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The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML.

Helios is a zinc-finger protein belonging to the Ikaros family of transcriptional regulators. It is expressed, along with Ikaros, throughout early stages of thymocyte development where it quantitatively associates with Ikaros through C-terminal zinc-finger domains that mediate heterodimerization between Ikaros family members. To understand the role of Helios in T-cell development, we used a retroviral vector to express full-length Helios or a Helios isoform that lacked the N-terminal DNA-binding domain in hematopoietic progenitor cells of reconstituted mice. Constitutive expression of full-length Helios resulted in an inhibition of T-cell development at the double-negative stage within the thymus. Although expression of the DNA-binding mutant of Helios did not contribute to developmental abnormalities at early times after transplantation, 60% of animals that expressed the Helios DNA-binding mutant developed an aggressive and transplantable T-cell lymphoma 4 to 10 months after transplantation. These results demonstrate a vital function for Helios in maintaining normal homeostasis of developing T cells and formally show that non–DNA-binding isoforms of Helios are lymphomagenic if aberrantly expressed within the T-cell lineage.

Chronic myelogenous leukemia (CML) is one of the most frequently diagnosed forms of leukemia. Recent advances in the understanding of the molecular mechanisms involved in the pathogenesis of this disorder led to new diagnostic and therapeutic approaches. From a disease that used to be diagnosed purely on morphologic grounds, CML is now an entity that cannot be diagnosed in the absence of relevant cytogenetic or molecular data. Identification of the characteristic t(9;22) cytogenetic abnormality or of the BCR-ABL molecular abnormality is mandatory, as current treatment approaches rely on the use of agents active against this specific molecular target. This article presents the evolution in the understanding of the pathogenic mechanisms involved in CML, as well as the methods and the criteria currently in use in its diagnosis and treatment.

BACKGROUND Bone marrow examination is essential in diagnosis, staging and monitoring of various hematologic disorders. The aspirate smears and core biopsy are complementary samples; current clinical benchmarks recommend an optimal core sample length of at least 15-20 mm. We assessed the core length in 2 cross-sectional cohorts from 2001 and 2011 in 32 academic medical centers from the US and Canada, the first such study to date. METHODS After IRB approval, participants collected data from pathology reports (including the preprocessing length) and measured aggregate postprocessing and evaluable marrow length using a uniform validated methodology on 100 consecutive marrow samples in 2001 and 2011 at each institution. Deidentified data was centralized at Roswell Park Cancer Institute (RPCI) and centers were anonymized. A total of 6374 samples were accepted for statistical analysis, performed using SAS (v. 9.4 or higher; SAS Institute, Cary, NC) at RPCI. Relationship between core length and NCCN status, geographic location, gender, age, and staging was assessed using the PROC MIXED and PROC GLIMMIX procedure using a random center effect and a nominal significance level of 0.05. RESULTS The study cohort included 56% men and 44% women, mean age 51 (range, 1-102) years, 88% adults (³18) and 12% children ( A core biopsy was obtained in 90% of 2001 and in 95% of 2011 samples. Most cores were unilateral (85%); bilateral sampling decreased from 10% to 4% between 2001 and 2011. An aspirate specimen was received in the pathology department in 86% of the cases; however, a clot section was prepared in only 56% of the cases. Preprocessing core length (PreCL; n=3141) documentation in pathology reports was missing in 9 centers for both years; in 3 centers it was available only for one year; its mean (standard deviation) was 16.9 (9.9) mm, decreasing from 2001 to 2011 [17.7 (11.9) to 16.2 (8); p=0.002]. Postprocessing core length (PostCL; n=5742) mean (SD) was 14.2 (8.5) mm, significantly shorter in women than in men (p Evaluable marrow space length (EML; n=5617) mean (SD) was 10.7 (7.5) mm and decreased from 2001 to 2011 [10.9 (8.5) to 10.6 (6.6); p=0.002]. Ninety-one samples (1.6%) with measureable PostCL were entirely devoid of evaluable marrow. PostCL was 15% shorter than PreCL. The EML measured 24% less than the PostCL and 33% less than the PreCL. Lymphoma staging samples (n=1222) were obtained from adults (96%) and children (4%); of these, only 45% and 28% reached the PostCL of 15 mm and 20 mm, respectively. Lymphoma involvement rate was 16%, 25%, 32%, 30%, and 23% when PostCL was There was no significant difference of PostCL mean based on geographic region or NCCN status. PostCL mean ranged from 8.8 to 29.6 mm and its median was ³15 mm in only 12 of 23, 9 of 32, and 2 of 32 centers for PreCL, PostCL, and EML, respectively (Fig 3). When compared to current benchmarks, only 53%, 38%, and 22% of all samples were ³15 mm and only 30%, 19%, and 10% were ³20 mm for PreCL, PostCL, and EML, respectively. CONCLUSIONS Current benchmarks for bone marrow core biopsy adequacy are met in only a minority of cases. Samples from women were shorter than from men. Bilateral sampling decreased and PreCL and EML diminished significantly between 2001 and 2011, while PostCL showed only minimal decrease. This discrepancy may be due to technical differences. Although staging cores were longer than non-staging ones, most did not meet the benchmarks. The wide variability among different centers suggests significant institutional differences and lack of standardization in bone marrow sampling. Over a third of all centers did not record preprocessing length, an important parameter which may guide operator assessment of adequacy at the bedside. PostCL and/or EML can be measured by the pathologist and integrated in the report to document adequacy. In staging samples, a minimal marrow space sampling should be required to avoid understaging. Consensus definition of an inadequate core biopsy is currently lacking but would be desirable for uniform pathology reporting, clinical protocols and management. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Taking a mechanistically unbiased approach to the discovery of new myeloma drugs we developed a three organ system assay that selected anti-myeloma compounds from a primary 30,000 small molecule ATP-based screen in one “sandwich” setup for tolerability by normal bone marrow, stability towards liver enzymes, and activity in the context of cell barriers, bone marrow stromal support, and short, kidney-clearance-like exposure (fig.1). The most promising compound (CCF642) emerging from this test had IC50s around 500 nM in all 8 MM cell lines tested including a proteasome inhibitor refractory line while the IC50 was not reached in 5 healthy bone marrow samples at doses up to 6750 nM. Similar sensitivity as in MM was seen in 7 lymphoma cell lines. Investigation of its mechanism of action revealed that CCF642 immediately stimulates protein ubiquitination which is followed by degradation of major myeloma survival factors (c-MYC, IRF4, NF-κB) and apoptosis, while normal bone marrow cells increase ubiquitination to a much lesser degree and only transiently without undergoing cell death. Experiments with an active biotinylated analog showed that it preferentially enters myeloma cells with cytoplasmic accumulation and over 10-fold lower entry into normal bone marrow. Immunoblots revealed that it becomes covalently attached to proteins in a time- and distribution pattern that parallels ubiquitination responses. In vitro, it bound to ubiquitin activating enzyme UBA1 at the active site cysteine in a similarly stable and reversible way as ubiquitin. Selective alkylation of cysteine sulfhydryl groups inhibited binding of biotinylated CCF642 to UBA1, while addition of ATP and ubiquitin led to its dissociation from UBA1. CCF642 may therefore at least in part act as a small molecule ubiquitin analog. Accordingly CCF642 binding to target proteins of UBA1 containing HeLa fraction II was increased by addition of ATP and blocked in the presence of EDTA. Injected twice a week IP at 30mg/kg it suppressed systemically engrafted luciferase expressing 5TGM1 mouse myeloma cells in syngeneic mice comparable to bortezomib given at the MTD for this mouse strain (0.5mg/kg SC twice a week) with equal prolongation of survival. Results support use of our “sandwich” assay to select myeloma drug candidates for in vivo testing and reveal a new UBA1 interacting small molecule ubiquitination enhancer with promise for clinical translation. Figure 1: Sandwich Three Organ System Assay Figure 1:. Sandwich Three Organ System Assay Disclosures No relevant conflicts of interest to declare.

Homeobox (Hox) gene mutations and their altered expressions are frequently linked to human leukemia. Here, we report that transforming growth factor beta (TGFbeta)/bone morphogenetic protein (BMP) inhibits the bone marrow transformation capability of Hoxa9 and Nup98-Hoxa9, the chimeric fusion form of Hoxa9 identified in human acute myeloid leukemia (AML), through Smad4, the common Smad (Co-Smad) in the TGFbeta/BMP signaling pathway. Smad4 interacts directly with the homeodomain of Hoxa9 and blocks the ability of Nup98-Hoxa9 to bind DNA, thereby suppressing its ability to regulate downstream gene transcription. Mapping data revealed that the amino-terminus of Smad4 mediates this interaction and overexpression of the Hoxa9 interaction domain of Smad4 was sufficient to inhibit the enhanced serial replating ability of primary bone marrow cells induced by Nup98-Hoxa9. These studies establish a novel mechanism by which TGFbeta/BMP regulates hematopoiesis and suggest that modification of Hox DNA-binding activity may serve as a novel therapeutic intervention for those leukemias that involve deregulation of Hox.

PU.1 is a member of the ETS family of transcription factors and is required for the development of multiple hematopoietic lineages. PU.1-/- mice die from hematopoietic failure at about embryonic day 18.5 (e18.5) and show a complete absence of B cells, mature T cells, and macrophages. This phenotype suggests that PU.1 may function at the level of the hematopoietic stem cell (HSC) or a multilineage progenitor. To investigate the role of PU.1 in the regulation of HSCs, PU.1-/- embryos were analyzed at various stages of embryonic development. The absolute number and frequency of HSCs were determined by flow cytometric analysis of c-Kit+Thy-1.1loLin-Sca-1+ (KTLS) cells. We found that KTLS cells were absent or severely reduced in PU.1-/- fetal liver from e12.5 to e15.5. Progenitor cells with a c-Kit+Lin-AA4.1+ and c-Kit+Lin-CD34+ phenotype were also severely reduced. In addition, PU.1-/- fetal liver at e14.5 lacked common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) but retained megakaryocyteerythroid progenitors (MEPs). Consistent with the loss of HSC activity, a 10-fold reduction in erythroid progenitors (mature erythroid burst-forming units [BFUEs]) was observed between e14.5 and e16.5. These data suggest that PU.1 plays an important role in the maintenance or expansion of HSC number in murine fetal liver. (Blood. 2004;104:3894-3900)

Since the mid-1980s, murine retroviral vectors have been used extensively by a number of investigators to clonally mark and genetically modify primitive hematopoietic stem cells (HSC) (1,2). During this period, both vectors and packaging systems used to generate virus have undergone considerable modification. This has led to increased production of high-titer, replication-defective retrovirus that is more resistant to gene inactivation following integration into hematopoietic cells. Current approaches to murine HSC transduction have become increasingly more standardized, although there remain numerous variations on a theme (see Chapter 15). This "classical" method utilizes preconditioned bone-marrow cells (typically from 5-fluorouracil [5-FU]-treated animals) and coculture of these cells with virus-producing packaging cells in the presence of exogenous cytokines. This approach generally yields high proportions of transduced cells that can repopulate lethally irradiated recipient mice for long periods of time, indicating that self-renewal activity is maintained-at least to some extent-in conditions that promote stem-cell cycling. With this approach, it is difficult to re-isolate transduced cells from packaging cells and from nontransduced bone marrow, which would be desirable in some clinically relevant cases.

Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.

Abstract 2736 Background: Epigenetic instability and aberrant repression of tumor suppressor genes are fundamental aspects of acute myeloid leukemia (AML) biology. Current prognostic assays for AML focus on the genetic aspects of disease biology, for example, chromosome abnormalities and recurrent mutations. We hypothesized that a simple assay for an epigenetic abnormality can address the epigenetic domain of AML, with implications for prognostication and for treatment selection with chromatin relaxing drugs. We focused on patients with intermediate risk cytogenetics (CG) and evaluated the association between histone (H4) acetylation and complete remission (CR), relapse-free survival (RFS), and overall survival (OS) in newly diagnosed AML patients with intermediate risk CG. Methods: We assessed histone H4 acetylation status in bone marrow biopsies from newly diagnosed AML patients with intermediate risk CG treated at our institution between the years 2003–2005. CG risk group was ascribed per Cancer and Leukemia Group B criteria. Association with CR, RFS and OS was assessed using logistic regression and Cox proportional hazard regression models. Variables used in the multivariate models included: age, WBC at diagnosis, gender, and history of antecedent hematologic disorder (AHD). B5-fixed bone marrow core biopsies were reviewed for areas with the highest blast concentration. Immunohistochemistry was performed for acetyl-H4 (1:200 dilution; polyclonal; Upstate Biotech, Lake Placid, NY) using automated stainers and heat-induced epitope retrieval. In each case, five hundred blasts were counted and only strong nuclear staining was classified as positive. Based on the distribution of cell counts, cases were classified as strongly positive if nuclear staining occurred in > 80% of the blasts. Results: Forty-two patients had intermediate risk CG and received cytarabine-based induction chemotherapy. The median age was 56.5 years (range 17–79) and median WBC count at diagnosis was 10.9 × 109/L (range 0.8– 227.9). Twenty two percent of patients had an AHD. Thirty three patients (78%) had normal cytogenetics. Chromosomal abnormalities +8,+21, and 11q23 abnormalities were present in 3 (7.1%), 1 (2.3%) and 4 (9.4%) patients, respectively. FLT3 status was available on only 9 patients; with 7 patients having no FLT3 mutations. Twelve patients (28.5%) received an allogeneic bone marrow transplant in first remission. The median follow-up time was 22.4 months (range 0.6–65.3). Thirty-four patients (81%) achieved CR. Using the 80% cut-off, 15 patients (35.7%) were positive for histone H4 acetylation. Histone (H4) acetylation was not associated with achievement of CR (OR=3.0, 95% CI 0.3–25.3, p=0.3). However, on multivariate analysis, a significant association was found between histone acetylation and risk of relapse (HR 0.34, 95% CI 0.11–0.99, p=0.05) and overall survival (HR=0.34, 95% CI 0.15–0.80, p=0.01). Inclusion of allogeneic transplant in the multivariate models did not change these estimates. Conclusion: Histone H4 acetylation is associated with improved RFS and OS in newly diagnosed AML patients with intermediate risk CG. These results will need to be validated in a larger population of uniformly treated patients. However, histone H4 acetylation has the potential as a simple assay to assess for the contribution of abnormal epigenetics to AML biology, with prognostic and potentially therapeutic implications, given the increasing role of epigenetically targeted therapy in myeloid malignancy management. Disclosures: No relevant conflicts of interest to declare.

Abstract 4667 Current concepts of the pathophysiology of primary immune thrombocytopenia (ITP) reflect a complex interplay between antiplatelet antibodies, cytotoxic T-lymphocytes (CTL), T regulatory cells, and impaired bone marrow megakaryopoiesis. The mechanisms of ITP in the presence of monoclonal gammopathy of unknown significance (MGUS), a form of secondary ITP, are less well understood, yet treatment has been essentially the same as for primary ITP. Inhibition of antiplatelet CTL responses through apoptosis induction in clonal antiplatelet-antibody producing plasma cells and proteasome inhibition in CTLs may be achieved with bortezomib. However, bortezomib may cause transient isolated thrombocytopenia via presumed inhibition of marrow thrombopoiesis or enhanced platelet clearance peripherally. We used bortezomib in a 55 year-old female with IgG lambda monoclonal gammopathy and ITP refractory to previous treatment with corticosteroids, intravenous immunoglobulin, splenectomy, danazole, and rituximab, none of which induced a durable increase in her platelet counts to hemostatic concentrations. She suffered from repeated episodes of prolonged epistaxis, and when the platelet count fell below 10K/mm3, she experienced a life-threatening gastrointestinal hemorrhage. Otherwise, she had no anemia, her creatinine and calcium remained normal throughout, and skeletal surveys showed no evidence for myeloma. Repeated bone marrow examinations, reviewed at our institution, demonstrated lambda monotypic plasma cells always below 10%, and a normal female karyotype on metaphase cytogenetics. Her thrombocytopenia and bleeding episodes improved transiently on dexamethasone/IMiD combinations (thalidomide followed by lenalidomide), with concurrent reduction in total IgG by almost 50% (M-spike was not measured prior to IMiDs), however she became intolerant to thalidomide due to peripheral neuropathy and experienced diarrhea on lenalidomide. Her platelets decreased and her epistaxis worsened as her m-spike increased on dexamethasone single agent. We thus focused our treatment approach on more robust suppression of the monoclonal protein and initiated a course of weekly bortezomib (1.3 mg/m2), with dexamethasone (20 mg) on the day of and the day after bortezomib. After one month of therapy, the platelet count normalized, and she had no further bleeding episodes. The bortezomib dose was reduced to 1 mg/m2 and dexamethasone reduced to 20 mg weekly after 2 months, with persistently normal platelet counts thereafter. Her ITP resolution on bortezomib was accompanied by a decrease of her serum M-protein from 1.04 to 0.39 g/dL. This case illustrates that bortezomib can be remarkably effective in refractory ITP with concurrent monoclonal gammopathy. Assuming similar pathophysiology, it might also have efficacy in primary refractory ITP. This case also raises the question of whether refractory ITP in the presence of a clonal plasma cell disorder should be considered as a myeloma defining symptom. Disclosures: No relevant conflicts of interest to declare.

Abstract 4681 Background Histone acetylation is a post-translational modification used by proteins to regulate specific chromatin functions. Histone deacetylases (HDACs) play a pivotal role in the pathogenesis of a subset of acute myeloid leukemias (AMLs). HDAC inhibitors are currently being evaluated in clinical trials. We have previously demonstrated an association between increased histone H4 acetylation and increased relapse-free survival (RFS) in patients with newly diagnosed acute lymphocytic leukemia (ALL) (Advani et al., ASH abstract #2798, 2007) and improved overall survival (OS) in ALL patients with first relapse (Advani et al., ASH abstract #1482, 2008). Here, we evaluated the association between histone H4 acetylation with achievement of complete remission (CR) and also with RFS and OS in patients with newly diagnosed AML. Methods In this retrospective cohort study, we assessed H4 acetylation status in bone marrow biopsies of newly diagnosed AML patients at the Cleveland Clinic between the years 2003-2005. Association with CR, RFS and OS was assessed using univariate and multivariate logistic regression and cox-proportional hazard regression models. Variables used in the models included: age and WBC at diagnosis, gender, cytogenetic (CG) risk group, and history of antecedent hematologic disorder (AHD). CG risk group was ascribed per Cancer and Leukemia Group B criteria. B5-fixed bone marrow core biopsies were reviewed for areas of highest blast concentration. Immunohistochemistry was performed for acetyl-H4 (1:200 dilution; polyclonal; Upstate Biotech, Lake Placid, NY) using automated stainers and heat induced epitope retrieval. In each case, five hundred blasts were counted and only strong nuclear staining was classified as positive. Based on the distribution of cell counts, cases were classified as strongly positive if nuclear staining occurred in > 80% of the blasts. Results Eighty-one patients had adequate tissue and clinical data available. We restricted the analysis to sixty patients who received standard induction chemotherapy with cytarabine and an anthracycline. The median age was 58 years (range 20-79) and median WBC was 8.85 × 109/ L (range 0.8-227.9). Seven patients (11.6%) had favorable CG, 41 (68.3%) had intermediate, and 12 (20%) had poor risk CG. Thirty two percent of patients had an AHD. Thirteen patients (21.6%) received an allogeneic bone marrow transplant in first remission. The median follow-up time was 25.5 months (range 2.1-70.6). Forty five patients (75%) achieved CR and the median OS was 12.7 months (range 0.4-70.6). Using the 80% cut-off, 19 patients (31.6%) were positive for H4-acetylation. Histone (H4) acetylation was not associated with achievement of CR (OR=4.1, 95% CI 0.7-24.5, p=0.1) or relapse-free survival (HR 0.62, 95% CI 0.18-2.08, p=0.44). However, in multivariate analysis, histone acetylation was associated with a significantly better OS (HR=0.51, 95% CI 0.29-0.88, p=0.01). Inclusion of allogeneic transplant in the multivariate models did not change these estimates. Conclusion Strongly positive histone (H4) acetylation is associated with better OS in multi-variate analysis. The results mimic our previous findings in ALL patients. Further studies will need to include FLT3 and NPM status in patients with normal cytogenetics in the multi-variate model. These results provide a rationale for AML regimens incorporating HDAC inhibitors, and use of this assay as a potentially relevant biomarker during HDAC inhibitor therapy. Disclosures: No relevant conflicts of interest to declare.