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Oncogenic alterations underlying B-cell acute lymphoblastic leukemia (B-ALL) in adults remain incompletely elucidated. To uncover novel oncogenic drivers, we performed RNA sequencing and whole-genome analyses in a large cohort of unresolved B-ALL. We identified a novel subtype characterized by a distinct gene expression signature and the unique association of 2 genomic microdeletions. The 17q21.31 microdeletion resulted in a UBTF::ATXN7L3 fusion transcript encoding a chimeric protein. The 13q12.2 deletion resulted in monoallelic ectopic expression of the homeobox transcription factor CDX2, located 138 kb in cis from the deletion. Using 4C-sequencing and CRISPR interference experiments, we elucidated the mechanism of CDX2 cis-deregulation, involving PAN3 enhancer hijacking. CDX2/UBTF ALL (n = 26) harbored a distinct pattern of additional alterations including 1q gain and CXCR4 activating mutations. Within adult patients with Ph− B-ALL enrolled in GRAALL trials, patients with CDX2/UBTF ALL (n = 17/723, 2.4%) were young (median age, 31 years) and dramatically enriched in females (male/female ratio, 0.2, P = .002). They commonly presented with a pro-B phenotype ALL and moderate blast cell infiltration. They had poor response to treatment including a higher risk of failure to first induction course (19% vs 3%, P = .017) and higher post-induction minimal residual disease (MRD) levels (MRD ≥ 10−4, 93% vs 46%, P < .001). This early resistance to treatment translated into a significantly higher cumulative incidence of relapse (75.0% vs 32.4%, P = .004) in univariate and multivariate analyses. In conclusion, we discovered a novel B-ALL entity defined by the unique combination of CDX2 cis-deregulation and UBTF::ATXN7L3 fusion, representing a high-risk disease in young adults.

FT819 is a first-of-kind, allogeneic, off-the-shelf CAR T-cell therapy derived from a clonal master induced pluripotent stem cell (iPSC) line precisely engineered to insert a novel 1XX anti-CD19 chimeric antigen receptor (CAR) under the regulation of the T-cell receptor alpha constant (TRAC) locus for optimized control of anti-tumor activity and to completely delete T-cell receptor (TCR) expression to eliminate the potential of graft-versus-host disease (GvHD). Unlike conventional allogeneic CAR T-cell therapies which require repeatedly sourcing of T cells from various donors as the starting material, the use of a clonal master engineered iPSC line serves as a renewable starting cell source and ensures routine mass production of a uniformly engineered, homogenous CAR T-cell product for broad patient access. T cell-derived iPSCs were generated using a proprietary non-integrating cellular reprogramming system and genetically modified to integrate a novel anti-CD19 1XX CAR into both alleles of the TRAC gene. After single cell subcloning, each engineered iPSC clone was screened for multiple critical quality attributes including pluripotency, identity, genomic stability, cassette integration, on/off-target integration, T-cell differentiation propensity, and CAR T-cell function. Accordingly, the ideal single cell-derived engineered iPSC clone was selected as the clonal master iPSC line for FT819 and was converted into a master cell bank (MCB). The iPSC MCB serves as a renewable source for the routine GMP manufacture of FT819 drug product. The FT819 production process consists of three stages: 1) generation of CD34-expressing hematopoietic progenitor cells from iPSCs (>90% CD34+ cells post enrichment); 2) lineage-specification to T cells followed by T-cell expansion (>5e5 fold expansion); and 3) fill/finish and cryopreservation of the drug product. As an example, in an initial small-scale manufacturing campaign, a total of 2.5 × 10 10 FT819 CAR T-cells were generated and filled and finished starting from one vial of the MCB. The FT819 drug product was tested on safety, identity, purity, and potency. The final product was comprised of CD45+CD7+ lymphocytes (>99%), with homogeneous CAR expression (>99% CAR+) and lacking expression of TCRαβ (not detected) on the cell surface. Importantly, there were no residual iPSCs detected in the FT819 drug product. The FT819 drug product exhibited potent and consistent effector function against NALM6 leukemia cells. The FT819 drug product is currently being used in a landmark Phase I study (NCT04629729), the first-ever iPSC-derived T-cell therapy to undergo clinical investigation, for the treatment of patients with relapsed/refractory B-cell lymphoma, chronic lymphocytic leukemia and precursor B-cell acute lymphoblastic leukemia. In summary, FT819 is a first-of-kind, off-the-shelf, CAR T-cell therapy uniquely derived from a clonal multiplexed-engineered master iPSC line. The novel manufacturing paradigm enables mass production of a uniformly engineered, homogenous cell therapy product that is available on-demand for broad patient access. A multi-center Phase 1 study of FT819 is currently ongoing for the treatment of B-cell malignancies. Key Words: cancer immunotherapy, cell therapy, CAR-T, CD19, allogeneic, induced pluripotent stem cell, iPSC, clonal master iPSC line, engineered, off-the-shelf, cGMP, production, manufacturing, FT819 Disclosures Yuan: Fate Therapeutics, Inc.: Current Employment. Clarke: Fate Therapeutics, Inc.: Current Employment. Lai: Fate Therapeutics, Inc.: Current Employment. Chang: Fate Therapeutics, Inc.: Current Employment. Yang: Fate Therapeutics, Inc.: Current Employment. Hsia: Fate Therapeutics, Inc.: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Lee: Fate Therapeutics, Inc.: Current Employment. van der Stegen: Fate Therapeutics, Inc.: Current Employment. Shaked: Fate Therapeutics, Inc.: Current Employment. Jalloh: Fate Therapeutics, Inc.: Current Employment. Moreno: Fate Therapeutics, Inc.: Current Employment. ORourke: Fate Therapeutics, Inc.: Current Employment. Sung: Fate Therapeutics, Inc.: Current Employment. Gutierrez: Fate Therapeutics, Inc.: Current Employment. Rezner: Fate Therapeutics, Inc.: Current Employment. Eberhart: Fate Therapeutics, Inc.: Current Employment. Magdaleno: Fate Therapeutics, Inc.: Current Employment. Farnan: Fate Therapeutics, Inc.: Current Employment. Plavsic: Fate Therapeutics, Inc.: Current Employment. Bressi: Fate Therapeutics, Inc.: Current Employment. Rivière: Centre for Commercialization of Cancer Immunotherapy: Other: Provision of Services; Fate Therapeutics: Other: Provision of Services, Patents & Royalties; The Georgia Tech Research Corporation (GTRC): Other: Provision of Services (uncompensated); FloDesign Sonics: Other: Provision of Services; Juno Therapeutics: Patents & Royalties. Valamehr: Fate Therapeutics, Inc.: Current Employment.

Introduction: Platelet glycoproteins are key contributors to platelet function but their glycans structure is unclear. Alterations in glycan composition have been reported to impact platelet clearance under physiological conditions and in the disease mechanism of immune thrombocytopenia (ITP). Therefore, this study sought to characterize glycan structures in human platelets from healthy control individuals and ITP patients using mass spectrometry (MS)-based glycomics approach, andto compare their glycomic profiles to facilitate understanding of glycan alterations in ITP. Methods: Glycan residues on platelet surface were determined by flow cytometry. Platelet lysates (1×10 8 platelets) from 4 healthy controls and from 4 ITP patients with a clear anomalous glycosylation pattern were characterized by MALDI-MS based glycomic approaches. N-linked glycans were released from platelet glycoproteins by PNGase F digestion and subsequently purified with a Sep-Pak C18 reverse phase cartridge. O-linked glycans were released by reductive elimination. Both pools of glycans were permethylated prior to MALDI-TOF MS to obtain an initial carbohydrate profile. Selected glycan molecular ion species were analyzed by MALDI-TOF-TOF MS/MS before and after digesting with exoglycosidases. Results: Glycans present in platelets from healthy controls and from ITP patients were largely consistent. The MS spectra for N-glycans showed a mixture of high mannose glycans (m/z 1579.8, 1783.9, 1988.0, 2192.1 and 2396.2); complex glycans (m/z 2966.5, 3776.9 and 4587.4); and bisected or truncated glycans (m/z 3211.6 and 4022.1). The spectra showed the presence of bi-, tri- and tetra-antennary complex glycans, which varied in their level of sialylation.Figure 1 shows the relative abundance ratio within eight families of core glycan structures. Platelets from ITP patients showed a consistent increase in desialylated structures such as Hex 5HexNAc 4Fuc 1. In addition to different amounts of attached sialic acid, varying levels of fucosylation were observed; ranging from the addition of a single core Fuc (m/z 2244.1), to the addition of up to three Fuc residues (m/z 3402.7). Collision-activated decomposition (CAD) MALDI-TOF/TOF analysis was performed to generate fragment ions from molecular ions detected in MALDI-TOF profiling for detailed sequencing of platelet N-glycans. This analysis suggested the presence of three isoforms: (i) sialylated tetra-antennary structure with core fucosylation and one Lewis x/a antenna; (ii) sialylated tetra-antennary structure with one Lewis y/b antenna; (ii) sialylated, core-fucosylated tri-antennary structure with one LacNAc extension and one Lewis x/a antenna. Sialidase S digestion was used to highlight the extent of desialylation in the presence of sialidase. Noteworthy, the ratio of non-sialylated bi-and tri-antennary N-glycans in ITP patients were higher than that in controls. This enzymatic digestion confirms the presence of α2,3-linked Neu5Ac on platelet glycans. Remaining sialylated structures may possess α2,6-linked Neu5Ac. O-glycan profiles obtained showed the predominance of core 1 and core 2 structures (Figure 2). The two most dominant glycan structures in core 1 were sialyl T antigen (GalNAc 1Gal 1NeuAc 1; m/z 895.5) and disialyl T antigen (GalNAc 1Gal 1NeuAc 2; m/z 1256.7), being the latter less abundant in ITP patients. The core 2 structure was modified by the addition of fucose and/or sialic acid residues (m/z 1157.7, 1344.8, 1518.9 and 1706.0). Conclusion: N- and O-glycan structures in human platelets were characterized by MALDI-TOF MS profiling to reveal interesting structural features including the presence of sialylLewis x/a epitope, Lewis x/a epitope, Lewis y/b epitope and LacNAc extensions in complex type N-glycans. Presence of terminal sialic acid and sialylLewis x/a on platelet N-glycan antenna also suggest their potentialfunction as ligands for siglecs that are associated with cell signaling functions. Siglec-1 and -2 have been suggested to have potential roles in ethiopathogenesis of autoimmune diseases. Desialylation observed in glycans of platelets from ITP patients, might trigger immune system activation in these patients. This research was funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Butta: Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; CSL-Behring: Research Funding; Novo-Nordisk: Speakers Bureau. Canales: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; Incyte: Consultancy; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead/Kite: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; Eusa Pharma: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Jiménez-Yuste: Pfizer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Alvarez Román: Octapharma: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

Programmed death 1 (PD-1) receptor and its ligand (PD-L1) facilitate immune evasion in multiple myeloma (MM). We hypothesized that pembrolizumab, PD-1-antibody, can enhance antimyeloma cellular immunity generated by pomalidomide, leading to improved clinical responses. In this single-center, phase 2 study, 48 patients with relapsed/refractory MM (RRMM) received 28-day cycles of pembrolizumab, 200 mg IV every 2 weeks, pomalidomide 4 mg daily for 21 days, and dexamethasone 40 mg weekly. Patients had a median of 3 (range: 2-5) lines of therapy, median age 64 (range: 35-83) years, and had received both an immune modulatory drug (IMiD) and proteasome inhibitor: (35 [73%] of 48) were refractory to both; (31 [70%]) had received an autologous transplant, and (30 [62%]) had high-risk cytogenetics. Adverse events grade 3 to 4 occurred in (19 [40%] of 48 patients), including hematologic toxicities (19 [40%]), hyperglycemia (12 [25%]), and pneumonia (7 [15%]). Autoimmune events included pneumonitis (6 [13%]) and hypothyroidism (5 [10%]), mostly ≤ grade 2. Objective responses occurred in (29 [60%] of 48) patients, including stringent complete response/complete response (4 [8%]), very good partial response (9 [19%]), and partial response (16 [33%]); median duration of response was 14.7 months. At median follow-up of 15.6 months, progression-free survival (PFS) was 17.4 months and overall survival was not reached. Analyses of pretreatment marrow samples revealed a trend for increased expression of PD-L1 in responding patients and longer PFS with increased T-lymphocyte infiltrates, irrespective of PD-1 expression. Pembrolizumab, pomalidomide, and low-dose dexamethasone have acceptable safety and durable responses in RRMM patients. This trial was registered at www.clincialtrials.gov as #NCT02289222.

The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galβ1,4GlcNAc) to create sialyl Lewis-X (sLe(X)) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe(X) epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34(+) hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a low platelet count (≤100x109/L) due to platelet destruction and insufficient platelet production. There is a wide variation in the presentation of the disease, the clinical course and the response to therapeutic treatments. It has been proposed that certain autoimmune diseases might be caused by changes in glycosylation of cell surface proteins which induce the immune system to recognize cells as "non-self". The aim of this work was to evaluate whether glycosylation might be involved in ITP ethiopathogenesis. So, in this preliminary study we aimed to evaluate the glycosylation pattern of platelets from ITP patients, its relationship with platelet features and with the response to treatments. Methods: Seventy eight ITP patients were recruited: 32 without treatment (UT-ITP) for at least six months, 35 responders to thrombopoietin receptor agonists (TPO-RA, 62.8% with eltrombopag, 27.2 % with romiplostim), and 11 refractory (r-ITP). Eighty one healthy controls were also included. Human peripheral blood samples were collected in 3.8% sodium citrate. Platelet activation was determined by flow cytometry (FCM) through binding of FITC-PAC1 (a mAb that recognizes the activated conformation of fibrinogen receptor) after activation with 100 mM thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland). Apoptosis was evaluated by measurement of active caspase-3, -8 or -9 by FCM with a specific kit (Millipore, Madrid, Spain). Platelet surface exposure of lectins was analyzed with 1 µg/ml FITC-conjugated Wheat germ agglutinin lectin (WGA) or 1 µg/ml FITC-conjugated Erythrina cristagalli lectin (ECA). WGA binds to sialic acid and N-acetylglucosaminyl residues and ECA is a galactose specific legume lectin. Glycoprotein derived glycans from a healthy control and from a r-ITP patient were characterized by MALDI-MS based glycomic approaches. N-linked glycans were released from platelet glycoproteins by PNGase F digestion whilst O-linked glycans were released by reductive elimination. Both pools of glycans were permethylated prior to MALDI-MS. Comparisons of quantitative variables were made with SPSS.22 software. Results: Platelet count (x109/L) was 269±55 in controls, 141±126 in UT-ITP, 111±85 in TPO-RA and 8±5 in r-ITP. ITP patients with the lowest platelet count showed the most pronounced binding of WGA and ECA (Table 1). Moreover, binding of these lectins showed a mild positive correlation with caspase activities (Table 1). No relationship was observed between platelet ability to be activated and glycosylation features. When lectins' binding was studied stratifying patients according to their response to therapeutic treatments we observed that platelets from all ITP patients bound more ECA and WGA (Figure 1). Despite no significant differences were observed between ITP groups, r-ITP seemed to bind more WGA. To increase our knowledge about protein glycosylation of platelet proteins in ITP patients we performed glycomic analyses of platelets from one healthy control and from one r-ITP patient. It was demonstrated that bi-, tri- and tetra-antennary complex type N-glycans are the dominant class of N-glycans with also some high mannose structures. Analysis of sialylated N-glycan, via digestion with sialidase, revealed lower levels of sialylation in r-ITP patient platelets compared to healthy control platelets. Platelet O-glycan data revealed the presence of mucin-type core-1 and core-2 structures. Conclusion: Glycomic analysis demonstrates that human platelet glycoproteins are modified with a complex array of both N- and O-linked glycans. Preliminary data indicates that there could be changes in glycosylation associated with immune thrombocytopenia. These changes in platelet protein glycosylation might be involved in an enhancement of apoptosis in platelets from ITP patients. Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). Disclosures Álvarez-Roman: Shire: Consultancy; NovoNordisk: Consultancy; SOBI: Consultancy. Jimenez-Yuste:Bayer: Consultancy, Research Funding; Sobi: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; NovoNordisk: Consultancy, Research Funding; Shire: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.

Acute myeloblastic leukemias (AML) are the most frequent acute leukemias in adults. Recent genomic and candidate gene studies identified novel recurrent somatic mutations in patients with AML with biological, clinical and therapeutic significance. In Uruguay, the characterization of AML is dependent on the integration of cytomorphology, immunophenotype, cytogenetics and molecular biology (mutations in FLT3, NPM1, CEBPA and cKIT). This allows stratification in prognostic risk groups and rationalization of therapeutic resources. However, with this molecular markers, complete stratification is not always succesful in patients with normal karyotype ( NK) . In order to expand the AML genetic markers analysis in Uruguayan patients ( 3.000.0000 persons country ) ; DNA samples from 49 adult patients at the onset of the disease and 3 healthy controls were analyzed by next generation sequencing (NGS, Illumina) using a customized panel of 30 cancer associated genes. After excluding synonymous variants and those with a population frequency >1%, we ended with 493 variants in promoters and intron ends, and 173 in the 28 genes coding regions. Notably, all patients had at least one variant. While none of the non-coding region variants were reported as pathogenic in COSMIC and/or ClinVar databases, 45 of those falling in coding regions were reported as pathogenic. The pathogenic variants were detected in 36/49 patients and 1 healthy control distributed in 16 genes. The most affected genes were TET2, NRAS, DNMT3, FLT3 and cKIT with 7, 6, 5, 4, 4 variants respectively. This preliminar findings in this study, highlight the relevance of detecting gene variability underlying prognostic risk subgroups to deliver patient tailored clinical decision support. The correlation between the genetic abnormalities, associated with the clinicopahtological features and the epigenetic studies have prognostic significance, and are continued to be analyzed. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Immunotherapy in MM is emerging as an effective modality in therapy of MM with the approval of several monoclonal antibodies and encouraging results for vaccines and T cell therapy. Programmed death 1 (PD-1) receptor and its ligand (PD-L1) is one mechanism of immune evasion by MM to suppress T cell function. In this trial, we hypothesized that pembrolizumab, a PD-1-blocking antibody, would enhance immune modulatory properties of pomalidomide in RRMM pts. METHODS: In this single center, phase II study, 48 patients with RRMM received 28-day cycles of pembrolizumab (at a dose of 200 mg IV) every 2 weeks (in a run in phase, first 6 patients received 200 mg IV every 4 weeks) plus pomalidomide (4 mg daily x 21 days) and dexamethasone 40 mg weekly. Study objectives were measurements of safety & efficacy and correlation of the CD3/PD-1 on T cells and PD-L1 on plasma cells with response. RESULTS: The median age was 64 years (range: 35-82); 38% were black and 65% were men, Patients had a median of 3 lines of prior therapy (range: 2-6); All patients had received both IMids and Proteosome inhibitors; 70% had prior auto-SCT. 80% were double refractory to both IMids (lenalidomide) and Proteosome inhibitors [bortezomib (n=18) or carfilzomib (n=20)] and an additional 20% were refractory to lenalidomide. The median time from MM diagnosis to study entry was 4 years (range: 1-25). Most common cytogenetic abnormalities were 1q+ (60%), hyperdiploidy (15%) and high-risk FISH [del 17p, t(4:14) and/or t(14:16)] in 38%. Six patients had soft tissue extramedullary plasmacytomas. There were no infusion-related reactions. Hematologic toxicities (≥ grade 3) were anemia (21%), neutropenia (40%), lymphopenia (15%) and thrombocytopenia (8%). Non-hematologic events Grade ≥3 were fatigue (15%), hyperglycemia (25%), upper respiratory tract infections (21%), rash (10%); and most frequent grade ≥2 were dyspnea (54%), dizziness (44%), increased creatinine 38%, edema (35%), rash (30%), constipation 30%) and arrhythmias (19%). Events of clinical significance, autoimmune mediated, included interstitial pneumonitis (13%), hypothyroidism (10%), transaminitis(6%), adrenal insufficiency (4%) and vitiligo (2%). Nine pts had pomalidomide dose reductions due to rash, neutropenia, palpitations and fatigue; one pt reduced pembrolizumab for pneumonitis. At a median follow up of 10 months (range: 2-18): 25 pts continue on the study and 23 pts discontinued therapy due to disease progression (n= 15), side effects (n=7) and protocol violation (n=1). Five pts died while on study due to progressive disease (n=3), sepsis (n=1, sAE), and one from a cardiac event. Three additional pts died off therapy. On intent to treat analysis; the overall response rate (ORR) with ≥ Partial response were observed in of 27 of 48 pts (56%) including: sCR (n=4, 8%), nCR (n=3, 6%), VGPR (n=6, 13%), PR (n=14, 29%). Additionally, 7 pts (15%) had minimal response, 9 (19%) had stable disease, 2 progressed and 3 were not evaluable for response. Of 38 double refractory pts ORR was 55% including, sCR (n=2, 5%), nCR (n=2, 5%), VGPR (n=4, 10%) and PR (n=13, 27%). Of 18 high-risk pts ORR was 33% including VGPR (n=2, 11%) and PR (n=4, 22%). Median duration of response for responding pts was 8.8 months and for pts ≥ VGPR, DOR was 10.7 months. Correlation of PD-1 and PD-L1 expression and response will be presented. CONCLUSION: Pembrolizumab, pomalidomide and dexamethasone shows promising durable therapeutic activity and an acceptable safety profile in RRMM pts. ClinicalTrials.gov number, NCT02289222 Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Several recent studies have linked the interactions of programmed death 1 (PD-1) receptor and its ligand (PD-L1) to immunologic control of MM. Expression of PD-L1 on myeloma cells and the abundance of PD-1 on various bone marrow microenvironment components contribute to tumor-mediated immune suppression. We hypothesized that pembrolizumab, a PD-1-blocking antibody, will activate myeloma specific cytotoxic T cells that can be enhanced by pomalidomide in RRMM patients to induce clinical responses. METHODS: In this ongoing single arm, phase II study, 24 patients with RRMM received 28-day cycles of pembrolizumab (at a dose of 200 mg IV) every 2 weeks (in a run off phase, first 6 patients received 200 mg IV every 4 weeks) plus pomalidomide (4 mg daily x 21 days) and dexamethasone 40 mg weekly. Study objectives were measurements of safety and efficacy and assessment of the PD-1 and PD-L1 protein expression in bone marrow samples. RESULTS: The median age was 65 years (range: 41-75); 35% were African American and 71% were men. Of the 24 patients, 75% had prior autologous transplantation and 96% were refractory to last therapy. All patients had received both IMids and Proteosome inhibitors; 75% were double refractory to both IMids and Proteosome inhibitors and additional 21% were refractory to lenalidomide alone. Patients had received a median of 3 lines of prior therapy (range: 1-6). The median time from MM diagnosis to study entry was 4 years (range: 1.2-15). All patients had abnormal cytogenetics: most common were 1q+ (72%) and high-risk FISH (40%) [del 17p, t(4:14) and/or t(14:16)]. There were no infusion-related reactions. Hematologic toxicities (≥ grade 3) were neutropenia (29%), lymphopenia (17%) and thrombocytopenia (8%). Non-hematologic adverse events included (Grade ≤2; ≥3): fatigue (n=12; 1), constipation (n=10; 0), dyspnea (n=9; 2), itching (n=6; 0), muscle spasms (n=6; 0), infection (n=4; 3), hyperglycemia (n=5; 0), edema (n= 4; 0), fever (n=3; 0), palpitation (n=2; 1), rash (n=3; 1) and hypotension (n=3; 0). Events of clinical significance, autoimmune mediated, included hypothyroidism (n=2), transaminitis (n=2), and pneumonitis (n=1). Four patients had pomalidomide dose reductions due to rash, neutropenia, palpitations and fatigue. Two patients died; one after cycle 1 (progressive disease) and one during cycle 2 (sepsis). Objective responses (modified IMWG criteria) were observed in 11 of 22 evaluable patients (50%) including: near complete response (n=3), very good partial response (n=2), partial response (n=6); additionally, 3 patients had minimal response, 6 had stable disease and 2 progressed. At a median follow up of 16 weeks; 17 of 22 patients continued on the study. Reasons for discontinuation included disease progression (n= 4) and protocol violation (n=1). Analysis of pretreatment and post-treatment tumor specimens for PD-1 and PD-L1 is in progress. CONCLUSIONS: Pembrolizumab in combination with pomalidomide and dexamethasone has promising therapeutic activity and an acceptable safety profile in heavily treated RRMM patients. ClinicalTrials.gov number, NCT02289222. Disclosures Off Label Use: Pembrolizumab.

To the editor: We routinely perform clinical exome sequencing on blood samples from patients without bone marrow transplants (BMTs). Moreover, we accept saliva samples due to the acceptable DNA quality, noninvasiveness, and improved percentage of participants.[1][1] We assess the quality at the wet

BACKGROUND: In patients with sickle cell disease (SCD), renal disease is strikingly high, with up to one third of adults developing chronic kidney disease. Renal disease is associated with considerable morbidity and mortality although it has not been as well studied as other SCD-related complications. Studies which could provide insight into clinically silent renal injury are limited. To date, no large studies of functional or structural renal changes have been published, particularly in the context of sickle cell crises. Magnetic Resonance Imaging (MRI) is safe and capable of providing quantitative assessments of disease without ionizing radiation. Clinically available MRI techniques have already been used to assess both acute and chronic kidney disease. This is the first study to evaluate these MRI techniques in SCD renal disease. OBJECTIVE: The objective of this pilot study was to evaluate the capability of quantitative MRI techniques to assess both structural changes and renal blood flow in patients with SCD. METHODS: Pediatric and adult patients were recruited to an IRB approved study from the Sickle Cell Anemia Centers at UH Rainbow Babies & Children’s Hospital and University Hospitals Case Medical Center. Six pediatric patients (age 12-18 years, 1 male, 5 female, eGFR 110-185 mL/min/1.73m2) have been recruited to date. Healthy controls were also recruited. Diffusion (DTI) and Arterial Spin Labeling (ASL) scans were performed to assess medullary microstructure and cortical perfusion (non-contrast), respectively. Apparent Diffusion Coefficient (ADC) and Fractional Anisotropy (FA) maps were calculated using established methods. Mean renal T2* was measured to assess iron burden. A Student’s t-test was performed to compare the mean renal DTI and ASL results between subjects and healthy controls. RESULTS: In the six pediatric subjects, medullary FA values were decreased, suggesting degradation of medullary microstructure. Both cortical ADC and medullary FA were significantly reduced, indicative of microstructural changes in SCD subjects compared to controls. ASL perfusion maps, likewise, showed reduced cortical perfusion for SCD subjects in comparison to controls. Preliminary differences in blood flow and microstructure were seen between SCD subjects on hydroxyurea (HU) and those not on HU. Analysis of the T2* data showed increased renal iron deposition that did not appear to correlate with serum ferritin. CONCLUSIONS: Our results suggest that quantitative diffusion and ASL MRI are sensitive to medullary microstructural and cortical blood flow changes in pediatric patients with SCD. Preliminarily, iron deposition in the kidney appears to relate to chronic hemolysis rather than transfusional iron. These sensitive techniques may be useful in assessing the impact of therapeutic interventions, such as HU, on renal function in children. There may also be utility in assessing the impact of acute hemolysis/sickling on renal microstructure and perfusion. Disclosures Piccone: Novartis Pharmaceuticals Cooperation: Speakers Bureau.

Analysis of patients with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling differentiation, maintenance, and decay of neutrophil granulocytes. Mutations in ELANE, GFI1, HAX1, G6PC3, WAS, and VPS45 are known to cause SCN. We here describe a new monogenetic SCN variant with biallelic mutations in the gene encoding Jagunal homolog 1 (JAGN1). We studied an index family from Northern Africa with a total of 5 children suffering from SCN. An Affymetrix SNP array-based genetic linkage analysis was performed and identified a single interval of perfect segregation with highly significant multi-marker LOD scores of at least 4.5spanning approximately 1.5Mbp from 9.52Mb to 11.04Mb on chromosome 3 of NCBI’s human genome build 36.3. This interval contained a total of 30 genes, including JAGN1 which encodes an ER-resident protein. Sanger sequencing revealed a homozygous mutation c.3G>A in exon 1 of the JAGN1 gene; this mutation leads to disruption of the defined start of translation. Systematic analysis of a cohort of 90 SCN patients identified 9 distinct homozygous mutations in the gene encoding Jagunal homolog 1 (JAGN1) in 14 SCN patients, thus accounting for approximately 10% of SCN patients. The clinical phenotype was variable and included failure to thrive, developmental delay and bone skeletal abnormalities. The only consistent finding in all JAGN1-deficient patients was SCN and partial or complete refractoriness to therapy with rh-GCSF. JAGN1 is the human ortholog of a gene originally identified in Drosophila melanogaster. Jagunal-deficient oocytes are characterized by defective ER reorganization and aberrant membrane trafficking during vitellogenesis. We found that JAGN1-mutant human granulocytes showed aberrantly enlarged ER structures and paucity of secretory vesicles. We hypothesized that that ER aberrations may be associated with defective N-glycosylation of multiple proteins in neutrophil granulocytes and found that JAGN1-mutant neutrophil granulocytes exhibited anomalous N-glycomic profiles characterized by a marked reduction in fucosylation of all their multi-antennary glycans. JAGN1-deficient neutrophil granulocytes showed increased apoptosis in response to TNFa and staurosporine, likely accounting for the lack of mature neutrophils in these patients. Additional studies in JAGN1-knockdown cells indicate that JAGN1 participates in the secretory pathway and is required for granulocyte-colony stimulating factor receptor-mediated signaling. Global proteomic analysis of the JAGN1-interactome identified a limited number of interaction partners including members of the Coat Protein I (COPI) complex (COPA, COPB2, and COPG2) which suggest a role for JAGN1 in vesicular trafficking from Golgi to ER. Taken together, JAGN1 emerges as a hitherto unrecognized factor necessary in differentiation and survival of neutrophil granulocytes and a novel gene implicated in SCN. Disclosures: No relevant conflicts of interest to declare.

Abstract 5032 Introduction. Combining bortezomib with pegylated liposomal doxorubicin for the treatment of Multiple Myeloma (MM) may reciprocally increase their efficacy in vitro and appears to offer higher overall response rates in vivo. With the aim to verify the synergistic interaction towards myeloma cells we planned a prospective study with bortezomib, non-pegylated liposomal doxorubicin and dexamethason in patients with relapsed/refractory multiple myeloma (R/R MM). In this setting of patients, showing a poor outcome because of multi-drug resistance, low-performance status and toxicity to previous chemotherapy, bortezomib, by inhibiting proteasome function, may enhance chemosensitivity to doxorubicin and overcome drug-resistance. Thus, to improve outcome minimizing therapy-related toxicity, bortezomib was added to non-pegylated liposomal doxorubicin and dexamethason (PAD regimen). Patients and Methods. From November 2005 and January 2012, 50 patients with R/R MM (relapsed n= 37 and refractory n= 13) referred to the our Institution received PAD regimen. Male/Female ratio: 27/23; Median age: 61 years (range 34–79); median time from diagnosis: 32 months (range 4–121); median of previous therapy lines: n= 3 (range 1–5); patients previously underwent to autologous and allogeneic hemopoietic stem cell transplantation (auto- and allo-HSCT): 22 and 5, respectively. Planned treatment: bortezomib 1, 3 mg/mq iv days 1, 4, 8, 11; non-pegylated liposomal doxorubicin 30 mg/mq on day 1 and dexamethason 40 mg days 1–4 of a 28-day cycle up to 6 cycles. Results. Forty patients (80%) received the planned treatment schedule whereas 10 patients did not complete it because of toxicity (1 patients) and resistant or progressive disease (9 patients). Median time to best response was 3 months (range 2–6). The overall response rate was 74% with 10 CR (20%), 15 vGPR (30%) and 12 PR (24%). Fifteen of the responder patients underwent HSCT (auto-HSCT: 9; allo-HSCT: 6). The previous use of anthracyclines (35 patients) and bortezomib (3 patients) did not seem influence the response. Median duration of response was 24 months (range 6–57 months). After a median follow-up of 58 months, 14 (28%) patients were alive and, of these, 7 (14%) in Continue CR. The safety profile was manageable: the hematologic grade 3/4 toxicity (neutropenia, thrombocytopenia and anemia) was 28%; non-hematologic grade 3/4 toxicity (sensory or motor neuropathy, infections, fever, fatigue, diarrhea) was 36%. Despite heavy previous treatments, including anthracycline-based, no significant cardiac toxicity was observed. Conclusion. According to our study, the PAD regimen appears feasible and effective in both elderly and heavily pre-treated R/R MM patients. In fact a significant improved clinical outcome in our cohort of patients was observed. Disclosures: No relevant conflicts of interest to declare.

Abstract 4642 Background BARD1 (BRCA1-associated RING domain protein 1) was first described as a BRCA1 partner and tumour suppressor protein, muted in most cases of breast and ovarian cancer. BARD1 functions are BRCA1-dependent, requiring formation of a stable heterodimer through interaction of the RING finger domains. BRCA1-independent functions for BARD1 have recently been described, making it an interesting and important molecule for study. BARD1 is expressed in almost all human tissues, including haematological cells, testis and breast tissue, but it is over-expressed in leukaemias, sarcomas and testis cancer, implicating BARD1 in development of cancer. Different BARD1 isoforms are up-regulated in breast, ovarian and uterine cancers but markedly down-regulated or absent in healthy tissues. Therefore, the presence of these isoforms might be a risk factor or causal event in the cancer pathogenesis. We investigated the role of BARD1 isoforms in leukaemia, through the study of the epigenetic mechanisms involved in regulation of its expression and function. As such, we determined the expression of a specific BARD1 isoform in different human leukaemia cell lines the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid, Vorinostat) of expression of BARD1. Results. Four human leukaemia cell lines (U937, NB4, K562 and HL60) express the BARD1 isoform of interest. Treatment of these cell lines with SAHA at 5 μ M resulted in decreased expression of this isoform (Fig. 1). Decreased expression of BARD1 by SAHA was also observed in in human breast cancer MCF7 cells, the usual model for BARD1 experiments and in the human neuroblastoma cell line Kelly (Fig. 2), but not in HeLa cells or an human epithelial carcinoma cell line (Fig. 2). The reduced expression of BARD1 was attributed to the action of 2 specific micro-RNAs (miRNAs), that directly bind its 3′untranslated region (UTR). SAHA-induced expression of miR-19a and miR-19b in primary human leukemia cells as demonstrated using miRNA microarray expression analysis and confirmed by Real-Time PCR (Fig. 3). These 2 miRNA were up-regulated after SAHA stimulation in human leukaemia cells. BARD1 is the predicted target for miR-19a and miR-19b based on miRBase database analyses. Transient transfection in NB4 cells with mimic miR-19a and mimic miR-19b confirmed expression of these miRNAs was associated with BARD1 down-regulation (Fig. 4 5.). The BARD1 3′UTR was cloned into a pGL3 vector with a downstream the luciferase gene reporting gene (Fig. 6). The plasmid was co-transfected in HeLa cells with each mimic miRNA and a luciferase assay was performed. We demonstrate that expression of miR-19a and miR-19b can directly bind BARD1 3′UTR, reducing luciferase activity in this system (Fig. 7), thereby confirming that BARD1 is a target of these miRNAs. These findings support our hypothesis that BARD1 is a promising target in leukemia and should be further investigated for its diagnostic and prognostic features. Disclosures: No relevant conflicts of interest to declare.

Abstract 1614 Poster Board I-640 Background Cytogenetic abnormalities in B-cell CLL are important prognostic indicators for predicting disease progression and treatment response. Until recently, only interphase cytogenetics has been readily applicable to clinical use in CLL due to the inability of traditional mitogens to promote effective metaphase cytogenetic identification. Recently, cytokine or CpG oligonucleotide stimulation has been utilized to promote efficient metaphase analysis. One concern with this approach is the actual clonality and reproducibility of such clones when examined by different cytogenetic laboratories. The CLL Research Consortium (CRC) conducted a prospective trial to test (1) whether CpG would enhance detection of abnormal clones and (2) whether the CRC cytogenetic laboratories would achieve similar results when testing the same patient samples. Methods Initially, one laboratory compared stimulation of CLL cells using CpG versus pokeweed mitogen (PWM) + 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the same samples. To test the reproducibility of CpG stimulation, fresh blood samples from 1 normal control and 12 CLL patients were collected at one site and distributed (blinded to phenotype and prior cytogenetic results) to 5 CRC cytogenetic laboratories where they were cultured for 3 days using CpG stimulation, harvested, banded and analyzed utilizing standard laboratory procedures. When possible, each laboratory analyzed 20 metaphases per case. Interphase FISH analysis for CLL (including probes for chromosomes 6q, 11q, 12, 13q, 17p, and IGH/CCND1) was also done on each sample. Results In the PWM+TPA versus CpG comparison, more cases were found to have clonal abnormalities with CpG stimulation (147 of 229, 64%) than with PWM+TPA (110 of 229, 48%; P=0.0005). All clonal abnormalities detected in the PWM+TPA cultures were also observed in the CpG cultures. In the 5-laboratory CpG comparison, the results were compiled and compared to the concurrent FISH results. The CpG karyotypes were concordant with the FISH results. The control case had a normal CpG-stimulated karyotype in all 5 laboratories. Three cases exhibited a complex karyotype; one of these also exhibited 11q-, one had 17p-, and one had neither. The cytogenetic descriptions varied modestly among labs, but all identified the complex clones. Every case including the control exhibited some nonclonal abnormal cells, but no pattern was detected among the 5 labs. When an abnormal clone was found by only 1 or 2 labs, it was present in only a very small number of cells. A small 13q- clone was observed in 2 cases in 1 and 2 labs (3 of 20 and 5 of 50 cells, respectively), and was confirmed by interphase FISH. A single lab found 2 of 30 cells with a t(12;15) in one case. Conclusions Abnormal clones in CLL are more readily detected with CpG stimulation than with traditional B-cell mitogens. The clonal abnormalities revealed by CpG stimulation are consistent with the “gold standard” interphase FISH results, and are reproducible among different cytogenetic laboratories. We have no evidence that CpG oligonucleotides create new clonal B-cell populations in vitro during 3 days of cell culture. CpG stimulation in vitro reveals clonal cytogenetic abnormalities and complexity in CLL that should be evaluated as potential independent prognostic parameters. Disclosures Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

Fluorescence in situ hybridization (FISH) probes and analysis methods for B-cell Chronic Lymphocytic Leukemia (CLL) vary extensively among cytogenetic laboratories. This is not unexpected, as neither national nor international standards have been established for most FISH studies. Lack of standardization is problematic when data collected at multiple institutions are used for clinical correlative studies. To circumvent such problems, the five participating laboratories in the CLL Research Consortium (CRC) designed and executed a joint CLL FISH validation study. Methods: Initially a survey was sent to assess equipment, methods and experience with FISH for CLL. In a pilot study to compare laboratory performance in scoring patient samples, slides from ten patients were prepared and sent to each participating lab to be hybridized with five probe sets (= 50 hybridizations) and analyzed according to their local protocol. In a second pilot study, slides from two patient samples and identical probe sets were sent to the participating labs where hybridization and analysis were carried out according to their local protocol. Next, technologists and directors from all participating labs attended a workshop where technologists working in pairs scored nuclei together, techniques and scoring criteria were established, and consensus reached on other concerns. In a proficiency test nine months after the workshop, slides from two patient samples (10 hybridizations) were hybridized and scored according to each lab’s protocol and results shared using a common reporting form. Results: Survey results indicated that four labs used the same commercially available CLL FISH panel, and one used a combination of probes from the same vendor plus several home-brew probes. Each lab scored between 100 and 200 nuclei per hybridization site, and each independently set normal cutoff values. The FISH panel included probes to detect 11q, 13q, and 17p deletions, trisomy 12, and IGH gene rearrangement. One lab included probes to detect 6q deletion. In the first pilot study each lab used their hybridization methods, probe sets, and scoring criteria. Differences among labs were observed due to variations in probe strategy, reporting of anomalies, and perhaps most important, scoring criteria. Probe strategy differences resulted in variable reporting of 11q- vs monosomy 11 and 12q duplication vs trisomy 12. Some participants reported 13q-x1 and 13q-x2 as subclones and some reported only 13q-. One lab reported an IGH rearrangement whereas the others scored IGH as normal. In the second pilot study each lab used the same methods and probe sets to facilitate comparison of scoring by the technologists. All labs correctly identified the abnormalities, and there were no false positive results. Minor scoring differences were attributed to variation in scoring criteria or inexperience with an unfamiliar FISH probe strategy. The proficiency test that followed the workshop demonstrated 100% concordance in identification of abnormalities. Inter-lab scoring was much improved compared to the first pilot study. The only exceptions were a 13q- range of 72–90% in one case, and a 17p- range of 38–67% in another case. Conclusion: The pilot studies identified a need to develop common scoring criteria. The subsequent workshop and proficiency test demonstrated that the collaborative effort resulted in more standardized scoring among the CRC laboratories. Our collaborative study emphasizes the need to establish rigorous standards and guidelines for FISH procedures and scoring criteria. Standardization of FISH methods among participating laboratories will enhance the confidence in FISH studies for both clinical applications and cooperative intergroup clinical research.

Introduction: The prevalence of pulmonary hypertension (PHT) is high in patients with sickle cell disease (SCD). Although most patients have only mild increases in their pulmonary artery systolic pressure (PASP), the presence of PHT is strongly associated with an increased risk of death. While PHT seen in SCD is thought to progress over time, both the rate of development of PHT and the factors that affect disease progression remain unknown. Methods: The 41 subjects in this study were drawn from an original cohort of 60 patients followed in the Sickle Cell Clinic at UNC-Chapel Hill. All patients were previously evaluated for PHT (defined using an age-, sex-, and BMI-adjusted reference range). Of the 60 patients in the original cohort, six are now deceased and 13 others were not available for repeat evaluation. The PASP was determined using Doppler echocardiography and then applying the modified Bernoulli equation (PASP = 4V2 + right atrial pressure). Individuals were not studied if they: 1) showed clinical evidence of left ventricular failure; 2) had a recent acute illness (e.g., vaso-occlusive crisis); or 3) had experienced an episode of acute chest syndrome within the preceding 4 weeks. Means and standard deviations were calculated for all measures at the time of initial evaluation and at the time of follow-up. Results: Of the 41 subjects in our study, PHT was originally present in 12, while no evidence of PHT was present in 29. Of the 29 subjects who initially had no evidence of PHT, 4 (or 14%) have now developed PHT (mean follow-up period of 3.3 ± 0.4 years). In these 4 subjects, the mean PASP at the time of initial and follow-up evaluations respectively were: 37.0 ± 2.0 mm Hg vs. 55.8 ± 11.0 mm Hg. The patients who developed PHT during the course of the study had lower systolic BP (143 ± 12 mm Hg vs. 128 ± 12 mm Hg), lower fetal hemoglobin levels (6.2 ± 5.7 % vs. 4.2 ± 3.7 %), and higher platelet counts (276 ± 119 X 103/μL vs. 426 ± 96 X 103/μL) at the time of their follow-up analyses. By contrast, 3 of the 12 subjects (or 25%) who were thought to have PHT at the time of their original evaluations were found to have normal PASP determinations at the time of their repeat echocardiograms (mean follow-up period of 3.2 ± 0.6 years). In these latter 3 subjects, the mean PASP values at the time of the initial and follow-up evaluations respectively were: 40.0 ± 4.6 mm Hg vs. 33.7 ± 4.7 mm Hg. Conclusion: In this small group of patients with SCD, we found that PHT developed in 14% of subjects who had no evidence of PHT 3 years earlier. Based on this observation, it seems that periodic echocardiograms to screen for the development of PHT would be appropriate. On the other hand, our observation that some patients initially classified as having PHT failed to have elevated PASP measurements at the time of follow-up illustrates the limitation of a single echocardiographic evaluation in establishing this diagnosis. Because of the increase in PASP that occurs during acute vaso-occlusive episodes, and the difficulty usually encountered in distinguishing steady state from crisis, the initial elevation of the PASP in these patients could have resulted from sub-clinical crisis states. For these reasons, a patient found to have an elevated PASP at the time of a screening echocardiogram should have a repeat study, and perhaps a right heart catheterization, before the diagnosis of PHT is firmly established.

Aim: To perform a systematic review of the efficacy of monotherapy with bortezomib versus thalidomide in patients with relapsed or refractory multiple myeloma. Methods: Published English literature from 1966 to June 2005 (MEDLINE, EMBASE, Cochrane library), publication reference lists, Janssen-Cilag Pty Ltd data-on-file, and abstracts from recent multiple myeloma conferences were reviewed. Prospective studies containing at least a single arm of any treatment group with n ≥ 30 and using continuing or variable thalidomide dosing were included. Studies adding dexamethasone for non-responders were excluded. Outcomes were analysed on an intent-to-treat basis. Statistical pooling was performed where possible for the following outcome measures: primary outcome of response rate, defined by a serum M-protein reduction ≥50% (A) and strict (e.g. EBMT) criteria (B), and for the secondary outcomes of overall survival and progression-free survival. Results: One bortezomib (n=333, APEX, NEJM2005, 352; 2487–98) and 15 thalidomide (n=1007) studies were included. Patient baseline characteristics including age, gender, IgG:IgA, disease duration and β2M were well matched, except that 48% of bortezomib patients had received prior thalidomide. On an intent-to-treat basis, the overall estimate for response rate (A) was 53% for patients receiving bortezomib versus 32% for thalidomide (p Conclusion: In patients with relapsed or refractory multiple myeloma, bortezomib achieved significantly higher response rates and longer one-year survival than thalidomide, despite 48% of bortezomib-treated patients having received prior thalidomide.

Introduction: Pulmonary hypertension (PHT) is a common complication of sickle cell disease (SCD). Most SCD patients who have PHT appear to have only mild elevations in their pulmonary artery systolic pressure (PASP). Although SCD patients with PHT have been reported to have an increased mortality, the effect of mild PHT on survival in patients with SCD remains uncertain. Methods: The study subjects represent a cohort of patients followed at the Sickle Cell Clinic at UNC, Chapel Hill. Doppler echocardiography was used to estimate the PASP. The PASP was determined by applying the modified Bernoulli equation (PASP = 4V2 + right atrial pressure), if an adequate tricuspid regurgitant velocity was observed. A subject was considered to have PHT if his/her PASP exceeded the upper limits of normal in the age- and body mass index-adjusted reference ranges. Subjects with PHT were sub-classified into mild (above normal values up to 44 mm Hg), moderate (45 – 74 mm Hg) or severe pulmonary hypertension (≥ 75 mm Hg). Patients’ data were censored at the time of their death or loss to follow-up. Results: The 60 subjects enrolled in this study have been followed for a period of 9 to 47 months (mean [± SD] of 29.8 ± 10.7 months). Pulmonary hypertension was observed in 18 subjects on initial evaluation, while the remaining 34 subjects had no evidence of PHT. The 18 subjects with PHT have been followed for 24.9 ± 10.6 months, while the 34 subjects without PHT have been followed for 31.8 ± 10.1 months. Six patients have died to date. All of these 6 patients had evidence of PHT, with estimated PASP values of 44, 44, 48, 54, 74 and 84 mm Hg (mean = 58 mm Hg) respectively. The presence of PHT was strongly associated with an increased risk of death (p = 0.0001). No other variables were associated with the risk of death in these patients. Although more patients with moderate and severe PHT died when compared to patients with mild PHT, the difference was not statistically significant. Conclusion: Pulmonary hypertension is strongly associated with an increased risk of death in patients with SCD. While our study did not find a significant difference in the risk of death when patients with mild PHT were compared to patients with more severe disease, the number of patients followed was relatively small and the duration of follow-up was short. More patients and a longer period of follow-up would be required to ascertain whether the effect of mild pulmonary hypertension on mortality is different from that of more severe PHT in patients with SCD. Figure Figure

Congenital dyserythropoietic anemia type II (CDA II) or HEMPAS is a genetic disease caused by plasma membrane abnormality. The enzymic defect of HEMPAS has been suggested to be the lowered activity of N- acetylglucosaminyltransferase II, resulting in lack of polylactosamine formation on proteins and leading to accumulation of polylactosaminyl lipids. In contrast to typical HEMPAS cases, cell-surface labeling of the erythrocytes of a HEMPAS variant G.K. showed an absence of polylactosamines either on proteins or on lipids. Fast-atom bombardment mass spectrometry analysis of G.K.'s erythrocyte glycopeptides detected a series of high mannose-type oligosaccharides, which were not detected in erythrocyte N-glycans of normal cells or of other HEMPAS cases: The former contains polylactosaminoglycans and the latter contains hybrid- type oligosaccharides. Keratansulfate (sulfated polylactosamines) in this patient's serum was abnormally low. The galactosyltransferase activity in microsomal membranes prepared from G.K.'s mononucleated cells was 24% of the normal level, whereas this enzyme activity in G.K.'s serum was comparatively higher than normal. Western blotting of G.K.'s membranes using antigalactosyltransferase antibodies showed that G.K. has reduced amounts of this enzyme present. The results collectively suggest that variant G.K. is defective in polylactosamine synthesis owing to the decreased quantity of the membrane-bound form of galactosyltransferase.