bluuebunny/crossref_metadata_2025 huggingface dataset:
Reducing the Risk for Transplant Related Mortality After Allogeneic Hematopoietic Cell Transplantation: How Much Progress Has Been Made?.
Authors: Horan John , Logan Brent R. , Agovi Manza-A. , Lazarus Hillard M , Bacigalupo Andrea , Ballen Karen , Martino Rodrigo , Juckett Mark B. , Khoury Hanna J. , Bredeson Christopher , Gupta Vikas , Smith Franklin O. , Hale Gregory , Carabasi Matthew , McCarthy Philip L. , Rizzo J. Douglas , Pasquini Marcelo C.
Published: October 2019

Abstract 649

TRM is among the major challenges for the success of HCT. Over the past two decades advances in the prevention and treatment of the major sources of TRM; regimen related toxicity, graft-versus-host disease (GVHD) and infections, have been made. To estimate the combined effect of these advances over time, we assessed changes in the incidence of TRM from 1985 through 2004 in 5,972 patients younger than 50 years, who received bone marrow (BM) or peripheral blood (PB) HCT with myeloablative conditioning for AML in first (CR1) or second (CR2) complete remission reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). The incidence of TRM was determined for four consecutive five-year periods for HLA-matched sibling donors (MRD) and the later three for unrelated donor (URD) separately by donor type and disease status at transplant. Cox proportional hazard regression models of TRM and overall survival outcomes were constructed with time periods as the main effect. Adjustments for patient and disease characteristics including age, performance score, coexistent diseases and cytogenetics were made in all multivariate models. Subgroup analyses were performed to account for the influence of major changes in transplant characteristics over time, i.e. GVHD prophylaxis, graft source and HLA matching. We observed a steady drop in the risk of TRM over time among patients in CR1 and CR2 receiving MRD transplants, which was associated with a significant reduction in risk of death (table below). Among URD recipients, TRM also improved with lower RR in 2000-2004 compared to earlier periods. No improvements in long term OS was observed in URD CR1 group. For patients in CR2, the RR for overall mortality was 0.74 (0.6-0.9, p=0.03) for 2000-2004 compared to 1990-1994. Subgroup analyses restricted to recipients of BM grafts, cyclosporine/methotrexate for all transplants and partially HLA-matched grafts for URD resulted in similar trends, suggesting that improvements in TRM were not solely related to utilization of PB, newer GVHD prophylaxis or better HLA matching. In conclusion, our results demonstrate lower risk for TRM over time in patients receiving HCT from MRD and URD for AML in CR1 and CR2. These reductions in risk of TRM have been accompanied by reduced risk of overall mortality in most groups of patients studied.

Disclosures:

No relevant conflicts of interest to declare.
Phase I Clinical Trial of the Anti-CD22 Immunotoxin CAT-8015 (HA22) for Pediatric Acute Lymphoblastic Leukemia (ALL).
Authors: Wayne Alan S. , Bhojwani Deepa , Jeha Sima , Stetler-Stevenson Maryalice , Pui Ching-Hon , McDevitt Jennifer , FitzGerald David J. , Kreitman Robert J , Kaucic Karen , Pastan Ira
Published: October 2019

Abstract 839

Although most children with ALL are cured, treatment is associated with multiple toxicities and outcome after relapse is poor. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. CD22 is a B-lineage differentiation antigen expressed on most B-lineage ALL blasts. The anti-CD22 immunotoxin RFB4(dsFv)-PE38 CAT-3888 (BL22) was recently shown to have clinical activity with an acceptable safety profile in children with ALL (Blood 2007;110:262a). We undertook a Phase I trial of a modified agent with higher CD22 binding affinity (CAT-8015 or HA22).

Methods:

Patients 6 months to 24 years of age with relapsed or refractory CD22 + B-lineage ALL or non-Hodgkin lymphoma were eligible for enrollment into this Phase I trial. CAT-8015 was administered at doses of 5, 10, 20, or 30 mcg/kg every-other-day for 6 doses every 21 days for up to 6 cycles. One patient was enrolled at each of the first 3 dose levels (5, 10, 20 mcg/kg) with standard 3+3 dose escalation commencing at 30 mcg/kg. All patients received acetaminophen, ranitidine and diphenhydramine to mitigate infusion-related symptoms, and prophylaxis for central-nervous-system leukemia with intrathecal hydrocortisone, cytarabine and methotrexate. Patients at high risk for tumor lysis syndrome received standard prophylaxis.

Results:

Seven patients with ALL (6 precursor-B, 1 mature B-cell) 5 to 17 years of age (median, 10) were treated on the clinical trial. All patients had been heavily pre-treated and had baseline cytopenias due to active malignancy and thus were not evaluable for hematologic toxicities. The most common adverse events observed to date have been hyperbilirubinemia, transaminase elevations, hypoalbuminemia, elevated creatinine, febrile neutropenia, abdominal pain, pyrexia, hypertension, microscopic proteinuria, hemoglobinuria, hypoxia and pleural effusion. Two of 4 patients treated at 30 mcg/kg experienced Grade 3 or greater toxicity consistent with capillary leak: 1 with Grade 3 pleural effusion and hypoxia and 1 with Grade 4 vascular leak syndrome. All toxicities attributed to CAT-8015 were reversible. Clinical activity was demonstrated in 4 of 7 subjects. One patient treated at 10 mcg/kg had a complete remission by morphology and flow cytometry. Three patients met the protocol definition for hematologic activity (blood count improvement). One of these patients developed high-titer neutralizing antibodies. Two patients met the protocol definition for stable disease. The patient treated at the lowest dose level had progressive disease.

Conclusions:

CAT-8015 appears to be active against chemotherapy-refractory ALL. Strategies to predict and/or prevent vascular leak syndrome are currently being developed.

Disclosures:

No relevant conflicts of interest to declare.
Recognition of B2GPI by the Lipoprotein Receptors.
Authors: Beglova Natalia , Lee Chang-Jin , De Biasio Alfredo
Published: October 2019

Abstract 851

Lipoprotein receptors of the LDLR family serve as clearance receptors for beta2-glycoprotein-I (B2GPI) and as signaling receptors for the B2GPI/antibody complexes in patients with antiphospholipid syndrome (APS). B2GPI binds lipoprotein receptors through its domain V (B2GPI-DV) and the lipoprotein receptors use the LA modules to interact with B2GPI. The LA modules, which are about 40 residues each, are arranged in sequence to form the ligand-binding domains of the receptors. The LA modules have little sequence homology both within a single receptor and between different receptors, but adopt a very similar three dimensional structure. We have investigated the structural features of the contact interface in the B2GPI-DV/LA complexes and the characteristic features of the LA modules required for efficient binding to B2GPI. Using solution NMR spectroscopy, we have demonstrated that three different LA modules bind to the same site on B2GPI-DV and that the binding site for B2GPI-DV on these LA modules is centered on the residues involved in coordination of the calcium ion. The dissociation constants measured for different LA complexes are in the range from 1 mM to approximately 100 mM. The difference in binding affinity is attributed to the auxiliary contacts that augment the interactions of the residues at the core of the binding interface. We have solved the structure of the B2GPI-DV/LA4 complex by molecular docking guided by NMR-derived restraints and have validated the structure by four independent experimental observations based on mutagenesis, solution NMR spectroscopy, fluorescence polarization and intrinsic tryptophan fluorescence measurements. The structure identified three lysine residues of B2GPI-DV, Lys308, Lys317 and Lys282, as critical for binding the LA modules. Our data shows that not all the LA modules can bind B2GPI-DV and that those LA modules capable of binding B2GPI-DV will interact with it in a similar fashion as observed in the B2GPI-DV/LA4 complex. Whether an LA module is capable of binding B2GPI could be distinguished based on the primary sequence in the vicinity of the calcium-coordinating residues. According to the current hypothetical model, the binding of B2GPI to anionic phospholipids in the presence of APS-related antibodies is the first event leading to the pathology of APS. However, we have shown that the LA modules interfere with binding of B2GPI-DV to cardiolipin indicating that B2GPI-DV cannot simultaneously bind to anionic phospholipids and lipoprotein receptors.

Disclosures:

No relevant conflicts of interest to declare.
The Receptor Tyrosine Kinase RON Is Constitutively Activated in Acute Myeloid Leukemia and May Represent a New Therapeutic Target.
Authors: Fialin Camille , De Mas Veronique , Manenti Stephane , Payrastre Bernard , Roche Serge , Recher Christian
Published: October 2019

Abstract 994

Poster Board I-16

Recepteur d'Origine Nantais (RON) is a receptor tyrosine kinase (RTK) expressed mostly on epithelial cells but also in normal hematopoietic stem cells and macrophages. RON is closely related to c-Met in terms of homology and function. Its oncogenic properties have been documented in solid tumors leading to the clinical development of small molecules inhibitors. However, the role of RON in haematological malignancies and specifically in AML, has received little attention. Interestingly, a truncated form of RON lacking most of the RON receptor extracellular domain but retaining the whole transmembrane and intracellular domains has been described in the leukemic cell line KG1 (Bardella C et al, Cancer Res 64, 5154–5161, 2004). Since RTK and TK play a crucial role in leukemogenesis, we have assessed the expression and the role of RON in both AML cell lines and patient samples.

The expression of both full length (fl) and short form (sf) of RON was assessed in 71 AML samples by RT-PCR. fl-RON and sf-RON were found in 48/71 pts (68%) and 26/71 (37%) respectively, whereas both forms were undetectable in 17 samples (24%). RON expression was further confirmed at the protein level by western blot analysis in 20 samples. Conversely, c-Met was not expressed at the protein level in 10 cases tested. In leukemic cell lines, fl-RON and sf-RON were expressed both at the mRNA and protein levels in KG1 and KG1a but not in HL60 and U937. Immunoprecipitation analysis showed that fl-RON and sf-RON were constitutively phosphorylated on tyrosine in KG1/KG1a cells. This phosphorylation was fully inhibited by the dual c-Met/RON inhibitor, SU-11274. Cell signaling induced by RON has also been explored. Specific down regulation by si-RNA to RON induced a significant decrease of Lyn phosphorylation. Conversely, AKT phosphorylation was not influenced by RON down regulation. We then assessed the activity of SU-11274 on the proliferation and survival of KG1 (RON+) and HL60 (RON-). The proliferation of KG1 (IC50=3.5μM) but not HL60 (IC50 not reached at 10μM) was strongly inhibited in a time and dose dependant manner. This inhibition of proliferation was mostly due to apoptosis induction. Accordingly using clonogenic and cytotoxic assays, we show that only RON positive samples from AML patients responded to SU-11274 (n=8). Moreover, specific down regulation of RON by si-RNA inhibited the clonogenic properties of KG1 cells.

Altogether, these data demonstrate that the tyrosine kinase RON is aberrantly deregulated in AML cells, control cell proliferation and could represent a new target for the treatment of AML.

Disclosures:

No relevant conflicts of interest to declare.
Ribosome Dysfunction and Ineffective Hematopoiesis.
Authors: Ebert Benjamin L.
Published: October 2019

Abstract SCI-11

The 5q- syndrome and Diamond Blackfan Anemia are related on a molecular level by ribosome dysfunction. The 5q- syndrome is a distinct subtype of myelodysplastic syndrome (MDS) associated with isolated, interstitial deletions of the long arm of Chromosome 5. Diamond Blackfan Anemia is a rare congenital disorder associated with bone marrow failure, craniofacial abnormalities, and limb bud defects. The hematologic phenotype of both diseases includes a severe refractory anemia, macrocytosis, and a deficiency of erythroid precursors. Recent evidence indicates that this erythroid defect is caused by RPS14 deletion in the 5q- syndrome, and by mutation of RPS19 or other ribosomal genes in at least 50% of patients with Diamond Blackfan Anemia. In both diseases, deletion or mutation of one allele of a ribosomal protein leads to defects in pre-rRNA processing and defective production of mature ribosomes. In murine and zebrafish models, haploinsufficiency for ribosomal genes phenocopies the erythroid failure characteristic of the human disorders. While the mechanistic consequences of ribosomal dysfunction have not been fully elucidated, the p53 pathway appears to play a central role. MDM2, an E3 ubiquitin ligase that promotes the degradation of p53, binds to several ribosomal proteins including RPL11. Deficiency of RPS6, and perhaps other ribosomal proteins, causes an accumulation of free RPL11, that binds to MDM2, preventing MDM2 from interacting with p53, thereby leading to an accumulation of p53. Several other genes that are mutated in hematologic disorders are involved in ribosome biogenesis and function, including SBDS, mutated in Shwachman Diamond syndrome; DKC1, mutated in some cases of dyskaratosis congenita; and NPM1, mutated in acute myeloid leukemia and deleted in some cases of MDS. The manner in which each of these genes disrupt ribosome function and cause distinct clinical phenotypes is currently under investigation.

Disclosures

Ebert: GlaxoSmithKline: Research Funding.
Bone Spared Myeloma.
Authors: Mundy Gregory R.
Published: October 2019

Abstract SCI-6

After many years, the precise role of the bone microenvironment in the initiation and progression of myeloma is still quite poorly understood. In part, this may be because it has such a complex but important role in the natural history of myeloma. Myeloma cells have dramatic effects on bone, which are well known. These effects are mediated by the effects of myeloma cells on the cells responsible for the normal bone remodeling cycle. What is less well appreciated is the effects of bone and its cellular constituents on the myeloma cells. It is apparent that myeloma cells interact closely with osteoclasts, osteoblasts and also marrow stromal cells in the bone microenvironment and these interactions probably lead to a change in the phenotype of the myeloma cells, and the subsequent production of factors that are associated with the bone destruction which is so common in myeloma. It is also becoming more appreciated that the actual quality of bone and its rigidity and integrity may have important effects on the myeloma cells. The bone loss associated with myeloma and the interaction of myeloma cells with host cells in the bone microenvironment seem to be local effects rather than generalized effects. The bone manifestations of myeloma, even at the clinical level, are still not well understood. For example, it is extremely likely that generalized osteoporosis is far more common in myeloma than is appreciated and occurs even in the presence of lytic bone lesions, and that bone mass may continue to decline in patients in remission. In experimental preclinical models of myeloma, it is quite apparent that modulation of the bone microenvironment has quite dramatic effects on the behavior of the myeloma cells and so the notion if the bone disease of myeloma could be abrogated or removed, the natural history of the neoplastic disease may be very different. Experiments are underway in the preclinical models to investigate this intriguing possibility.

Disclosures

No relevant conflicts of interest to declare.
Regulation of White Cell Development.
Authors: Newburger Peter E. , Weissman Sherman M.
Published: October 2019

Abstract SCI-9

During hematopoiesis, determination of lineage and maturation to functional leukocytes depend upon cytokine-mediated changes in the transcriptional programs of progenitor and precursor cells. The classic binary branching tree of hematopoiesis now appears to be a more subtle series of gradual changes in differentiation probabilities, with competitive promotion and inhibition of lineage pathways by regulatory transcription factors such as (among others) PU.1, C/EBPα, GFI-1, EGR1/2, and NAB2 for the myeloid lineages and RUNX1, Notch-1, E2A, GATA-3, EBF, and PAX5 for lymphoid cell development. In addition, the recent discovery of regulatory non-coding RNAs (ncRNAs) has revealed another, important layer of control of hematopoiesis. The best studied members of this diverse group are the microRNAs, which often down-regulate multiple target transcripts. miRNAs involved in the regulation of myeloid development and function include miR-155, miR-223, and miR-17-19 cluster members. In addition, miR-9, miR-146a, miR-155, and miR-181a regulate the responses of immunocytes of the innate and acquired immune systems. Most recently, increasing numbers of long ncRNAs have been identified and found to regulate expression of other genes, both in cis and in trans. EGO (eosinophil granule ontogeny), a 500 base pair spliced, polyadenylated transcript regulates eosinophil granule protein gene expression. HOTAIRM1 (Hox antisense intergenic RNA, myeloid-1), a ∼500 base pair spliced polyadenylated ncRNA, affects neutrophil expression of both contiguous and distant HoxA cluster genes, as well as transcripts for CD18 integrin. Thus the control of white cell development depends not simply on a small number of key transcription factors, but rather on a complex network of interacting protein and ncRNA regulators of the transcriptional and translational programs of cell differentiation and function.

Disclosures

No relevant conflicts of interest to declare.
Array Comparative Genomic Hybridization of Ribosomal Protein Genes In Diamond-Blackfan Anemia Patients; Evidence for Three New DBA Genes, RPS8, RPS14 and RPL15, with Large Deletion or Duplication
Authors: Gazda Hanna , Landowski Michael , Buros Christopher , Vlachos Adrianna , Sieff Colin A. , Newburger Peter E. , Niewiadomska Edyta , Matysiak Michal , Glader Bertil , Dobson Lori , Atsidaftos Eva , Lipton Jeffrey Michael M. , Beggs Alan
Published: October 2019

Abstract 1007

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, usually presenting during infancy or in early childhood. Although anemia is the most prominent feature of DBA, the disease is also characterized by cancer predisposition, growth retardation and congenital malformations, in particular craniofacial, upper limb, heart and urinary system defects, which are present in ∼30-50% of patients. We completed our large scale sequencing of 80 ribosomal protein (RP) genes and found eight of them mutated in DBA. In total, together with three RP genes identified by others, there are 11 genes mutated in ∼54% of DBA patients; RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, RPL19 and RPL26. To search for moderate and large RP gene deletions and duplications we performed high resolution array comparative genomic hybridization on 80 DNA samples from DBA patients who did not have mutations in the 11 known RP genes. We found a deletion of exon 2 and 3 (4800 bp), deletion of the coding region, and duplication of exons 2 and 3 (488 bp) in RPS19 gene in three probands; three deletions of exons 1, 2 and 3 in RPS17 in three probands (2920 bp, 2886 bp and 3018 bp); and deletion of exons 1, 2 and 3 of the RPS26 gene. We also identified two deletions and a duplication in three RP genes previously not found mutated in DBA; RPS8 duplication of exon 3 (764 bp), RPS14 deletion of exons 2, 3, 4 and 5 (2568 bp) and RPS15 deletion of exon 4 (1995 bp). The deletions and duplications are being confirmed by multiplex PCRs. Interestingly, RPS14 was previously identified as a 5q- syndrome gene demonstrating that abnormality of this protein can cause both DBA and 5q- syndrome. These data bring to 14 the total number of RP genes mutated in DBA.

Disclosures:

No relevant conflicts of interest to declare.
Phase II Study of the Combination of Clofarabine Plus Idarubicin and Cytarabine (CIA) In Patients with Relapsed/Refractory Acute Myeloid Leukemia (AML).
Authors: Naqvi Kiran , Ravandi Farhad , Kantarjian Hagop , Borthakur Gautam , Ferrajoli Alessandra , Thomas Deborah A , Koller Charles Asa , Konopleva Marina , Byrd Anna , Kwari Monica , Faderl Stefan
Published: October 2019

Abstract 1061

Clofarabine is a second generation nucleoside analog with activity in patients with acute leukemias. Previous studies with the combination of clofarabine plus cytarabine have reported a complete remission rate (CR) of 24% in relapsed AML and 52% in previously untreated AML patients. To explore further combinations with drugs active in AML, we conducted a phase II study of CIA in patients with relapsed and primary refractory AML. Patients were treated at the maximum tolerated dose (MTD) determined from a preceding phase I study: clofarabine 22.5mg/m2 IV daily × 5, idarubicin 6 mg/m2 IV daily × 3, and cytarabine 0.75 g/m2 IV daily × 5 days. A total of 63 patients were enrolled. Patients with prior exposure to clofarabine were not eligible. The median age was 51years (18 - 80years). Twenty-four patients were primary refractory and 39 had relapsed disease. All the patients received CIA in 1st salvage. For patients with relapsed AML, the median duration of the first CR was 36 weeks (3 – 215 weeks). Three patients underwent stem cell transplant (SCT) prior to treatment with CIA and 28 patients had received at least intermediate doses of cytarabine prior to relapse. Fifteen patients had secondary AML following a preceding phase of MDS. Twenty-five percent of patients had -5/-7 and 6% had trisomy 8. Thirteen patients (21%) achieved CR, 7 (11%) CRp (complete remission without platelet recovery), and 4 (6%) a partial remission (PR) for an overall response rate of 38%. The response rate in patients with primary refractory disease was 33% while the response rate in patients with relapsed disease was 41%. Five patients (8%) died during induction (</= 28 days); 4 from septic shock and 1 from diffuse alveolar hemorrhage. Median duration of follow-up was 65 weeks (range 6–161). Median overall survival was 34 weeks (range 1–161). Disease free survival for those patients who achieved CR/CRp was 66 weeks (range 4–156). A total of 24 (38%) patients underwent SCT following CIA. The most common toxicity was nausea/vomiting in 49 (78%) patients followed by hepatic dysfunction in 43 (68%) patients and diarrhea in 42 (67%) patients. In summary, the CIA combination has activity in patients with relapsed/refractory AML. Further evaluation in better prognosis patients is ongoing.

Disclosures:

Ravandi: Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kantarjian:Genzyme: Research Funding. Faderl:Genzyme: Research Funding.
Phase II Study of All-Trans Retinoic Acid (ATRA), Arsenic Trioxide (ATO), with or without Gemtuzumab OzogamIcin (GO) for the Frontline Therapy of Patients with Acute Promyelocytic Leukemia (APL).
Authors: Ravandi Farhad , Estey Elihu H. , Cortes Jorge E. , O'Brien Susan , Pierce Sherry A. , Brandt Mark , Ferrajoli Alessandra , Borthakur Gautam , Konopleva Marina , Verstovsek Srdan , Garcia-Manero Guillermo , Faderl Stefan , Kantarjian Hagop
Published: October 2019

Abstract 1080

Background:

The role of arsenic trioxide (ATO) in the frontline treatment of patients with acute promyelocytic leukemia (APL) remains unclear with a number of studies reporting high and durable responses with single agent ATO. We have conducted a trial combining all-trans-retinoic acid (ATRA) with ATO with or without gemtuzumab ozogamicin (GO) in patients with previously untreated APL.

Patients and methods:

From July 2002 to June 2010, 104 patients with newly diagnosed APL were treated with a combination of ATRA plus ATO in two studies. The first cohort of 47 patients received ATRA (45 mg/m2 daily) and ATO (0.15 mg/kg daily beginning on day 10 of ATRA). High-risk patients (White blood cell count [WBC] > 10 × 109/L) received GO 9 mg/m2 on the first day of induction. From July 2007, the second cohort of 57 patients received ATRA (45 mg/m2 daily) and ATO (0.15 mg/kg daily) concomitantly on day one of induction. They also received GO 9 mg/m2 on day 1, if high risk, and any time during induction if the WBC rose to > 30 × 109/L (and more recently if > 10 × 109/L). Monitoring for PML-RARA fusion gene using reverse transcriptase-polymerase chain reaction (RT-PCR) was conducted after induction and throughout consolidation and follow up. The median age for the 104 patients was 46 years (range, 14–81). Their median presenting WBC was 2.7 × 109/L (0.4-131.4 × 109/L) and their median platelet count was 36 × 109/L (range, 7–261 × 109/L). Seventy three (70%) had low risk and 31 (30%) high risk disease (based only on the presentation WBC ≤ or > 10.0 × 109/L).

Results:

Overall, 102 patients (98%) achieved complete remission (CR) and 2 died during induction. With a median follow-up of 115 weeks (range, 4 to 397 weeks), 94 patients remain alive. The estimated 5-year survival rate was 88% and event-free survival 86%; only 5 of the patients achieving a CR (5%) have relapsed. The median overall survival, remission duration and event-free survival have not been reached (Figure). Thirty six patients have been alive and in remission for more than 3 years and 21 for more than 5 years. Two late deaths (beyond 300 weeks) occurred in CR from unrelated causes.

Conclusion:

The combination of ATRA and ATO (with or without GO) as initial therapy for APL is highly effective and safe; it can potentially substitute for chemotherapy containing regimens in high and low risk patients.

Disclosures:

Ravandi: Cephalon: Honoraria, Research Funding, Speakers Bureau. Off Label Use: Off-label use of arsenic trioxide in frontline therapy of APL; off label use of gemtuzumab ozogamicin in APL. Verstovsek:Incyte Corporation: Research Funding.
Rapid One-Tube Long PCR-Sequencing for ABO Genotyping From Exons 2 to 7.
Authors: Park Geon , Jang Sook Jin , Moon Dae Soo , Park Young Jin , Huh Ji Young
Published: October 2019

Abstract 1116

Background:

ABO is the most clinically important blood group system in transfusion and transplantation medicine. DNA-based methods for determining the ABO genotype have markedly advanced in recent years and have been increasingly introduced in clinical laboratories. Most studies using sequencing methods for ABO genotyping have focused on exons 6 and 7, ignoring the other exon and intron sequences. Although these approaches usually can distinguish among common ABO alleles, erroneous or ambiguous typing results might be obtained in cases of hybrid and subgroup alleles with various missense or nonsense mutations. For more accurate ABO genotype results, the entire exon sequence including the introns and regulatory regions should be analyzed, but such sequencing methods are time-consuming, labor intensive, and expensive.

Methods:

To investigate the entire sequence of ABO exons 2–7 including introns 2–6 and excluding non-coding region of exon 7, long shuttle one-tube PCR-sequencing method was designed. We used a rapid DNA polymerase with high fidelity (Phusion® Flash II DNA Polymerase; Finnzyme OY, Espoo, Finland). ABO genotyping was performed using this technique in peripheral blood of two unrelated families.

Results:

Time requirement of the PCR amplification and purification processes were about 1.8 h and 20 min, respectively. Also, this technique could read the entirety of exons 2–7 including introns, required one PCR tube, one purification step, and 27 sequencing reactions for the entire sequencing. A total of seven different ABO alleles from two families were analyzed. All genotyping results agreed with serologic findings and results expected by Mendelian inheritance. The ABO phenotypes (genotypes) of family A members were A1 (A105/A105), O (O01/O02), A1 (A105/O01), A1 (A105/O02), and A1 (A105/O02) in the husband, wife, and three sons, respectively. The ABO genotype of husband in family B was cis-ABO1/O01 and his serologic ABO typing result was ABweak. His wife had the common AB phenotype (A1B); however, her genotype revealed a hybrid of A and B alleles and the B101 allele. The hybrid allele comprised the consensus ABO allele in exons 2–5 (including introns 2–4), the B101 allele in exon 6 (with introns 5 and 6), and the Al01 allele in exon 7.

Conclusion:

This method is suitable for the fast analysis of ABO genotypes and may be valuable in clinical transfusion or forensic applications.

Disclosures:

No relevant conflicts of interest to declare.
FXII Antisense Oligonucleotide Mediated Depletion Results In Effective Anticoagulation without Bleeding Risk.
Authors: Crosby Jeff R. , Gao Dacao , Zhao Chenguang , May Chris , MacLeod Robert , Monia Brett P.
Published: October 2019

Abstract 1149

Human clinical efficacy has been demonstrated with antisense oligonucleotides (ASOs) in several disease indications, including dyslipidemia, cancer, diabetes and multiple sclerosis. Coagulation factors are attractive targets for antisense therapeutics for several reasons. The liver is principally responsible for production of coagulation factors and is an organ which is highly sensitive to ASO drugs. ASOs are highly specific, and thus we can selectively evaluate multiple coagulation factors to determine their roles in thrombosis, bleeding, and other processes. Employing antisense technology we selectively reduced levels of several individual clotting factors and compared the relative risk/benefit profiles. Antithrombotic activity was determined following ferric chloride induced thrombosis in the inferior vena cava and bleeding tendency was measured by blood volume loss following tail nick. ASOs targeting several clotting factors were studied, including factors FII, FVII, FIX, FXI and FXII. Previously we characterized the beneficial effects of targeting FXI, demonstrating antithrombotic effects without increased bleeding risk. Here, we describe the effects of targeting another member of the intrinsic pathway, FXII. FXII ASO treatment resulted in a dose-dependent and specific reduction in FXII mRNA levels in liver. These reductions in FXII levels correlated well with a prolongation of aPTT with no effects on PT prolongation. The anticoagulant effect of FXII ASO treatment also correlated well with antithrombotic activity in a FeCl3 induced IVC thrombosis mouse model across a wide dose range. In addition to the venous thrombosis model, FXII ASO treatment was effective across a wide dose range in a model of arterial thrombosis. Furthermore, FXII ASO treatment was well tolerated and no prolongation of tail bleeding time was observed at any dose tested, indicating a broad safety margin for FXII ASO targeting (in mice). In addition, FXII inhibition was protective in a mouse TF driven pulmonary embolism model. Inhibiting coagulation factors with ASOs is highly specific and offers an attractive approach to identifying optimal antithrombotic targets as well as human therapeutics for the treatment of coagulation related disorders with the potential for improved safety profiles.

Disclosures:

No relevant conflicts of interest to declare.
Association of Interferon-Gamma +874(T/A) Single Nucleotide Polymorphism with Response to Immunosuppressive Therapy In Patients with Severe Aplastic Anemia.
Authors: Chang Hong , Zhang Jia-You , Mu Xiao-Yan , Meng Wen-Tong , Liu Ting
Published: October 2019

Abstract 1157

The involvement of IFN-γ in the pathophysiology of AA has been well documented, and overexpression of IFN-γ in the marrow and blood of patients with AA has been reported. T to A single nucleotide polymorphism (SNP) at position +874 of intron 1 of the IFN-γ gene (IFN-γ +874) can directly affect the expression of the IFN-γ gene since position +874 of intron 1 coincides with a putative nuclear factor (NF)-κB binding site. Thus far, no study has reported the correlation between the IFN-γ gene polymorphism and responsiveness of patients with SAA to immunosuppressive therapy. Therefore, we investigated the distribution of the IFN-γ +874 T/A gene by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and analyzed the correlation between the IFN-γ +874 T/A gene and responsiveness of patients with SAA to IST. ARMS-PCR was used to amplify the polymorphic segments of the IFN-γ +874 T/A gene in the samples obtained from 54 patients with aplastic anemia and 51 healthy adults. Further, enzyme linked immunosorbent assay was used to assay IFN-γ levels in the blood plasma of 35 patients with severe aplastic anemia (SAA) before IST and 20 healthy blood donors. The results showed that the frequencies of IFN-γ +874 TT genotype in patients with aplastic anemia were significantly higher than the corresponding frequencies in the healthy adults (42.6% vs. 17.6%, χ2 = 13.780, p = 0. 01) (Table 1). The response rate in SAA patients with increased IFN-γ levels in the blood plasma was higher than that in SAA patients with decreased IFN-γ levels in the blood plasma (73.7% vs. 25.0%, p < 0.05). Of the 35 patients with SAA, 15 showed the IFN-γ +874 TT genotype, whereas response in 11 patients, the high response rate was significantly in the favor of the IFN-γ +874 TT genotype (73.3% vs. IFN-γ +874 non-TT 35%, p < 0.05) (Table 2). In conclusion, the IFN-γ +874 T/A gene polymorphism may be correlated with response to immunosuppressive therapy. Our study showed that the frequencies of IFN-γ +874 TT genotype in patients with AA were significantly higher than the corresponding frequencies in the healthy adults, thereby suggesting that IFN-γ +874 A/T gene polymorphism is correlated with the susceptibility of aplastic anemia. Our study also showed that a high concentration of IFN-γ in the blood plasma before receiving IST and IFN-γ +874 TT genotype were predictors of a good response to IST; this finding was consistent with that of other studies.

Disclosures:

No relevant conflicts of interest to declare.
The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.
Authors: Park Chun Shik , Suppipat Koramit , Lacorazza H. Daniel
Published: October 2019

Abstract 1209

Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis.

Disclosures:

No relevant conflicts of interest to declare.
The Gamma Catenin/CBP Complex Maintains Survivin Transcription In β-Catenin Deficient/Depleted Cancer Cells.
Authors: Kim Yong-Mi , Ma Hong , Oehler Vivian , Jiang Enzi , Nguyen Cu , Masiello David P. , Liu Han , Zhao Yi , Radich Jerald P. , Kahn Michael
Published: October 2019

Abstract 1218

Survivin is a member of the inhibitor of apoptosis (IAP) gene family, which plays essential roles in prevention of apoptosis, cell division, and cancer development. Aberrant expression of Survivin is associated with essentially all tumors compared to their normal tissues. A number of signaling pathways are involved in the transcriptional regulation of Survivin. Previously, we demonstrated that Survivin expression is CBP/β-catenin/TCF-dependent and that ICG-001, a specific inhibitor of binding to the N-terminus of CBP, effectively attenuates Survivin expression. Now, using NCI-H28 cells, which harbor a homozygous deletion of β-catenin, we demonstrate that Survivin transcription can similarly be mediated by nuclear γ-catenin. In this study, we confirm that γ-catenin, like β-catenin, accumulates in the nucleus and that it can increase TOPFLASH reporter activity upon Wnt3a stimulation in NCI-H28 cells. In addition, overexpression of γ-catenin increases the expression of the TCF/catenin target genes Survivin and cyclinD1. This effect can be diminished by overexpression of dominant negative TCF or transactivation domain deleted γ-catenin. We demonstrate that g-catenin by binding to TCF family members and specifically recruiting the coactivator CBP drives Survivin transcription particularly in β-catenin-deficient cells indicating that γ-catenin can participate in the canonical Wnt signaling cascade. We also examined the relative expression of γ-catenin and β-catenin in 90 cases of chronic myeloid leukemia (CML) in a published gene expression microarray data base. A statistically significant negative correlation between γ-catenin and β-catenin was found in AP/BC cases (-0.389, P = 0.006). Furthermore, in subsequent independent validation studies by qPCR in 28 CP and BC patients increased g-catenin expression predominated in BC cases and was associated with concomitantly increased Survivin expression. Gene expression was 3- and 6-fold greater in BC patients as compared to CP patients, for γ-catenin and survivin, respectively. Consistent with this observation, nuclear γ-catenin accumulation was evident in this population consistent with a potential transcriptional role. Combined treatment with imatinib mesylate (IM) and ICG-001 significantly inhibited colony formation in sorted CD34+ CML progenitors (survivin+/γ-cateninhigh/β-cateninlow) isolated from one BC and one AP patient resistant to IM. Our data indicates the potential of ICG-001 to block both the CBP/γ-catenin interaction and the CBP/β-catenin interaction and this may have clinical significance in cancers in which γ-catenin plays a significant transcriptional role.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Virus-Specific T Cells with HLA Alloreactivity Do Not Produce Graft-Versus-Host Disease In Human Subjects
Authors: Melenhorst J. Joseph , Leen Ann M. , Bollard Catherine M. , Quigley Máire F , Price David A , Rooney Cliona M , Brenner Malcolm K. , Barrett A. John , Heslop Helen E.
Published: October 2019

Abstract 1252

We and others have recently established that T cell reactivity with non-self HLA (HLA alloreactivity) arises not only from naive T cells but also from the antigen-experienced T cell pool, including Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific T cells. Virus-specific T cells could therefore mediate graft-versus-host disease (GvHD) if infused into partially HLA mismatched recipients. We reviewed our clinical experience with adoptive transfer of allogeneic hematopoietic stem cell transplant donor-derived virus-specific T cell lines in 153 recipients who received donor-derived EBV-specific CTLs (N=114), bivirus CTLs specific for adenovirus and EBV (n=14), and trivirus CTLs specific for CMV, adenovirus and EBV (n=25). Seventy three donor-recipient pairs were partially HLA-mismatched, with the degree of HLA mismatching varying from one allele to a full haplotype. De novo GvHD did not develop after infusion of cytotoxic T lymphocytes (CTL), and the incidence of GvHD reactivation was 6.5% and not significantly different between recipients of HLA matched or mismatched CTL. Thus, virus-specific CTL did not mediate GvHD, even in recipients of partially matched CTL. Next we analyzed the HLA alloreactivity of four donor-infused bivirus-specific T cell lines, using activated T cells, that are known to lack CMV and EBV antigen expression, as antigen presenting cells (TAPC). We used a panel of 44 TAPC covering the most frequent HLA class I and II alleles. The CTL lines were labeled with CFSE and stimulated with TAPC for 6 hours, after which production of TNFα and IFNγ/IL-2 by CD4+ and CD8+ T cells in the CFSE-positive fraction was analyzed by flow cytometry. All CTLs responded to a number of TAPC, with some APC being recognized strongly. The majority elicited only weak or no response from the CTLs. We then assessed whether the CTLs recognized TAPC expressing the recipient's HLA alleles. We found moderate reactivity of the CTL with 1–5 TAPC expressing recipient HLA alleles. Taken together, our data indicate that reactivity of virus-specific CTLs with hematopoietic APC does not correlate with the risk of developing GvHD, and that virus-specific CTL can safely be infused into HLA class I and/or II mismatched recipients.

Disclosures:

No relevant conflicts of interest to declare.
Immune Phenotyping and Naive T Cells as a Predictor of Response to Therapy In Chronic Lymphocytic Leukemia (CLL)
Authors: Zent Clive S. , Abraham Roshini , Lin Yi , Gustafson Michael , Wu Wenting , Dietz Allan
Published: October 2019

Abstract 1362

The role of anti-tumor immunity is not well understood in CLL but does have the potential to be manipulated to improve treatment responses. We studied the predictive values of 43 immune phenotypes in patients enrolled in a clinical trial of early treatment of high risk CLL. The study enrolled 33 patients with CLL diagnosed using standard NCI-WG96 criteria who had previously untreated early - intermediate stage disease and did not meet NCI-WG96 guidelines for conventional treatment. High risk disease was defined as at least one of the following: 17p13 deletion; 11q22 deletion or unmutated IGHV together with either expression of ZAP-70 (>20%) or CD38 (>30%). Patients were treated with GM-CSF 250 mcg sc 3 × week × 5 weeks, alemtuzumab sc with dose escalation over 3 days then 30 mg/d 3 × week × 4 weeks, and rituximab 375 mg/m2 weekly × 4 weeks for a total duration of therapy of 5 weeks. We measured immune phenotypes (representing Tregs, naïve, effector memory, central memory T cells, B cells, NK cells, monocytes, among others) at the time of enrollment, through treatment and at 1 year after enrollment in 28 patients. We identified pre-treatment immune phenotypes that correlated with disease burden (as measured by the number of circulating B cells). Increased disease burden was correlated with increased absolute numbers of CD3+, CD8+, CD4+ T cells, NK (CD56+CD16+), Dr+CD8+ T cells, and Tregs (CD4+CD25+CD127+); p<0.001. The disease burden was inversely related to the number of circulating monocytes (p<0.01) and unrelated to the circulating number of naïve T cells. We repeated the pre-treatment phenotype correlation studies using the CD19+ cell counts 12 months after treatment for 13 patients who have completed the final analysis. Interestingly, the number of naïve CD4 and CD8 cells (CD4+RA+62L+CD27+ and CD8+RA+62L+CD27+) of the patient prior to treatment correlated with CD19+ cells 12 months after treatment (p=0.024 and p=0.002 respectively; n=13). Finally, we correlated pre-treatment immune phenotypes with time to progression. Again, an elevated T cell phenotype (CD4+RA+62L+CD27- as a percent of CD4+ cells) correlated to increased time to progression (p=0.04; n=28). It is noteworthy that the naïve T cell phenotype did not correlate with tumor burden prior to therapy. Thus, while pre-treatment lymphocyte expansion is commensurate with tumor burden in CLL, the frequency and numbers of naïve T cells prior to treatment has some predictive value to response to treatment. These data suggest that CLL induces the expansion of lymphocytes, suppresses monocytes with an induction in naïve T cells acting as an indicator of higher B cell counts at 12 months in patients with durable remissions.

Disclosures:

Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.
Dependency of CLL Cell Survival on Direct Physical Interaction with Bone Marrow Stromal Cells In Vitro Is Largely Outweighed by Secretion of Soluble Factors with Time In Culture
Authors: Gehrke Iris , Poll-Wolbeck Simon Jonas , Hallek Michael , Kreuzer Karl-Anton
Published: October 2019

Abstract 1365

The major pathophysiology feature of chronic lymphocytic leukemia (CLL) cells is their extended life span due to a pronounced resistance towards apoptotic stimuli in vivo. Despite this, CLL cells die within a few days when isolated from their natural microenvironment and are placed under cell culture conditions. That is why the bone marrow microenvironment has been ascribed an essential role in maintenance of the apoptotic resistance of the CLL cell. Thereby both, the physical interaction between bone marrow stromal cells and CLL cells and the secretion of soluble factors have been described to be essentially involved. We analysed the survival capacity of CLL cells in monoculture and in coculture with the bone marrow-derived stromal cell line HS5 with and without physical separation using transwells for up to 7 days by flow cytometric determination of Annexin-V/PI status. As expected, in vitro CLL cell survival was significantly reduced when physical contact between CLL cells and bone marrow stromal cells was prevented. Interestingly, this was only the case for short term cultivation for up to three days. With time under culture conditions CLL cell survival became less dependent on direct physical contact with the HS5 feederlayer, suggesting the secretion of soluble factors to compensate for the loss of pro-survival signals obtained from direct cell-cell interactions over time. This was further supported by the fact of reduced survival support for CLL cells when HS5 proliferation, hence production and secretion of soluble factors, was prevented by mitomycin treatment or formaldehyde fixation. The use of an expanded human Cytokine Antibody Array (Affymetrix), which analyses the presence of the most common 36 cytokine proteins, might offer information about the composition of soluble factors present in the supernatant which are essential for CLL cell survival in vitro. In conclusion, while direct cell-cell contact between CLL cells and bone marrow stromal cells provides an immediate protection against in vitro apoptosis of CLL cells, the secretion of soluble factors, most likely by both, CLL and bone marrow stormal cells, leads to the creation of an in vitro environment which can to a certain extent compensate for the loss of prosurvival signals obtained by direct physical interactions.

Disclosures:

No relevant conflicts of interest to declare.