferase-expressing 697 B-ALL cells and treated with UNC569 (15 mg/kg/d) had significantly decreased leukemia burden compared to vehicle-treated control mice as measured by bioluminescence imaging (86.1×106±19.2×106 photons/second (n=15) vs 29.6×106±9.0×106 photons/second (n=10), p=0.04). Similarly, in the zebrafish T-ALL model, responses to UNC569 treatment (defined as 〉25% decrease in disease burden) were observed in 84% of animals treated with 4 μM UNC569 for 14 days (n = 16/19) and 77% of animals treated with 3 μM UNC569 for 21 days (n = 7/9). Conclusion: UNC569 is an effective Mer TKI that inhibits activation of Mer in ALL cells, mediates anti-leukemia activity against ALL cells in culture, and decreases colony-formation in methylcellulose. In addition, UNC569 has an anti-leukemia effect in primary ALL patient samples that express Mer protein, in a murine xenograft B-ALL model, and in a transgenic zebrafish model. Taken together, these data support further development of Mer TKI as a novel and effective ALL therapy. Disclosures: Wang: WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Kireev:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Liu:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Yang:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Frye:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties.

Description: Abstract 4912 Introduction: Cytomegalovirus reactivation (HCMV) occurs frequently after hematopoetic stem cell transplantation and is often associated with an increased treatment-related mortality. Recently we have demonstrated that a HCMV-reactivation is associated with a reduced relapse risk and improved overall survival rate for patients with acute myeloid leukemia, but not for patients with chronic myeloid leukemia after allogeneic transplant. Now, we aimed to evaluate the effects of a HCMV-reactivation in patients transplanted for lymphoma. Methods: We performed a retrospective study in a cohort of 67 patients (44 male/23 female) with lymphoma, who were transplanted with an unmanipulated graft from an unrelated donor (URD) after conditioning with a reduced-intensity regimen (n=9) or myeloablative regimen (n=58) in our centre. 48 patients were transplanted from HLA-matched donors, whereas 19 patients received a transplant from an URD with a HLA-mismatch. ATG was given to 19 of 67 patients. The median age of patients was 42 years (range 18–67). 25 patients were diagnosed with diffuse large B-cell lymphoma, 14 pts with follicular lymphoma, 13 pts with T-cell lymphoma, 10 pts with mantle cell lymphoma, 2 ptswith transformed B-cell lymphoma, 3 pts with Hodgkin's lymphoma. The median IPI score was 2 (n=50) prior to transplant, the median score for FLIPI was 2 (n= 14). A HCMV-reactivation occurred in 27 of 67 transplanted patients (40%) and was documented by CMV-related matrix protein pp65 antigenemia test and routinely accompanied by a CMV preemptive therapy with valganciclovir or ganciclovir. In in-vitro experiments we exposed the human T cell lymphoma cell line Karpas 299 to HCMV and measured the apoptosis rate by FACS and HCMV copy numbers by real-time RT-PCR 14 days after exposure. Results: We found an improved 4-year progression-free-survival rate (PFS) in patients with a HMC reactivation. The cumulative incidence of 4-year PFS was 55.9% in patients with HCMV-reactivation versus 38.7% in patients without a HCMV reactivation after transplant (p=0.049). The 4-year overall survival was 64.9% versus 46% without a HCMV reactivation (p=0.16). The incidence of acute GVHD grade 2–4 and chronic GVHD was not different in both groups (42 and 43% for acute GVHD and 46% and 45% for chronic GVHD). In in-vitro experiments we could not detect the induction of apoptosis by HCMV in the T cell lymphoma cell line Karpas 299 nor an increase of HCMV DNA after HCMV exposure demonstrating that the lymphoma cellline was not infected by HCMV. Conclusion: The pathway how HCMV induces an anti-lymphoma effect is not clear yet and seems to be different from that in AML, in which HCMV infects AML cells directly as shown earlier. Nevertheless, a HCMV reactivation seems to improve the outcome for patients with lymphoma after transplant, which is of clinical interest. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1635 Background: We have previously showed that a CMV- reactivation after allogeneic HSCT is associated with a reduced risk for leukemic relapse and improved overall survival in patients with AML (Elmaagacli et al Blood 2011). Further, experimental data in a lymphoma mice model reported from Erlach et al. showed that coinfection with murine CMV revealed a strong anti-lymphoma effect by induction of apoptosis in lymphoma cells and improving rate of overall survival in mice after transplant. Methods: This prompted us to investigate the influence of early replicative CMV infection in 94 (median age [years]: 45, 18 – 70) patients with lymphoma, who received transplants from unrelated (n=67) or related (n=27) donors. Patients were transplanted from HLA-identical (n=74), HLA-mismatched (n=20). 13 patients were transplanted for indolent lymphoma (FL n=11, CLL n=2), 67 patients for aggressive lymphoma (B-lineage n=35, T-lineage n=27, transformed n=5), 11 patients for MCL and 3 patients for HD. The disease stages of patients at HSCT were CR in 20 patients, PR in 40 patients, refractory in 30 patients and untested in 2 patients. 55 patients (59%) received previous autograft and 82 patients (87%) were treated prior to transplant with at least 3 chemotherapy lines. The hematopoietic cell transplantation specific comorbidity index (HCT-CI) were 0–2 in 76 patients (81%) and 3+ in 18 patients (19%). Myeloablative preparative regimen was applied in 60 patients (64%) while 34 patients (36%) received a RIC. Sixty-eight % of patients (n=48) were at risk for CMV reactivation based on either patient or donor pretransplant CMV serostatus. CMV replication as detected by pp65 antigenemia assay occurred in 34 patients (36%). Results: Taking all competing risks into account, the cumulative incidence of progressive free survival (PFS) at 5 years after alloSCT was 38 % (95 % confidence limit [95 % CL]: 31 – 45) in patients without as compared to 20 % (95 % CL: 9 – 31) in patients with early pp65 antigenemia (p

Description: Abstract 5503 Introduction Early detection of inapparent replicative human cytomegalovirus (HCMV) infection together with its preemptive antiviral treatment has led to a marked reduction of life-threatening HCMV disease after allogeneic hematopoietic stem cell transplants (alloSCT). A new aspect of HCMV reactivation and pretransplant HCMV serostatus has recently emerged by an earlier retrospectively performed report from us showing that the occurrence of a HCMV-reactivation after transplant reduces the risk for relapse in patients with AML and NHL. This idea was supported by a study by Scheper and co-worker (Leukemia, 2013) reporting recently, that gamma-delta T cells elicited by HCMV reactivation after alloSCT cross-recognize HCMV and leukemia. Here we evaluate the potential impact of early HCMV replication in a prospectively performed observational study about the occurrence of a HCMV- reactivation after T cell repleted alloSCT on the risk for leukemic relapse in patients with AML (Registration Trial DRKS00004300). Patients and Method Between January 2012 and March 2013 we enrolled in this trial 83 patients with AML who were consecutively transplanted at the University Hospital of Essen. 48 of 83 patients received a myeloablative (TBI based conditioning n=27, chemotherapy based conditioning n=23) and 35 patients a RIC regimen. Patients were transplanted in 1.CR (n=40), 2.CR (n=23) or more progressive disease stages (n=20) from HLA-identical sibling donor (n=17) or HLA-identical unrelated donor (URD) (n=42) or mismatched unrelated donor (n=24). The median age of patients was 53 years (range 18-72) and that of the donors 38 years (range 12-61). GVHD prophylaxis was performed with MTX and CSA, or CSA and MMF with or without ATG (n=64) (30-60mg total dose). The incidence of acute GVHD grade 2-4 was statistically not different in both groups (78% versus 85%). Results HCMV status of recipient (R) or donors (D) were in 29% R-/D-, 8% R-/ D+; 34% D+/R- and 29% R+/D+. Patients with a documented HCMV-reactivation (HCMV-R) had an estimated relapse incidence (CIR) at 1-year after transplant of only 8% compared to 43% in patients without a HCMV-R (p=0.03). Patients in more progressive disease phase of AML (N=43) benefit more from a HCMV-R in regard of CIR than patients in 1.CR of AML (0% versus 55% estimate for relapse at 1-year after transplant for patients with HCMV-R compared to patients without HCMV –R, p=0.028). One-year overall survival was statistically not different in both groups. Non relapse mortality was greater in patients with HCMV reactivation 37.8% versus 12.5%, p=0.1) Conclusion The first result of this prospective study confirms an independent advantageous effect of early HCMV replication on the leukemic relapse risk in patients with AML after transplant, which was more pronounced in patients in  progressive disease phase of AML than patients in 1.CR of AML. Disclosures: Off Label Use: The off-label use of HCG will be presented here for the first tiem for treatment of chronic GVHD and will clearly marked as off-label use.

Description: Abstract 1049 Elevated pretransplant serum ferritin levels have been associated with an increased susceptibility for opportunistic infections and increased incidence of morbidity and mortality after allogeneic haematopoietic stem cell transplantation (HCT). We studied in 81 patients who underwent myeloablative allogeneic HCT for acute myeloid leukemia pre- and posttransplant serum ferritin levels and correlated the serum ferritin levels with the TLR9 expression and the cellular immune reconstitution 3 and 12 months post transplant. Further, we studied in vitro-experiments in Kasumi-1 cells the TLR1, TLR2, TLR3, TLR5, TLR7, TLR9 and TLR10 expression after overwhelming iron and ferritin exposure. The average pretransplant serum ferritin level was 1245 μg/ml (mean) in all AML-patients (mean 1100μg/l for patients with AML in 1.CR and mean 1820μg/l for patients with AML 〉 1.CR). Post transplant serum ferritin level increased up to 2080 μg/ml (mean) for all AML patients (mean 1290mg/l for AML in 1.CR and mean 2350 μg/l for patients with AML 〉 1.CR). The application of 300ng iron to acute leukaemia cell lines SD1, and Kasumi-1 cells increased significantly TLR1, TLR2, TLR3, TLR5, TLR7 and TLR9 expression in relation to the housekeeping gene abl measured by real-time RT-PCR. In Kasumi-1 cells TLR1 increased up to 50,6% (p=0.014) TLR2 up to 35.5% (p=0.046), TLR3 up to 57,8% (p=0.006), TLR5 up to 62.9% (p=0.005), TLR7 up to 46.2% (p=0.02), TLR9 up to 44.2%(p=0.026) and TLR10 up to 54,7% (p=0.07) compared to untreated Kasumi-1 cells. The application of 2000μg/L ferritin increased TLR9 expression even more extensively in Kasumi-1 cells: TLR1 increased up to 332% (p

Description: Abstract 2589 Introduction: Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. Pediatric cure rates for ALL have dramatically increased in recent years as intensified therapeutic regimens have been developed. However, intensified therapy is associated with a significant and increased risk of short- and long-term toxicities. In addition, patients who relapse, do not reach remission, or have certain cytogenetic abnormalities have a poor prognosis and limited treatment options. Therefore, novel and less toxic therapies are needed. Tyrosine kinases are frequently abnormally regulated in cancer cells. Mer receptor tyrosine kinase is ectopically expressed in ALL cell lines and patient samples. Inhibition of Mer expression reduces pro-survival signaling, dramatically increases the sensitivity of leukemia cells to cytotoxic agents, and significantly delays development of leukemia in a mouse model. Thus, Mer tyrosine kinase inhibitors (TKIs) are excellent candidates for targeted therapies. We report here the first small molecule selective for Mer TK (UNC569) and characterization of its biochemical and anti-tumor activities in cell culture models of ALL. Methods: UNC569 is a substituted pyrazolopyrimidine that has been developed by a structure-based design and iterative medicinal chemistry from the known Mer/C52 cocrystal structure. Inhibition of Mer kinase activity by UNC569 was determined by a microfluidic capillary electrophoresis (MCE) assay in which phosphorylated and unphosphorylated substrate peptides were separated and analyzed through a LabChip EZ Reader. Western blot analysis of phosphorylated and total Mer protein was used to determine Mer inhibition by UNC569 in 697 (B-ALL) and Jurkat (T-ALL) cell lines. UNC569-mediated anti-leukemia activity was determined by detection of metabolically active cells using MTT reagent after 48 hours of exposure and by determining colony-formation in methylcellulose medium in the presence of UNC569. To investigate interactions with standard ALL therapies, apoptotic and dead cells were identified by flow cytometric analysis of cells stained with YO-PRO-1 and propidium iodide dyes after treatment with a chemotherapeutic agent alone or in combination with UNC569. Results: UNC569 is a novel small molecule Mer TKI with potent activity against Mer kinase (IC50 = 2.9 nM). In cell-based assays, UNC569 inhibited accumulation of phospho-Mer in both 697 and Jurkat ALL cells (IC50

Description: Introduction Although many novel therapeutic agents have been explored, multiple myeloma (MM) remains an incurable hematopoietic malignancy. Mer receptor tyrosine kinase (Mer TK) is a member of the TAM (Tyro3, Axl and Mer) family and is involved in the progression of several human malignancies. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity in vitro, and delays tumor progression in vivo. These observations make Mer TK inhibitors to excellent candidates for targeted therapies. We show here for the first time that Mer TK is ectopically expressed in MM cells and test the response of MM cell lines to our previously described small molecule Mer inhibitor, UNC1062, a substituted pyrazolopyrimidine. Here we demonstrate the biochemical and biologic effects of treatment with UNC1062, which suggests efficacy of Mer inhibitors as a novel strategy for the treatment of MM. Methods: Western blot analysis was used to determine expression of the TAM receptors and to evaluate inhibition of Mer TK activation and downstream signaling by UNC1062 in MM cell lines. UNC1062-mediated anti-myeloma activity with or without bortezomib was determined using short-term (MTT) and long-term (colony-formation) assays. Cleaved PARP and caspase 3 were detected by western blot analysis as indicators of apoptosis. Results: Western blot analysis of protein extract from MM cell lines showed that four (RPMI8226, U226, AMO-1 and KMS-12BM) out of six lines (66.6%) express Mer TK protein at varying levels, with AMO-1 expressing the least and U226 expressing the most. The MM cell lines OPM-2 and NCI-H929 did not express Mer TK. UNC1062 potently inhibits Mer TK activity in vitro (Mer IC50= 1.1 nM, Morrison Ki = 0.33 nM). In cell-based assays, treatment with UNC1062 inhibited accumulation of phospho-Mer and downstream signaling through ERK and Stat5 in U226 cells. UNC1062 also reduced proliferation and/or survival in Mer TK-expressing RPMI8226 cells (IC50 = 0.98 ± 0.14 μM, n=7) and U226 (IC50 = 0.28 ± 0.09 μM, n=3) cells. Treatment with UNC1062 did not lead to a meaningful reduction of metabolically active cells in Mer-negative NCI-H929 and OPM-2 cells. In contrast, UNC1062-treated Mer-positive MM cells exhibited increased levels of cleaved caspase 3 and cleaved PARP confirming apoptosis induction. Treatment with UNC1062 (100 nM) resulted in a statistically significant, dose-dependent decrease in colony-formation in methylcellulose compared to control cultures in Mer-positive RPMI8226 cells (418 ± 23 vs. 291 ± 37 colonies, p = 0.04, n = 3). No significant reduction of colony formation was observed in Mer-negative OPM-2 cells. Treatment of U226 cells with UNC1062 (800 nM) in combination with bortezomib resulted in a statistically significant reduction in relative cell number compared with bortezomib alone (0.61 ± 0.06 vs 0.41 ± 0.06, p = 0.04, n =3). Conclusion: Mer TK is ectopically expressed in the majority of investigated MM cell lines. UNC1062 treatment of Mer TK positive MM cells inhibits Mer TK activity and downstream signaling, mediates anti-neoplastic activity in liquid culture, and decreases colony-formation in methylcellulose. In addition, UNC1062 treatment sensitizes MM cells to bortezomib. Taken together, these data support further development of Mer TK inhibitors as novel MM therapy alone and in combination with bortezomib. Disclosures: No relevant conflicts of interest to declare.

Description: The impact of early human cytomegalovirus (HCMV) replication on leukemic recurrence was evaluated in 266 consecutive adult (median age, 47 years; range, 18-73 years) acute myeloid leukemia patients, who underwent allogeneic stem cell transplantation (alloSCT) from 10 of 10 high-resolution human leukocyte Ag-identical unrelated (n = 148) or sibling (n = 118) donors. A total of 63% of patients (n = 167) were at risk for HCMV reactivation by patient and donor pretransplantation HCMV serostatus. In 77 patients, first HCMV replication as detected by pp65-antigenemia assay developed at a median of 46 days (range, 25-108 days) after alloSCT. Taking all relevant competing risk factors into account, the cumulative incidence of hematologic relapse at 10 years after alloSCT was 42% (95% confidence interval [CI], 35%-51%) in patients without opposed to 9% (95% CI, 4%-19%) in patients with early pp65-antigenemia (P 〈 .0001). A substantial and independent reduction of the relapse risk associated with early HCMV replication was confirmed by multivariate analysis using time-dependent covariate functions for grades II to IV acute and chronic graft-versus-host disease, and pp65-antigenemia (hazard ratio = 0.2; 95% CI, 0.1-0.4, P 〈 .0001). This is the first report that demonstrates an independent and substantial reduction of the leukemic relapse risk after early replicative HCMV infection in a homogeneous population of adult acute myeloid leukemia patients.