bluuebunny/crossref_metadata_2025 huggingface dataset:
Splicing Mechanisms That Generate Distinct Isoforms of Protein 4.1R During Terminal Erythroid Differentiation.
Authors: Parra Marilyn K , Mohandas Narla , Conboy John G.
Published: October 2019

Abstract 4036

Poster Board III-972

The protein 4.1R gene is a large complex gene with two translation initiation sites (AUG1 and AUG2) that encode protein isoforms with distinct N-terminal structure and function. Expression of these isoforms is regulated by alternative splicing at either of two splice acceptor sites that flank exon 2' (E2'), in which AUG1 is located. In late erythroblasts E2' is excluded, ensuring translation at AUG2 and synthesis of 80kDa protein 4.1R isoforms. Our earlier studies (EMBO J. 27:122-31, 2008) described a two-step intrasplicing pathway that enforces this splicing outcome exclusively in 4.1R pre-mRNA initiated at exon 1A (E1A), the major transcription start site in late erythropoiesis. The downstream exon 1B (E1B) first splices to the proximal acceptor site at E2' to generate an intermediate structure, which is then re-processed by splicing of E1A to the internal acceptor at E2, removing E1B as well as E2'/AUG1. Experiments with minigenes suggested that intrasplicing is likely independent of specific promoter elements at E1A, but absolutely requires 5' splice site and branch point motifs associated with E1B. Here we sought evidence for functionality of these latter elements in the more physiological context of the endogenous 4.1R gene. The intrasplicing model predicts that morpholino oligonucleotides complementary to key regulatory motifs will block the first step of the pathway in natural 4.1R pre-mRNA transcripts, and yield inappropriate splicing of E1A to the first acceptor at E2'. Antisense morpholinos directed against the E1B branchpoint or E1B 5'splice site were transfected into cells and 4.1R splicing was examined 48hrs later by RT-PCR analysis. Both anti-4.1 morpholinos, but not a control morpholino, resulted in a concentration-dependent shift of E1A splicing to the proximal E2' acceptor site. In other studies we explored whether intrasplicing could occur internally within a gene, using a model pre-mRNA in which a constitutive exon was engineered between E1A and E1B. Analysis of this experiment suggested that any exon upstream of active E1B would follow the intrasplicing pathway and delete E2'. We speculate that internal E1B-like elements in other genes could be selectively activated or silenced by splicing regulatory motifs in order to control downstream splice acceptor choice. Preliminary experiments indicate that weakening the E1B 5' splice site and its upstream pyrimidine tract might permit such regulation as observed for other alternative exons. Together these results provide additional support for the intrasplicing model and suggest that it could function more widely in human genes to coordinate splicing events and generate multiple protein isoforms with distinct functions.

Disclosures:

No relevant conflicts of interest to declare.
Non-Competitive Protein Phosphatase-1 Inhibitors Prevent Sickling of SS RBCs.
Authors: Hairston Jerod , Combi Keon , Foster Altreisha , Kim Bak , Gordeuk Victor R. , Nekhai Sergei
Published: October 2019

Abstract 4038

Poster Board III-974

Protein phosphatase-1 (PP1) has been implicated in the regulation of KCC (K:Cl) transporters, which transport K+ and Cl- ions from red blood cells (RBCs) and in the setting of sickle cell disease may contribute to RBC dehydration and sickling. We have studied host cell protein phosphatase-1 (PP1) in the context of HIV-1 replication and designed novel small molecule non-competitive inhibitors of PP1 that are efficient in the inhibition of HIV-1 but not toxic for cultured cells. We analyzed the effect of our novel non-competitive PP1 inhibitors and the conventional competitive PP1 inhibitor, ocadaic acid, on the sickling of hemoglobin SS RBCs in vitro. We cultured hemoglobin SS RBCs overnight at 1% O2 in the presence of the PP1 inhibitors and then photographed the RBCs and counted the percentage of sickled RBCs. We found that the non-competitive PP1 inhibitor, 1E7-04 prevented RBC sickling by 40% at 10 mM concentration. The 1E7-04 was not toxic at 10 mM concentration for cultured CEM T cells as determined by trypan blue exclusion assay using an automatic cell counter. Our study suggests that small molecular inhibitors of PP1 might be candidates for the future design of anti-sickling drugs.

Acknowledgments.

This work was supported by NHLBI grant U54HL090508-02; NHLBI grant R25 HL003679-08 from the National Institute of Helath and The Office of Research on Minority Health and by U.S. Civilian Research & Development Foundation grant.

Disclosures:

No relevant conflicts of interest to declare.
ACUTE PROMYELOCYTIC LEUKEMIA (APL) IN PATIENTS AGED > 70 Years.
Authors: Finsinger Paola , Breccia Massimo , Minotti Clara , Carmosino Ida , Cannella Laura , Volpicelli Paola , Vozella Federico , Stefanizzi Caterina , Loglisci Giuseppina , Cimino Giuseppe , Foà Robin , Latagliata Roberto
Published: October 2019

Abstract 4160

Acute Promyelocytic Leukemia (APL) is a rare subtype of Acute Myeloid Leukemia (AML) more common in younger adults, with a median age of 45 – 50 years at onset. The use of All-trans Retinoic Acid (ATRA) as tailored treatment has made APL a very curable disease also in patients aged > 60 years; however, there are only few case reports in very elderly APL patients. To address this issue, we revised clinical data and treatment results in 12 patients aged > 70 years with newly diagnosed APL followed at our Institution from 1/91 to 12/2008. Clinical characteristics at onset were as follows: M/F 7/5, median age 74.7 years (range 70.0 – 80.8), M3/M3v 11/1, median WBC 1.3 × 109/l (range 1.0 – 7.4), median PLTS 53 × 109/l (range 12 – 302), BCR1/BCR3 6/6. According to Sanz risk score, 7 patients were at low-risk and 5 at intermediate-risk; 6/12 patients had arterial hypertension, 4/12 a concomitant cardiologic disease, 3/12 a cerebro-vascular disease and 2/12 a previous neoplasia. Induction therapy consisted of ATRA + Idarubicin in 8 patients (2/8 with reduced Idarubicin dosage) and ATRA alone in 4 patients; in this latter group, however, 2/4 needed to add chemotherapy (CHT) based on Mitoxantrone + AraC due to hyperleukocytosis during ATRA treatment. All patients achieved both morphological and molecular Complete Remission (CR) after a median time of 50 (range 29 – 65) and 105 (range 51 – 239) days, respectively. Infective complications were observed in 10/12 patients (4 episodes of FUO, 6 sepsis, 2 cystitis and 1 oral abscess) while ATRA syndrome occurred in 2/12 patients; in addition, there were 3 episodes of cardiac ischemia and 3 episodes of paroxystic atrial fibrillation. All but one patient received consolidation therapy (based on CHT alone in 7 patients, CHT + ATRA in 3 patients and ATRA alone in 1 patient), followed by maintenance treatment in 8 patients. Four patients had a relapse (hematological in 3 cases and molecular in 1 case) after 8, 11, 35 and 56 months respectively. At present, 6 patients are still alive, 4 died due to disease progression (3) or senectus while in CR (1) and 2 were lost to follow-up while in CR: mean event-free survival and overall survival were 89.2 months (95%CI 52.6 – 125.8) and 99.9 months (95%CI 65.0 – 134.7), respectively. In conclusion, ATRA-based treatment of APL is safe and effective also in very elderly patients, with long-lasting disease-free and overall survival.

Disclosures:

No relevant conflicts of interest to declare.
Fondaparinux for the Treatment of Recurrent Venous Thromboembolism During Low Molecular Weight Heparin Therapy: A Retrospective Case Series.
Authors: Ramasubbaiah Rashmi , Kiel Patrick J , Mehta Rakesh , Greist Anne , Jones Kellie L
Published: October 2019

Abstract 4179

Introduction:

Fondaparinux is a synthetic pentasaccharide that exerts its anticoagulant effects by indirectly inhibiting Factor Xa. Large randomized controlled multicenter trials have shown that daily administered subcutaneous fondaparinux at specified weight based dosing is as effective as unfractionated heparin (UFH) and low molecular weight heparin (LMWH) for the initial treatment of venous thromboembolic events (VTE), in conjunction with warfarin. In the case of recurrent DVT or PE, however, there is no standard of care and optimal anticoagulation still remains a challenge. Based on limited data, most patients on warfarin with recurrent VTE would be switched to LMWH or UFH, but the use of fondaparinux in this setting is limited to anecdotal experience.

Methods:

We report the Indiana University experience with the use of fondaparinux for recurrent DVT or PE, failing therapy with LMWH. This is a retrospective review of 13 patients with recurrent thromboembolism, studied in two hospital settings. The mean age was 37 years (range 23-65), there were 8 females and 5 males, and all the patients had failed enoxaparin (two patients had also progressed on tinzaparin). The underlying medical conditions that predisposed to thromboses varied: sickle cell disease (3), antiphospholipid syndrome (3), cancer (3), May Thurner syndrome (1), and 3 patients with inherited thombophilia (Antithrombin III deficiency, Factor V Leiden and Prothrombin 20210 Gene Mutation). Mean weight was 90.7 kg (range 52-150). Mean time to recurrence on LMWH was 330 days. Fondaparinux was prescribed at 7.5mg daily or 10mg daily, based on body weight. Anti-Factor Xa levels were measured in at least half of the patient population, and response was assessed by clinical improvement or progression.

Results:

Thirty one percent (4/13) of patients had recurrent thromboembolic episodes (response rate 69%). Time to progression during therapy with fondaparinux was available on 2/4 patients, and was 17 months (range 13-21). No adverse events including bleeding complications or death were reported.

Conclusions:

Fondaparinux may have efficacy in the treatment of recurrent VTE that has progressed on LMWH. These results are hypothesis generating and should be evaluated prospectively.

Disclosures:

No relevant conflicts of interest to declare.
Decreased Immunogenicity of Purified Topical Bovine Thrombin Preparations.
Authors: Zhu He , Hoppensteadt Debra , Wahi Rakesh , Fareed Jawed
Published: October 2019

Abstract 4209

It has been reported that severe coagulopathy following exposure to topical bovine thrombin may be attributed to the impurities in bovine thrombin preparations. Prepared through the activation of bovine prothrombin by thromboplastin, a crude thrombin preparation was further purified using ion-exchange chromatography and membrane filtration steps to yield thrombin 4A and 4B preparations. The aim of this study was to compare the relative purity of these bovine thrombin preparations by virtue of the detection of bovine prothrombin-related antigens. Bovine prothrombin was administered to three individual rabbits on days 0, 21, 42, 91, 123 and 151 using standard immunologic method. Blood was drawn on 30, 50, 105, 137 and 165 days and the pooled antisera from three rabbits were purified to obtain IgG using protein G affinity columns. Utilizing western blotting, serial diluted bovine crude thrombin, 4A and 4B preparations were probed using the prothrombin IgGs from each time point to explore prothrombin-related antigens in these preparations. Compared with the IgG collected on day 30, the prothrombin IgGs collected from day 50 to 165 showed a time-dependent increased detecting ability for the prothrombin antigens in the bovine thrombin preparations. The lowest amount of crude thrombin, 4A and 4B preparations that prothrombin IgG could detect was 0.125U, 10U and 20U, respectively. The rank order of the number of immunoreactive bands in three bovine thrombin preparations probed by the prothrombin IgGs collected from any given time point was consistant and reproducible: crude thrombin > thrombin 4A > thrombin 4B. The results indicate that thrombin 4B represents the most purified thrombin preparation and relatively non-immunogenic form among three thrombin preparations studied.

Disclosures:

No relevant conflicts of interest to declare.
Stem Cell Mobilization Using 5 Pm Dosing of Plerixafor.
Authors: Cooper Dennis L , Baker Julie , Medoff Erin , Pratt Kathryn , Conkling-Walsh Ann , Snyder Edward L. , Seropian Stuart , Foss Francine
Published: October 2019

Abstract 4233

Introduction

Based on the superiority of plerixafor plus G-CSF (PG) compared to G-CSF (G) alone for mobilizing autologous stem cells, PG has been approved for stem cell mobilization in patients with multiple myeloma and NHL. PG may also be useful for mobilizing stem cells in patients who have failed mobilization with G alone or chemotherapy plus G. Although pivotal studies in myeloma and NHL employed evening (10 pm) dosing of P in order to capture peak mobilizing activity of PG 10-11 hours later, this schedule is inconvenient. Methods: A regimen using 5 pm dosing of P was employed in 17 patients (9 NHL, 7 myeloma, 1 germ cell tumor) including 10 patients who received PG and 7 pts who transitioned to PG after chemo plus G. Apheresis was performed at 8:30 am after P. Four patients had failed previous mobilization with chemo plus G and 4 pts were expected to be poor mobilizers because of prior Rx (lenalidomide 3, fludarabine 1). In nine patients, P was added to G (2 pts) or chemo plus G (7 pts) during the mobilization episode because of inadequate stem cell collections.

Results

An engraftable dose (> 2.5 × 10E6CD34/kg) of stem cells was achieved in all eight pts who either failed prior mobilization or were anticipated to be poor mobilizers because of past Rx and in 6 of 9 pts who had P added during suboptimal mobilization with either G (2/2) or chemo plus G (4/7). In successfully mobilized pts (14/17), the median number of CD34 cells collected was 6 × 10E6/kg in two aphereses. In the latter group, the median CD34 count/μl on the first day after P was 29 (R: 11-161) and had increased a median of 4.5 fold (R: 2.75 – 40.2) compared to one day earlier. Six pts have had transplant with a median WBC and platelet recovery of 10 and 11 days, respectively. Conclusions: PG with 5 pm dosing of P and apheresis at 8:30 the next morning was effective in pts who failed prior mobilization and in pts expected to be poor mobilizers because of prior Rx. Further studies are required to determine the efficacy and optimal scheduling of P when added during suboptimal chemo plus G mobilization.

Disclosures:

Off Label Use: Plerixafor is a stem cell mobilizing agent that is approved for multiple myeloma and NHL. One patient with a germ cell tumor has been included in this study of an alternative mobilization strategy. Foss:Eisai : Speakers Bureau.
High-Dose Etoposide with Granulocyte Colony-Stimulating Factor for Mobilization of Autologous Peripheral Blood Stem Cells in Patients with Hematologic Malignancies.
Authors: Zhang Xiao-yan , Li Jianyong , Qian Sixuan , Qiu Hongxia , Wu Han-xin , Lu Hua , Xu Wei , Hong Ming
Published: October 2019

Abstract 4235

We hoped to explore the efficacy and safety of high-dose of etoposide with granulocyte colony-stimulating factor (G-CSF) for mobilization of peripheral blood stem cells. 43 patients (25 males and 18 females, 15-71years old) with hematologic malignancies, including 17 patients with multiple myeloma, 16 with non-Hodgkin's lymphoma, 1 with Hodgkin's lymphoma, 8 with acute non-lymphocytic leukemia, 1 with acute lymphocytic leukemia, received an etoposide dose of 1.6g/m2. One of the patients had failed to be mobilized by cyclophosphamide and G-CSF before. The total dose of undiluted etoposide was given on day 1 as a continuous intravenous infusion via a central vein for 10 hours. G-CSF 5μg/kg was used on day 3 and given daily subcutaneously until leukopheresis was completed. The results showed that leukopheresis was started at days 10 (range 9-13 days) following etoposide therapy. the mean number of CD34 positive cells collected in all 43 patients was 9.71×106/kg (range 0.5-53.26×106/kg),by an average of 2.15 (range 1-5) leukophereses times. Mobilization procedure that produced yields of greater than 2 .0×106/kg were achieved in 40 patients (including the one failed to be mobilized by cyclophosphamide and G-CSF). Toxicity showed oropharyngeal mucositis, faucitis, urethritis and upper respiratory tract infection in some patients. It is concluded that high-dose etoposide with G-CSF is an effective and safe mobilizing regimen for autologous peripheral blood stem cells in majority patients with hematologic malignancies.

Disclosures:

No relevant conflicts of interest to declare.
Finding of Kinase Domain Mutations at Diagnosis IN PATIENTS with Chronic PHASE Chronic Myeloid Leukemia (CP-CML) MAY Identify Those at High RISK of Disease Progression.
Authors: Carella Angelo Michele , Cirmena Gabriella , Catania Gioacchino , Pica Gianmatteo , Beltrami Germana , Ballestrero Alberto , Patrone Franco , Garuti Anna
Published: October 2019

Abstract 4251

Kinase domain (KD) mutations are associated with resistance to Imatinib in CP-CML but their incidence and prognostic significance before any treatment are unclear. To assess if KD mutations at diagnosis may have prognostic significance, we have recently reviewed (before treatment with Imatinib) the mutation status of 45 patients,of whom low risk: 24 patients; intermediate risk: 8 patients; high risk: 13 patients, according to Sokal/Euro. We found that a) no patient with low and intermediate risk showed KD mutations at diagnosis; b) 4/13 high risk patients showed the following mutations at diagnosis: S265R, E255K, F359Y and E255V/E258V; other 5 patients developed mutations during Imatinib treatment (E255V, T315I, E255V/E258V, H396R and D248-274). All patients with low-intermediate risk are alive and well at a median of 44 months (range, 15-71 months). On the contrary, 3/4 high risk patients with KD mutations at diagnosis progressed and died of blastic evolution at 23, 33 and 69 months despite Nilotinib and Dasatinib therapy. Other 3/5 high risk patients who developed mutations under Imatinib, died of blastic evolution at 22, 23 and 43 months despite Nilotinib and Dasatinib treatment. Seven high risk patients are alive under Imatinib between 4 and 51 months. In conclusion, the KD mutations at diagnosis were frequent in high Sokal/Euro risk group supporting the concept that such mutations could be related to the basic biology of the disease. These data, if confirmed, could modify our approach to high risk patients with KD mutation at diagnosis, i.e. utilizing second/third TKI generation earlier during the disease and, in selected cases, reconsidering allografting earlier before leukemic evolution make such procedure useless.

Disclosures:

No relevant conflicts of interest to declare.
LOW SPARC EXPRESSION IN CML and ITS Upregulation DURING Imatinib TREATMENT.
Authors: Palumbo Giuseppe Alberto , Giallongo Cesarina , Cava Piera La , Tibullo Daniele , Stagno Fabio , Romano Alessandra , Conticello Concetta , Chiarenza Annalisa , Raimondo Francesco Di
Published: October 2019

Abstract 4258

SPARC, also known as osteonectin, is a highly conserved between species phosphoprotein that has been recently identified as a tumor suppressor gene. Besides its involvement in bone metabolism, where it mediates collagen deposition, it has been shown that SPARC plays several roles, ranging from cell adhesion to inhibition of cell-cycle progression and VEGF activation. In mice, it has been reported that implanted tumors grow faster in SPARC-null animals. In humans, patients affected by MDS 5q-syndrome responsive to lenalidomide, have an increase of SPARC expression.

Chronic myeloid leukemia (CML) is a disease characterized by uncontrolled proliferation and adhesion defect. In our study we investigated the expression of SPARC mRNA by quantitative PCR in 22 CML patients. mRNA SPARC relative expression in mononuclear cells isolated from peripheral blood at diagnosis was significantly lower (range 0.004-0.98, mean 0.3, median 0.21, SD 0.3) than in normal control subjects assumed as a reference (range 0.31-2.5, mean 1, median 0.8, SD 0.8).

During imatinib treatment, SPARC mRNA increased of 2 logs at 3 months and of 3 logs at 1 year. In one patient that had to stop imatinib treatment a decrease of expression to levels comparable to normal subjects, around 1 log higher than pretreatment levels, was noticed; when the patient restarted treatment with the tyrosine kinase inhibitor, SPARC level raised of 2 logs again.

Bcr/Abl+ K562 cell line has a low level of SPARC mRNA (0.4 relative expression compared to pooled normal controls), probably due to promoter hypermethylation. Treatment of K562 with an hypomethylating agent, such as 5-azacytidine, for 72 h resulted in an increased expression of SPARC mRNA of 1 log. Moreover, K562 proliferation at 72 h was inhibited by adding esogenous SPARC to colture medium with an IC50 of 50 mcg/ml. In the presence of imatinib 1 μM, SPARC IC50 was lowered to 30 mcg/ml.

Taken all together these data suggest that SPARC deficiency in CML could play a role in excess growth, cell adhesion and angiogenesis impairment. Furthermore, its upregulation after imatinib treatment may be related to the alteration of bone metabolism that is observed in CML patients treated with this tyrosine kinase inhibitor.

Disclosures:

No relevant conflicts of interest to declare.
Preliminary Study On Influence of Imatinib-Induced Apoptosis in Chronic Myeloid Leukemia Cells Co-Culture with Fibronectin Adhesion.
Authors: Xiaoli Liu , Xu Na , Du Qingfeng , Liu Zhi , Li Rong , Yang Jun , Wang Shuang , Zhong Min , Wang Yu
Published: October 2019

Abstract 4264

The bone marrow microenvironment supports growth, differentiation and proliferation of normal hematopoietic cells and can also contribute to malignant growth. Recently,it is considered that except for the point mutant of BCR-ABL kinase contribute to imatinib-resistant therapy for patients with chronic myeloid leukemia(CML), environment-mediated drug resistance (EM-DR) is a potential factor in imatinib resistance. Our previous studies found that Integrin, focal adhesion kinase(FAK), RhoA(a small GTPase) are important adhesion molecules,and related to imatinib resistance. But how and what they crosstalk with each other is still open to debate.

In order to simulated bone marrow microenvironment, we used the major components of bone marrow microenvironment- Fibronectin (Fn) co-cultured with human leukemia K562 cells.and then K562 cells were inoculated with Fn, collagen-coated plate(Co) and suspended cultures as control(mask) group,and then treated with 0.4μM,0.8μM,1.6μM,3.2μM,6.4μM imatinib for 24h,48h and 72h, detected cell apoptosis and proliferation by MTT and AnnexinV-PI assay, examined p-FAK and Rho-GTP by Wersten Blotting and Pull down-Wersten Blotting. The data showed that compared to the Co and mask groups, the cells growth inhibition and apoptosis in Fn co-culture group was significantly reduced. The protein expression of p-FAK and Rho-GTP was higher in the Fn group,and in time-dependent manner. When K562 cells in Fn group were transfected with 150nM siRNA-RhoA for 48h, there was no significant difference compared with the Co and mask groups. Furthermore, the above groups treated with anti-integrin monoclonal antibody (anti-CD29 mAb), we found that p-FAK was significant lower compared with without anti-CD29 mAb in the Fn group; but there was no significant difference of Rho-GTP compard with without anti-CD29 mAb in Fn group. These results indicate that Fn adhesion co-culture could reduce imatinib-induced cell growth inhibition and apoptosis, and this mechanism may be correlated to Rho-GTP activity,and anti-integrin monoclonal antibody could not completely block the integrin binding to Fn on K562 cells, or there was other pathway activated RhoA. The mechanism of EM-DR is complex,and which is well worth us to speculate and study.

Disclosures:

No relevant conflicts of interest to declare.
No Direct Effect of NOD2/CARD15 Variants On Clinical Outcome After Non-Myeloablative Allogeneic Stem Cell Transplantation.
Authors: van der Straaten Hanneke M. , Paquay Martine M. , Tilanus Marcel G.J. , Verdonck Leo F. , Huisman Cynthia
Published: October 2019

Abstract 4322

Background

Single nucleotide polymorphisms (SNPs) in the innate immunity receptor NOD2/CARD15 have been demonstrated to modulate the outcome of allogeneic haematopoietic stem cell transplantation. The effect of the NOD2/CARD15 polymorphism seems to be associated with donor source as well as type of conditioning regimen.

Methods

We reviewed NOD2/CARD15 mutations in all donor/recipient pairs of 192 consecutive patients who received non-myeloablative allogeneic stem cell transplantation(SCT) at our institution between 2002 and 2006. All patients were treated uniformly with fludarabine 30 mg/m2/day for 3 days followed by 200 cGy TBI (n=154) or TBI alone (n=38) and received grafts from HLA-matched related (n=132) or unrelated (n=60) donors.

Results

Mutated alleles were observed in 36 of 192 (19%) patients and in 35 of 192 (18%) donors. These SNPs, however, did not have a significant impact on clinical outcome data (P > 0.05, Kaplan Meier and Fine & Gray's test).

Acute graft-versus-host disease (GVHD) occurred in 24 of 61 (39%) patients with the polymorphism and in 66 of 131 (50%) patients without the polymorphism. Chronic GVHD developed in 28 of 55 (51%) patients with SNP pairs and in 79 of 121 (65%) patients with the wild type. The incidence of transplant-related mortality was 21% in both groups, 13 of 61 patients in the group with the polymorphism and 27 of 131 without the polymorphism. Relapse was seen in 23 of 61 (38%) patients with the SNP pairs and in 48 of 131 (37%) wild type patients. Finally, overall survival was 43% (26/61) in patients with the polymorphism and 39% (51/131) in patients without the polymorphism.

Conclusion

These data indicate that mutations in the NOD2/CARD15 genes do not influence the clinical outcome of non-myeloablative allogeneic SCT directly. Since NOD2/CARD15 variants are not recognized as a single significant prognostic factor, screening for NOD2/CARD15 when selecting a donor does not seem to have additional value in patients undergoing non-myeloablative SCT.

Disclosures:

No relevant conflicts of interest to declare.
Short Progression Free Survival Post Non-Myeloablative Sibling Allogeneic Stem Cell Transplantion for Multiple Myeloma.
Authors: Song Kevin , Sutherland Heather J. , Shepherd John D. , Abou Mourad Yasser , Barnett Michael J , Forrest Donna L. , Hogge Donna E. , Narayanan Sujaatha , Nantel Stephen H. , Power Maryse M , Toze Cynthia L. , Smith Clayton , Nevill Thomas J.
Published: October 2019

Abstract 4323

Introduction

Allogeneic stem cell transplant has been considered the only potentially curative treatment for patients with myeloma. Due to the high treatment related mortality associated with myeloablative allogeneic stem cell transplantation, non-myeloablative allogeneic stem cell transplantion is being investigated.

Methods

Between May 2003 and June 2008, 23 patients received a non-myeloablative allogeneic stem cell transplant (NMT) using a fully matched sibling as the donor. All had received a previous autologous stem cell transplant (ASCT). 17 received the NMT as a part of a planned tandem transplant post ASCT. Six received the NMT after relapse post ASCT. Conditioning chemotherapy was a combination of cyclophosphamide 1000 mg/m2 daily x 2 days and Fludarabine 25mg/m2 daily for 5 days. Survival was measured from the date of allogeneic stem cell infusion.

Results

Median age at NMT was 52 years. Eight were female. Immunoglobulin isotype was 13 IgG; 5 IgA; 1 IgD; 4 light chain. ISS stage was 10 – stage 1; 8 – stage 2; 3- stage 3; 2 – insufficient information. 9/21 had del 13q; 3/9 t (4;14); 1/7 del 17p. Disease status at the time of NMT were 5 CR/nCR, 11 PR, 1 SD, 3 relapse chemo-sensitive, 3 relapse chemo-resistant. Median follow-up is 29 months (7 – 65 months). Median event free survival (EFS) for all patients is 17 months (95 % CI 8-26 months). Median EFS for the 17 patients who received NMT as a part of a planned tandem procedure was 18 months. Median overall survival (OS) for all patients is 29 months (95% CI unable to calculate). At the time of analysis 17 patients remain alive. Five patients are alive in continuous remission at a median of 40.9 months from NMT (13.5-47.6 months). Twelve patients have active disease requiring treatment. Three patients have died of myeloma, one of graft-versus-host-disease (GVHD) and two of other causes. Nineteen patients (83%) developed GVHD at some time post-transplant. Of 14 patients who have relapsed, eleven patients had GVHD at the time of relapse. Of the 6 patient who received the NMT after relapse post ASCT, 5 have relapsed post NMT and one died of GVHD within 9 months.

Conclusion

Non-myeloablative sibling allogeneic stem cell transplantation for myeloma produces short progression free survival in-spite of the presence of graft-versus-host-disease and only a limited number of patients benefit. Patient who receive this treatment after relapse from prior autologous stem cell transplant do particularly poorly. Improved overall survival is primary due to improvements in post-relapse myeloma therapy.
Comparison of Outcomes Between Transplantation From Unrelated Donor Umbilical Cord Blood and From Unrelated Donor Bone Marrow or Peripheral Blood in Children with Acute Leukemia.
Authors: Lee Soo Hyun , Chueh Heewon , Yoo Keon Hee , Sung Ki Woong , Koo Hong Hoe
Published: October 2019

Abstract 4340

Introduction

Although umbilical cord blood (UCB) is an accepted alternative to bone marrow or peripheral blood (BM/PB) for transplantation, allele-matched marrow or peripheral blood is generally regarded as the preferred graft source. Our aim is to compare leukemia-free survival after transplantation of unrelated donor UCB and allele-matched BM/PB, and to provide guidelines for selection of an appropriate donor and graft source in children with acute leukemia.

Patients and Methods

Outcomes of 22 children with acute leukemia and transplanted with single unit UCB were compared with outcomes of 34 BM/PB recipients. All transplantations were performed in Samsung Medical Center between May 2003 and June 2009. Recipients of UCB were transplanted with grafts that were HLA-matched (n = 3) or HLA-mismatched for one (n = 15) or two antigens (n = 4). BM/PB recipients were transplanted with grafts that were HLA-matched (n = 20) or mismatched (n = 14). While allele-level typing for HLA-A, HLA-B, HLA-DRB1 and antigen-level typing for HLA-C was used for BM/PB transplantation, antigen level typing for HLA-A, HLA-B and HLA-DRB1 was used for UCB transplantation. Myeloablative conditioning regimens were used for both BM/PB and UCB transplantation.

Results

The median times to neutrophil and platelet recovery were 15 (range 10–21) and 22 (12–29) days after BM/PB transplantation, and 17 (13–44) and 34 (24–148) days after UCB transplantation, respectively. The 4-year probabilities of leukemia-free survival were 58.0% after HLA-matched BM/PB transplants, 65.9% after HLA-mismatched BM/PB transplants, 66.7% after HLA-matched UCB transplants, 67.5% after one-mismatched UCB transplants with high cell dose (NC > 3.0 × 107/kg), 40.0% after one-mismatched UCB transplants with low cell dose (NC < 3.0 × 107/kg), and 33.3% after two-mismatched UCB transplants. The incidence of grade 3-4 acute graft-versus-host disease (GVHD) were 12.1% after BM/PB transplantation and 5.3% after UCB transplantation, respectively (P = 0.64). Rates of extensive chronic GVHD were 53.8% after BM/PB transplantation and 23.1% after UCB transplantation, respectively (P = 0.09). Transplant-related mortality rates were higher after UCB transplantation (21.7%) than BM/PB transplantation (2.9%) (P = 0.03). Relapse rates were similar after BM/PB transplantation (29.4%) and UCB transplantation (21.7%) (P = 0.56).

Conclusion

These results support the use of HLA-matched or one-antigen HLA mismatched UCB in children with acute leukemia who need transplantation. Faster identification of a suitable cord blood unit may result in a timely transplantation avoiding delay that could expose the patient to the risk of losing the eligibility for transplant due to relapse or other complications.

Disclosures:

No relevant conflicts of interest to declare.
The Transplant Outcomes of a 10/10 Matched Unrelated Donor (MUD) Stem Cell Transplant (SCT) Is Similar to a HLA Identical Related Donor (RD) Transplant.
Authors: Veeder Catherine , Davies Sheila , Havey Christina , Maciejewski John J. , Nathan Sunita , Gregory Stephanie A. , Venugopal Parameswaran , Shammo Jamile , Fung Henry C.
Published: October 2019

Abstract 4346

Background

Allogeneic SCT is a potential curative treatment for many hematological malignancies. Unfortunately, approximately 70% of patients who may benefit from an allogeneic SCT can not find a HLA identical related donor. Through National Marrow Donor Program, about 60-70% patients will be able to identify a suitable donor for transplant. However, prior studies suggested that MUD-SCT was associated with increased transplant related morbidity and mortality when compared with RD-SCT. Hence, many hematologists/oncologists were hesitant to recommend a MUD-SCT even when a matched related donor is not available. We hypothesized that through optimal HLA typing and using a 10/10 molecularly matched unrelated donor, the outcomes for a MUD-SCT will be similar to RD-SCT.

Method

Beginning in 2004, we adopted a new policy in HLA typing and all patients and donors will be tested for HLA: A, B, C, DR, and DQ using molecular techniques. Here, we report the outcomes on 24 consecutive patients who revived a 10/10 MUD-BMT at our institution.

Results

Between February, 2004 and April, 2008, 24 patients (8 males & 16 females) with various hematological malignancy and life threatening hematological disorders underwent a 10/10 molecularly matched MUD-SCT at Rush University Medical Center. Median age at SCT was 43 (range 18-67). Diagnoses included acute myeloid leukemia:11, myelodysplasia:3, non-hodgkin lymphoma:3, chronic myeloid leukemia:2, acute lymphoblastic leukemia:2, severe aplastic anemia:2 and hodgkin lymphoma:1. While 9/24 (38%) has early disease, 15/24 (62%) had advanced disease at SCT. All patients received a myeloablative conditioning regimen. Four patients (4/24, 17%) died before day 100 post-SCT from treatment related toxicity; all 4 pts > 55 years old. The 1-year survival was 62.5% with a minimal follow-up of 1 year for surviving patients. At last follow-up, 9 patients survived greater than 3 years post-SCT. The cumulative incidences of acute GVHD Grades ≥ 2 and 3 - 4 were 42% and 13% respectively.

Conclusion

We conclude that the transplant outcomes of 10/10 matched unrelated donor stem cell transplant appears similar to matched related donor stem cell transplant. Thus, matched related donor stem cell transplant should be considered for patients lacking a HLA identical donor whenever an allogeneic SCT is clinically indicated.

Disclosures:

No relevant conflicts of interest to declare.
PAD Induction Therapy Followed by Autologous Peripheral Blood Stem Cell Transplant (APBSCT) as Upfront Treatment for Myeloma (MM) in Chinese Patients.
Authors: Lie Albert Kwok Wai , Wu DePei , Wang Chun , Chim Chor Sang , Liang Raymond , Fu Zhengzheng , Wan Liping
Published: October 2019

Abstract 4367

Introduction

The anti-myeloma effect of bortezomib, used as a single agent or in combination chemotherapy, is well recognized in various clinical studies. The aim of this study is to determine the feasibility of using the PAD combination (bortezomib, doxorubicin and dexamethasone) as upfront treatment in Chinese patients with newly diagnosed MM, followed by APBSCT.

Method

Patients between the ages of 18 to 65 year-old with newly diagnosed MM were recruited. PAD (bortezomib 1.3 mg/m2 on days 1, 4, 8 & 11; doxorubicin 9 mg/m2 on days 1 & 4; dexamethasone 40 mg on days 1-4, 8-11 & 15-18 during cycle 1 and days 1-4 during cycles 2-4) induction therapy was administered for four 28-days cycles, followed by autologous peripheral blood stem cell mobilization with cyclophosphamide 3 g/m2 plus G-CSF 5 mg/kg/BD. Subsequently, APBSCT was performed with high dose melphalan 200 mg/m2 conditioning.

Results

A total of 25 patients has been recruited so far (male:female = 14:11). The age ranged from 44 to 65 year-old (median 53). Eleven patients were of IgG subtype, 6 were IgD, 4 were IgA and 4 were kappa light chain disease. By Durie-Salmon staging, 22 were stage III, 2 were stage II and 1 was stage I. Seventeen (68%) patients have completed the four cycles of PAD induction therapy, while 3 (12%) are still having on-going PAD induction. Five (20%) patients withdrew from the PAD therapy. One patient developed disease progression after 2 cycles of PAD, while the other 4 developed serious adverse events with the first cycle of PAD, including 1 patient each with severe bradycardia, atrial fibrillation, severe sensory neuropathy and pulmonary haemorrhage. Of the 18 patients who had evaluable response to PAD (i.e. 17 patients who completed four cycles of PAD and 1 who progressed on PAD induction), the results were 3 CR, 7 VGPR, 5 PR and 3 NR. Fourteen of the patients with at least PR received APBSCT. Post-APBSCT responses were so far evaluable in 11 patients with 6 CR, 4 VGPR and 1 PR. Hence, PAD is a feasible and effective upfront induction therapy for Chinese patients with MM resulting in a satisfactory response rate. Nevertheless, significant adverse events may occur even early on with the treatment. Subsequent APBSCT further improved the response rate but long-term survival rate remains to be established.

Disclosures:

No relevant conflicts of interest to declare.
The Significance of Elevated Beta 2-Microglobulin (b2-m) in B-CLL: Evidence of in Vitro b2-m Secretion Following Activation of B-CLL Cells.
Authors: Berrebi Alain , Shvidel Lev , Arditti Fabian David , Bassous Lucette , Haran Michal , Shtalrid Mordechai
Published: October 2019

Abstract 4380

Beta 2-microglobulin (b2-m) is an 11-kDa protein recognized as the light-chain component of the MHC-I molecule. It is produced by nucleated cells membranes and is detectable in the serum and other body fluids. The link between the b2-m/ MHC-1 molecules and membrane structure has been associated with lymphocyte activation. Serum b2-m is elevated in a variety of disorders and has a prognostic value in lymphoprolifera! tive diseases such as multiple myeloma and CLL. High b2-m in CLL, has been found to correlate mainly with the advanced stage of the disease and high lymphocyte count and is routinely used as a bad prognostic factor (Keating, 1995, Montillo, 2005). Previous studies from our Institute have shown significantly higher level of b2-m in CLL/PL and PLL and in cases associated with autoimmune disorders close to levels found in active myeloma (Shvidel, 1996, Duek, 2006). We suggested that b2-m levels are increased during B-cell activation which not necessarily correlates with aggravation of CLL. In an attempt to further evaluate the correlation of bm-2 and B-cells activation we stimulated B-CLL cells in vitro with pokeweed mitogen (PWM) and compared the results with CLL controls after 3 and 7 days of culture. Level of b2-m in supernatants was tested using a microparticle enzyme immunoassay. B-CLL cells from 8 patients (4 in stage Binet A, 3 B and 1 C) were studied. Except for stage A, the serum b2-m was mildly increased. The results showed a significant in vitro secretion of b2-m after three and seven days in supernatants of PWM-activated cells compared to unstimulated controls: mean values after 3 days were: 188 ng/ml (range 96-279) in activated B-cells versus 57ng/ml (25-103) in controls; and after 7 days 599 ng/ml (401-914) versus 164 (67-267) in controls. Interestingly, CD8+ T-cells from a T-large granular leukemia patient stimulated with PHA do not secrete significant amount of b2-m in vitro (156 ng/ml versus 87 at day 7). Our results demonstrate that activation of B-CLL cells in vitro induced a marked secretion of b2-m. Thus, the significance of b2-m in CLL may correlate with degree of activation of B-CLL cells. This hypothesis is supported by the increase of CD38 (an activation marker) in CLL cases with high b2-m serum level. Moreover, the results of our study suggest that progression of disease may be the results of in-vivo activation of the cells and lends support to previous studies showing that CLL cells are G0 cells with a potential to progress in the cell cycle after stimulation.

Disclosures:

No relevant conflicts of interest to declare.
Plasma Circulating Caspase-3 Index as a Biomarker in Patients with Chronic Lymphocytic Leukemia.
Authors: Bruey Jean-Marie , Estrov Zeev , Kantarjian Hagop , O'Brien Susan , Keating Michael , Wierda William , Albitar Maher
Published: October 2019

Abstract 4381

Because of the accumulation of lymphocytes and relative paucity of proliferating cells characteristic of B-cell chronic lymphocytic leukemia (CLL), defective apoptosis has been proposed as a key step in the pathogenesis of this disease. Activity levels of the proapoptotic enzyme caspase-3 have been used as a simple, quantitative measure of ongoing apoptosis in various cancers. Here we explored the clinical value of assessing apoptosis levels in patients with CLL, using caspase-3 activity in plasma as a surrogate marker for apoptosis. The study included 194 patients with CLL and 96 normal control subjects. Caspase-3 activity was measured in plasma samples by incubation with the substrate DEVD. Circulating caspase-3 activity was detectable in the plasma of all CLL patients and normal control subjects, but was significantly (P=0.005) lower in CLL patients (median=7.49; range=4.2-19.68 pmol/min/μL) than in controls (median=8.27; range=4.54-34.30 pmol/min/ul. Absolute levels of caspase-3 levels in plasma in CLL did not correlate with any of the laboratory variables examined (WBC, platelets, HGB, B2M), Rai stage, or performance status. To assess the extent of apoptosis in relevance to level of the disease or tumor load, the caspase-3 index was calculated by normalizing plasma caspase-3 activity to the number of circulating lymphocytes in peripheral blood. The circulating caspase-3 index correlated negatively with bone marrow cellularity (P<0.001), spleen size (P= 0.002), and number of sites of enlarged lymph nodes (P<0.001). Interestingly, the circulating caspase-3 index correlated positively with Rai stage (P=0.03, Kruskal-Wallis) but not IgVH mutation status (P=0.74) or performance status (p=0.72). More importantly, higher circulating caspase-3 index values (>8 pmol/min/1000 lymphocytes/ul) were significantly associated with poor overall survival (P=0.005). However, in multivariate analysis incorporating caspase-3 index along with beta-2 microglobulin level and IgVH mutation status showed that caspase-3 was not predictor of survival (p=0.7). In conclusion, apoptosis as determined using plasma caspase-3 activity is low in CLL. However, high circulating caspase-3 activity index values appear to reflect more aggressive disease. Further studies are needed to explore the possibility that this circulating caspase-3 index reflects the proportion of cells that are transformed into larger cells, with consequently higher proliferation and apoptosis rates, in patients with CLL.

Disclosures:

No relevant conflicts of interest to declare.
Alpha-Type 1-Polarized Dendritic Cells Pulsed with Apoptotic Allogeneic Myeloma Cell Line Could Induce the Strong CTL Responses against Autologous Myeloma Cells.
Authors: Yang Deok-Hwan , Kim Mi-Hyun , Hong Cheol-Yi , Ahn Jae-Sook , Kim Yeo-Kyeoung , Chung Ik-Joo , Kim Hyeoung-Joon , Lee Je-Jung
Published: October 2019

Abstract 4931

Cellular therapy using dendritic cells (DCs) is emerging as the one of useful immunotherapeutic modality in multiple myeloma (MM). In order to generate potent functional DCs, alternative approaches for using appropriate and effective tumor antigens, such as myeloma cell lysates, myeloma apoptotic bodies, DC-myeloma cell hybrids or DC-transfected with myeloma-derived RNA, in steady of idiotype protein having with weak antigenicity are tried in MM. However, it is laborious work to obtain sufficient autologous tumor cells as a source of tumor antigen in the clinical setting. We investigated the feasibility of immunotherapy in patients with MM using myeloma-specific CTLs, which were generated in vitro by alpha-type 1-polarized DCs (αDC1s) pulsed with UVB-irradiated allogeneic myeloma cell line, ARH77 cells.

Material and

Methods

Peripheral blood (PB) was collected from MM patients (IgG, Ü-type) or normal healthy donors with HLA-A0201+ with informed consent according to the protocol approved by the institutional ethical committee of the Chonnam National University Hwasun Hospital. Phenotypic analysis of aDC1s was performed using a FACS Aria cell sorter. Tumor antigen uptakes (CD83+/PKH67+), allogeneic mixed lymphocyte reaction (MLR) and induction of myeloma-specific cytotoxic T lymphocytes (CTLs) were analyzed.

Results

αDC1s were significantly increased the expression of several molecules without differences by loading tumor antigens. αDC1s showed high production of IL-12 during maturation and after subsequent stimulation of CD40L, but were not significantly affected by loading tumor antigens. Myeloma-specific CTLs against autologous myeloma cells from MM patients were induced by αDC1s pulsed with allogeneic apoptotic ARH77 cells. Autologous CTLs generated by pulsed αDC1s displayed a larger number of IFN-g secreting cells against autologous myeloma cells as a target cell for ELISPOT assay. Pre-incubation of the target cells with an anti-MHC class I or II mAb inhibited the IFN-g release, indicating MHC class molecule-dependent cytotoxicity (figure)

Conclusion

We suggested that autologous DCs pulsed with allogeneic myeloma cell line could generate potent myeloma-specific CTLs against autologous myeloma cells and could be practicable method for approaching cellular immunotherapy in MM.

Disclosures

No relevant conflicts of interest to declare.