bluuebunny/crossref_metadata_2025 huggingface dataset:
Higher Plasma Cell Burden Predicts for Early Death In Patients with AL Amyloidosis
Authors: Chee Cheng E. , Rajkumar S. Vincent , Gertz Morie Abraham A , Lacy Martha , Zeldenrust Steven , Kumar Shaji , Hayman Suzanne R. , Buadi Francis , Russell Stephen , Kyle Robert , Greipp Philip R. , Dispenzieri Angela
Published: October 2019

Abstract 1893

Background:

Recent data has shown that the level of immunoglobulin free light chain (FLC) is a prognostic factor for patients with AL amyloidosis. Approximately 25% of patients with AL amyloidosis have coexistent multiple myeloma (MM) with myeloma-related end-organ damage and patients may also present with 310% plasma cells (PC) in the bone marrow (BM) in the absence of any MM features. The goal of this study was to determine the effect of increased BM plasmacytosis in the absence of MM end-organ damage on early mortality.

Methods:

We performed a retrospective study of 263 consecutive patients with AL amyloidosis seen at Mayo Clinic within 30 days of diagnosis from July 2002 – April 2009, to compare the effect of PC burden among those who died within 90 days of diagnosis (n=88) and those who survived more than 90 days after diagnosis (n=175). Only those patients with documented BM PC were included in this study. MM end-organ damage was defined according to the CRAB criteria.

Results:

In those who died <90 days after diagnosis and patients who lived beyond 90 days, the proportion of patients with 310% BM PC were 57% and 55%, respectively. Overall survival (OS) was significantly shorter in the early mortality group when there was 310% BM PC present at diagnosis (25 vs. 54 days, p=0.006), but this was not observed in the group who survived beyond 90 days (Figure 1). In patients with 310% BM PC, troponin-T, ejection fraction (EF), and hemoglobin (Hgb) were significantly worse in the early mortality cohort but patients who survived beyond 90 days had significant increase in intraventricular septal (IVS) thickness and beta-2 microglobulin (Table 1). As expected, patients with 310% BM PC in both cohorts had significantly higher serum M-spike and involved FLC (Table). The same cohort of patients were analyzed substituting MM-related end-organ damage for 310% BM PC and similar results were observed with the exception of uric acid and calcium being significantly higher in the early mortality cohort with MM (results not shown).

Conclusion:

This study demonstrated that an excess of early—but not late—deaths occur in AL amyloidosis patients with 310% BM PC at diagnosis. In our cohort, this finding can be attributed to higher troponin-T levels, indicating more severe cardiac involvement as observed by worse cardiac function. These findings warrant additional investigation.

Disclosures:

Lacy: Celgene: Research Funding. Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding. Dispenzieri:Celgene: Honoraria, Research Funding; Binding Site: Honoraria.
Strong Antibody Responses Against a Unique Region of CT-Antigen SSX-2 Are Induced After Allogeneic Stem Cell Transplantation and Correlate with Clinical Remission In Patients with Multiple Myeloma
Authors: Kobold Sebastian , Luetkens Tim , Cao Yanran , Eberhardt Corinna , Tams Sinje , Bartels Britta Marlen , Bartels Katrin , Meyer Sabrina , Haag Friedrich , Bokemeyer Carsten , Zander Axel R. , Kröger Nicolaus , Atanackovic Djordje
Published: October 2019

Abstract 1900

Cancer-testis (CT) antigens show an expression restricted to malignancies and the human germline among healthy tissues and, therefore, represent attractive targets for cancer vaccines. CT antigen SSX-2 is expressed in about 20% of multiple myeloma patients (MM), however, little is known about the occurrence of spontaneous humoral immune responses against SSX-2 in these patients. Moreover, no information is available regarding the functionality of anti-SSX2 antibody responses and a possible impact on the course of the disease. Screening 1098 peripheral blood samples and 200 bone marrow samples of 194 MM patients, as well as 100 healthy donors serving as controls, we found six patients (3%) to present with SSX-2 specific antibodies. When we mapped the target epitopes using overlapping peptides for the whole SSX-2 sequence, we found the antibodies to be exclusively directed against amino acids 81–90. Remarkably, all antibodies were of the IgG3 subtype. In addition, SSX-2 antibodies were strictly antigen-specific and represented potent activators of the complement system. Correlating the antibody responses with clinical events, we found that the majority (5 out of 6) of seropositive patients had been antibody-negative before allogeneic stem cell transplantation (alloSCT) and had only developed anti-SSX2 antibodies after transplantation. Donor-derived antibody responses increased with depth of remission and, in case of recurrence of the disease, dropped to low or undetectable levels. Our data suggest that alloSCT is able to induce immune responses against myeloma specific SSX-2 antigen which is of low immunogenicity under normal conditions. Such antibody responses are of an effective phenotype and seem to correlate with protection from disease recurrence. To further characterize the biological role of transplantation-induced anti-SSX2 immune responses, we will analyze our patients for the presence of SSX-2-specific T cells which might be induced together with serological responses in the framework of an integrated immune response. Active immunotherapy might contribute to amplifying and shaping anti-SSX2 immune responses in myeloma patients, particularly after alloSCT.

Disclosures:

No relevant conflicts of interest to declare.
Aberrant Expression of Regulatory miRNAs and Transcripts for IRS-PI3K Growth and Survival Signaling In Waldenstrom's Macroglobulinemia
Authors: Hunter Zachary , Cao Yang , Lewicki Megan , Sun Jenny , Tseng Hsiuyi , Hanzis Christina , Brodsky Philip , Manning Robert , Xu Lian , Yang Guang , Zhou Yangsheng , Liu Xia , Jiang Jingrui , Patterson Christopher , Treon Steven P.
Published: October 2019

Abstract 1912

Background:

Waldenstrom's Macroglobulinemia (WM) is a rare low grade non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells in (LPC) the bone marrow, an elevated serum IgM, and frequently accompanied with hyperviscosity syndrome. The insulin receptor substrates (IRS) are important mediators of the insulin like receptor family and PI3K signaling leading to PKC and AKT activation.

Methods:

TaqMan low density arrays were used to evaluate the relative levels of 667 miRNAs in 11 WM patient and 5 age matched healthy donor (HD) CD19+ bone marrow cells. The results of this screen were validated using individual stem loop RT-PCR assays. Additional mRNA targets were identified using an existing gene expression profiling (GEP) data set of 22 WM patients and 8 HD using the Affymetrix U133 plus 2 platform. GEP findings were validated using an independent cohort of 18 WM patients and 7 HD.

Results:

Aberrant miRNAs identified were miR-21 (+3.27 fold p=0.035), miR-29c (+3.17 fold; p=0.003), miR-155 (+5.53 fold; p=0.082), miR-9* (-3.94 fold; p=0.001), miR-27b (-4.94 fold; p=0.001), miR-126 (-21.52 fold; p=0.006), miR-126* (-25.55 fold; p=0.039), miR-145 (-34.27 fold; p<0.001), miR-223 (-24.25 fold; p=0.041), and miR-886-5p (-3.01 fold; p=0.004). Importantly, 5 of these 10 miRNAs targeted members of the IRS-PI3K signaling pathway, a pathway important for growth and survival of WM cells: miR-29c (PIK3R1); R-155 (SHIP1); miR-21 (PTEN); miR-145 (IRS1); miR-126 (IRS1 and PIK3R2), and predict for increased protein levels for PIK3R2 and IRS1 with lower protein levels for PIK3R1, PTEN and SHIP1. Moreover, GEP and confirmatory RT-PCR revealed down-regulation of the IRS-PI3K pathway members IRS2 (-2.0 fold; p=0.004) and PIK3R1 (-2.0 fold; p=0.04).

Conclusions:

The results of this demonstrate aberrant expression of regulatory miRNAs and transcripts for IRS-PI3K growth and survival signaling in WM, and provide support for the development of IRS/PI3K targeted therapeutics in WM.

Disclosures:

No relevant conflicts of interest to declare.
Association of Age with Fluorescence In Situ Hybriditization (FISH) Abnormalities In Multiple Myeloma Patients Reveals Higher Rate of Igh Translocations Among Older Patients
Authors: Butler Charles M , Alge Joseph L , Stuart Robert K , Costa Luciano J
Published: October 2019

Abstract 1913

Introduction:

Molecular cytogenetic evaluation of chromosome abnormalities has proven to be an important tool to determine prognosis in newly diagnosed multiple myeloma (MM) patients. The majority of MM patients will have one or more cytogenetic abnormality detectable by fluorescence in situ hybridization (FISH). Deletion of 17p13, t(4;14)(p16;q32) and t(14;16)(q23;q32) define high-risk MM, while abnormalities common in standard risk MM are hyperdiploid karyotype and t(11;14)(q13;q32). The prognostic importance of deletion of 13q14 detected by FISH is controversial with worse outcome noted in some but not all series. Even though a strong correlation between age and patter of chromosome abnormalities has been demonstrated in other hematologic malignancies (e.g. acute leukemias), such association has not been clearly defined in MM. We utilized a cohort with high frequency of young patients to further explore the correlation between FISH abnormalities and age in MM.

Methods:

This is a retrospective analysis of all patients seen at our institution with diagnosis of MM from 2002 to 2010 and for whom comprehensive FISH analysis of MM cells was available. Patients were divided in two groups according to age. The proportions of specific FISH findings were compared between the two groups using chi-square test.

Results:

One hundred patients were identified with a median age of 59 years (range 20–81). We defined two groups containing younger (age < 60 years, N=51) and older patients (age ≥ 60 years, N=49). FISH abnormalities were detected in 66% (99%C.I. 56.7–75.3%) of patients. Older MM patients had a significantly higher incidence of l t(4:14) abnormalities (16.3% vs 3.9% p=0.039) and IgH rearrangements in general (36.7% vs 15.7% p=0.02). There were no significant differences between the two groups for the frequency of any of the remaining FISH abnormalities (see Figure). Similarly, no pattern of association was seen between FISH panel and initial International Staging System.

Conclusions:

Even though there does not seem to be a strong association between age and MM FISH abnormalities, our findings suggest a higher frequency of translocations involving IgH, particularly t(4;14), among older patients. It is unlikely however that the differences in outcomes between younger and older MM patients are due to intrinsic biological differences.

Disclosures:

No relevant conflicts of interest to declare.
Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells
Authors: Fagerli Unn-Merete , Stühmer Thorsten , Holien Toril , Holt Randi Utne , Bruland Ove , Chatterjee Manik , Shaughnessy John , Mathas Stephan , Sundan Anders , Bargou Ralf C , Dörken Bernd , Borset Magne , Janz Martin
Published: October 2019

Abstract 1915

Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target.

Disclosures:

No relevant conflicts of interest to declare.
Brain-Derived Neurotrophic Factor Is a Potential Osteoclast Stimulatory Factor In Multiple Myeloma
Authors: Sun Chun-Yan , Hu Yu , She Xiao-Mei , Qin You , Zhang Lu , Chen Lei , Ai Li-Sha , Fang Jun
Published: October 2019

Abstract 1917

Background and Objective:

Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain-derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In the present study, we investigated whether BDNF was present in marrow from patients with MM and possibly involved in MM cell-induced osteolysis.

Methods and Results:

Levels of bone marrow plasma BDNF was measured by ELISA in a cohort of individuals with MM and controls. The concentration of BDNF was found to be significantly elevated in patients with MM (879 ± 93) pg/ml when compared with bone marrow plasma derived from normal control subjects (186 ± 52) pg/ml (p < 0.001). Moreover, bone marrow plasma levels of BDNF positively correlated with plasma cell burden and extent of bone disease in MM patients. In osteoclast formation assay, bone marrow plasma from 31 of 37 patients with MM tested significantly stimulated the formation of osteoclast when compared to controls (61.8 ± 7 [mean ± SEM for the 31 patients] versus 25.2 ± 6 TRAP+ multinucleated cells/well [mean ± SEM for the 12 controls]; p < 0.01). The effect was significantly blocked by a neutralizing antibody to BDNF (p < 0.05), suggesting a critical role for BDNF in osteoclast activation. Furthermore, BDNF was found to dose-dependently increased the formation of multinucleated, TRAP+ osteoclast. The direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. Using reverse-transcriptase polymerase chain reaction analysis and western blotting assay, we demonstrated that BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF. These data suggested that TrkB is the functional receptor mediating BDNF's effect on osteoclast formation. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein (MIP)-1α (r = 0.45, p < 0.005) and receptor activator of nuclear factor-κB ligand (RANKL) (r = 0.68, p < 0.0001), two major osteoclast stimulatory factors in MM.

Conclusion:

Taken together, our results demonstrate the ability of MM cells to secret BDNF correlates with the severity of osteoclastic bone resorption, and provide evidence that BDNF play a causal role in the development of MM bone lesions through TrkB receptor.

Disclosures:

No relevant conflicts of interest to declare.
Combined Bendamustine, Prednisolone and Lenalidomide (RBP) In Refractory or Relapsed Multiple Myeloma. First Results of a Phase I Clinical Trial
Authors: Pönisch Wolfram , Heyn Simone , Wagner Ina , Mohren Martin , Hoffmann Franz-Albert , Schüler Frank , Schmalfeld Marion , Al-Ali Haifa K. , Jäkel Nadja , Niederwieser Dietger W
Published: October 2019

Abstract 1971

Introduction:

While the role of lenalidomide monotherapy in the treatment of relapsed/refractory patients with multiple myeloma (MM) is established, combination therapies with Lenalinomide are still under investigation. Bendamustine is a bi-functional alkylating agent with a purine-like benzimidazole ring effective in combination with steroids, thalidomide and bortezomib for the treatment of patients with MM. In the current trial, combination therapy of bendamustine, lenalinomide and prednisolone (RBP) was tested for feasibility and safety in patients with relapsed or refractory MM.

Patients and Methods:

This is a phase I trial examining dosing of lenalidomide in combination with bendamustine and prednisolone. The first cohort of patients received a starting dose of 10mg/d d1-21 lenalidomide, 60mg/m2/d d1-2 bendamustine and 100mg/d d1-4 prednisolone. Escalation steps in the next cohorts included 15, 20 and 25mg of lenalidomide followed by an escalation step of 75 mg/m2 bendamustine. Three patients were enrolled at each dose level and the first two cycles were evaluated for maximum tolerable dose. Patients received RBP in 4-week cycles for a maximum of 8 cycles in order to evaluate efficacy. Patients with stable or responding disease following 8 cycles of RBP received single-agent oral lenalidomide 10 mg once daily on days 1–21 of each 28-day cycle as maintenance.

Results:

: Nine patients (3 at each dose level of 10 mg, 15 mg or 20 mg lenalidomide) have been enrolled to date and 9 patients have completed at least 2 cycles. Response was assessed using modified EBMT criteria to include near complete remission (nCR) and very good partial remission (VGPR). 8 of 9 patients responded after at least 2 cycles with 2 VGPR, 4 PR, 1 MR and 1 stable disease. One patient experienced progressive disease. None of the 9 patients developed dose-limiting hematoxicity as defined by an ANC < 1,0 × 109/l with fever for > 3 days or an ANC <0,5 × 109/l for > 7 days or platelet count < 25 × 109/l for > 3 days. Neutropenia was reported in 4 patients (CTC grade ≥ 3) but no thrombocytopenia (CTC grade ≥ 3) was observed. No grade 3 or 4 non hematological toxicity was encountered and no dose modification was required.

Conclusions:

RBP with a dose of 20 mg lenalidomide d 1–21 and 60 mg/m2 bendamustine d 1–2 is well tolerated in patients with relapsed or refractory MM. Maximum tolerable dose was not reached. Further dose increase according to the protocol is in progress.

Disclosures:

Niederwieser: Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.
Filamin A Deficiency Leads to Premature Platelet Formation and Increased Clearance
Authors: Begonja Antonija Jurak , Weber Sarah E. , Hoffmeister Karin M. , Hartwig John H. , Falet Herve
Published: October 2019

Abstract 2027

Filamin A (FlnA) is a large cytoplasmic protein that crosslinks and stabilizes actin filament networks and links membrane glycoproteins and signaling proteins to the underlying cytoskeleton. FlnA loxP-PF4-Cre mice that specifically lack FlnA in their platelets and megakaryocytes (MKs) have a macrothrombocytopenia with increased tail bleeding time. FlnA KO platelets have decreased surface expression of the von Willebrand factor receptor (vWfR) and are cleared rapidly from the circulation of WT mice, indicating an inherent surface defect that leads to detection and removal. FlnA loxP-PF4-Cre mice have a marked increase in MK numbers in bone marrow and spleen. Detailed analysis of platelet production by FlnA KO MKs in vitro reveals an altered maturation program and defects in vWfR stability, compared to WT. Surprisingly, the surface expression of vWfR components (Ibα, Ibβ, IX and V) on FlnA KO MKs is normal or increased compared to WT MKs, although the total amount of GPIbα is decreased and GPIbα is not associated with the actin cytoskeleton. Analysis of the GPIbβ subunit shows increased degradation in FlnA KO MKs and platelets. Although FlnA KO and WT MK cultures contain comparable cell numbers, FlnA KO MKs more rapidly convert their proplatelets into large CD61+ platelet-sized particles. These findings suggest that aberrant platelet maturation by FlnA KO MKs results in enlarged platelets that are cleared rapidly because of altered vWfR expression.

Disclosures:

No relevant conflicts of interest to declare.
Role of the Krüppel-Type Zinc Finger Transcription Factor ZBP-89 In Human Globin Gene Regulation and Erythroid Development
Authors: Woo Andrew J. , Kim Jonghwan , Xu Jian , Huang Hui , Cantor Alan
Published: October 2019

Abstract 2067

The molecular mechanisms underlying developmental globin gene regulation remain incompletely understood. Prior studies have identified key cis-regulatory elements within the beta globin locus that contain core regions of closely spaced functional binding sites for GATA, NF-E2p45/maf and GT/GC box binding transcription factors. We recently identified the GT/GC-box binding transcription factor ZBP-89 as a novel GATA-1 interacting partner, and showed that it is involved in erythroid development in mice (Woo et al. 2008. Mol. Cell Bio. 28:2675-2689). Brand et al. independently isolated ZBP-89 in NF-E2p45/mafk complexes from induced mouse erythroid leukemia (MEL) cells (Brand et al. 2004. Nat. Struct. Mol Biol. 11:73-80). In the current study, we show that ZBP-89 protein levels increase during in vitro erythroid differentiation of human bone marrow derived CD34+ cells. This correlates with the onset of alpha and beta globin gene transcription. ChIP-chip studies using ENCODE v2.0 arrays demonstrate that ZBP-89 occupies key cis-regulatory elements within both the beta globin (locus control regions HS3, HS2; delta and beta proximal promoters; and an intergenic region between gamma1 and delta globin) and alpha globin (HS-48, HS-40, HS-10 and alpha globin proximal promoters) loci in primary human erythroid precursors. Comparative analysis across the entire ENCODE array reveals a strong positive correlation between ZBP-89 occupancy, RNA polymerase II occupancy, and the activating histone marks acetylated histone 3 (AcH3) and trimethylated histone 3 lysine 4 (H3K4me3); and a negative correlation with the repressive mark trimethylated histone 3 lysine 27 (H3K27me3). Motif analysis under the ZBP-89 occupancy peaks indicates a preference for GGGG(G/A)NGGGG in vivo binding sites. Lentiviral shRNA mediated knock down of ZBP-89 in the in vitro differentiated CD34+ cells results in 30–50% reduction of alpha-, gamma-, and beta-globin gene expression, as well as modestly decreased expression of a number of additional erythroid-specific genes. Co-immunoprecipitation experiments demonstrate physical association between ZBP-89 and the GCN5/Trapp histone acetyltransferase complex. Based on these findings, we propose that ZBP-89 participates with GATA-1 and NF-E2 in the final epigenetic changes required for high-level expression of globin and other erythroid genes in terminally differentiating human erythroid cells.

Disclosures:

No relevant conflicts of interest to declare.
Antigen Presenting Cell-Mediated Ex Vivo Expansion of Human Umbilical Cord Blood Cells Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity
Authors: Shah Nina , Xing Dongxia , Lee Dean A. , Cooper Laurence J.N. , Decker William , Li Sufang , Robinson Simon , Yang Hong , Parmar Simrit , Tung Shawndeep , Wang Michael , Champlin Richard , Bollard Catherine M , Shpall Elizabeth J.
Published: October 2019

Abstract 2100

Background:

Multiple myeloma (MM) is the second most common hematologic malignancy in adults and, to date, is incurable. Allogeneic natural killer (NK) cells are active in various hematologic malignancies and may have a role against MM. Umbilical cord blood is a potential source for allogeneic NK cells and ex vivo expanded umbilical cord blood-derived NK (UCB-NK) cells demonstrate activity comparable to that of peripheral blood-derived NK cells. Here we demonstrate the anti-myeloma activity of UCB-NK cells expanded to clinical grade by a novel technique using artificial antigen presenting feeder cells (“K562 Clone 9” cells) modified to express IL-21 (“K562-cl9-mIL21”).

Methods:

Cord blood mononuclear cells (CBMCs) were cultured in 10% human serum albumin media with IL-2 (500 IU/ml) and K562-cl9-mIL21 feeder cells (2:1 feeder: CBMC ratio) for 21 days. Thereafter, cells were subjected to CD3-immunomagnetic depletion. CD3-negative cells were then used as effector cells in functional assays. Flow cytometry was used to confirm NK cell purity (C56+/CD3- cells) and a standard chromium-51 assay was performed to determine NK cell cytotoxicity. Targets included K562 cells, MM cell lines RPMI 8226, ARP-1 and U266, autologous non-neoplastic UCB cells (negative control) and bone marrow-derived CD138+ plasma cells from myeloma patients.

Results:

Expansion of CBMCs with K562-cl9-mIL21 yielded a >2000 fold expansion of NK cells, compared with 47 fold expansion of CD56-selected cells cultured with IL-2 alone (p <0.05). After CD3 depletion, UCB-NK cultures were comprised of 92% CD56+/CD3- cells. K562-cl9-mIL21-expanded UCB-NK cells demonstrated cytotoxicity against the classic NK cell target K562 as well as MM cell lines RPMI 8226, ARP-1 and U266 (Fig 1). In addition, these UCB-NK cells were also active against primary, bone marrow-derived CD138+ plasma cells from myeloma patients, an effect which was augmented by pre-incubation of UCB-NK cells with lenalidomide (Figure 2, NK=UCB-NK cells, NKR=UCB-NK cells+lenalidomide).

Conclusions:

UCB-NK cells can be expanded ex vivo to clinically relevant doses for allogeneic NK cell therapy via co-culture with K562-cl9-mIL21 feeder cells. Expanded UCB-NK cells are cytotoxic to myeloma cell lines and primary myeloma cells. Further development of UCB-NK cells as an adjunct therapy in stem cell transplantation for myeloma is warranted.

Disclosures:

Wang: Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding.
Genome-Wide Linkage Analysis Reveals Novel Loci Modifying Plasma Von Willebrand Factor Undetected by Genome-Wide Association
Authors: Desch Karl , Li Jun Z , Ozel Ayse Bilge , Thornburg Courtney , Siemieniak David , Ginsburg David
Published: October 2019

Abstract 2116

Plasma levels of von Willebrand Factor (VWF) are highly variable among healthy people and ~70% of this variability is heritable. Elevated levels of VWF are associated with thrombosis while low levels of VWF cause the common bleeding disorder, von Willebrand Disease (VWD). The genome-wide association study (GWAS) is a powerful tool for identifying common genetic variants contributing to complex traits, though less common variants are likely to be missed by this approach. We performed combined GWAS and linkage analysis in a cohort of healthy young college students. The study was approved by the University of Michigan IRB (IRBMED #2005-0080). 1189 healthy individuals (14-35 yrs of age) were recruited, comprising 507 sibships. VWF levels were determined by alphalisa assay (mean 109%, range 26 – 410%). VWF level heritability (h2) was 73% among siblings and 82% among monozygotic twins. Samples were de-identified and DNA was extracted from peripheral blood, quantified and genotyped using the Illumina Omni-1 quad SNP chip at the Broad Institute (MIT, Boston). 1182 individuals passed genotyping quality control with >97% call rates for 979,855 SNPs. Genotyping quality control was performed as part of the NIH's GENEVA program. GWAS on log transformed, age and sex adjusted VWF levels identified a major peak over the ABO locus (p<1×10-36), [Figure 1] confirming the known influence of ABO blood type on plasma VWF. Preliminary genome-wide linkage analysis (MERLIN) performed on plasma VWF levels in the 507 sibships identified 5 novel loci with peak LOD scores greater than 5 (chromosomes 2, 7, and 21), in addition to the ABO locus on chromosome 9 [Figure 2]. Other than ABO, none of these loci were detected in our GWAS analysis, or the previously reported CHARGE Consortium VWF GWAS (Smith, N et al. Circulation, 2010). Our analysis is now being extended to additional hemostasis and other related phenotypes using the same SNP data. In conclusion, linkage analysis identified novel loci contributing to the variation of VWF in healthy siblings that were undetected by GWAS. Though GWAS is an effective tool for the analysis of many complex traits, including plasma VWF variation, the majority of the heritable component of these traits remains unexplained. However, the missing heritability may be due, at least in part, to multiple rare variants at a limited number of loci. Our results suggest that genome-wide linkage analysis in multiple sibships may identify such loci and that this approach may be broadly applicable to hemostasis and other complex traits. Identification of the major loci regulating plasma VWF levels may lead to an improved prediction of bleeding and thrombotic risk and could suggest novel targets for therapeutic intervention.

Disclosures:

No relevant conflicts of interest to declare.
CD34+/CD38- Leukemia Stem Cells Aberrantly Express CD82 Adhesion Molecule
Authors: Ikezoe Takayuki , Nishioka Chie , Yang Jing , Serada Satoshi , Naka Tetsuji , Yokoyama Akihito , Udaka Keiko
Published: October 2019

Abstract 2168

To identify molecular targets in leukemia stem cells (LSCs), this study compared the protein expression profile of freshly isolated LSCs (CD34+/CD38- compartment) with that of non-LSC (CD34+/CD38+ compartment) counterparts from individuals with acute myelogenous leukemia (AML) using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 98 proteins were overexpressed, while six proteins were underexpressed in LSCs compared with their non-LSC counterparts. Proteins overexpressed in LSCs included a number of proteins involved in DNA repair, cell cycle arrest, gland differentiation, anti-apoptosis, adhesion, and drug resistance. Aberrant expression of CD82, a family of adhesion molecules, in LSCs was noted in additional clinical samples (n=6) by flow cytometry. In addition, we found that imatinib-resistant chronic eosinophilic leukemi EOL-1R cells expressed a greater amount of CD82 and remained in a dormant state compared to the parental EOL-1 cells. Interestingly, down-regulation of CD82 in EOL-1R cells by a small interfering RNA stimulated their migration capacity, as assessed by the transwell assay. These observations suggested that the aberrant expression of CD82 probably played a role in adhesion of hematopoietic cells to bone marrow microenvironment. Targeting CD82 could detach LSCs from bone marrow niche and sensitized these cells to anti-leukemia agents.

Disclosures:

No relevant conflicts of interest to declare.
B-Cell Depletion Using IgG1 Anti-CD20 Spares MZ B Cells and Promotes Tolerance to Human FVIII In a Murine Model of Hemophilia A
Authors: Zhang Ai-Hong , Skupsky Jonathan , Scott David W.
Published: October 2019

Abstract 2206

Preventing and reversing inhibitor formation remains one of the major challenges for hemophilia A therapy. Anti-CD20 mAb (Rituximab) has been reported to be beneficial for hemophilia A patients who failed immune tolerance induction (ITI). However, the evaluation of anti-CD20 therapy often is complicated in the clinical setting by concomitant use of other immune modulating drugs, such as hydrocortisone and IVIG. In this study, we tested the effect of B-cell depletion per se on tolerance induction to FVIII in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential effects were used. We previously showed that IgG1 anti-CD20 selectively depleted follicular (FO) B cells and spared marginal zone (MZ) B cells, while IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice (inhibitor titer = 30.7 ± 4.8 BU/ml), a single dose of IgG1 anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice given daily, high dose FVIII i.v. injection as a model for ITI. Surprisingly, only a marginal effect was achieved when we repeated the same protocol using IgG2a anti-CD20 for B-cell depletion, which efficiently depletes both FO and MZ B cells. To examine tolerance to FVIII, we re-challenged the treated mice with 2 μg FVIII intraperitoneally three months after the initiation of B cell depletion using IgG1 anti-CD20 when the number of peripheral B cells had recovered 60 % or more. The inhibitor titers remained significantly lower in the IgG1 anti-CD20 group after this FVIII boost injection (60.9 ± 33.2 versus 190.3 ± 33.5 BU/ml in control IgG1 group; p = 0.02). Importantly, after the mice were subcutaneously challenged with an unrelated antigen, OVA in CFA, there was no significant difference in anti-OVA IgG titers between the two groups. Taken together, these results suggested that selectively depletion of FO B cells by IgG1 subtype anti-CD20 mAb treatment may facilitate the tolerance induction to FVIII. (Supported by NIH R01 HL061883 and a fellowship from the American Heart Association)

Disclosures:

No relevant conflicts of interest to declare.
De-Immunization of Immunodominant HLA-DR Epitopes In the C2 Domain of Factor VIII
Authors: Song Chang , Moise Leonard , Tassone Ryan , McClaine Elizabeth , Martin William D. , DeGroot Annie S. , Scott David W.
Published: October 2019

Abstract 2207

Development of anti-factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle of replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce binding of FVIII sequences to HLA, based on predicted anchoring residues. This process is known as ‘de-immunization’. Previously, we found that immunogenic peptide epitopes in the C2 domain of FVIII could be modified to reduce MHC class II binding resulting in lower antigenicity in vitro in hemophilic E16 mice (H-2b). Here, we extended these studies to HLA-DR transgenic mice and identified immunodominant C2 peptides in E16, DR3 and DR4 mice as well as in DR3 mice crossed to E16 mice (DR3×E16). E16 and DR transgenic mice were immunized with the designated original high class II-binding peptides (ORG) or modified peptides (MOD) emulsified in CFA, and lymph node (LN) cells were tested in T-cell proliferation and ELISpot assays against the ORG and MOD peptides. We found that while p2191-2210 dominantly stimulates H-2b, p2271-89 is immunodominant in DR3 transgenic animals; DR3×E16 T cells recognized both epitopes as would be predicted. On the other hand, p2231-51 and p2310-30 were immunodominant in DR4 mice. We also found that modifications of dominant epitope peptides 2191, 2271, 2231, 2310 showed lower HLA class II binding and T-cell proliferation in corresponding mouse strains; stimulation of IL17-a and IFNγ cytokine production were reduced with modified 2191 in DR3xE16 mice. Direct immunization with rFVIII containing modified peptides is required to validate potential immunogenicity. These results will be utilized to design and produce a functional full-length modified rFVIII that can be used as a less immunogenic therapeutic protein for hemophilia A patients. (Supported by NIH grant R43 HL088834-01 to ADG.)

Disclosures:

Moise: EpiVax: Employment. Tassone: EpiVax: Employment. McClaine: EpiVax: Employment. Martin: EpiVax: Employment, Equity Ownership, Patents & Royalties. DeGroot: EpiVax: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Scott: EpiVax: Consultancy.
Characterization of p53-Dependent Pathways In RPL11 Mutant Zebrafish
Authors: Danilova Nadia , Sakamoto Kathleen M. , Lin Shuo
Published: October 2019

Abstract 2227

Deficiency of ribosomal protein (RPs) is associated with Diamond Blackfan Anemia, a congenital syndrome with bone marrow failure and variable malformations. Recent studies have demonstrated that deficiency of several RPs leads to activation of p53 and related family members, ΔNp53 and ΔNp63. In addition, P53 inhibition rescues hematopoiesis in RP-deficient mice and zebrafish. p53 activation, however, results in the alteration of many pathways and specific mechanisms leading to bone marrow failure and other defects in DBA remain unidentified. To understand the global changes in gene expression downstream of RP insufficiency, we took a system biology approach to characterize the molecular pathways involving p53. We studied pathways downstream of p53 that contribute to the RPL mutant phenotype in zebrafish. Changes in p53-dependent pathways in RPL11 mutants included dysregulation of cell cycle and apoptosis, shift in energy production from glycolysis to aerobic respiration, suppression of biosynthesis of structurally important proteins, downregulation of detoxifying enzymes, and upregulation of factors leading to activation of adrenal-pituitary axis and inflammation. Among multiple metabolic changes were those that led to increased levels of insulin and glucose. Both differentiation and production of blood were affected. Expression of genes that regulate the development and function of neural system were notably decreased, in particular, those involved in the development of anterior brain structures and eyes. The production and migration of neural crest cells were disrupted suggesting possible explanation for craniofacial and other defects observed in patients with DBA. The RPL11 mutants also had abnormal development of hematopoietic stem cells and shortened life span of mature red blood cells. Dexamethasone treatment led to downregulation of p53 expression; suppression of p53 targets mediating cell cycle arrest and apoptosis; and rescue of expression of HSC markers such as runx1. The life span of erythroid cells was also increased in response to dexamethasone treatment. These data contribute to our understanding of the molecular pathophysiology of DBA and suggest new potential targets for therapeutic intervention.

Disclosures:

Sakamoto: Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.
Fecal Calprotectin In Allogeneic Stem Cell Trasplantation as Surrogate Marker of Gastrointestinal Graft Versus Host Disease
Authors: Oreiro Mariana Bastos , de la Guia Ana Lopez , Nieto Jose B , De Paz Raquel , Baltasar Patricia , Hernandez Dolores , de la Rua Ana Rodriguez , Gallardo David , Canales Miguel
Published: October 2019

Abstract 2327

Background:

Calprotectin is a major cytosolic protein of neutrophils that have showed to be a sensitive marker of intestinal inflammation. The aim of our study has been to evaluate fecal calprotectin (FC) as a diagnosis tool in patients with acute gastrointestinal graft versus host disease (GI GVHD).

Methods:

Since March 2009, patients with suspicion of acute GI GVHD were consecutively included. Patients were tested for FC (reference range: 0–30 mg/kg) before starting treatment. Infections by Clostridium difficile, cytomegalovirus, fecal bacteria and intestinal parasites were also excluded. Colonoscopy was performed in all patients and biopsy samples were taken for histopathological examination.

Results:

To date, eleven patients have been included. The median age was 48.2 (r: 21–67). Indications for transplantation included acute myeloid leukemia (6 patients), myelodysplastic syndrome (2 patients), acute lymphoid leukemia, severe aplastic anemia and refractory follicular lymphoma (1 patient, respectively). Ten patients received myeloablative conditioning and one received reduced intensity conditioning regimen. Five patients were histologically diagnosed with acute GI GVHD. The median for FC in this group was 510.5 (r: 107.4–629). The median of FC in patients without GI GVHD was 117 (r: 13.9–205). Patients with GI GVHD had higher values of FC than patients without GI GVHD (p:≤0,009) For a cut-off point value of 205, sensitivity for the test was 83.3% (IC 61.3–100%), specificity was 100%, positive predictive value was 100%, and negative predictive value was 83.3% (IC 61.3–100%). In 3 patients who were diagnosed with cytomegalovirus enteritis and had no criteria for GI GVHD, the FC values were 15.4, 105, and 100.7. In a patient with Candida spp. infection FC was 13.9. The FC was higher in patients with grade IV GI GVHD, with median of 590 (r: 502–629).

Conclusion:

FC appears to be a promising non-invasive biomarker of acute GI GVHD. If these findings are confirmed, it may provide a useful non-invasive test for the diagnosis of GI GVHD in patients following allogeneic transplantation.

Disclosures:

No relevant conflicts of interest to declare.
Successful Treatment of High-Risk and Refractory Acute Myeloid Leukemia with haploidentical Stem Cell Transplantation Plus NK cell therapy
Authors: Uharek Lutz , Friedrichs Birte , Nogai Axel , Gentilini Chiara , Basara Nadezda , Niederwieser Dietger , Thiel Eckhard
Published: October 2019

Abstract 2370

Regardless of the treatment applied, the outcome for patients with refractory hematological malignancies is extremely poor. For haploidentical stem cell transplantation (haploSCT), which has been evaluated especially in high-risk AML patients lacking an HLA-identical donor, registry data show an overall survival below 10% for AML not transplanted in remission. In order to improve immune competence and to reduce the risk of relapse, we have combined the early transfer of NK cells with the haploSCT approach. PATIENTS AND METHODS: Twenty-five patients (AML = 16, ALL = 5, CML = 2, Hodgkin = 1, MDS = 1) received a haploSCT followed by transfer of purified CD56+CD3-NK cells at day +2. Conditioning consisted of total body irradiation, thiotepa, fludarabine, and OKT3. NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. No other immunosuppression was routinely delivered. RESULTS: Patients received a mean of 13.3 × 106 CD34+ cells/kg (range 6.12–26.97) with 2.89 × 104/kg (0.95-7.4) contaminating CD3+ cells. A mean of 9.8 × 106 CD56+CD3- NK cells/kg (range 1.61–32.2) was adoptively transferred. Most of the patients developed early GVHD of the skin (median onset day=12), which promptly resolved after short term treatment with steroids and CSA. The main cause of death consisted of infections (n = 10), chronic GvHD (n= 2), and relapse (n = 4). After a median follow-up of 1442 days (3.9 years) (range 0.1 to 7.1 years) 9 of 25 patients are alive and in complete remission resulting in a 2-year overall survival (OS) estimate of 29%. Out of 16 high-risk patients with AML, 10 had refractory or active disease prior to transplantation. For a matched pair analysis, these patients were matched for age, disease status, conditioning regimen and year of transplantation using the EBMT database. AML patients treated with haploSCT plus NK cell transfer had a superior 2-year overall survival (40 vs 11%, 95% CI) in comparison to matched controls with haploSCT only (p=0.02) CONCLUSIONS: HaploSCT plus NK cells is feasible and shows promising survival rates for a group of patients lacking other treatment options. Best results were achieved in patients with AML not only in remission but also with refractory disease.

Disclosures:

No relevant conflicts of interest to declare.
Association of HLA-G Haplotype Risk Model with Treatment Free Survival In Chronic Lymphocytic Leukemia Patients
Authors: Kapp Johanna , Ochsenfarth Crista , Switala Magdalena , Frey Ulrich , Sellmann Ludger , Duerig Jan , Horn Peter A , Duehrsen Ulrich , Rebmann Vera , Nuckel Holger
Published: October 2019

Abstract 2418

Introduction:

The human leukocyte antigen G (HLA-G) molecule exhibits limited tissue distribution and exerts multiple immunoregulatory functions. Since the net result of these effects is immunosuppression, HLA-G expression in tumor cells may favor their escape from antitumor immune responses, thus allowing tumor progression. The HLA-G protein polymorphism is extremely restricted in comparison to the classical HLA antigens. However, specific HLA-G polymorphisms in exons 3, 4, 5, 7 and parts of the 3`UTR encode for different haplotypes. Demonstrating the extensive and proven prognostic role of HLA-G in various diseases and especially in B-CLL we investigated the role of the HLA-G haplotypes in B-CLL.

Methods:

Genotyping was performed on the basis of examining exons 3, 4, 5, 7 and parts of the 3`UTR by pyrosequencing in 190 patients with B-CLL. In total, we found 17 different haplotypes in the cohort.

Results:

The haplotype distributions between 190 B-CLL patients and 190 healthy controls were not different, arguing that the haplotypes of HLA-G do not increase the susceptibility for B-CLL. However, combining four different haplotypes (*01:06, *01:12; *01:12 variant and *01:13N) we evaluated a risk score (0=no risk allele; 1=one risk allele; 2=two risk alleles) for TFS. 77 patients showed no risk allele, 90 patients one risk allele and 23 patients two risk alleles. The TFS for those patients with two risk alleles were significantly shorter (median TFS 33 months) than for those with one risk allele (median TFS 49 months) and those with no risk alleles (median TFS 88 months)(log rank test: p=0.03). In multivariate analysis we could show that the stage according to Binet (HR 1.7, 95% CI 1.2–2.6, p=0.005), CD38 status (HR 1.8, 95% CI 1.0–3.2, p=0.04) and the haplotype risk model (HR 1.7, 95% CI 1.1–2.6, p=0.011) were independent predictors for first therapy.

Conclusion:

Our study is the first study demonstrating that the combination of different alleles of the HLA-G gene is associated with the risk of first therapy in B-CLL patients. This fact emphasizes the extensive role of this gene by the tumor escape mechanism and could be responsible for progress in other cancers.

Disclosures:

No relevant conflicts of interest to declare.