ALBERT

Description: Abstract 659 Background: NPM1 mutations and WT1 expression are frequently identified in acute myeloid leukemia (AML). Both markers have been reported as suitable molecular target for minimal residual disease (MRD) monitoring. Recently, Gemtuzumab Ozogamicin (GO) has been shown to improve event free survival and overall survival in adult AML. Aims: To follow MRD in AML patients with NPM1 mutations (NPM1mut) and/or WT1 expression (WT1+) and treated in the randomized ALFA 0701 GO trial conducted by the Acute Leukemia French Association (Castaigne et al, Lancet 2012), in order to validate their prognostic significance and to evaluate the effect of GO on MRD response. Methods: The ALFA 0701 trial included 278 patients aged 50–70 years with newly-diagnosed de novo AML, randomized to receive or not five doses of 3 mg/m2 GO, on day 1, 4 and 7 of standard induction chemotherapy and on day 1 of two consolidation chemotherapy courses, respectively. A total of 77 NPM1mut (35 in control arm, 42 in GO arm) and 178 WT1+ (87 in control arm, 91 in GO arm) AML patients were studied. WT1 + at diagnosis was defined as a ratio WT1/ABL transcript above 25% in bone marrow (BM) or 5% in peripheral blood (PB) samples. PB and BM samples were collected at diagnosis, after induction and after each consolidation course. MRD levels were assessed using cDNA-based real-time quantitative PCR (RQ-PCR) and reported as the ratio NPM1mut or WT1 transcript/100 ABL transcript. Occurrence of a MRD level below the threshold (NPM1mut

Description: Key Points In ET, a CALR mutation correlates with a monoclonal X chromosome inactivation pattern, which differs from JAK2V617F mutant disease. The presence of a CALR mutant is associated with suppression of wild-type myelopoiesis.

Description: Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders characterized by the abnormal production of various myeloid cells. Aberrant JAK2 signaling (e.g. induced by JAK2-V617F) plays an etiological role in MPN formation. While JAK2 inhibitors improve patient symptoms, they do not induce cell death as neoplastic cells appear to be rather insensitive to JAK2 inhibition and effectively rapidly become resistant to treatment. Therefore, the development of additional therapeutic approaches for MPNs is needed. Pim kinases are serine/threonine kinases that protect hematopoietic cells from apoptosis and also play a role in regulating hematopoietic stem cell growth. In mouse models, elevated Pim expression contributes to the development of lymphoma. Pims are constitutively active and thus regulated by protein expression, which is controlled by Pim gene expression and Pim protein stability. Pim1 gene expression is normally induced by JAK2/STAT5 signaling in response to extracellular growth factor stimulation, as Pim1 is a direct transcriptional target of STAT5. The deregulated JAK2 signaling in MPNs also induces Pim expression. STAT5 is required for MPN disease in mouse models, suggesting genes transcriptionally regulated by STAT5 are required for MPN disease formation. Together with the anti-apoptotic signaling and transforming properties of Pims, this suggests Pims may play a role in MPNs. We hypothesized that Pim kinases may offer a therapeutic target for MPNs and that Pim kinase inhibitors in combination with JAK inhibitors may cause neoplastic cytotoxicity, improving on current JAK2-inhibitor mono-therapy for MPNs. JAK2-V617F-dependentMPN model cells (HEL, SET2, Uke1, and BaF3/JAK2-V617F, including cells that are resistant to the JAK2 inhibitor ruxolitinib) as well as MPN patient cells, were treated with Pim kinase inhibitors, SGI-1776 and AZD1208, and the JAK2 inhibitor, ruxolitinib. The effects on cell growth, cell cycle, viability, and cell signaling were studied. High concentration SGI-1776 (10 μM) inhibited cell growth and viability of MPN model cells while lower doses (1 and 3 μM) had little effect on the growth and viability of these cells. Combination of 3 μM SGI-1776 with low dose ruxolitinib significantly enhanced growth inhibition and cell death of HEL and SET2 cells. Similar results were obtained with the much more effective and selective Pim inhibitor, AZD1208. We show that ruxolitinib inhibits Pim expression in MPN cells, and Pim expression is restored in ruxolitinib-resistant cells. Importantly, low dose SGI-1776 or AZD1208 (100 nM) re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib treatment. Significantly, as single agents, both SGI-1776 and AZD1208 inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but not erythroid colonies of normal controls. The combination of AZD1208 and ruxolitinib exhibited enhanced inhibition of colony formation of primary cells from MPN patients compared to treatment with either drug alone. These data indicate that Pim kinase inhibitors in combination with a JAK2 inhibitor may offer a more efficacious therapeutic approach over JAK2 inhibitor mono-therapy for MPNs. Disclosures No relevant conflicts of interest to declare.

Description: Deep venous thrombosis (DVT) is a common postoperative complication in patients undergoing major orthopaedic surgery of the lower limbs, such as total hip replacement (THR), total knee replacement (TKR), or hip fracture surgery (HFS). In the absence of thromboprophylaxis, subclinical venous thrombosis rates as high as 60% have been reported when using systematic bilateral phlebography after orthopaedic surgery. As a result, routine pharmacological thromboprophylaxis with low-molecular-weight heparin (LMWH) or a new oral anticoagulant agent is strongly recommended in patients undergoing these procedures. With the availability of efficient and safe clotting factor concentrates, THR, TKR, as well as ankle arthrodesis are frequently performed in subjects with haemophilia suffering from chronic haemophilic arthropathy. Yet, pharmacological prophylaxis of venous thromboembolism (VTE) in this patient group remains controversial. With the exception of retrospective case reports and small series, the incidence of VTE disease in haemophilic patients after major orthopaedic surgery is still unclear. Surveys suggest that more than half of hemophilia treatment centers in the United States and Europe use thromboprophylaxis in patients with haemophilia (PWH) who require major orthopedic surgery. However, this is not supported by evidence or surgical practice guidelines, and simply increases the risk of bleeding in such patients. Given the aging nature of the haemophilia population and the incidence of joint disease, the question of the thrombotic risk associated with major orthopaedic surgery and joint replacement surgery in particular is highly relevant. Few studies have addressed this issue and more information is needed on which to base optimal preventive strategies for venous thrombosis in patients with haemophilia undergoing elective major orthopedic surgery. We here report the results of 2 parallel and independent prospective studies from Belgium and Norway currently evaluating by systematic US-Doppler imaging the incidence of subclinical deep venous thrombosis in consecutive haemophilic patients referred for major orthopaedic surgery. In Belgium, the study has so far included 36 different patients (32 HA, 4HB) undergoing 50 major orthopaedic procedures of the lower limbs. In Norway, the study has involved 29 patients (26 HA, 3 HB, all with severe haemophilia) undergoing 29 orthopaedic procedures, most of the lower limbs. In both countries, most patients were treated with continuous infusion of clotting factor concentrates and none of them did receive antithrombotic pharmacological prophylaxis. In the 2 studies that included in total 65 patients undergoing 79 major orthopaedic procedures, no case of clinically patent DVT or PE was detected (Table 1). In total there were 5 cases of DVT evidenced by US imaging which were all distal, not complicated by PE and treated with a short course of low-molecular weight heparin in most cases. The overall incidence of subclinical DVT calculated on the whole population was 6%. In conclusion, these data provide for the first time multicentric and imaging-based evidence that the risk of DVT following major orthopaedic surgery among patients with haemophilia undergoing major orthopaedic surgery is very low and that systematic pharmacological thromboprophylaxis in this specific population is for most patients not required. The two studies are still ongoing and should include a larger number of participants in the future. Table 1Characteristics of patients with haemophilia undergoing major orthopaedic surgeryHTC BrusselsHTCOsloTotalPatients362965Haemophilia A322658Haemophilia B437Severe252954Moderate909Mild202Procedures502979Knee301141Ankle81220Hip729Femur404Lumbar101Elbow044Venous thrombosis415HTC: Haemophilia Treatment Center Disclosures No relevant conflicts of interest to declare.

Description: Introduction Non-Hodgkin’s lymphomas with secondary involvement of the central nervous system (CNS) typically carry a poor prognosis. There is limited data in the literature regarding the optimal therapy for these patients. IDARAM is a cytotoxic regimen which penetrates the CNS. It has demonstrated efficacy in early studies in secondary CNS lymphoma [Moreton et al, Cancer Chemother Pharmacol (2004) 53: 324–8]. We present here the largest series of patients with secondary CNS involvement treated with IDARAM+/- rituximab at University College Hospital London between June 2005 and January 2013. Methods Thirty-eight patients were treated with 1-4 cycles of IDARAM, consisting of methotrexate 12.5mg by intrathecal injection day 1; idarubicin daily 10mg/m2 IV days 1 and 2; dexamethasone 100mg daily IV infusions of 12h duration days 1-3; cytosine arabinoside daily 1000mg/m2 IV over 1 hour days 1 and 2; and methotrexate 2000mg/m2 IV over 2 hours day 3. CSF was analysed for lymphomatous involvement by flow cytometry on D1. Patients with a clear CSF from the outset only received one intrathecal injection on day 1 of cycle 1. Most patients with B-cell lymphoma additionally received 1-2 doses of rituximab 375 mg/m2 IV on day 1 (+/- day 8) of each cycle. Results Baseline characteristics are presented in table 1. 16/38 (42%) patients had secondary CNS involvement at the time of initial diagnosis of NHL or within 2 months (‘New Diagnosis’). 22/38 (58%) had refractory (CNS involvement diagnosed in latter part of therapy or within 3 months of completion of primary therapy) or relapsed (CNS involvement diagnosed at relapse - either combined with a systemic relapse or isolated to CNS) disease. 84 cycles of IDARAM were delivered to 38 patients. Patients received a median of 2 cycles. 20/37 responded (overall response rate (ORR) = 54%) with 12/37 (32%) achieving a complete response and 8/37 (22%) a partial response (PR). Median progression-free survival (PFS) was 6.8 months and overall survival (OS) was 27.5 months. In patients with a ‘New’ diagnosis ORR was 10/15 (67%) CR 7/15 (47%), PR 3/15 (20%), median PFS was 48 months, median OS has not been reached (see figure 1). Neither age nor performance score predicted outcome in these patients. Grade 3 or 4 haematological toxicity was observed in 100% of cycles. Non-haematological grade 3 or 4 toxicities included infection 81%, stomatitis 11.3%, diarrhoea 4.3%, other 6.5%. 4/84 (5%) of cycles were complicated by ITU stay and treatment-related mortality was 2/38 (5%). 8 (21%) of patients received consolidation radiotherapy (4 patients in CR, 4 in PR), 4 patients (10%) received salvage radiotherapy for disease refractory to IDARAM, and 3 patients (8%) received palliative radiotherapy. 8 patients (20%) received an autologous stem cell transplant; 6 (75%) as consolidation and 2 (25%) for disease relapsing after IDARAM. Conditioning was with TBI in 6 (75%) and LEAM/ BEAM in 2 (25%). 2 patients received reduced-intensity conditioned allogeneic stem cell transplants from HLA-matched unrelated donors: one for primary refractory disease and one for relapsed disease after autograft in 1st CR. Of 10 newly diagnosed patients achieving CR/PR with IDARAM, 5/5 who received consolidation autograft, and 4/5 who did not, remain alive (median follow-up 30 months). 1 patient in each group has relapsed. Conclusions IDARAM +/- R is a well-tolerated regime with encouraging response rates in patients with poor prognosis lymphoma. The short duration of survival of patients with relapsed or refractory disease indicates the urgent need to identify efficacious new treatments for these patients. Patients in whom CNS involvement is detected early in the disease course fare reasonably well, including older patients and those with a poor performance status. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points An anti-CD37 antibody-drug conjugate can kill B-lymphoma cells via direct inhibition, effector function, and payload delivery. Targeting CD37 with an antibody-drug conjugate results in selective depletion of malignant human B cells.

Description: Virtually all recurrent molecular alterations in acute myeloid leukemia (AML) functionally converge to cause signal transduction pathway dysregulation that drives cellular proliferation and survival. The mammalian target of rapamycin complex 1 (mTORC1) is a rapamycin-sensitive signaling node defined by the interaction between mTOR and raptor. Constitutive mTORC1 activity is nearly universal in AML. However, pharmacologic inhibition with rapamycin or second-generation mTOR kinase inhibitors has shown limited anti-leukemic activity in both preclinical models as well as patients, suggesting that addiction to this oncogene is not a recurrent event in AML. Here we report that sustained mTORC1 activity is nonetheless essential for the cytotoxicity induced by pharmacologic activation of AMP-activated protein kinase (AMPK) in AML. Our studies employed a novel AMPK activator called GSK621. Using CRISPR and shRNA-mediated silencing of the AMPKa1 catalytic subunit, we showed that AMPK activity was necessary for the anti-leukemic response induced by this agent. GSK621-induced AMPK activation precipitated autophagy, as demonstrated by western blotting, immunofluorescence, flow cytometry and electron microscopy. Blocking autophagy via shRNA-mediated knockdown of ATG5 and ATG7 protected AML cells from cytotoxicity resulting from treatment with GSK621, suggesting that autophagy promotes cell death in the context of active AMPK. GSK621 cytotoxicity was consistently observed across twenty different AML cell lines, primary AML patient samples and AML xenografts in vivo. GSK621-induced AMPK activation also impaired the self-renewal capacity of MLL-ENL- and FLT3-ITD-induced murine leukemias as measured by serial methylcellulose replating assays. Strikingly, GSK621 did not induce cytotoxicity in normal CD34+ hematopoietic progenitor cells. We hypothesized that the differential sensitivity to GSK621 could be due to the difference in amplitude of mTORC1 activation in AML and normal CD34+ cells. In contrast to most reported cellular models in which AMPK inhibits mTORC1 both directly (through raptor phosphorylation) and indirectly (through TSC2 phosphorylation), sustained mTORC1 activity was seen following GSK621-induced AMPK activation in AML. Inhibition of mTORC1 either pharmacologically (using rapamycin) or genetically (using shRNAs targeting raptor and mTOR) abrogated AMPK-induced cytotoxicity in AML cells, including primary AML patient samples. This protective effect was mediated by mTORC1-dependent modulation of the ATF4/CHOP stress response pathway. The ultimate functional consequence was that, rather than diminishing GSK621-induced cytotoxicity, persistent mTORC1 activity was in fact synthetically lethal with AMPK activity in AML cells. This synthetic lethality could be recapitulated in normal CD34+ progenitors by constitutive activation of mTORC1 using a lentivirally-transduced myrAKT construct. It could also be enhanced in AML cells by mTORC1 overactivation induced by CRISPR-mediated deletion of TSC2. Taken together, these data show that the magnitude of mTORC1 activity determines the degree of cytotoxicity triggered by AMPK activation. This finding may have important implications for AMPK and mTORC1 signaling pathways in cancer biology more broadly. Context-dependent permissiveness towards mTORC1 activation may amplify the response to cytotoxic stress, such as that resulting from AMPK activation by GSK621. Our results therefore support AMPK activation as a promising therapeutic strategy in AML and other mTORC1-active malignancies which warrants further investigations in clinical trials. Disclosures Brusq: GSK: Employment. Nicodeme:GSK: Employment.