ALBERT

Description: Abstract 4013 Background: In recent years, azacytidine (AZA) has become the standard of treatment for high risk myelodysplasia (MDS). The approved schedule of AZA uses a 75mg/m2/d s.c. regimen for 7 days based on the CALGB-9221 (Silverman, JCO 2002) and the AZA-001 studies (Fenaux, Lancet Oncol 2009). The clinical response rates after AZA have been extensively presented but there is only limited data on the rates of cytogenetic response (CyR). Based on the review of the literature, there are no specific cytogenetic data published on prospective trials. Methods: We based our analysis on a randomized phase 2 study from the US Leukemia Intergroup (E1905 study, NCT00313586) testing 10 days of AZA (50mg/m2/d s.c.) vs 10 days of AZA+ the histone deacetylase inhibitor entinostat (4 mg/m2/d PO days 3 and day 10). MDS, CMML, and AML with myelodysplasia-related changes were included. This analysis includes all patients with cytogenetic abnormalities (at baseline or acquired following treatment) with available cytogenetic follow-up (cycle 6). Of 150 patients, 70 demonstrated baseline cytogenetic abnormalities. To date, forty patients (27 MDS and 13 AML) were evaluable for both time points. Karyotypes were performed at local laboratories, and reviewed centrally (RPK and GH). Cytogenetic response was assessed using IWG 2000 (Cheson et al, Blood 2000) criteria. Results: The clinical response rate (CR+PR+ trilineage HI) according to IPSS cytogenetic risk stratification were of 20%, 33%, and 35% for favorable, intermediate and poor cytogenetic risk groups respectively (p=NS). Patients with Chr 7 abnormalities (i.e. -7 or -7q, n=18) had a response rate of 28% including 17% CR. Of patients with complete cytogenetic data, the rate of overall CyR was 52% (n=21): 22% (n=9) complete CyR, 30% (n=12) partial CyR. This represents a complete CyR of 13% and a partial CyR of 23% as a proportion of all treated patients with initial cytogenetic abnormalities (including those who did not receive six cycles of therapy). To date, confirmatory FISH analyses were available for 4 patients with CyR (2 CCyR and 2 PCyR). All four had complete clearance of their cytogenetic clone. Among the cytogenetic responders, 15 had MDS and 6 had AML (p=NS). CyR did not differ between the two treatment arms. CyR and clinical response were highly correlated (p

Description: Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of lymphoma in adults. Gene expression profiling has demonstrated that DLBCL can be classified into two distinct subgroups – activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL. These subgroups arise through distinct normal cells of origin, activate different oncogenic pathways and display markedly different clinical outcomes. Deregulation of the transcriptome is believed to play a key role in the malignant transformation of B cells that culminates in the development of either ABC or GCB DLBCL. Here we describe global differences in RNA expression, mutation and splicing in relation to the pathogenesis of these subgroups of DLBCL. Methods: RNA sequencing (RNAseq) has emerged as a powerful tool for defining the cancer transcriptome. While mRNA sequencing is the most widely applied method for RNAseq, it overlooks non-coding RNAs, requires high-quality RNA and lacks strand-specificity. To overcome these limitations, we developed a method for strand-specific total RNA sequencing (ssRNAseq) to characterize the transcriptomes of 112 DLBCL tumors. Results: Through this work, we defined the entire spectrum of coding and non-coding RNAs expressed in DLBCLs including hundreds of lincRNAs, snoRNAs and microRNAs in addition to mRNAs. We found that the strand-specificity of our method was greater than 95% in all cases. This strand-specific sequencing strategy allowed us to maintain the orientation of the transcript to enable more accurate transcript annotation and better prediction of novel transcripts. Furthermore, we showed that our method had equal efficacy on frozen and FFPE tumor specimens from the sample patient in 24 cases. In addition, through simultaneous measurement of expression of diverse RNA types combined with mutations in MYD88, GNA13, EZH2, and BCL2, we demonstrated that we could distinguish the clinically important subgroups of DLBCL. Finally, we applied ssRNAseq to distinct training and validation sets of DLBCL cases (N=86 and N=112) to define alternative splicing events in DLBCL and found 1,021 genes that were preferentially spliced in a subgroup-specific manner. These alternatively spliced genes were selectively enriched in a number of different pathways important in lymphomas including those related to immune function, cell cycle progression and focal adhesion pathways, suggesting that alternative splicing regulates a number of important oncogenic processes in DLCBL. Conclusions: Strand-specific total RNA sequencing is a powerful method for defining the transcriptome and alternative splicing events in DLBCL. Here we define a complete coding and non-coding transcriptome of DLBCL and report the first characterization of subgroup-specific alternative splicing in DLBCL using high throughput sequencing. Our data demonstrate the power of our ssRNAseq method in defining the molecular patterns underlying DLBCLs and provide a starting point for defining the role of alternative splicing in this complex and heterogeneous disease. Disclosures Mann: Quiagen: Research Funding.

Description: Chronic myelomonocytic leukemia (CMML) is a hematopoietic malignancy with hybrid myeloproliferative and myelodysplastic features. The diagnostic criteria for CMML are evolving with the progress of our knowledge on various genetic lesions involved in the pathogenesis of myeloid neoplasms. This shift, including molecular genetic lesions in the diagnosis process, is highlighted in updated 2008 WHO classification system, which excludes myeloproliferative neoplasms with PDGFRB rearrangement, monocytosis and eosinophilia from CMML category. Despite these recent advancements, CMML remains a heterogeneous group of diseases with variable patient outcomes and no well-defined targeted therapy. To further investigate the biological diversity of this disorder, we studied microRNA (miRNA) expression profiles, their relation to the diagnostic and clinical parameters in CMML, and compared these profiles to global miRNA expression in normal reference bone marrow samples. MicroRNAs are a class of non-coding RNA molecules that alter gene expression by targeting and blocking mRNA. The role of miRNAs in carcinogenesis is related to their targeting of messenger RNAs encoding for oncogenes and tumor suppressor genes. Bone marrow samples from 22 patients with CMML were included in the study. Median age of the patients was 71 years with a range from 39 to 92 years. There were 15 males and 7 females. Seventeen patients presented with CMML-1 (blasts less than 5% in peripheral blood and less than 10% of bone marrow differential count). The remaining patients showed CMML-2. Nine patients had WBC below 13×109/L defining a myelodysplastic type of CMML. Cytogenetic results were available in 20 patients. Fourteen patients demonstrated a normal karyotype. Normal pooled bone marrow samples were used as a reference. The total RNA was isolated using RecoverAll RNA extraction kit. Micoroarray studies were performed using Agilent human miRNA microarrays (version 1.0) containing probes for 470 human and 64 human viral miRNAs cataloged in the Sanger database v9.1. The results were analyzed using BRB array tool and Genesis software. Unsupervised hierarchical clustering discovered two different groups of CMML samples with patterns of miRNA expression distinct from normal bone marrows (oneway ANOVA). Twenty seven miRNAs were differentially expressed in normal bone marrow reference samples vs. CMML-1 and -2. There was an overlap in miRNA profiles between groups of CMML based on blast percentage (CMML-1 vs. CMML-2), WBC count (

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 556 Bone and bone marrow are dynamic tissues with specialized niches that support numerous bone derived stem and progenitor cells with hematopoietic, mesenchymal and endothelial potentials. These niches are comprised of osteoblasts, stromal cells and an extracellular matrix of collagens, fibronectin and proteoglycans. Osteoblasts (OB) lining the endosteum are a significant regulatory component of hematopoiesis and numerous studies have demonstrated that stem cells reside in close proximity to OB. Prostaglandin E2 (PGE2) is an abundant lipid mediator that exerts numerous activities including inhibition of myelopoiesis and regulation of hematopoietic stem cell self-renewal, and is also a positive regulator of OB number and activity. We investigated the therapeutic potential of inhibiting PGE2 biosynthesis via NSAID administration to expand progenitor cells and disrupt the bone marrow niche in order to facilitate stem and progenitor cell mobilization. Mice were treated with NSAID for 1–4 days, and femurs analyzed for OB and stem and progenitor content. Endosteal OB lining both bony trabeculae and cortical bone in the diaphyseal and epiphyseal regions of the femur demonstrated a time dependent “flattening”, as determined by width measurement of 50 random OB per femur (2.82 to 1.45 microns after 4 days, P

Description: Transition to CML blast crisis is characterized by the continued presence of the Philadelphia chromosome and the acquisition of additional molecular and chromosomal alterations. Loss of heterozygosity at chromosome region 1p36 is a frequent finding in CML blast crisis. RIZ1 is located at 1p36 and frequently undergoes deletion, rearrangements, and loss of heterozygosity in a variety of cancers. Taken together, these data suggest that decreased RIZ1 expression may contribute to CML progression. We used immunohistochemistry to analyze RIZ1 expression in matched bone marrow biopsy specimens from CML chronic phase patients that progressed to accelerated phase or myeloid blast crisis. Similar to control bone marrow, strong cytoplasmic and nuclear RIZ1 expression was observed during chronic phase in all cases. However, RIZ1 expression was found to be markedly decreased in matched bone marrow biopsy specimens obtained during myeloid blast crisis in five patients. RIZ1 expression was maintained in the immature cells of two CML patients, one in accelerated phase with 15% myeloid blasts and the other in myeloid blast crisis, indicating that low RIZ1 expression is not an inherent property of immature hematopoietic cells. To confirm this, we analyzed G-CSF mobilized peripheral blood by flow cytometry and found RIZ1 to be expressed in mature myeloid and CD34+ cells. Transient transfection of myeloid blast crisis cell lines K562, YN-1, ERY-1, and JURL-MK1 with a RIZ1 expression plasmid (pRIZ1) increased the number of cells undergoing early and late apoptosis and reduced viability by 20–80% within 24 hours. RIZ1 effects on erythroid differentiation were assessed in K562, YN-1, and ERY-1 as these cell lines express low levels of hemoglobin, reflecting their myeloid/erythroid progenitor phenotype. As transient RIZ1 expression in these cells is too toxic to measure erythroid differentiation, we modified K562 to express less toxic levels of RIZ1 by stably integrating RIZ1 under the control of a CMV promoter (K562+RIZ1). As determined by benzidine staining, stable expression of RIZ1 in K562+RIZ1 increased erythroid differentiation compared to K562 alone. To confirm that RIZ1 is responsible for the enhanced erythroid differentiation, we transfected K562+RIZ1 as well as ERY-1 and YN-1 (which have higher endogenous RIZ1 levels than K562), with a plasmid that expresses RIZ1 shRNA (pRIZ1shRNA). Expression of pRIZ1shRNA in K562+RIZ1 reduced RIZ1 protein expression and erythroid differentiation to levels similar to that observed in K562 and decreased erythroid differentiation in ERY-1 and YN-1. RIZ1 effects on JURL-MK1 differentiation were assessed by measuring CD33 and CD117 as the expression of these proteins decreases during myeloid differentiation. Transient transfection of JURL-MK1 with pRIZ1 decreased CD33 and CD117 expression as assessed by flow cytometry. In summary, our study demonstrates that RIZ1 expression is frequently reduced in myeloid blast crisis. Furthermore, RIZ1 expression decreases cell proliferation, increases apoptosis, and enhances differentiation in cell line models of CML myeloid blast crisis. Taken together, our results build upon previous observations that a putative CML tumor suppressor gene is present at 1p36 and suggest that deregulation of RIZ1 expression may contribute to CML progression.

Description: Introduction: Cyclophosphamide/Fludarabine (Cy/Flu) nonmyeloablative conditioning for the treatment of acute myeloid leukemia (AML) in remission and myelodysplastic syndrome (MDS) permits engraftment with minimal tissue injury. Unrelated donor (URD) recipients experience rapid neutrophil engraftment, quicker rates to full donor T cell and peripheral blood mononuclear cell (PBMC) chimerism and significantly more effective disease control. Following allogeneic PBMC infusion on day 0, peripheral blood leukocyte counts on day (+) 1 are uniformly less 〈 200/mm3. Mixed chimeric, predominately donor T and NK lymphocytes are detectable on day (+) 6-8 prior to neutrophil engraftment. The lack of respiratory symptoms or splenic enlargement post-infusion suggested that cells may be detectable in the marrow at a much earlier time point post-transplant than is classically recognized after myeloablative allotransplant. We hypothesized that mature lymphocytes would be detectable in a cellular marrow environment by day (+) 8. To begin to test this hypothesis, we collected marrow between day (+) 7-9 (avoiding weekends) from 6 consenting participants with AML, MDS or chronic lymphocytic leukemia (CLL) who participated in a single institution, phase II clinical trial at Indiana University and the IU Melvin and Bren Simon Cancer Center. Methods: Marrow aspirate H and E stains, biopsies, and flow cytometric analyses (including CD2, CD3, CD56 and CD19) were obtained pre-transplant and on day (+) 7-9 following PBMC infusion in the setting of minimal intensity cyclophosphamide/fludarabine conditioning. PMBC chimerism was analyzed on day (+) 7-9. Clinical characteristics and peripheral blood cell counts were recorded. Results: Three patients had AML or MDS and 3 patients had CLL. All patients achieved full engraftment. In the AML/MDS subgroup day (+) 7-9 cellularity ranged from less than 5 to 70% with PBMC chimerism from 39.2 to 93.4% donor. In the CLL subgroup day (+) 7-9 cellularity ranged from 50 to 70% with PBMC chimerism from 0.9 to 5.6% donor. Baseline cellularity, corresponding peripheral cell counts, and marrow flow cytometric analyses from day (+) 7-9 are included in Table 1. Conclusion: Cy/Flu conditioning appears to be associated with early marrow cellularity at a time when peripheral blood chimerism is mixed. Day (+) 7-9 marrow examinations after Cy/Flu are generally quite cellular. Patients with AML/MDS and CLL appear to differ substantially in histological appearance, cell differentials and flow cytometric analyses. This suggests a unique engraftment kinetic that has yet to be appreciated and may be a useful platform for the study of cellular immune anti-cancer activity. Abstract 5851 Table 1 Disease Disease state Baseline cellularity Cellularity Day 7-9 WBC Day 7-9 BM CD2% BM CD3% BM CD19% BM CD56% PBMC chimerism (donor%) AML CR1 60 20 300 75 12 1 86 93.4 MDS active 70 70 900 6 17 3 17 39.2 Secondary AML active 80

Description: Diamond-Blackfan anemia (DBA) is a rare disorder of impaired erythropoiesis belonging to a broader family of inherited and acquired anemias known as pure red cell aplasias (PRCA). The cardinal feature of DBA is aregenerative anemia accompanied by macrocytosis, reticulocytopenia and decreased or absent erythroid precursors in an otherwise normocellular marrow. Classic DBA is associated with mutations of genes encoding ribosome subunits. Advancements in molecular techniques have prompted discussion regarding the genetic basis of the disease and its prognostic implications as non-ribosomal genes are beginning to be implicated in DBA. Acquired mutations within the X-linked transcription factor GATA1 have been described in Down syndrome-related transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AKML), while germline GATA1 mutations affecting both isoforms are associated with congenital thrombocytopenia. Normal alternative splicing of GATA1 produces a full-length GATA1 isoform (flGATA1) as well as a shortened GATA1 isoform (GATA1s), which lacks the N-terminal domain that activates GATA1-driven erythropoiesis program and recruits flGATA1 to a subset of megakaryocyte and erythroblast genes. Rare germline defects known as "GATA1s mutations" are characterized by an unbalanced production of the GATA1s isoform at the expense of a flGATA1 transcript due to disrupting exon 2 splice sites or the ATG initiation codon. GATA1s mutations had been reported to phenocopy Diamond-Blackfan anemia by causing isolated red blood cell aplasia in some patients, although thrombocytopenia with structural platelet abnormalities and dyserythropoiesis as well as DBA-like picture progressing into MDS had been described in other GATA1s families. Therefore, while GATA1s mutations uniformly disrupt erythropoiesis, the impact of defective flGATA production on megakaryopoiesis in non-trisomy 21 individuals is less clear. Here we describe a child with dyserthropoietic anemia, marked megakaryocyte dysplasia, peripheral thrombocytosis and platelet dysfunction due to a novel disease-causing mutation within the 5' UTR of GATA1. DBA was initially suspected due to progressive macrocytic anemia beginning in infancy. Bone marrow analysis revealed not only a paucity of erythroid precursor cells and dyserythropoiesis, but also prominent megakaryocytosis and megakaryocyte dysplasia, not typically associated with classic DBA. Ribosome mutational analysis was unremarkable, as were cytogenetics and MDS-FISH analysis. Given aregenerative anemia mimicking DBA as well as megakaryocyte dysplasia, the question was raised whether the findings could be associated with a germline mutation in GATA1. Indeed, sequencing revealed a novel, pathogenic mutation in the 5'UTR of GATA1 (c.-21 A〉G). We explored the impact of this novel mutation in silico, found it to disrupt a consensus splice site, and further demonstrated that this mutation conferred a striking predominance of the GATA1s isoform at the transcript and protein level. Two-color immunohistochemistry confirmed loss of full-length GATA1 protein in the patient's bone marrow and showed that full-length GATA1 expression is restricted to megakaryocytes and erythroblasts in healthy bone marrow, consistent with dysmegakaryopoiesis and decreased erythropoiesis in our patient. Our findings support a role for the N-terminus of GATA1 in both erythroblast and megakaryocyte maturation and function in vivo. Furthermore, the findings of prominent megakaryocytes in the bone marrow and resultant functional platelet abnormalities may provide subtle clinical clues to differentiate Diamond-Blackfan anemia due to ribosomopathy from dyserythropoiesis secondary to GATA1s mutations. We conclude that GATA1 sequencing, including non-coding GATA1 regions, should be considered in males with congenital multi-lineage dysplasia and/or DBA-like clinical presentation. Disclosures No relevant conflicts of interest to declare.