ALBERT

Description: Key Points TP53 3′UTR variations demonstrate prognostic value in DLBCL.

Description: Oncogenic c-Myc is known to balance excessive proliferation by apoptosis that can be triggered by p53-dependent and p53-independent signaling networks. Here, we provide evidence that the BH3-only proapoptotic Bcl-2 family members Bcl-2 modifying factor (Bmf) and Bcl-2 antagonist of cell death (Bad) are potent antagonists of c-Myc–driven B-cell lymphomagenesis. Tumor formation was preceded by the accumulation of preneoplastic pre-B and immature immunoglobulin M–positive (IgM+) B cells in hematopoietic organs of Eμ-myc/bmf−/− mice, whereas Eμ-myc/bad−/− mice showed an increase of pre-B cells limited to the spleen. Although the loss of Bad had no impact on the tumor immunophenotype, Bmf deficiency favored the development of IgM+ B cell over pre-B cell tumors. This phenomenon was caused by a strong protection of immature IgM+ B cells from oncogene-driven apoptosis caused by loss of bmf and c-Myc–induced repression of Bmf expression in premalignant pre-B cells. Steady-state levels of B-cell apoptosis also were reduced in the absence of Bad, in support of its role as a sentinel for trophic factor-deprivation. Loss of Bmf reduced the pressure to inactivate p53, whereas Bad deficiency did not, identifying Bmf as a novel component of the p53-independent tumor suppressor pathway triggered by c-Myc.

Description: Context and objective Approximately 5-10% of diffuse large B-cell lymphoma (DLBCL) patients carry MYC gene translocations (MYC-translocation+) with a poor prognosis after standard chemotherapy. MYC-translocation+ DLBCL patients carrying BCL2 translocations (MYC+/BCL2+ double-hit lymphoma) have a worse survival. The efficacy of adjuvant radiotherapy in this setting is unknown. The purpose of this study is to evaluate the efficacy of radiotherapy as a part of the therapeutic regimen for patients with MYC-translocation+ DLBCL. Patients and methods From the International DLBCL R-CHOP consortium program, we selected 581 patients with de novo DLBCL treated with standard R-CHOP immunochemotherapy (diagnosed and treated from 2000 to 2010). We excluded patients with transformed DLBCL, primary mediastinal, cutaneous, testicular or central nervous system large B-cell lymphomas, patients with HIV infection, and patients not treated with R-CHOP. The median follow-up was 54.9 months. Fluorescence in situ hybridization assessing MYC was performed for all the patients (n=581) and results were correlated with available clinical data to identify clinicopathologic features associated with MYC translocation, and to evaluate the prognostic significance of MYC translocations regarding overall survival (OS, from the time of diagnosis to death from any cause) and progression-free survival (PFS, from the time of diagnosis to relapse or death from any cause). In the MYC-translocation+ DLBCL group, 38 patients received chemotherapy alone and 21 patients received chemotherapy with adjuvant radiation therapy. The clinicopathologic features and survival of MYC-translocation+ DLBCL patients treated with (n=21) and without radiotherapy (n=38) after immunochemotherapy were compared to in order to evaluate the radiotherapy efficacy and other confounding factors. Results MYC translocations were detected in 59 DLBCL patients (10.2%). Patients with MYC-translocation+ DLBCL more often had bulky tumors, involvement of multiple extranodal sites, and poorer OS (hazard ratio [HR]: 2.0, 95% confidence interval [CI]: 1.20 - 3.35, P= .0083) and PFS (HR: 1.96, 95% CI: 1.22 - 3.13, P= .0005) independent of the International Prognostic Index score. Poor survival was primarily attributable to patients with MYC+/BCL2+ double-hit DLBCL who were predominantly of germinal center B-cell subtype. Among MYC-translocation+ DLBCL patients, a better survival was achieved in patients who received radiotherapy (for OS, HR: .32, 95% CI: .15 - .71, P= .0049; for PFS, HR: .35, 95% CI: .17 - .73, P= .0043). Conversely, radiotherapy abolished the adverse impact of MYC translocations. In addition, radiotherapy was associated with better survival in the subset of patients with MYC+/BCL2+ double-hit lymphoma (P = .017 for OS, and P = .05 for PFS). However, owing to the common use of radiotherapy as consolidation therapy, the favorable prognoses in the group of patients who received radiotherapy could also be attributed to limited-stage disease and more frequent complete remission (CR) after first-line treatment and therefore these factors confound interpretation of the data. To address these issues, we evaluated radiotherapy efficacy in separate patient groups: patients who achieved CR, non-CR (PR/SD/PD) patients, and patient with stage I/II, or stage III/IV disease. The efficacy of radiotherapy appeared more apparent in patients with advanced disease who did not achieve CR after first-line chemotherapy. Multivariate analysis after adjustment for stage and IPI score validated that radiotherapy significantly improved OS (HR: .28, 95% CI: .10 - .81, P= .018) and PFS (HR: .32, 95% CI: .13 - .80, P= .015) of MYC-translocation+ DLBCL patients. Conclusions The presence of MYC translocations in DLBCL is an important biomarker that facilitates prognostic prediction and treatment stratification independent of the IPI score. For chemoresistant MYC-translocation+ DLBCL patients, radiotherapy seems to be an effective adjuvant regimen likely due to the higher frequency of extranodal involvement and bulky disease in MYC-translocation+ DLBCL patients. Our results provide a rationale for larger scale studies to assess the potential role of radiotherapy in the management of MYC-translocation+ DLBCL patients, particularly patients with MYC+/BCL2+double-hit DLBCL. Disclosures: Winter: Millenium: Research Funding; Novartis : Research Funding; Pfizer (Wyeth): Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Janssen (Pharmacyclics): Research Funding; Spectrum (Allos): Consultancy; Sanofi Aventis: Consultancy; Tgen: Consultancy; AMBIT Biosciences (Spouse): Research Funding; Celgene (Spouse): DSMB, DSMB Other, Research Funding; Ariad Pharmaceuticals (Spouse): Research Funding; Novartis (Spouse): Consultancy, Research Funding; Amgen (Spouse): Consultancy, Research Funding; Astellas (Spouse): Research Funding; Caremark/CVS: Consultancy; Pfizer (Spouse): Consultancy; Sanofi Aventis (Spouse): DSMB, DSMB Other; Bristol Myers Squibb (Spouse): DSMB, DSMB Other; UptoDate, Inc.(Spouse): Patents & Royalties.

Description: Myeloproliferative neoplasms (MPNs) are originated by mutations in a hematopoietic stem cell (HSC), frequently in the Janus kinase 2 (JAK2) gene. Different outcomes of this common event and limited efficacy of JAK2 inhibitors suggest the contribution of other factors. Additional HSC mutations and HSC-niche interaction might influence MPN progression, characterized by sequential expansion of HSCs, blood cells and megakaryocytes. Ensuing bone marrow (BM) fibrosis and osteosclerosis, which are contributed by osteoblastic lineage cells in a BCR/ABL CML model (Schepers et al Cell Stem Cell 2013), impede normal hematopoiesis. We have previously shown that BM nestin+ mesenchymal stem cells (MSCs) innervated by sympathetic nerve fibers regulate HSCs (Méndez-Ferrer et al Nature 2008 & 2010). Here we demonstrate that damage to this regulatory network is required for MPN manifestation. Nestin+ MSCs and NESTIN mRNA expression were rapidly reduced in the BM of MPN patients and mice expressing the human JAK2-V617F mutation. This reduction was not due to nestin+ MSC differentiation into fibroblasts or osteoblasts, as shown by 25-week lineage-tracing studies using Nes-CreERT2;RCE-loxP mice, but instead caused by early MSC apoptosis. In turn, nestin+ cell reduction stimulated MPN progression; selective nestin+ cell depletion using Nes-CreERT2;iDTA mice increased peripheral white and red blood cells, megakaryocytic invasion of spleen germinal centers and BM osteosclerosis. Our recent results indicate that the neural crest contributes during development to BM MSCs with specialized HSC niche function and that postnatal murine BM Nestin-GFP+ cells do not only contain MSCs but also Schwann cell precursor-like cells (Isern et al, ISSCR Annual Meeting 2013). BM Nestin-GFP+ cells from MPN mice showed reduced expression of HSC maintenance and mesenchymal genes, and increased expression of genes related to axon guidance and Schwann cell differentiation. Principal component analyses of independent biological samples further showed that control BM nestin+ cells clustered together with MSCs, whereas MPN BM nestin+ cells resembled Schwann cell precursors. These data suggested alterations to the neural component of the BM HSC niche in MPN. Indeed, BM sympathetic nerve fibers and Schwann cells, closely associated but different from Nestin-GFP+ cells, were rapidly reduced in the BM of diseased animals. Symptomatic MPN mice were treated with selective β3-adrenergic agonists to compensate for the loss of sympathetic stimulation of nestin+ MSCs. Treatment with BRL37344 or the recently FDA approved drug Mirabegron prevented MPN-associated neutrophilia and thrombocytosis, while it did not affect peripheral blood counts of wild-type mice. While vehicle-injected animals showed severe BM fibrosis, long-term BRL37344 treatment led to virtual absence of focal reticulin deposits or excessive fibroblasts. To further confirm the contribution of BM neural damage to MPN pathogenesis, diseased mice were treated with a neuroprotective agent. Sympathetic nerve-ensheathing Schwann cells were strongly reduced in the BM of vehicle-injected animals but preserved in 4-methylcatechol-treated mice. Like in BRL37344-treated animals, this was associated with prevention of very early MPN events, including neutrophilia and BM overproduction of the pro-inflammatory cytokine interleukin-1β. Since MPN is originated by a mutant HSC, we reasoned that sympathetic neuropathy might contribute to MPN pathogenesis through early disruption of the HSC niche. The chemokine Cxcl12 regulates HSC migration and proliferation. At early MPN stage, HSC expansion and mobilization correlated with decreased BM Cxcl12 expression and protein levels. Concomitantly, BM nestin+ MSC number and their Cxcl12 expression were significantly reduced. BRL37344 treatment completely restored the number of BM nestin+ cells and improved Cxcl12 BM levels. Treatment with 4-methylcatechol or BRL37344 prevented the early expansion of mutant hematopoietic progenitors, whereas long-term BRL37344 treatment efficiently reduced mutant hematopoietic progenitor numbers in BM and peripheral blood. These results demonstrate that damage of the niche, induced by the mutated HSCs, critically contributes to JAK2-V617F+ MPN pathogenesis. They also unravel HSC niche-forming MSCs and their neural regulation as promising novel therapeutic targets in MPN. Disclosures: Arranz: Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties. Off Label Use: Beta-3-adrenergic agonists (e.g. FDA-approved Mirabegron) and neuroprotective drugs for the treatment of myeloprolifeative diseases. Isern:Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties. Méndez-Ferrer:Centro Nacional de Investigaciones Cardiovasculares (CNIC): National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013, National patent application number 201330677 entitled “Neuroprotective/neurocompensatory therapy for the treatment of myeloproliferative diseases”, with priority date May 10, 2013 Patents & Royalties.

Description: Key Points AP-1 complexes of the Jun/ATF type promote growth of ABC DLBCL cell lines. High expression of ATF3 is a hallmark of samples from patients with non-GC/ABC DLBCL.

Description: Key Points MALT1 protease activity stabilizes MYC. The MALT1-MYC network might represent a therapeutic target for MCL patients.

Description: Key Points Cases of cHL may express TCA on the neoplastic cells. TCA-cHL have nodular sclerosis histology and lack T-cell genotype, with worse outcome compared with TCA-negative cHLs.

Description: Abstract 949 Introduction: Diffuse large B cell lymphoma (DLBCL) has a highly variable outcome, and individual risk assessment is largely based on clinical features. Gene expression profiling (GEP) stratifies patients into those with germinal center B-cell (GCB) and activated B-cell subtype (ABC) subtype with different prognoses. These groups have been shown to predict prognosis in patients treated with CHOP or R-CHOP. Conversely, the role of other recognized prognostic markers, such as BCL2 gene abnormalities or Bcl2 expression has been questioned in the new therapeutic era. Materials and Methods: In 438 patients treated with R-CHOP for de novo DLBCL, we analyzed the tumors by immunohistochemistry for Bcl2 protein expression and by interphase fluorescence in situ hybridization (FISH) for BCL2 translocation and other abnormalities. All cases were successfully studied by GEP. The cutoff for Bcl2 protein expression, 60%, used as prognostic factor was determined using receiver operating characteristic curves. Progression-free survival (PFS) and overall survival (OS) were assessed. Results: The t(14;18)(q32;q21) was detected in 82 cases (18.7%) and BCL2 gains occurred in 63 cases (14.3%). Both t(14;18) and BCL2 gains strongly correlated with higher levels of Bcl2 protein expression (p

Description: Abstract 3461 Expansion of CD3+ large granular lymphocytes (T-LGL) can be observed in situations such as viral infection, autoimmune disease, malignant neoplasm, and following allogeneic hematopoietic stem cell transplantation (HSCT). We sought to evaluate in patients treated with allogeneic HSCT incidence, characteristics, and clinical significance of persistent post-transplant T-LGL expansion. In this single center retrospective cohort study, we included all patients seen between January/08 and December/09 in our out-patient clinic for their regular follow-up. In patients with persistent lymphocytosis (〉3 G/l for 〉3months) and an abnormal CD4/CD8 ratio, an extensive immunophenotyping was assessed; in case of an abnormal expansion of T-LGL cells a TCR gene rearrangement was performed. In 14 (7%) out of 215 evaluated patients a T-LGL expansion was diagnosed. Patients' characteristics with and without T-LGL expansion are summarized in Table 1. The median time between HSCT and diagnosis of lymphocytosis was 12 months (1-58). The median lymphocyte count was 4.24 G/l (3.0-26.5). The median duration of lymphocytosis was 29 (4-176) months. In 13/14 cases there was a CD3+/CD8+ immunophenotype, in 1 case was CD3+CD4+. In 5/14 patients a clonal TCR-gene rearrangement was observed. None of the patients presented neutropenia. Mild anemia was observed in 8/14 patients (57%), and thrombocytopenia in 2/14 patients; both changes were most probably not related with the T-LGL expansion. None of the patients had typical clinical signs of a T-LGL leukemia. In the univariate analysis acute GvHD and CMV reactivation were the only variables associated with T-LGL expansion, In the multivariate the relative risk (RR) of CMV reactivation was 5.063 (95%CI: 1.586–16.160; p=0.006) and the RR of acute GvHD grade 2–4 was 2.831 (95%CI: 0.831–9.648; p=0.096). Conclusion: we detected a T-LGL expansion in 7% of patients after HSCT. No symptoms or clinical signs related to T-LGL leukemia were observed. The T-LGL expansions, even when monoclonal, showed a chronic but indolent course. They have to be considered rather as an expression a chronic stimulation, triggered by causes such CMV reactivation or acute GvHD rather than as a malignant transformation. The question whether a T-LGL expansion plays a GvL role could not be answered in this study due to the small number of patients and the study design. Disclosures: No relevant conflicts of interest to declare.

Description: The t(11;19) translocation leading to the MLL-ENL fusion is recurrently found in pediatric and adult de novo and therapy related mixed-lineage acute leukemia and is often associated with a poor prognosis. Previous studies have shown that (retroviral) overexpression of MLL-ENL potently immortalizes bone marrow cells in vitro and induces a lethal acute myeloid leukemia (AML) in mice. To establish a mouse model that phenocopies more closely the human disease, we generated conditional transgenic mice in which the expression of MLL-ENL is controlled by doxycycline (DOX) through a stably integrated reverse tet-responsive transactivator (rtTA). Induction of MLL-ENL expression in newborn or adult mice resulted in a leukemic phenotype that phenocopied pre-B- and myeloid mixed lineage leukemia as observed in most patients with MLL-ENL. The diseased mice displayed excessive splenomegaly, massive lymph node as well as multiple organ infiltration by two co-existing types of blasts mostly expressing higher or lower levels of B220 and Gr1/Mac1 and similar levels of c-kit. Expression of the fusion gene and disease induction was DOX dosage dependent and reversible upon DOX removal. Despite significantly lower fusion gene expression levels as we observed in retroviral systems the median latency for the development of the disease in this model (104.3±16.9 days) was comparable to them (62±10.4 days) and much shorter than any of the previously reported MLL-ENL knock-in mouse models (〉 1 year). Continuous ex vivo expression of MLL-ENL provided bone marrow and fetal liver hematopoietic cells with a strong self-renewal capacity and caused the accumulation of immature blast-like cells upon serial replating in methylcellulose cultures. In the presence of factors favoring myelopoiesis, like IL-3, DOX removal resulted in a complete differentiation towards the granulocytic-monocytic, lineages expressing high levels of Mac1/Gr-1, whereas IL-7 favored differentiation towards the B-cell lineage characterized by the expression of high levels of B220. In addition, MLL-ENL induced a DOX dependent aberrant self renewal capacity and a differentiation block in methylcellulose cultures of hematopoietic stem cells (Lin- c-kit+ Sca-1+, LSK) and various progenitors including common lymphoid progenitor (CLP) and granulocyte-macrophage progenitor (GMP) cells. Interestingly, MLL-ENL expression preferentially expanded LSK- rather than GMP-derived cells as assayed by growth curves in long-term (〉 1 month) liquid cultures in the presence of cytokines in vitro. In line with this observation, in vivo, expression of MLL-ENL in long-term hematopoietic stem cells (LT-HSC) induced an aggressive mixed lineage leukemia characterized by the presence of two distinguishable populations of blasts, whereas induction in GMPs never induced a disease. These data suggest that MLL-ENL preferentially transforms hematopoietic stem cells rather than more differentiated progenitors. Thus, this novel transgenic mouse model for MLL-ENL induced acute leukemia closely recapitulates the human disease and combines the advantages of the existing knock-in and retroviral models. This model allowed us to demonstrate that in contrast to the MLL-AF9 fusion, that preferentially immortalizes GMPs, MLL-ENL preferentially transforms HSCs. We anticipate that our model will be a valuable tool to study the cellular origin and to search for and/or validate novel therapeutic targets for MLL-ENL induced acute leukemia. Disclosures: No relevant conflicts of interest to declare.