bluuebunny/crossref_metadata_2025 huggingface dataset:
Antisense Strategies to Specifically Deplete Levels of Factors II, VII and IX in Mice To Assess Their Potential as Targets for Anticoagulation.
Authors: Zhang Hong , Zhao Chenguang , Crosby Jeff , Siwkowski Andy , Freier Sue , Witchell Donna , Monia Brett P.
Published: October 2019

Numerous potential intervention points exist within coagulation pathways for development of novel anticoagulant agents. To help determine the most suitable target for the discovery of novel antithrombotic agents, we have implemented an antisense strategy to specifically deplete levels of various coagulation factors in mice. Here we report the effects of antisense oligonucleotides (ASOs) targeting factors II, VII, and IX on target RNA and protein levels in mice, as well as PT/aPTT. ISIS 401025 targeting factor II, ISIS 403102 targeting factor VII, and ISIS 402618 targeting factor IX dose-dependently suppressed their respective target transcript levels in a specific manner in primary mouse hepatocytes. Following subcutaneous administration to mice, each ASO reduced target RNA levels in liver and target protein levels in plasma in a specific and dose-dependent fashion (ED95 of approximate 25 mg/kg for each ASO). In conjunction with these effects, clotting times were also prolonged in a similar dose responsive fashion. Both PT and aPTT were increased following factor II inhibition and a PT-INR of 2.5 was achieved at 25 mg/kg, corresponding to a reduction in prothrombin transcript of 95%. As expected following factor VII ASO treatment, PT was significantly increased, while aPTT was unchanged. Conversely, following factor IX ASO treatment, aPTT was marginally but significantly prolonged, while PT was unchanged. These results demonstrate the capability of ASOs to dose-dependently and specifically modulate coagulation factor activity depletion and support the ASO approach as a novel strategy for the discovery of novel anticoagulation agents. Studies are in progress to assess relative safety and to expand the anticoagulation profile in mice for each of the factors under study.
Outcome after Autologous Stem Cell Transplantation for Multiple Myeloma in Patients with Preceeding Plasma Cell Disorders.
Authors: Kumar Shaji , Dingli David , Dispenzieri Angela , Lacy Martha Q. , Hayman Suzanne R. , Buadi Francis K. , Rajkumar S. Vincent , Litzow Mark R. , Gertz Morie A.
Published: October 2019

Background: Nearly a third of patients with newly diagnosed myeloma (MM) have a preceding diagnosis of plasma cell proliferative disorder (PCPD), mostly monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) or plasmacytoma. While high dose therapy improves survival in patients with myeloma, it is not clear if patients with preceding PCPD have a different outcome.

Methods and Results: We identified 151 patients with preceding PCPD from among 804 patients undergoing high dose therapy at our institution. These included 59 patients (7.3%) who had a preexisting diagnosis of MGUS, 88 (11%) patients, in whom the diagnosis of MM was preceded by SMM, including 23 patients who also had a preceding MGUS or plasmacytoma. In addition, 27 (3.4%) patients gave a preceding history of plasmacytoma. The median duration from the first diagnosis of a PCPD to that of myeloma was 32.4 months (range, 6.1 to 31 years), and was longer for patients with an initial diagnosis of MGUS compared to those with out preceding MGUS. While response rates including complete responses were generally similar between the two groups of patients, those with any preceding PCPD had a median TTP of 24.2 months (95% CI; 20, 28.3) compared to 17.3 (95% CI; 15.7, 18.8) months for those with no previous history (De novo Myeloma); p = 0.01 (Figure 1). The median OS from transplant for patients with previous history was 63.7 months (95% CI; 50.2, 77.4) compared to 48.3 months (95% CI; 39.7, 56.9) for the de novo group, p = 0.01 (Figure 2). These differences were most striking for those with a preceding diagnosis of MGUS; who had a longer time to progression (27.6 months vs. 17.7 months; p = 0.02), and longer OS from transplant (80.2 months vs. 49.3 months, p = 0.046) compared to those without any preceding MGUS. In a multivariate analysis, transplant within 12 months of diagnosis, lower serum M-protein at transplant, PCLI <1%, absence of cytogenetic abnormalities, achievement of CR, and presence of any preexisting monoclonal disorder prior to diagnosis of myeloma were independently prognostic for risk of post transplant relapse.

Conclusion: Patients with preexisting PCPD at the time of myeloma diagnosis undergoing HDT has a better outcome reflecting more indolent disease and a favorable biology than those presenting with de novo myeloma and no antecedent plasma cell dyscrasia.

Figure Figure Figure Figure
Polyclonal Hypergammaglobulinemia in a Randomized Sample of Ghanaian Adult Males.
Authors: Buadi Francis , Katzmann Jerry A. , Pfeiffer Ruth M. , Hsing Ann W. , Yeboah Edward D. , Biritwum Richard B. , Dispenzieri Angela , Kyle Robert , Rajkumar Vincent , Landgren Ola
Published: October 2019

Introduction Diffuse or polyclonal hypergammaglobulinemia has been associated with multiple medical conditions such as autoimmune diseases, infections, and chronic liver diseases. However, the possibility of differences in gamma globulin levels in different races and its association with environmental and socioeconomic factors is not well studied.

Method A randomized sample of 917 adult males (50–74 years) in an urban community in Ghana underwent in-person interview and health examination. Serum from all persons was analyzed by electrophoresis performed on agarose gel. Those found to have a monoclonal protein (n=54) were excluded from further analysis. Gamma globulin levels were measured in the remaining 863 samples. Associations between selected characteristics and gamma globulin levels were assessed by Chi-square test and logistic regression models.

Results The median gamma globulin level was 1.86 gm/dl. Seventy three percent of the study population had gamma globulin levels above the 1.6 gm/dl reference level and will be considered to have hypergammaglobulinemia. Surprisingly, 25% had gamma globulin levels above 2.17 gm/dl. In univariate analysis increased gamma globulin levels were associated with older age within the study population. Only 54% of the males aged 60 yrs or younger had gamma globulin levels above 1.6 gm/dl compared to 69% in those older than 60 yrs (p=0.007). Higher education level was associated with a low prevalence of hypergammaglobulinemia 51% compared with 74% in those with less than high school education (p <0.0001). Smoking was not associated (p=0.16) with gamma globulin levels. Although a higher proportion (71%) of people with history of alcohol use had hypergammaglobulinemia compared to the control (63%) this was not statistically significant (p=0.07). Self-reported history of genital warts, genital herpes and meningitis was associated with hypergammaglobulinemia, whereas syphilis, malaria, cholera, gonorrhea, hepatitis, measles and tuberculosis had no association with gamma globulin levels in univariate analysis.

Conclusion Three quarters of this randomized sample of Ghanaian adult males has hypergammaglobulinemia based on current laboratory definitions. More work is needed to uncover underlying causes for the observed high gamma globulin levels in this population. Further evaluation of a younger population with less environmental influence may help explain whether this is due to genetic differences, environmental exposures, or a combination.
MicroRNA Expression Contributes to Hypoxia-Inducible Factor-1a-Induced Differentiation of Myeloid Leukemic Cells.
Authors: He Ming , Zhang Bo , Yin Qianqian , Guo Meng , Zhao Qian , Chen Guo-Qiang
Published: October 2019

Abstract 1007

Poster Board I-29

In our previous study, we showed that the hypoxia or hypoxia-mimetic agents induces differentiation of myeloid leukemic cells. During this event, hypoxia-inducible factor-1a (HIF-1a) exerts a critical role in its transcriptional activity-independent manner. MicroRNAs (miRNAs) regulate the expression of 30% of the transcripts of human genome on a posttranscriptional level. Recent work on miRNA has shed light on the possible involvement of miRNA genes in biological processes such as cell proliferation, differentiation and hematopoiesis. To reveal the miRNA expression profile and related roles in HIF-1a-induced differentiation of myeloid leukemic cells, miRNA expression profiling was analyzed by microarray in HIF-1a-expressing U937T cells (U937Tclone) and HIF-1a-absent cells (U937Tempty) in 0 day, 2 days and 4 days after tetracycline removal. The differentially expressed miRNAs, as verified by quantitative real-time RT-PCR and Northern blot, were subjected to gene ontology (GO) analysis and pathway analysis. The upregulated and downregulated miRNAs in U937Tclone cells in three time points after tetracycline removal were 11 miRNAs and 7 miRNAs, respectively. The differential expression of miR-26a, -107, -19b, -20a, -17, -92a, -92b and -106a was validated. On the basis of bioinformatic analysis, we focused on the changed components of miRNA 17-92 cluster and miRNA 106a-92 cluster, mostly considered as oncomirs, which have the similar downregulated expression profile. The results showed that miR-17 was significantly depressed by 40% and 70% in 2 and 4 days after tetracycline removal in U937Tclone. And miR-20a was significantly depressed by 60% and 80%, respectively. High-enrichment GOs containing differentiation-related targeted genes and miRNA-gene networks revealed STAT3 as one of the highest degree of target genes, which was targeted by the three changed components of miR-17 family (miR-17, -20a and -106a). STAT3 protein was validated to be upregulated in U937Tclone after tetracycline removal by Western blot, and no fluctuation in STAT3 mRNA levels was observed, which may uncover the critical role of miR-17 family in the cell differentiation induced by HIF-1a. In summary, significantly different miRNA profiles were seen in U937Tclone cells as compared to U937Temptycells. The downregulation of the components of miRNA 17-92 and 106a-92 cluster may contribute to HIF-1a-induced myeloid leukemic cell differentiation by targeting STAT3 or other signaling pathways. Further functional study of these miRNAs will be carried out by overexpression and knockdown experiments. These data would shed new insights for understanding mechanisms underlying leukemogenesis and designing the new therapeutic strategy for differentiation induction of acute myelogenous leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Differential Down-Regulation of RPL35a in Human Bone Marrow Progenitors Demonstrates a p53 Independent Mechanism Mediating the Diamond Blackfan Anemia Phenotype.
Authors: Caywood Emi , Farrar Jason E. , Lipton Jeffrey Michael , Arceci Robert J.
Published: October 2019

Abstract 1096

Poster Board I-118

We have previously reported the first known mutations in a gene that encodes a large ribosomal subunit protein, Rpl35a that are linked to Diamond Blackfan Anemia (DBA), a congenital pure red cell aplasia. We have demonstrated that down-regulation of RPL35a results in decreased cellular proliferation, increased apoptosis, impaired ribosomal RNA processing and defective ribosome biogenesis. In order to examine the mechanisms by which defects in RPL35a lead to the development of DBA, we have developed a lentiviral system containing shRNA to down-regulate RPL35a and RPS19, a gene encoding a small ribosomal protein known to be defective in about 25% of DBA cases. Defects in these cellular processes have been linked to disruption of cell cycle progression through activation of nucleolar associated regulatory mechanisms. Cell cycle analysis of the normal human bone marrow progenitors transduced with the specific shRNA demonstrates a G1 arrest when compared to cells transduced with a luciferase shRNA control construct. Similar findings were made in the p53-mutant cell line, TF-1. In contrast, cell cycle analysis of the p53-mutant UT-7/Epo cell line demonstrates a primarily G2/M arrest. To further investigate the mechanism of the cell cycle arrest in bone marrow progenitors, p53 expression was analyzed by Western Blot. Only a nearly complete knockdown resulted in increased p53 expression; shRNA affecting a haploinsufficient state had no evidence of increased p53 expression. However, clonogenic studies of human bone marrow progenitors transduced with shRNA constructs that approximate haploinsufficiency demonstrate a selective absence of erythroid colonies compared to myeloid colonies, thus mimicking the phenotype of DBA. In contrast, bone marrow progenitors transduced with shRNA that causes near complete absence of expression of RPL35a have neither myeloid nor erythroid clonogenic potential consistent with a stem cell catastrophe. These data may suggest a p53 independent pathway can mediate the effects of ribosomal protein deficiency in DBA as well as demonstrate a model for further understanding abnormalities in ribosomal proteins that result in an in vitro phenocopy of altered DBA hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Exposure to Novel Agents Increases Post Relapse Survival in Patients with High Risk Myeloma Defined by Early Relapse (<12 months).
Authors: Venner Christopher P. , Connors Joseph M. , Sutherland Heather J. , Shepherd John D. , Hamata Linda , Mourad Yasser Abou , Barnett Michael J , Forrest Donna L. , Hogge Donna E. , Nantel Stephen H. , Narayanan Sujaatha , Nevill Thomas J. , Nitta Janet , Power Maryse M , Toze Cynthia L. , Smith Clayton , Song Kevin
Published: October 2019

Abstract 2872

Poster Board II-848

Intro:

Relapse within 12 months of stem cell transplant is an important clinically relevant prognostic factor predicting poor outcome for patients with multiple myeloma (MM). Little is known about the use of novel agents (NA) in this setting and their impact on survival. In British Columbia autologous stem cell transplantation (ASCT) as a part of initial treatment is the standard of care for patients who are eligible. The use of NA is largely restricted to the relapsed setting thus providing a large cohort of patients to assess the impact of these drugs.

Patients:

We reviewed our experience with 418 patients with MM treated with ASCT diagnosed between 1985 and 2008. 307 have relapsed. Although thalidomide was available starting in 1999, the other novel agents, bortezomib and lenalidomide, were not available until after 2004, permitting division into two groups before and after Dec 2004.

Methods:

The cohort was separated into four groups based upon year of relapse (pre and post 2004) and remission duration post ASCT. Early relapse was defined as recurrence within 12 months (m) of ASCT. Four groups were defined: Group A, late relapse after 2004 (n=138); B, early relapse after 2004 (n=70); C, late relapse before 2004 (n=58); D, early relapse before 2004 (n=41). We examined the impact of NA exposure on post relapse survival (PRS).

Results:

Median overall survival from diagnosis of myeloma of the whole cohort was 79.9 months (95% CI; 70, 89). Median PRS was 31.0m (95% CI; 25, 36). Exposure to novel agents conferred a better PRS (35.7m, 95% CI; 30, 42 vs. 9.1m, 95% CI; 4, 13; p < 0.001). Results comparing the various groups are summarized in table 1. Kaplan-Meier curves of the four groups are shown in figure 1.

Conclusion:

Post relapse survival for patients with MM treated with ASCT has improved with the introduction of novel agents. Although relapse within 12m of ASCT remains an important clinical prognostic factor predicting worse outcome, the introduction of bortezomib and lenalidomide after 2004 correlates with a 24 month improvement in post relapse survival in these patients.

Disclosures:

Connors: Roche Canada: Research Funding.
Recurrent Thromboembolism in a Tertiary Care Pediatric Center.
Authors: Rajpurkar Madhvi , Zia Ayesha N. , Sabo Cynthia , Thomas Ronald , Chitlur Meera B. , Frey Mary Jane , Lusher Jeanne M.
Published: October 2019

Abstract 2987

Poster Board II-963

Background:

Thrombosis in children, though rare, is being increasingly recognized in pediatric tertiary care centers. Most data on children have largely been extrapolated from adults. Comprehensive data about the incidence and risk factors for recurrence of pediatric thrombosis are scarce.

Aim:

1) To estimate the incidence of recurrence of thrombosis at a single pediatric tertiary care center and 2) To determine the risk factors for recurrent thrombosis in children.

Methods:

After local IRB approval, charts of patients (pts) with documented venous or arterial thrombosis admitted or referred to the hematology service from 2001-2008, were reviewed and pertinent data obtained. Data were analyzed using the 17.0 version of SPSS.

Results:

Preliminary analysis revealed 238 pts (ages 0-30 years) with 183 deep venous (DVT, 75%), 53 arterial (22%) and 2 combined (0.8%) episodes of thrombosis at initial presentation. Of the 183 pts with DVT there were 110 females, 128 males; of pts with arterial strokes there were 21 females, 32 males. Overall 11 pts (4.6%, 6 females, 5 males) had a recurrence, with 91% venous and 9% arterial. Three (27%; 2 females, 1 male) out of the 11 pts had a third recurrence, which were all venous. Sites for recurrence were lower extremity (82%), neck (9%) and thorax (9%).

Risk factors for recurrence were positive family history of DVT (7/11; 63.6%), elevation in factor VIII (5/11; 45.5%), proximal lower extremity thrombosis as primary site (5/11; 45.5%), obesity (4/11; 36.4%), the presence of PTTLA (4/11; 36.4%) and the presence of inflammatory bowel disease (27.3%). Heterozygous factor V Leiden and congenital AT III deficiency was present in one pt each (9%) with recurrence. Other congenital thrombophilia traits were not seen in patients with recurrent events. Interestingly, gender, duration of treatment and residual clot after treatment (partial resolution) were not significantly associated with risk of recurrence.

Conclusion:

Recurrent thrombosis is infrequent in children and a positive family history, elevated factor VIII, and other acquired factors such as obesity and inflammatory bowel disease were identified as risk factors for recurrence. Inherited thrombophilia and partial resolution of thrombosis were not associated with recurrence in this series of patients. Larger multi-center trials are needed to identify risk factors for recurrence in children. To our knowledge this is the first pediatric study that has systematically evaluated the incidence and risk factors associated with recurrent pediatric thrombosis.

Disclosures:

No relevant conflicts of interest to declare.
PAX5-PML Acts as a Dual Dominant-Negative Form of Both PAX5 and PML.
Authors: Kurahashi Shingo , Hayakawa Fumihiko , Miyata Yasuhiko , Minami Yosuke , Abe Akihiro , Naoe Tomoki
Published: October 2019

Abstract 3070

Poster Board III-7

PAX5 is a transcription factor expressed in B lymphoid lineage from pro-B cell to mature B cell, and is required for B-cell development and maintenance. De-regulated and reduced PAX5 activity has been implicated in B cell malignancies both in human disease and mouse models. Recently, approximately 30% of childhood and adult B cell acute lymphoblastic leukemia have aberrancies in PAX5 gene such as deletions, point mutations, or chromosomal translocations. Chromosomal translocation t(9;15)(p13;q24) was found in 2 cases of childhood acute lymphoblastic leukemia and resulted in an in-frame fusion of PAX5 to PML gene. PAX5 moiety of PAX5-PML retains its DNA binding domain but loses its transactivation domain suggesting PAX5-PML will be a dominant negative form of PAX5. PML is originally found as a fusion partner of RARαa in PML-RARαa, an oncoprotein found in acute promyeloid leukemia (APL), and is now thought to be a tumor suppressor and a pro-apoptotic factor. PML-RARαa dominant-negatively affects PML function by disrupting PML nuclear bodies (NBs) where PML exerts its function and gives APL cells survival advantage. These findings give rise a speculation that PAX5-PML not only causes differentiation block by transcriptional repression of PAX5 target genes but also confers a survival advantage by inhibition of PML function. However, no functional analysis has been done for PAX5-PML.

Here, we demonstrate that PAX5-PML had a dominant negative effect on both PAX5 and PML. PAX5-PML inhibited transcriptional activity of PAX5 in luciferase reporter assay. PAX5-PML expression also suppressed expression of CD19, one of the transcriptional targets of PAX5, in B-lymphoid cell line. Surprisingly, PAX5-PML hardly showed DNA binding activity in electro mobility shift assay although it retains DNA binding domain of PAX5, suggesting that inhibition of PAX5 DNA binding by occupation of PAX5 binding sites would not be the mechanism for PAX5-PML to inhibit PAX5 transcriptional activity. On the other hand, co-expression of PAX5-PML inhibited PML sumoylation, an essential post-translational modification for PML to form NBs, and altered PML localization from NB pattern to diffuse nuclear pattern. Furthermore, treatment with arsenic trioxide, a therapeutic reagent for APL which induces enhancement of PML sumoylation, reconstitution of PML NBs, and apoptosis in APL cells, induced recovery of PML sumoylation and reconstitution of PML NBs also in cells expressing PAX5-PML. More importantly, arsenic trioxide treatment of PAX5-PML expressing HeLa cells, which showed resistance to PML dependent apoptosis, overcame anti-apoptotic effect of PAX5-PML. These data suggest the involvement of this fusion protein in the leukemogenesis of B-ALL in dual-dominant negative manner and the possibility that some cases of ALL can be treated with arsenic trioxide.

Disclosures

Naoe: Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.
A Prospective Study of Iron Overload in Patients Undergoing Ablative Stem Cell Transplantation.
Authors: Armand Philippe , Rhodes Joanna , Kim Haesook , Cutler Corey , Ho Vincent T. , Koreth John , Alyea Edwin P , Soiffer Robert J , Antin Joseph H
Published: October 2019

Abstract 3332

Poster Board III-220

Introduction

Iron overload is a recently recognized problem for patients undergoing hematopoietic stem cell transplantation (HSCT). Hyperferritinemia is common and is associated with significantly increased treatment-related mortality (TRM) and poorer overall survival after HSCT. However, serum ferritin may be a poor surrogate for total body iron burden, and no prospective study of parenchymal iron overload has yet been reported. We initiated a prospective study of adult patients with acute leukemia or myelodysplastic syndrome (MDS) undergoing myeloablative HSCT, in order to estimate the prevalence of iron overload in this population. We measured pre-HSCT serum ferritin, C-reactive protein (CRP), iron, total iron binding capacity, and genotyped patients for HFE mutations. All patients also underwent liver and cardiac MRI with measurement of T2*, from which liver iron content (LIC) and cardiac iron loading were inferred.

Results

41 of 45 planned patients have been enrolled to date. Median age was 46 years (range, 18-63). 24 patients had AML, 11 had ALL, and 6 had MDS. They had received a median of 2 prior chemotherapy courses (range, 0-6). Among the 39 patients with available transfusion history, the median number of prior RBC transfusions was 19 (range, 0-59). 88% of patients had a pre-HSCT serum ferritin above normal; the median value was 1432 (range, 20-6989). Higher ferritin values were associated with more advanced disease stage, number of prior chemotherapy regimens, and number of transfusions. The median LIC was 3 g/g dry weight (g/gdw) (range, 0.6-12.9). 85% of patients had an LIC above the upper limit of normal (1.8 g/gdw), and 17% had an LIC above 7 g/gdw. Only 1 patient had cardiac iron overload (cardiac T2*<20 msec). Pre-HSCT LIC correlated best with serum ferritin (r=0.7), although it also correlated with transfusion history (r=0.56) and with transferrin saturation (r=0.50). The correlation improved further (r=0.76) when ferritin was divided by log(CRP), when CRP was above normal (see Figure).

Median follow-up after HSCT is 4.5 months (range, 0.2-13.5), precluding full analysis of clinical outcomes at this point.

Conclusion

Hepatic iron overload is very common in patients with acute leukemia or MDS undergoing HSCT, and is strongly correlated with transfusion history. Cardiac iron overload is rare. Pre-HSCT serum ferritin, adjusted for CRP, is a good surrogate marker for hepatic iron overload. This close relationship lends support to prior studies that used serum ferritin to assess the impact of pre-HSCT iron overload on transplantation outcome. Moreover, it provides the basis for the assessment of iron chelation strategies to reduce TRM.

Disclosures

Armand: Novartis: Consultancy, Research Funding.
Unrelated Cord Blood Transplantation for Severe Acquired Aplastic Anemia Using a Reduced-Intensity Conditioning: High Graft Rejection but Good Survival
Authors: Liu Huilan , Geng Liangquan , Wang Xingbing , Ding Kaiyang , Tang Baolin , Tong Juan , Yao Wen , Wang Zuyi , Sun Zimin
Published: October 2019

Abstract 2368

We report a single center experience in treating 16 consecutive patients (nine male and seven female) with severe aplastic anemia (AA) who received unrelated cord blood transplantation (UCBT) between 2006 and 2010. The main outcomes of interest were the tolerability and toxicity of UCBT, hematopoietic reconstitution and survival. The first two patients using a conditioning regimen consisting of cyclophosphamide (total dose 120°<<200 mg/kg), rabbit anti-thymocyte globulin (ATG, total dose 10°<<15 mg/kg) and methylprednisolone (total dose 1.5g). The other 14 cases received a reduced-intensity regimen composed of cyclophosphamide (total dose1200mg/©O), ATG (total dose 30 mg/kg) and fludarabine (total dose 120°<<180 mg/m2). Cyclosporine and mycophenolate mofetil were used as GVHD prophylaxis. The median age was 17 years (range 5–61 years) and the median weight was 48 kg (range 16–65 kg). Ten of sixteen were very SAA (VSAA). The median interval between diagnosis and UCBT was 30 days (range, 15–180). All except one did not received ATG-based immunosuppressive therapy before UCBT. Twelve patients received single UCB unit, four cases received double units. Donor/recipient HLA was at least four of six loci matching. The median nucleated cell dose infused was 4.27×107 cells/kg (range 2.34–13.41×107 cells/kg) and CD34+ cell dose infused was 2.02×105 cells/kg (range 0.71–4.35×105 cells/kg). Two patients were not evaluable for engraftment because of early death on day +21 and +22 due to severe infection and intracranial hemorrhages, respectively. Only one of the fourteen cases had engraftment with complete chimerism of single UCB unit from day 7, whose ANC© f 0.5 ×109/L and platelet© f20 ×109/L occurred on day 12 and day 31 post double UCBT, respectively. But she experienced secondary graft failure after three months post transplantation and attained survival by successful haplo-identical related transplantation. Thirteen patients experienced primary graft rejection, but all of them acquired autologous recovery. The 3-month and 6-month cumulative incidence of response was 61.5% and 85.7%, respectively. Currently, 14 patients are alive, having survived for 189 to 1712 days (median, 544 days) after their transplantations. The probability of overall survival at 4 years was 87.5%.Our data indicate that UCBT for new diagnosed SAA using no irradiation but fludarabine-based conditioning still seems to be inevitable to led to the high risk of rejection, but may facilitate autologous recovery and improve survival with low risk of transplant-related mortality.

Disclosures:

Sun: Fund of 11th Five-Year Science and Technology Key Project of Anhui Province (06013128B): Research Funding; Fund of Anhui Provincial “115” Industrial Innovation Program: Research Funding.
DEK/NUP214 Fusion Gene Predicts Good Outcome In Acute Myeloid Leukemia In Complete Remission After Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Ishiyama Ken , Takami Akiyoshi , Nakao Shinji , Hidaka Michihiro , Maeda Tetsuo , Kawa Keisei , Nagamura Tokiko , Tabuchi Ken , Atsuta Yoshiko , Sakamaki Hisashi
Published: October 2019

Abstract 2386

Introduction

Acute myeloid leukemia (AML) with chromosomal translocation (6;9)(p23;q34) results in a chimeric fusion gene between DEK/NUP214 (formerly DEK/CAN), and is a rare disease, accounting for approximately 1% of cases. This entity is considered to be a good indication for allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to its poor prognosis and non-responsiveness to chemotherapy. However, whether the prognosis of patients with exclusive DEK/NUP214 abnormality is improved by allo-HSCT has not been demonstrated. We conducted a retrospective study to examine the outcomes of allo-HSCT in AML patients with the DEK/NUP214 gene using the databases of the Japan Society for Hematopoietic Cell Transplantation (JSHCT) and the Japan Cord Blood Bank Network (JCBBN).

Methods

We identified de novo AML patients who underwent transplant between January 1996 and December 2007 with no prior treatment by allo-HSCT. Clinical features and outcomes of the patients with DEK/NUP214 abnormality and 3 different control groups (patients with chromosome 7 abnormalities, complex karyotype or normal karyotype) were compared.

Results

Of 4517 AML cases meeting the above criteria, the number of patients with normal karyotype, chromosome 7 abnormalities, complex karyotype and DEK/NUP214 abnormality was 2227, 139, 416 and 64, respectively. The median age of each group was 41, 37, 41 and 34 years, respectively; patients with the DEC/NUP214 abnormality were significantly younger than those with the complex karyotype, and patients with normal chromosomes. Gender was not biased among the groups. Overall survival (OS) of the patients with DEC/NUP214 abnormality at 5 years was 49%, which was superior to the OS of the patients with normal karyotype, chromosome 7 abnormalities and complex karyotype (44%, 30% and 27%, respectively; P < 0.0001; Figure). This trend was significant in HSCT grafts with bone marrow and peripheral blood stem cells. In patients with DEK/NUP214 abnormality, none of the following factors improved survival; combination of gender between patient-donor, dose of total body irradiation prior to HSCT; and use of prophylactic immunosuppressants for graft-versus-host disease. However, the status at HSCT had a significant impact on outcome (5-year OS: 66% in patients in complete remission (CR) at HSCT vs. 31% in non-CR patients; P < 0.001). Analysis exclusively for the patients in CR at HSCT showed similar results as above.

Conclusion

Our results suggest that patients with the DEK/NUP214 abnormality are more likely to respond to allo-HSCT. Allo-HSCT should be particularly recommended for patients experiencing CR after prior remission-induction chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
4-1BB Ligand Mediates a “Vicious Cycle” of Immune Evasion Upon Reciprocal Interaction of Chronic Lymphocytic Leukemia and NK Cells
Authors: Buechele Corina , Baessler Tina , Schmiedel Benjamin J , Kanz Lothar , Salih Helmut R.
Published: October 2019

Abstract 2416

NK cells play an important role in tumor immunosurveillance. Their reactivity is governed by various immunoregulatory molecules, which influence both direct anti-tumor immunity and NK responses induced by therapeutic antibodies like Rituximab. Various members of the TNF/TNFR family modulate differentiation, proliferation, activation, and death of both tumor and immune effector cells including NK cells. Recently we reported that the TNFR family member 4-1BB/CD137 is expressed on human NK cells following activation. In contrast to the stimulatory role of its murine counterpart, we found that human 4-1BB impairs NK anti-tumor reactivity upon interaction with its ligand 4-1BBL expressed on blasts of a substantial proportion of acute myeloid leukemia patients (Blood 115: 3058-69; 2010). In addition, we found that expression of 4-1BBL is general feature of leukemic cells of chronic lymphocytic leukemia (CLL) patients causing impaired direct and Rituximab-induced NK cell reactivity (Blood 114: 279; 2009). Here we report that reverse signaling via 4-1BBL into CLL cells following interaction with 4-1BB, which is absent on NK cells of healthy donors, but expressed at substantial levels on NK cells of CLL patients, induced pronounced production of immunoregulatory cytokines like TNF, IL-6 and IL-8 by the CLL cells. Moreover, we found that sera of CLL patients contained elevated levels of these immunoregulatory cytokines as compared to healthy controls. When PBMC of healthy donors were exposed to supernatants of in vitro cultured CLL cells or sera from CLL patients, this resulted in pronounced 4-1BB expression on the NK cells. This effect could be prevented by addition of the TNF blocker Infliximab to patient sera. The 4-1BB expression induced by CLL sera resulted in impaired NK reactivity specifically against 4-1BBL-expressing targets as revealed by functional analyses with 4-1BBL transfectants and the respective mock-controls. Moreover, the induced 4-1BB expression also impaired NK cell reactivity against primary CLL cells constitutively expressing 4-1BBL, thus closing a cycle of immune evasion. Taken together, our data demonstrate that 4-1BBL enables CLL cells to evade NK anti-tumor reactivity, and disruption of the “vicious 4-1BB-4-1BBL cycle” in CLL - NK interaction e.g. by TNF- or 4-1BB-blockade may serve well to enhance NK reactivity in therapeutic strategies like antibody treatment or allogenic stem cell transplantation in CLL, which rely on sufficient NK cell function.

Disclosures:

No relevant conflicts of interest to declare.
PKA Mediated Phosphorylation of TAL1 Regulates Its Interaction with LSD1 During Hematopoiesis.
Authors: Li Ying , Deng Changwang , Hu Xin , Fu Xueqi , Qiu Yi , Huang Suming
Published: October 2019

Abstract 2599

TAL1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages. TAL1 is a phosphorylated protein and its activities are mediated by the corepressors and coactivators that associate with TAL1. However, the functional link between phosphorylation and the recruitment of co-regulators by TAL1 is currently unknown. We showed that TAL1 dynamically interacts with LSD1 complex containing both histone H3K4 demethylase and deacetylase activities during hematopoiesis (Proc. Natl. Acad. Sci. USA 106: 10141–10146). To further understand the molecular mechanism that regulates the TAL1 and LSD1 interaction during hematopoiesis, we determined whether TAL1 directly interacts with LSD1 and characterized the domains required for this interaction. TAL1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142–185 proximal to the bHLH domain, which contains a serine 172 residue that becomes phosphorylated by Protein kinase A (PKA) during the transcriptional activation of TAL1. The PKA inhibitor, H89, stimulated TAL1 interaction with LSD1 in hematopoietic cells, while Forskolin, an activator of PKA, completely abolished TAL-LSD1 interaction. We further mutated serine 172 of TAL1 to Alanine (Ala) or to Aspartic acid (Asp) to mimic unphosphorylated or phosphorylated TAL1, respectively. While the TAL1Ser172Ala mutant remains interacted with LSD1, TAL1Ser172Asp specifically loses its interaction with LSD1 indicating that serine 172 phosphorylation of TAL1 by PKA destabilizes the TAL1 and LSD1 interaction. Our previous results indicate that LSD1 inhibits TAL1 mediated erythroid differentiation. To further test whether the activity of TAL1 is mediated through an interaction with LSD1, we expressed the TAL1 mutant that deleted the LSD1 interacting domain in erythroid progenitor cells and showed that the deletion of the LSD1 interacting domain of TAL1 leads to a promotion of erythroid differentiation and H3K4 hypermethylation of the P4.2 promoter. In contrast, the expression of the TAL1Ser172Ala mutant and TAL1-LSD1 chimerical fusion enhanced cellular proliferation and colony formation ability of the hematopoietic progenitor cells while these constructs inhibited erythroid differentiation. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and erythroid differentiation. The results suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of Exercise Capacity In Children with SCD by Six Minute Walk Test
Authors: Minniti Caterina , Alam Shoaib , Kato Gregory J. , Nouraie Mehdi , Sable Craig , Ensing Gregory , Campbell Andrew , Rana Sohail , Darbari Deepika , Gladwin Mark , Castro Oswaldo , Gordeuk Victor R.
Published: October 2019

Abstract 2664

Background:

The six-minute walk (6MW) test is used in pediatrics in clinical practice and research to determine cardiopulmonary functional status. A change in 6MW may be affected by factors not strictly related to cardiopulmonary function, which may be different in different patient populations. In children and adolescents, age and height has been found to be a strong predictor of 6MW distance. We set out to study the effects of hematological and echocardiographic variables on 6MW distance in children with sickle cell disease (SCD) and its changes over time.

Methods:

We reviewed prospectively collected hematological, 6MW distance, and echocardiographic data from four hundred children with SCD (including 311 Hb SS or β0) and 69 controls (including 21 Hb AS) enrolled in PUSH (Pulmonary Hypertension and the Hypoxic Response in SCD). Subjects were evaluated at baseline and after 18–24 months, as per protocol. An un-encouraged 6MW was performed in children 5 years or older by trained personnel as per the guidelines of the American Thoracic Society. Subjects were studied at steady state, at least three weeks after any acute exacerbation of SCD. We used ANOVA for univariate analysis and stepwise linear regression for multivariate analysis.

Results:

Median (interquartile range) 6MW in severe SCD genotype (SS and S-β0) was 444 (399-508) and in controls was 495 meters (435-539) (P = 0.0002), while it was 461 meters (408-518) in milder SCD genotypes (P=0.2 for comparison with severe genotypes) (Table 1). In multivariate analysis, Hb, WBC and history of frequent pain episodes were significantly associated to distance of 6MW.

Follow up 6MW obtained in 174 SCD subjects revealed a decline of 10% or more in distance in 22% of subjects with severe genotypes and 33% of other genotypes. The decline was more frequent in the subset of SS subjects with TRV>2.59 (40% vs 19%).

CONCLUSION:

Six minute walk distance is significantly shorter in children with SCD, even as young as 5 years of age, when compared to age and race appropriate controls, indicating early compromise of exercise capacity. SS and S-β-0 genotype subjects have more impairment of exercise capacity compared to milder genotypes. Predictors of 6MW distance are similar in SCD and non SCD subjects, which validates the use of this test in this patient population.

Longitudinal changes in subjects with SCD are similar, with declines in about a quarter of the subjects. Patients with SS who have an elevated TRV have the highest rate of decline in 6MW.

These results validate the use of 6MW as a tool for assessing exercise capacity in children with SCD.

Disclosures:

No relevant conflicts of interest to declare.
Unrelated Umbilical Cord Blood Transplantation for Hematological Malignancies Using Fludarabine/BUCY2 Conditioning Regimen
Authors: Sun Zimin , Liu Huilan , Geng Liangquan , Wang Xingbing , Ding Kaiyang , Wu Yun , Zhou Chenyang , Tong Juan , Song Kaidi , Liu Hongjun , Li Chun , Wang Zuyi
Published: October 2019

Abstract 4572

Cord blood transplantation (CBT) is largely used to treat patients affected by hematological malignant disorders. Myeloablative TBI-based conditioning appears to provide reliable engraftment after CBT for malignancies. However, the toxicity of TBI limits their widespread use. So far, a standard non-TBI based regimen has not been firmly established. In order to overcome graft failure, we investigated a strategy using Fludarabine (FLU)/BUCY2 regimen in CBT for patients with hematologic malignancies. Seventeen patients(children 16, adult 1) with hematologic malignancies who underwent single-unit CBT used a conditioning regimen comprising FLU 120 ‡r/‡u, intravenous busulfan (BU) 12.8‡r/kg and cyclophosphamide (CY)120 mg/kg (FLU/BUCY2). All patients were given a combination of cyclosporine A and mycophenolate mofetil for graft-versus-host disease (GVHD) prophylaxis. Seventeen patients with acute leukemia (n=13), chronic myelogenous leukemia (n=4) were treated, thirteen of whom were high risk diseases and two were advanced-stage at CBT. Seventeen patients with a median age of 8 years (range,2.5–46 years) and a median weight of 32 kg (range, 12–55 kg)received the median number of nucleated cells and CD34+cells infused were 5.70× 107/kg (range: 3.15–9.60×107/kg) and 3.84× 105/kg (range:1.27–5.24 ×105/kg), respectively. The cumulative incidence of primary donor engraftment was 94% (16 patients); one patient had secondary graft failure. Median time to neutrophil≥0.5×109/L was 17 days (range 12–30) and platelet engraftment (≥20×109/L) was 35 days (range 14–56). Preengraftment syndrome (PES) developed in 71% of the patients at a median of 7days (range: 5–13).9 cases developed acute GVHD (56%), more than grade II in three cases. Two of fourteen patients who survived more than 100 days developed chronic GVHD. 12 cases are alive at a median follow-up of 7 months (range 3~ 11).The probability of overall survival at 100 days and 1 year are 88.2% and 67.9%, respectively. Two cases had extramedullary relapsed. Five cases died of severe GVHD (n=3), pulmonary toxicity (n=1) and secondary graft failure (n=1). Preliminary evidence of the small study suggests successful engraftment and decreasing relapse rate following FLU/BUCY2 regimen for CBT in patients with hematologic malignancies. But it had a tendency towards increasing the incidence of GVHD-related morbidity and mortality. Whether this regimen offers a survival benefit for patients with poor-risk leukemia has to be tested in larger prospective trials.

Disclosures:

No relevant conflicts of interest to declare.
CD9 Alters Migration and Morphology of CD9-Positive B Acute Lymphoblastic Leukemia Cells
Authors: Vallée Audrey , Dondi Elisabetta , Bonneau Jacinthe , Leroy Christine , Rio Anne-Gaelle , Troadec Marie-Bérengère , Varin-Blank Nadine , Galibert Marie-Dominique , Gandemer Virginie
Published: October 2019

Abstract 4623

CD9 is a member of the tetraspanin family and has been involved in various pathways. The expression of CD9 has been correlated to the risk of metastases or a poor clinical outcome in various types of cancer.

We have previously shown that TEL/AML1-positive acute lymphoblastic leukemia (ALL) is characterized by deregulation of 14 genes, including CD9 gene.

Here, we investigated the role of CD9 in cell motility in the context of TEL/AML1-positive ALL.

Leukemic blasts isolated from bone marrow of patients and B lineage ALL cell lines that expressed or not CD9 (REH CD9+, Raji CD9- and Raji transfected with CD9) were used for the following assays. For adhesion assay, plates were coated with 1mg/ml superfibronectin. Cells were allowed to attach for 1h30 with or without blocking CD9-antibody (Ab). Adherent cells were quantified using MTS. For migration assay, cells were seeded in the upper chamber on 5mm transwell microporous polycarbonate membranes, treated with different blocking Abs or drugs (CD9-Ab, IgG control, AMD3100) while 100 ng/ml CXCL12 (B-ALL specific chemokine CXCR4 ligand) was added to the medium of the lower chamber. After 5 hours, the migrated cells were recovered from the lower chamber, numbered and analysed by FACS.

We showed that the expression of CD9 decreased the ability of lymphoblasts to attach to fibronectin both in cell lines and in patients cells. This effect was reversed by CD9 blocking antibody. Conversely, migration induced by CXCL12 was reduced by anti-CD9 antibody. We also demonstrated an alteration in cell morphology in CD9-positive cells compared to their CD9-negative control cells. We thus provide novel evidence that CD9 is a key player of the CXCR4-mediated migration pathway by promoting actin rearrangement in response to CXCL12. As CXCR4/CXCL12 axis is central for supporting normal and pathological hematopoiesis, our data could highlight the impact of CD9 expression on the abilities of blasts to disseminate and may explain the specific outcome of TEL/AML1 B-ALL, especially in the perspective of late relapses.

This work was supported by la Ligue Régionale contre le cancer (Ille et Vilaine) et la Fondation pour la Recherche Médicale.

Disclosures:

No relevant conflicts of interest to declare.
Cerebral Aneurysm Bleeding Due to Disseminated Fusarium Infection in a Patient with Acute Monocytic Leukemia
Authors: Qian Shenxian , Gao Da-quan , Jun-feng Tan
Published: October 2019

Abstract 4736

Fusarium is a saprophytic organism that is found widely distributed in soil, subterranean and aerial plants, plant debris and other organic substrates. The organism can cause local tissue infections in immunocompetent patients such as onychomycosis, bone and joint infections, or sinusitis. Since the first case of disseminated Fusarium was described, the incidence of disseminated disease has increased significantly, particularly affecting those immunocompromised with hematological malignancies. We report here a disseminated Fusarium infection with skin manifestations and cerebral aneurysm bleeding in a patient of acute monocytic leukemia. A 49 year old female with acute monocytic leukemia (M5) underwent three courses of therapy with FlAG(cytarabine puls Fludarabine) in second relapse. At the time of hospital admission, she was receiving G-CSF for severe granulocytopenia post-intensification with FLAG, and had also received broad-spectrum antibiotics and fluconazole prophylaxis during previous episodes of febrile neutropenia. Despite treatment with liposomal amphotericin B for five days, She presented persistent fevers, he developed, nonpruritic erythematous Skin Eruption or bleb on her right shin, right shoulder (Figure 1), scalp and foot. The lesions developed central necrosis after four days. The Exudatin in erythematous skin bleb on foot culture was positive for Fusarium solani(Figure 2), which was also confirmed on two different skin lesional drainage culture. Although the patient was treated with liposomal amphotericin B, the clinical symptoms worsened. After liposomal amphotericin B was replaced with voriconazole (VRCZ), Recovery was observed after voriconazole introduction and resolution of neutropenia. Thrombocyte count increased up to 50×109. she suddenly lost consciousness duing lunch. Brain CT scan revealed the hemorrhage in brain. Magnetic Resonance Imaging(MRI) and CTA revealed rupture of brain aneurysm(Figure 3 A,B). Cerebral aneurysm caused by infections differ from congenital brain aneurysm, which often occur the junctions where these arteries come together and form weak spots. The clinical presentation, diagnosis, prognosis and therapeutic options of fusariosis in immunocompromised patients are briefly discussed.Figure 1:Skin EruptionFigure 1:. Skin EruptionFigure 2:The Exudatin in erythematous skin bleb culture was positive for Fusarium solaniFigure 2:. The Exudatin in erythematous skin bleb culture was positive for Fusarium solaniFigure 3:Magnetic Resonance Imaging(MRI) and CTA revealed rupture of brain aneurysm(Figure 3 A,B).Figure 3:. Magnetic Resonance Imaging(MRI) and CTA revealed rupture of brain aneurysm(Figure 3 A,B).

Disclosures:

No relevant conflicts of interest to declare.