ALBERT

Description: BACKGROUND: Primary mediastinal B-cell lymphoma (PMBCL) are a subtype of diffuse large B cell lymphoma (DLBCL), that represent 2-4% of all non-Hodgkin lymphoma (NHL) and 6-12% of DLBCL. They affect young adults with a median age at diagnosis of 35 years old and are more frequent in females (2:1). Approximately 80% of patients have stage I or II disease at the time of diagnosis, with B symptoms, bulky mass and superior vena cava syndrome (SVCS) in 50 %. There is no standard initial therapy regimen, anthracyclines with Rituximab are the most used. Local consolidation radiotherapy (RT) is reserved for bulky disease and to complete partial remissions (PR) after chemotherapy. AIMS: To analyze prognostic and epidemiological factors, treatment administered and response and survival rates of patients diagnosed with PMBCL in our center. METHODS: Retrospective cohort study between September 2003 and June 2014. The treatment received was 6 to 8 cycles of CHOP-like chemotherapy (Cyclophosphamide, Adriamycin, Vincristine, Prednisone ± methotrexate / Intrathecal triple therapy MTX- TIT ), with or without Rituximab, and subsequent radiotherapy if necessary. The following results were evaluated by univariate analysis: overall survival (OS), disease-free survival (DFS) and incidence of relapse after diagnosis. DFS was defined as the interval of time from complete remission (CR) to relapse or last visit. Response to treatment was assessed by PET-CT. RESULTS: We observed 14 patients diagnosed with PMBCL in our hospital with a median age of 33 years (r, 21-58 years) and female predominance (Š:‰ 10:4). At diagnosis 42.8 % had B symptoms and 42.8% elevated lactic dehydrogenase. Fifty percent of patients presented with SVCS. Central nervous system and bone marrow infiltration was not observed. Early-stage disease at diagnosis (I 30% and II 53%) was observed in 83%, with IPI 1 in 78.5% of patients. The number of cycles received was 6 in 78.5 %, 8 cycles in 14.2 %, and only 1 cycle of CHOP in one patient. Altogether 71.4 % received Rituximab and 71.4 % received MTX-TIT. Ten patients (71.4%) received RT (median 36 Gy) after chemotherapy, 9 of which had initial bulky disease. We observed an overall response rate of 92.8 % after chemotherapy (57.1 % CR, 35.7% PR). After a median follow-up of 60 months (r, 4.4 to 130.8 months) 12 patients had responded to treatment, were alive, without relapse and in complete response, and one patient is currently receiving the 6º cycle of CHOP pending reevaluation (awaiting last MTX-TIT and RT consolidation) and obtained CR after the third cycle of R-CHOP-MTX-TIT. Median overall survival was not reached and median DFS was of 54.8 months. Only one patient died (mortality 7.1 %) due to influenza A in the context of postchemotherapy aplasia after the first cycle of CHOP. CONCLUSIONS: We observed prognostic and epidemiological factors similar to those described in literature, although in our series, CHOP -like chemotherapy ( ± MTX- TIT ) with or without Rituximab and RT has shown improved survival rates and 100% of CR. Consolidation radiotherapy was successfully used to complete treatment in patients that only achieved PR after chemotherapy. Rescue chemotherapy followed by autologous hematopoietic cell transplantation was not required. It is difficult to draw conclusions on the impact of the therapeutic regimen received, we believe multicenter analysis with larger numbers of patients are necessary. Disclosures No relevant conflicts of interest to declare.

Description: Severe bacterial sepsis often leads to a systemic procoagulant and proinflammatory condition that can manifest as disseminated intravascular coagulation, septic shock, and multiple organ failure. Because activation of the contact proteases factor XII (FXII), prekallikrein, and factor XI (FXI) can trigger coagulation and inflammatory responses, the contact factors have been considered potential targets for the treatment of sepsis. However, the pathogenic role of contact activation in severe infections has not been well defined. We therefore investigated whether an anticoagulant antibody (14E11) that selectively inhibits prothrombotic FXI activation by activated FXII (FXIIa) modifies the course of bowel perforation-induced peritoneal sepsis in mice. Early anticoagulation with 14E11 suppressed systemic thrombin- antithrombin complex formation, IL-6, and TNF-α levels, and reduced platelet consumption in the circulation and deposition in the blood vessels. Treatment with 14E11 within 12 hours after bowel perforation significantly improved survival compared with vehicle treatment, and the saturating dose did not increase tail bleeding. These data suggest that severe polymicrobial abdominal infection induces prothrombotic FXI activation, to the detriment of the host. Systemic anticoagulation by inhibiting FXI activation or FXIIa procoagulant activity during sepsis may therefore limit the development of disseminated intravascular coagulation without increasing bleeding risks.

Description: Introduction: The incidence of acute myeloid leukemia (AML) increases with age, however treatment efficacy and tolerability in older patients are poor compared to younger patients. Without treatment, patients succumb to their illness within a few months of diagnosis. Further, disease relapse is inevitable in the majority of cases without additional post-remission therapy after successful induction of remission. The use of allogeneic hematopoietic stem cell transplantation (HSCT) is considered a potential cure for AML but its use is limited in older patients because of significant comorbidities and increased transplant-related morbidity and mortality. This retrospective study assessed outcomes of older AML patients treated with chemotherapy with or without HSCT. Methods: The linked Surveillance, Epidemiology, and End Results (SEER)-Medicare database, was utilized in this retrospective cohort analysis of 3327 first primary AML patients. Patients were diagnosed between January 1, 2000 to December 31, 2009, were 〉66 years, continuously enrolled in Medicare Part A and B with no HMO coverage in the year prior to diagnosis and received treatment with chemotherapy with or without HSCT. Chi-square test for categorical variables and ANOVA or t-test for continuous variables was used to compare patient characteristics between treated patients with and without HSCT. Kaplan-Meier curves and Cox proportional hazards regression assessed overall survival. Date of last follow-up was December 31, 2010. Results: There were 276 (8%) patients who underwent HSCT therapy and 3051 (92%) who did not. HSCT patients were younger at diagnosis with mean age of 73 compared to the non-HSCT group (75 years; p75 years (n=1470) HR 95% CI HR 95% CI HR 95% CI No ref ref ref Yes 0.80 0.69-0.92 0.63 0.53-0.75 1.22 0.97-1.54 Disclosures Satram-Hoang: Genentech, Inc.: Consultancy. Hurst:Genentech, Inc.: Employment. Reyes:Genentech, Inc.: Employment.

Description: Corticosteroids and lenalidomide decrease red blood cell transfusion dependence in patients with Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome (MDS), respectively. We explored the effects of dexamethasone and lenalidomide, individually and in combination, on the differentiation of primary human bone marrow progenitor cells in vitro. Both agents promote erythropoiesis, increasing the absolute number of erythroid cells produced from normal CD34+ cells and from CD34+ cells with the types of ribosome dysfunction found in DBA and del(5q) MDS. However, the drugs had distinct effects on the production of erythroid progenitor colonies; dexamethasone selectively increased the number of burst-forming units-erythroid (BFU-E), whereas lenalidomide specifically increased colony-forming unit-erythroid (CFU-E). Use of the drugs in combination demonstrated that their effects are not redundant. In addition, dexamethasone and lenalidomide induced distinct gene-expression profiles. In coculture experiments, we examined the role of the microenvironment in response to both drugs and found that the presence of macrophages, the central cells in erythroblastic islands, accentuated the effects of both agents. Our findings indicate that dexamethasone and lenalidomide promote different stages of erythropoiesis and support the potential clinical utility of combination therapy for patients with bone marrow failure.

Description: Lenalidomide is a highly effective drug for the treatment of multiple myeloma and has activity in additional B cell lymphomas. Lenalidomide has been shown to bind the CRBN-DDB1 E3 ubiquitin ligase, but it is unknown how lenalidomide alters the activity of this enzyme complex, and how this leads to therapeutic efficacy.  We used a combination of quantitative proteomic approaches to demonstrate that lenalidomide acts by a novel mechanism of action for a therapeutic agent: in multiple myeloma cells, lenalidomide increases the binding of two substrates, IKZF1 (Ikaros) and IKZF3 (Aiolos), to the CRBN substrate adaptor; increases the ubiquitination of these substrates; and causes the targeted degradation of these transcription factors that are essential for the differentiation and survival of plasma cells including multiple myeloma cells. To identify targets of the CRBN-DDB1 ubiquitin ligase that are altered by lenalidomide, we applied SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative mass spectrometry studies to globally assess changes in ubiquitination and proteome levels in the multiple myeloma cell line MM1S. Two members of the Ikaros transcription factor family, IKZF1 and IKZF3, were differentially ubiquitinated and decreased after lenalidomide treatment.  Subsequent validation experiments in various cell lines demonstrated that lenalidomide, thalidomide, and pomalidomide cause a decrease of endogenous and ectopically expressed IKZF1 and IKZF3 protein levels but not mRNA levels. Furthermore, we confirmed that IKZF1 and IKZF3 bind CRBN in the presence of lenalidomide, supporting CRBN’s role as a substrate adaptor. Consistent with this, shRNA mediated knockdown or overexpression of a CRBN mutant (CRBNYWAA) that does not bind lenalidomide abrogated lenalidomide’s effect on IKZF1 and IKZF3. Moreover, CRBN promoted IKZF3 ubiquitination in vitro in the presence of lenalidomide, demonstrating that it is an enzymatic substrate. Using deletion mutants of IKZF3 we identified a 58-amino-acid degron in the N-terminal zinc finger domain that is sufficient for lenalidomide-induced degradation. Based on sequence alignment of that region between lenalidomide responding Ikaros proteins IKZF1 and IKZF3 vs. non-responding IKZF2, IKZF4 and IKZF5 we substituted a single amino acid (IKZF3Q147H) that prevented binding of IKZF3 to CRBN and conferred resistance to lenalidomide induced degradation. IKZF1 and IKZF3 are essential transcription factors for terminal B cell differentiation. We evaluated the biological effects of IKZF1 and IKZF3 loss using shRNAs in a variety of cell lines. IKZF1 and IKZF3 specific shRNAs inhibited the growth of multiple myeloma cell lines while lenalidomide insensitive cell lines derived from other hematopoietic neoplasms were unaffected. Similarly, a dominant negative IKZF3 mutant resulted in growth inhibition of MM1S cells. In contrast, expression of IKZF3Q147Hconferred lenalidomide resistance to MM1S cells. Lenalidomide induces IL-2 expression and release in T cells. We found that lenalidomide induced a dose-dependent decrease of IKZF1 and IKZF3 protein levels in primary human T cells. Previous studies have shown that IKZF3 is a transcriptional repressor of IL-2. To further evaluate the effect of IKZF3 loss, we transduced primary human T cells with shRNAs targeting either IKZF3 or control. IL2 RNA levels increased 3.3 fold after lenalidomide treatment in T cells expressing control shRNAs. In contrast, the baseline IL2 RNA level in T cells transduced with IKZF3 specific shRNAs was 3.7 fold higher compared to controls and this effect could not be further stimulated by lenalidomide. In conclusion, selective targeting of two lymphoid transcription factors, IKZF1 and IKZF3, explains lenalidomide’s selective growth inhibition in multiple myeloma and likely other B cell lymphomas as well as its immunomodulatory effects in T cells. Furthermore, selective ubiquitination and degradation of specific targets provides a novel mechanism of therapeutic activity for proteins that are not otherwise amenable to small-molecule inhibition. Disclosures: Ebert: Celgene: Membership on an entity’s Board of Directors or advisory committees.

Description: Abstract 1257 Haploinsufficiency of ribosomal proteins (RP) has been shown to be the common basis for the anemia observed in ribosomopathies such as Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q (del(5q) MDS). DBA is a congenital bone marrow failure syndrome characterized by a profound macrocytic anemia. More than half the patients with DBA have been shown to have a heterozygous loss of an RP gene, with RPS19 being the most frequently mutated. The “5q- syndrome” is a subtype of myelodysplastic syndrome (MDS) also characterized by severe anemia that is caused by heterozygous loss of the RPS14 gene on chromosome 5q. The p53 pathway is known to play a critical role in the pathophysiology of the ribosomopathies. The leading hypothesis is that ribosomal haploinsufficiency leads to disrupted ribosome biogenesis with an accumulation of free ribosomal proteins that bind MDM2. MDM2 is an E3 Ubiquitin ligase that normally binds to and targets p53 for proteosomal degradation. The consequent accumulation of p53 leads to cell cycle arrest and apoptosis, which ultimately results in anemia. Several animal models have shown that the anemia associated with RP haploinsufficiency is almost completely alleviated in a p53 null background. However, we and others have shown that p53-independent pathway(s) also contribute to the anemia associated with RP haploinsufficiency. We have previously modeled DBA and del (5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14 respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. We have further demonstrated that treatment of Rps19 and Rps14 deficient embryos with the amino acid L-Leucine, a known activator of mRNA translation, results in a marked improvement in anemia. This observation was confirmed in primary human CD34+ cells, following shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that L-leucine treatment activates the mTOR pathway in zebrafish embryos deficient in Rps19 or Rps14. In order to determine if the effect of L-Leucine on RP deficient erythroid cells is p53 dependent, we injected rps19 and rps14 morpholinos into zebrafish embryos and treated them with L-Leucine. Total RNA was collected 48hpf and evaluated for expression of p53 by qPCR analysis. As expected, the expression of p53 and its downstream targets (p21 and PUMA) were upregulated in Rps14 and Rps19 deficient embryos. P53 expression levels remained elevated even after L-Leucine treatment. Levels of p21, a direct transcriptional target of p53, remained unchanged in L-Leucine treated RP deficient zebrafish embryos; however, expression of PUMA increased following L-Leucine treatment. The expression of the PUMA gene has previously been shown to have a p53-independent regulatory component. Preliminary studies in the A549 cell line, which harbors wild type p53, also showed increased levels of p53 expression upon shRNA mediated downregulation of both RPS19 and RPS14, which remained unaltered following L-Leucine treatment. These observations are currently being confirmed in primary human CD34+ cells, following shRNA knockdown of RPS19 and RPS14. Our preliminary studies show that the effect of L-Leucine in improving the anemia in models of DBA and del(5q) MDS occurs independently of p53. This supports our hypothesis that the erythroid phenotype in these disorders has a p53-independent component. Our finding that L-Leucine treated RP deficient cells are likely to express elevated rather than diminished levels of p53 in spite of improved anemia also has important implications for the clinical management of patients, since p53 inactivation is associated with tumor growth. A trial using L-Leucine for patients with DBA will be opening soon in the United States. Disclosures: Ebert: Celgene: Consultancy; Genoptix: Consultancy.