bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous Oncogenic Nras Signaling Promotes Proliferative Exhaustion of Hematopoietic Stem Cells Through a Dose-Dependent Hyperactivation of ERK,
Authors: Wang Jinyong , Liu Yangang , Du Juan , Ryu Myung-Jeom , Ranheim Erik A. , Zhang Jing
Published: October 2019

Abstract 3997

Both monoallelic and biallelic oncogenic NRAS mutations are identified in human leukemias, suggesting a dose-dependent role of oncogenic NRAS in leukemogenesis. We recently characterized conditional mouse models that express one or two copies of constitutively active Nras G12D in the hematopoietic compartment (Nras G12D/+ and Nras G12D/G12D). Nras G12D/G12D results in stronger downstream signaling than Nras G12D/+ and consequently distinct hematopoietic phenotypes. In particular, we found that somatic expression of Nras G12D/G12D but not Nras G12D/+ leads to an acute myeloproliferative neoplasm (MPN). The development of acute MPN is associated with cytokine-evoked hyperactivation of ERK but not Stat5 and Akt in hematopoietic stem/progenitor cells (HSPCs). Interestingly, genetically altered HSCs appear to be required for initiation and maintenance of chronic and acute MPD phenotypes mediated by Nras G12D/+ and Nras G12D/G12D, respectively. Furthermore, Nras G12D/G12D-mediated signaling promotes excessive HSC proliferation and leading to HSC exhaustion. Exhaustion of HSCs is tightly associated with diminished MPN phenotypes in non-engineered recipient mice into which Nras G12D/G12D HSCs are transferred by bone marrow transplantation. To investigate the molecular mechanisms underlying the exhaustion of Nras G12D/G12D HSCs, we performed a microarray analysis using highly purified control, Nras G12D/+, and Nras G12D/G12D HSCs. To our surprise, neither p16INK4a and p15INK4b, critical cell senescence genes, or other genes or pathways known to involve in HSC self-renewal were differentially expressed in Nras G12D/G12D HSCs. In contrast, gene ontology analysis revealed significant differential expression of genes in the MEK/ERK pathway in Nras G12D/G12D HSCs. This result is consistent with our phospho-flow study showing that Nras G12D/G12D selectively hyperactivates ERK in HSPCs. It has been documented that prolonged ERK signaling is a potent inducer of differentiation in cultured PC12 cells and inhibition of normal ERK signaling by a chemical inhibitor increases self-renewal in mouse embryonic stem cells. Thus, we are currently testing whether hyperactivation of ERK promotes depletion of Nras G12D/G12D HSCs through promoting HSC differentiation and whether blocking hyperactivation of ERK restores the normal HSC function and prevents MPN development.

Disclosures:

No relevant conflicts of interest to declare.
Adoptive Immunotherapy with CMV Specific Cytotoxic T Lymphocytesfor Stem Cell Transplant Patients with Refractory CMV Infections,
Authors: Bao Lei , Cowan Morton Jerome , Dunham Kimberly , Horn Biljana N. , McGuirk Joseph , Gilman Andrew , Lucas Kenneth
Published: October 2019

Abstract 4033

Adoptive immunotherapy with cytomegalovirus (CMV) specific cytotoxic T lymphocytes (CTL) is an effective strategy for preventing and treating viral reactivation following allogeneic stem cell transplantation (SCT). We have previously shown that CMV CTL can be generated in 1–2 weeks by stimulating donor lymphocytes with peptide mixes derived from full length pp65 and IE1. We conducted a multi-institutional study of CMV specific CTL for patients with persistent or anti-viral resistant CMV infections following allogeneic SCT, to determine the safety, feasibility, and immunologic effects of this approach. We were successful in stimulating CTL from 10/10 donors with pooled CMV overlapping peptide mixes. Five of the 7 subjects who met infusion criteria had new onset CMV specific CTL activity detected within 4–6 weeks post infusion. Of the two subjects who did not have immunologic responses post-infusion, one received CTL with a low viability post-thawing, and the other patient was receiving cyclosporine A and systemic corticosteroids at the time of the infusion, achieving only a low, transient increase (10%) in pp65 specific activity. There was no GVHD attributable to these infusions. These findings indicate that the infusion of CTL stimulated over 1–2 weeks with overlapping CMV peptides can result in virus specific immune reconstitution in SCT recipients, without exacerbations of GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Microenvironment Shields Myeloma From Adoptive Immunotherapy by Cell Adhesion Mediated Immune Resistance (CAM-IR),
Authors: de Haart Sanne J , Minnema Monique C. , Aarts-Riemens Tineke , Bovenschen Niels , Lokhorst Henk M. , Mutis Tuna
Published: October 2019

Abstract 4039

Cellular immunotherapy can induce long term remissions in multiple myeloma (MM) patients. Despite reported successes in a limited number of patients, the immune system fails to completely eradicate MM in the majority, proving the ability of MM cells to evade cellular immunity. The nature and mechanism of this immune escape remains not fully understood. Most studies have focused on mechanisms by which tumor cells down-regulate their antigenicity or suppress immune effector cells. Here we show a critical role for the tumor microenvironment in inducing a cell-adhesion mediated ability in MM cells to resist T cell mediated killing; a mechanism similar to cell-adhesion mediated drug resistance (CAM-DR).

We used a compartment-specific bioluminescence-based viability assay, in which luciferase transduced MM cell lines were co-cultured with MM-reactive CD4+ and CD8+cytotoxic T cells (CTLs) in presence and absence of accessory cells. In these co-culture assays, we demonstrated the effective CTL-mediated lysis of MM cells. However, this lysis was significantly decreased in the presence of accessory cells, such as bone marrow stromal cells, including patient derived stromal cells, activated endothelial cells and fibroblasts.

We identified that the reduction of CTL-mediated lysis was predominantly due to the induction of a cell-cell contact mediated resistance in MM cells against CTL killing, proving for the first time the existence of a cell-adhesion mediated immune resistance (CAM-IR). In support to this, blocking the adhesion of MM cells to accessory cells with an ICAM-1 antibody or with RGD peptide, which is a ligand for cell surface integrins, abrogated the resistance of MM cells against CTL-mediated lysis. Intracellular mechanisms of this immune resistance were evaluated by western blot. Adherence of MM cells to accessory cells significantly increase the expression levels of survivin in MM cells, a well known inhibitor of apoptosis protein (IAP), which blocks the CTL mediated apoptotic events via inhibiting the phosphorylation of caspase-3. Our current focus is the restoration of MM cell sensitivity to T cell mediated killing via downregulation of survivin expression.

In conclusion, we report for the first time the potentially important role of microenvironment in compromising immunotherapy by the induction of CAM-IR. Our results suggest that immunotherapy may be improved by modulating the interaction of MM with its microenvironment

Disclosures:

No relevant conflicts of interest to declare.
The Immature Reticulocyte Fraction Predicts Marrow Recovery and Can Obviate the Need for Bone Marrow Examination in Persistently Neutropenic Cancer Patients
Authors: Haider Sajjad , Katragadda Lakshmikanth , Muzaffar Jameel , Grazziutti Monica , Akbar Muhammad R , Cottler-Fox Michele H. , Barlogie Bart , Anaissie Elias J.
Published: October 2019

Abstract 4526

Introduction:

Prolonged and persistent neutropenia (white blood cell (WBC) ≤1000/μL) after chemotherapy is associated with increased risk for infection in cancer patients and is often an indication for a diagnostic bone marrow aspirate and biopsy. A test that predicts WBC recovery ≥ 1000/μL and that obviates the need for marrow sampling would be of clinical value. The immature reticulocyte fraction (IRF) reflects erythroid production and hence a recovering marrow.

Materials and Methods:

We identified 17 myeloma patients with prolonged pancytopenia after either myeloablative or non-myeloablative chemotherapy between March 2010 and February 2011, and compared the time of occurrence of IRF doubling (IRF-D) to the findings on bone marrow examination.

Results:

The time to IRF doubling preceded increase of WBC ≥ 1000/μL in 15 of 17 patients by a mean of 4.5 days (range 0–18 days). In two patients, the IRF-D coincided with WBC ≥ 1000/μL. The IRF doubled 2–4 days before the bone marrow examination in four patients and at a mean of 3.7 days (range 1–13 days) after the marrow examination in the remaining 13.

Conclusion:

We conclude that the IRF-D is a simple, inexpensive and widely available test that can precede marrow recovery by several days and may therefore obviate the need for a diagnostic bone marrow aspirate and biopsy in patients with prolonged and persistent neutropenia.

Disclosures:

No relevant conflicts of interest to declare.
Comparison of Sirolimus and Mycophenolate Mofetil As Salvage Treatment for Acute Graft-Versus-Host Disease
Authors: Nishihori Taiga , Pidala Joseph , Kim Jongphil , Tomblyn Marcie , Anasetti Claudio
Published: October 2019

Abstract 4548

Acute graft vs. host disease (aGVHD) refractory or dependent on glucocorticoid therapy is a major source of mortality following allogeneic hematopoietic cell transplantation (HCT). Comparative studies to evaluate the efficacy of available salvage immune suppressive agents are lacking. We retrospectively compared the efficacy of sirolimus (SIR) and mycophenolate mofetil (MMF) as salvage aGVHD therapy for glucocorticoid refractory, dependent or intolerant patients. Of 281 consecutive patients who received HCT at Moffitt Cancer Center from 07/2004 to 09/2009, we identified 84 patients who received tacrolimus/methotrexate (Tac/MTX) as GVHD prophylaxis, were treated with systemic glucocorticoids as first line therapy for acute GVHD grades 2–4, were refractory (n=72) or dependent (n=12) to glucocorticoids, and received second line GVHD treatment with MMF (n=56) or SIR (n=28). Patient demographics and treatment variables were similar between the two groups except for year of transplant, which was earlier for MMF. Median age of the entire group was 50 (range 17 – 68). Disease diagnoses included AML (n=27), NHL (n=14), MDS (n=12), ALL (n=9), CML (n=7), CLL (n=4), SAA (n=2), MPD (n=5), MM/PCL (n=3), and HL (n=1). Conditioning regimens were busulfan/fludarabine for 71, and other regimens for 13. Except for 1 bone marrow graft in each group, all received peripheral blood stem cells. Graft sources were from HLA-matched siblings (35), or 8/8 HLA-matched unrelated donors (49). Overall grade distribution of aGVHD at time of salvage for MMF vs. SIR was the following: grade 1 (13 vs. 2), grade 2 (31 vs. 16), grade 3 (9 vs. 5) and grade 4 (3 vs. 5). Median steroid dose at the time of salvage was 1 (range 0.17 – 2.28) mg/kg for MMF group and 1 (range 0.12 – 2.0) mg/kg for SIR group. Median time from steroid to salvage was 20 (range 1 – 208) days for MMF and 19 (range 1 – 275) days for SIR (p=0.84). Complete response (CR) rates following initiation of MMF or SIR did not significantly differ at the following time points: 1 week (MMF 30%, SIR 21%), 4 weeks (MMF 44%, SIR 46%), and 6 weeks (MMF 60%, SIR 58%). Overall response rates (ORR) also did not differ significantly: 1 week (MMF 57%, SIR 42%), 4 weeks (MMF 57%, SIR 77%), and 6 weeks (MMF 72%, SIR 75%). Flare or progression of aGVHD while on salvage regimen was noted in 50% (MMF) and 36% (SIR) of patients (p=0.64). Median overall survival from the time of salvage therapy for MMF vs. SIR did not significantly differ, 11.6 (95% CI 7.0 – 28.1) vs. 9.7 (95% CI 5.4 – 15.9) months, log-rank p = 0.88. These retrospective data suggest that MMF and SIR have comparable activity in the treatment of steroid refractory or dependent acute GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation of Enteral Versus Parenteral Feeding As Nutritional Support In Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Lemal Richard , Guièze Romain , Moluçon-Chabrot Cécile , Hermet Eric , Ravinet Aurélie , Combal Cécile , Cabrespine Aurélie , Bay Jacques-Olivier , Corinne Bouteloup
Published: October 2019

Abstract 4576

Introduction

Allo-HSCT procedure is associated with a frequent and potentially severe malnutrition which could highly participate to the transplant-related morbidity (TRM). Optimal nutritional management is still poorly known while both enteral nutrition (EN) and parenteral nutrition (PN) are effective. We proposed to retrospectively evaluate the impact of EN versus PN as nutritional support on early outcome of allo-HSCT.

Patients and methods

We retrospectively analyzed all the successive patients who needed a nutritional support during their first allo-HSCT in our center from January 2009 to October 2010, excepting whose who had a progressive disease at time of transplant. Datas were compared in an intent to treat analysis according the EN or PN initial nutritional support strategy.

Results

We analysed early outcome of 56 successive patients. Twenty of them received a myeloablative conditioning regimen and 36 a reduced intensity one. A total of 28 agreed to receive EN via a nasogastric feeding tube and the remaining 28 received PN. No significant difference in terms of age, diagnosis, disease status at transplant, conditioning regimens, stem cell source, GVHD nor antifungal secondary prophylaxis could be observed between the EN and PN groups. We found a lower median duration of fever in EN (2[0–8] vs. 5[0–17] (days); p=0.0026) and a lower need for antifungal therapy in EN group (7/28 vs. 17/28; p=0.0069), with a lower median duration of intravenous antifungal use (0 day [0–99] in EN vs. 7 days [0–93] in PN; p=0.00034) while incidence of bacteriemiae was not different. We observed a lower rate of replacement of central veinous catheter in EN group (3/28 in EN vs. 9/28 in PN; p=0.05) and a lower rate of transfer to ICU in the EN group (2/28 in EN vs. 8/28 in PN, p=0.036) but early death rate (<100 days) was the same in each group (4/28 vs. 4/28, p=NS). Median neutropenia and thrombopenia duration and median transfusion requirements were not significantly different. Fourteen patients in EN group and 18 in PN group presented a grade 3–4 oral mucositis (p=NS). Grade III-IV GVHD incidence was comparable in both groups (4/28 vs. 8/28; p=0.19).

Conclusion

Compared with PN, EN directly decreases the infectious risk, particularly the fungal risk, and its complications in allo-HSCT, without influencing hematopoietic toxicity nor GVHD incidence. Based on these encouraging results, we are now conducting a prospective, multicentric and randomized trial to confirm EN benefice in allo-HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Morphological Atypia Represents Favorable Prognosis of Chronic Lymphocytic Leukemia in Japanese Patients
Authors: Suzumiya Junji , Aoki Sadao , Isobe Yasushi , Ohta Masatsugu , Takizawa Jun , Izutsu Koji , Suzuki Ritsuro , Tsukasaki Kunihiro , Kuriyama Kazutaka , Ohshima Koichi , Tamura Kazuo
Published: October 2019

Abstract 4594

Background:

B-chronic lymphocytic leukemia (B-CLL) is rare in Japan. Therefore, only a few data are available on the clinical characteristics of B-CLL for Japanese patients (pts). We reported that the rate of atypical CLL appeared to be higher in Japanese pts than that of Caucasians.

Patients and Methods:

This nation-wide study was performed from Oct. 2003 to March 2008, and was supported by Japanese Elderly Leukemia and Lymphoma Study Group. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Boards of the core members' hospitals. The pts with CLL and CLL-like disease were registered. Precursor leukemia and adult T-cell leukemia/lymphoma were excluded. The smears of peripheral blood and bone marrow of all the registered pts were examined by the Diagnosis Committee by using the 4th edition of WHO classification and FAB classification. FAB classification defines typical CLL as prolymphocytes≤10% and atypical CLL including CLL/PLL as 11%≤prolymphocyte<55% and CLL mixed as prolymphocytes<10% and pleomorphic lymphocyte>15%. Mantle cell lymphoma was excluded by means of cytogenetics, fluorescent in situ hybridization of CCND1-IGH, and immunohistochemistry of CCND1.

Results:

One hundred and thirty-one evaluable pts were registered. There were 96 pts with B-CLL, 2 SLL, 1 B-PLL, 3 hairy cell leukemia, 16 leukemic phase of other B-cell lymphomas. Thirteen pts had NK or T cell leukemia/lymphoma with 7 T-LGL, 4 T-PLL, 1 NK-LGL, and 1 Sezary syndrome. Among B-CLL, there were 62 pts with typical morphology (typical CLL), 18 with atypical morphology (atypical CLL), 16 with intermediate morphology between typical and atypical CLL (intermediate CLL). The differences among 3 subgroups are shown in Table. No differences were found between the 3 morphological subgroups for age, sex, stage (modified Rai and Binet classification), hemoglobin level, platelet counts, the presence of hepatomegaly, splenomegaly and lympoadenopathy, ECOG-PS and the rate of treatment requirement. However, the incidence of CD5 and CD23 expression was higher in typical CLL than in atypical/intermediate morphology groups (P<0.001), i.e. 7% of the pts with atypical CLL showed co-expression of CD5 and CD23. The overall survival (OS) of typical CLL pts was lower than that of intermediate or atypical CLL pts (Figure).

Conclusion:

Morphological atypia was more frequent in Japanese B-CLL pts, partly because of the low incidence of typical CLL. The atypical/intermediate CLLs were associated with atypical immunophenotype (absence of CD5 and/or CD23) and favorable prognosis. Prospective study is ongoing to evaluate other prognositic factors including the implication of IgVH gene mutation status and ZAP-70 expression in Japan.

Disclosures:

No relevant conflicts of interest to declare.
Primary Localized Laryngeal Amyloidosis (LA)
Authors: Richards Matthew , Le Kim , Lorenzo-Medina Mercedes , Dane Langsjoen , Arango Carmen , Gupta Anuradha , Garcia Rafael , Campos-Gines Angel F , Landeros Mark M , Herrada Juan
Published: October 2019

Abstract 4596

Background:

Localized Laryngeal Amyloidosis (LA) is a rare condition. There is limited understanding of its natural history. Our aim is to report the clinical evolution of a patient diagnosed with localized LA.

Methods:

Case report.

Results:

In May 2002, a 43-year-old gentleman presented with hoarseness and a CT scan of the neck showed a thickening of the right vocal cord with a mass that measured 2.9 × 1.2 cm in widest diameter. The patient had this mass removed surgically and the pathology showed a positive Congo red staining and a positive birefringence under polarized light, diagnostic of amyloidosis. No evidence of systemic amyloidosis or an overt B-cell lymphoma was found in this patient.

Patient lost follow-up until October 2008 when he was found to have soft tissue induration around the left false vocal cord area. The biopsy showed again amyloidosis.

Conclusions:

LA recurrences can manifest several years after initial diagnosis. Long-term follow-up is essential.

Disclosures:

No relevant conflicts of interest to declare.
Common Fragile Site Gene FRA16D WWOX (WW domain containing oxidoreductase) Is Frequently Altered in Plasma Cells of Multiple Myeloma and MGUS, Possibly Related to Disease Development
Authors: Sasaki Yoshiko , Handa Hiroshi , Takahashi Noriyuki , Hattori Hikaru , Saitoh Takayuki , Osaki Yohei , Matsumoto Morio , Isoda Atsushi , Sawamura Morio , Saito Akio , Yokohama Akihiko , Koiso Hiromi , Mitsui Takeki , Tsukamoto Norifumi , Nojima Yoshihisa , Murakami Hirokazu
Published: October 2019

Abstract 4646

The common fragile sites (CFSs) are regions of profound genomic instability, and hot-spots for deletions and other alterations in cancer cells. We have previously shown that promoter of FHIT is frequently methylated in multiple myeloma (MM) and correlated with worse prognosis. WWOX is located at a CFS region on chromosome 16q23.3, this gene is also a target of alterations in multiple cancers. The objective of current study is to find if this tumor suppressor gene is altered in MM and MGUS. Six myeloma cell lines, bone marrow mononuclear cells (BMMNC) of 165 MM and 25 MGUS patients were subjected to this study after obtaining informed consent. Isolated CD138 positive plasma cells (PC) of 24 MM were examined to see if the alteration occurred really in myeloma cells. Using methylation specific PCR, WWOX promoter methylation was detected in 2 of 6 cell lines, 35% of MM patients and 8% of MGUS patients (p=0.01). The overall survival of the MM patients with methylated WWOX tended to be worse than with unmethylated WWOX (p=0.2). Using nested RT-PCR, aberrant short transcripts of WWOX lacking exons coding SDR domain were detected in 4 cell lines, 68% of MM and 60% of MGUS (p=0.51) In isolated PC, aberrant transcripts of WWOX were detected in 58% of MM and 25% of MGUS (p=0.02). Real time PCR demonstrated higher WWOX expression in isolated PC of MM and MGUS than of BMMNC other than plasma cells (p=0.0001), and those higher WWOX were aberrant type (p=0.001). As aberrant WWOX is known to function as dominant negative for wild type, our results imply that this aberrant WWOX is an oncogene as well as losing tumor suppressor function. Since WWOX is demonstrated to regulate beta-catenin and NF-kB pathway, high frequency of aberrant WWOX expression indicates an important role in myeloma development.

Disclosures:

No relevant conflicts of interest to declare.
Expression of PIM-2 and NF-κB Is Increased in Patients with Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) and Correlates with Complete Remission Rate
Authors: Kapelko-Slowik Katarzyna , Urbaniak-Kujda Donata , Wolowiec Dariusz , Dybko Jaroslaw , Jazwiec Bozena , Slowik Miroslaw , Jakubaszko Jacek , Owczarek Tomasz , Grzymajlo Krzysztof , Kuliczkowski Kazimierz
Published: October 2019

Abstract 4648

Background.

PIM-2 is a proto-oncogene that encodes for serine/threonine kinase which interacts with various signalling molecules. PIM-2 is highly expressed in neoplastic tissues and in leukaemic and lymphoma cell lines which is consistent with a its role during oncogenic transformation. The nuclear factor kappa B (NF-κB) pathway appears to be deregulated in variety of tumors, with sustained activity of NF-κB leading to apoptotic resistance in tumor cells

The aim.

The aim of this study was to investigate whether the PIM-2 and NF-κB expression is altered in acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL). Patients and methods. One hundred fourty-three patients were included: 91 with AML and 52 with ALL (42 with B-ALL and 10 with T-ALL), aged 18–84 (median=41). Seventy-five patients reached complete remission (CR): 50 in AML and 25 in ALL. Bone marrow samples were collected at the time of diagnosis. Control samples were obtained from 24 healthy donors. We analysed PIM-2 and NF-κB expression by RQ-PCR analysis.

Results.

Expression of both PIM-2 and NF-κB in all leukaemic patients and in subgroups: AML and ALL was significantly higher than in controls. In AML group patients who reached CR expressed PIM-2 and NF-κB at significantly lower levels than patients with primary resistance to chemotherapy (with no CR, NCR). Moreover in AML, we have found the correlation between PIM-2, NF-κB expression and blasts in myelogram and PIM-2 and patients‘ age.

Our data indicate that PIM-2 and NF-κB genes expression is increased in patients with AML and ALL and correlates with CR rate in AML patients.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Diagnosis of Haemophilia B Leyden In a Chinese Pedigree: C>G Transition At Position +9 of the FIX Gene
Authors: Zhou Rong-Fu , Zhang Xian , Ouyang Jian , Tao Hong , Shao Xiao-Yan , Xu Jingyan , Li Ping
Published: October 2019

Abstract 4651

Objective

To investigate the molecular change causing Haemophilia B in a Chinese family.

Methods

The peripheral blood samples had been extracted from proband and his family members. The activated partial thromboplastin time (APTT), prothrombin time (PT) and FIX activity (FIX:C) were adopted for phenotype diagnosis. All the 8 exons and their flank boundaries were amplified by polymerase chain reaction (PCR). The PCR products were sequenced directly. The corresponding DNA fragments of their famliy members were amplified by PCR and then screeded by direct sequencing.

Results

The 3-year-old Chinese boy had persistent nose swelling for 10 days from an injury. He had a long APTT of 96.6s which could be corrected to 30.2s with mixed normal plasma. PT, TT and fibrogen level was in normal range, respectively. FVIII:C was 54%, FIX:C was 1.6% and AT:C was 115%. DNA sequencing of the proband’s F9 gene revealed that there was a C>G mutation at nt +9 in the promoter region. His mother, maternal grandmother and two maternal grandmother’s sisters all had the same C/G genotype, with FIX:C 44%, 89%, 81% and 67%, respectively. His maternal uncle, aged 30, had the C>G mutation with FIX:C 40% and another maternal uncle, aged 12, had the same mutation with FIX:C 4.2%.

To date, there are only two cases with Haemophilia B Leyden reported from Asia, one from Thailand (nt +8 T>C) and the other from HongKong (nt +6 T>A). The mutation at nt +9 C>G lies in the binding sites for transactivating factors, such as CCAAT/enhancer binding protein (C/EBP), affects the first (site 1: nt +1 to +18) of five known cisacting sites in the FIX promoter. The clinical picture of the proband reported herein and his maternal uncle with the same genetic defect conforms to the Leyden phenotype, because the uncle now has a relatively normal FIX level (40%) and no recent bleeding episodes.

Conclusion

The mutation at nt +9 C>G in FIX promoter might be the causative one leading to FIX deficiency in this Chinese pedigree.To the best of our knowledge, this is the first time to report the mutation at this site of FIX promoter.

Disclosures:

No relevant conflicts of interest to declare.
Acute Hemolytic Reactions Reported in ITP Patients Treated with Rho (D) Immune Globulin: Symptom Onset and Does Prior Exposure Lessen the Risk?
Authors: Muller Carmen , Little Gregg , Hess Kristen , Sinclair Chris
Published: October 2019

Abstract 4670

BACKGROUND:

Acute hemolytic reactions (AHR) associated with administration of Rho (D) Immune Globulin (RhIG; WinRho® SDF, Cangene Corporation, Winnipeg, Canada) have been infrequently reported during the previous 16 years. AHR were defined as any case reported with signs of hemolysis (hemoglobin decrease, haptoglobin decrease, LDH increase, elevation of bilirubin, etc.) occurring within hours to a number of days after RhIG administration irrespective of the degree of change from baseline. Intravascular hemolysis (IVH) was defined as a specific type of AHR in which plasma or urinary hemoglobin were positive. The reporting rate of AHR (including IVH) among RhIG-treated patients was previously estimated as 1/946 doses (0.11%), while the estimated reporting rate of IVH was 1/3,271 doses (0.03%).1 Reports surrounding the clinical monitoring of such patients have led to the analysis of AHR post-marketing data.

AIMS/PURPOSE:

Identify the onset of AHR/IVH symptoms relative to RhIG and determine if prior exposure to RhIG mitigates the risk of such events.

METHODS:

Retrospective evaluation of AHR (AEs reported with signs of hemolysis; including IVH) from the Cangene post-marketing surveillance database.

RESULTS:

CONCLUSION:

Of the ITP patients who developed IVH, 40% reported an onset of symptoms within 4 hours of RhIG infusion; an additional 10% reported symptoms within 8 hours. The symptom onset was unidentified in 18% of IVH cases. In addition, previous uneventful administration of RhIG did not preclude the possibility of an occurrence of IVH and its complications following subsequent administration of RhIG; however, the majority (approx 84%) of reports of IVH were among RhIG naïve patients.

Disclosures:

Muller: Cangene: Employment. Little:Cangene: Employment. Hess:Cangene: Employment. Sinclair:Cangene: Employment.
HIV-Associated Thrombotic Thrombocytopenic Purpura
Authors: Boro Jordana , Pavenski Katerina , Ambler Kimberley L.S. , Leitch Heather A , Hicks Lisa K
Published: October 2019

Abstract 4677

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening form of microangiopathic hemolysis that can be associated with HIV infection, and has previously been reported to be associated with low CD4 counts. The existing literature on HIV-TTP is very small and largely made up of small case series generated through databases of patients with HIV. As a result, reports have tended to focus on HIV parameters with limited information available regarding the presentation, management and outcome of the TTP itself. We conducted a retrospective review of TTP cases referred to the pheresis units St. Michael’s Hospital and Vancouver General Hospital in Vancouver between July 1993 and May 2011. Ten cases of HIV associated TTP were identified (8 male; 2 female). Median age at presentation was 38 years. One patient had been previously diagnosed and treated for TTP at a different institution. Average duration of HIV infection prior to TTP diagnosis was 5 years (range 0 to 11). Median CD4 count at TTP diagnosis was 79 × 106/ml (range 2 to 326). Median platelet count at presentation was 14 × 106/ml (range 5 to 233), median haemoglobin was 74 g/L (range 61 to 133), all patients had an LDH > 2x the upper limit of normal, and all for whom data was available (8/10) had fragmentation on blood film. Creatinine was elevated in 9 of 10 patients. ADAMTS13 was assessed in 3 of 10 patients and was deficient in one. Five of 10 patients had fever. At presentation, 6 of 10 patients had neurological symptoms (most commonly seizures and/or confusion), but none suffered permanent neurological deficits. All patients were treated with plasmapheresis and received a median of 16 exchanges (2-56). Four patients received concurrent steroids, two patients received IVIG, and one patient received pheresis, steroids IVIG, vincristine and rituximab. Eight of 10 patients achieved complete remissions, one patient achieved a partial response, and one died on treatment. Four of the responding patients subsequently relapsed (0.6 to 13.8 months after the initial episode of TTP); two achieved second remissions, and two died on treatment. In conclusion, our series of HIV associated-TTP confirms previous reports that HIV-TTP tends to occur in patients with CD4 counts less than 200. Complete remissions can be achieved with standard management. However, based on our small series, relapses may be more common and mortality greater than in the general TTP population.

Disclosures:

Leitch: Novartis Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees.
Generation of Induced Pluripotent Stem Cells From the Peripheral Blood of a Pyruvate Kinase Decifient Patient
Authors: Quintana-Bustamante Oscar , Garate Zita , Segovia Jose Carlos , Cerrato Laura , Güenechea Guillermo , Bueren Juan
Published: October 2019

Abstract 4713

The generation of induced pluripotent stem cells (IPSCs) from patients with specific diseases has improved the possibilities of dissecting the etiology of human pathological conditions and has opened potential new therapeutic options for the treatment of human diseases. One of the major safety concerns in the generation of hiPSCs is the persistence of copies of the reprogramming genes in the genome of iPSCs. Additionally, the use of cells from accessible tissues, like peripheral blood, constitutes preferential options for cell reprogramming, compared to other cells whose collection would require a more aggressive intervention. Here we have selected Pyruvate Kinase Deficiency (PKD) as a human disease affordable to be modeled and genetically corrected using hiPSC technology. PKD is an autosomal recessive disease that affects the erythrocyte R-type Pyruvate Kinase (RPK) enzyme by mutations in the PKLR gene, causing chronic nonspherocytic hemolytic anemia. Deficiency in the RPK, the last enzyme of the glycolisis, reduces the energetic balance of red blood cells and therefore affects the viability and function of the erythrocytes. Clinical symptoms of PKD can progress from mild to severe anemia and can be lethal in the most severe cases. Peripheral blood mononuclear cells (PB-MNC) from healthy donors and from a PKD patient were infected with five independent recombinant non integrative RNA Sendai viruses (SeV) encoding four human reprogramming factors (OCT3/4, SOX2, KLF4, c-MYC) and the Azami green protein as reporter. After infection with SeVs, no differences in the reprogramming efficiency of healthy and PKD PB-MNC were observed (1 hES-like colony per 6,000 transduced healthy or PKD PB-MNC). Integrated SeVs were not found in PBMNC-hiPS lines, as deduced from the lost of Azami green expression and also from RT-PCR analyses of the reprogramming factors. Healthy and PKD PBMNC-hiPSCs could be cultured for more than 15 passages, indicating their self-renewal ability. The pluripotent characteristics of these PBMNC-hiPS were confirmed by the analysis of expression of endogenous pluripotent genes by Q-RT-PCR and immunofluorescence. This study confirms the feasibility to generate integration-free hiPS lines from healthy and PKD PB-MNC. These hiPSC lines constitute an invaluable tool for the development of a human model of PKD and for applying new gene therapy strategies.

Disclosures:

No relevant conflicts of interest to declare.
Severe Neutropenia in Patients with Chronic Hepatitis C Not on Antiviral Therapy At the Memphis Veteran's Affairs Medical Center
Authors: Sheehan Vivien , Waters Bradford , Weir Alva B.
Published: October 2019

Abstract 4733

Abstract: We reviewed the records of 685 patients evaluated for treatment of chronic hepatitis C virus (HCV) at the Veteran's Affairs Medical Center in Memphis, TN in 2010. We identified six cases of severe neutropenia (defined as an absolute neutrophil count <500 x103/dL), in the absence of medications known to cause neutropenia such as interferon. None had concomitant HIV infections. Bone marrow biopsies were performed in three of the six patients and were negative for malignancy or abnormal myelopoesis. In this case series report, we compare laboratory parameters and clinical histories of these six patients to a similar cohort of chronic hepatitis C patients with at least one documented absolute neutrophil count (ANC) between 1000 x103/dL and 1500 × 103/dL. The two groups differed only in ANC and frequency of anti-nuclear antibody positivity. There was no significant difference in race, MELD scores, viral load, viral type, hemoglobin levels or platelet levels. Neither group suffered serious systemic infections. There was no evidence that the severe neutropenia was related to portal hypertension or splenomegaly. In our series, patients with severe neutropenia in the setting of chronic HCV had a benign course that did not lead to severe infections and responded to granulocyte colony stimulating factor. This opens the question of whether these patients may be candidates for antiviral therapy.

Disclosures:

No relevant conflicts of interest to declare.
Quantification of Human Equilibrative Nucleoside Transporter 1 in Blood Leukocytes Using Flow Cytometry
Authors: Davis Bruce H. , Curtis Adam , Booij Birgitte , Sandvold Marit Liland
Published: October 2019

Abstract 4737

We have developed an assay intended to measure the human Equilibrative Nucleoside Transporter 1 (hENT1) level in leukemic blast cells in blood or bone marrow samples from patients with acute myeloid leukemia or myelodysplasia (MDS). This is ultimately intended as companion diagnostic assay for hENT1 to identify those patients with poor treatment prognosis with cytarabine (ara-C) or other cytotoxic therapies that rely on hENT1 transport into abnormal cells. We optimized an intracellular staining assay incorporating a patent pending technique of utilizing fluorescent labeled monoclonal antibodies, internal fluorescent calibration beads and analytical software that allows identification of leukocyte subpopulations using a novel quantitative flow cytometric assay incorporating antibody binding inhibition by the immunizing peptide to demonstrate specific antigen binding by the anti-hENT1 monoclonal antibody. Initial validation studies were performed on blood samples from both healthy adults and some clinical samples, including samples from HIV+ individuals (samples 4A* and 4B*), infection/sepsis patients (samples 9–11**), and MDS as shown in figures. These preliminary studies showed two novel observations. Firstly expression differs in the major leukocyte populations with expression highest in eosinophils (hENT1 index range of 17.36 – 61.52), followed by neutrophilic granulocytes and monocytes and lymphocytes with the lowest level. Secondly while intra-assay imprecision was low with a CV <7%, some day to day variation was seen in healthy donors and greater variation seen with patient samples, thus indicating significant modulation of cellular expression within myeloid and monocytic cells. These preliminary results suggest retroviral therapy may decrease hENT1 expression, infection or its treatment increases hENT1 expression in myeloid cells, and MDS myeloid cells are also altered in hENT1 levels from the healthy state. These findings indicate potential clinical utility for a quantitative assay of hENT1 expression in hematolymphoid cells in a variety of conditions.

Disclosures:

Davis: Clavis Pharma: Consultancy; Trillium Diagnostics, LLC: Equity Ownership. Curtis:Trillium Diagnostics, LLC: Employment. Booij:Clavis Pharma: Employment. Sandvold:Clavis Pharma: Employment.
Tumor Lysis Syndrome As a Major Risk Factor of Posterior Reversible Encephalopathy Syndrome in Patients with Lymphoid Malignancies of Childhood
Authors: Suzuki Daisuke , Kobayashi Ryoji , Iguchi Akihiro , Sano Hirozumi , Yasuda Kazue , Kobayashi Kunihiko
Published: October 2019

Abstract 4754

Background

Posterior reversible encephalopathy syndrome (PRES) is a neurological complication that is caused by various factors such as hypertension, the use of calcineurin inhibitor(cyclosporin or tacrolimus). Its initial symptoms usually are seizure, headache, visual disorder. Although PRES associated with lymphoid malignancies has been reported, it only consits of case reports or small series. Furthermore, its risk factors have never been well-documented.

Patients and methods

A total of 126 pediatric patients with lymphoid malignancies received chemotherapy in our hospital between January 2000 and October 2010. Of these patients 84 were boys and 42 were girls. Age of patients at the onset of their respective diseases was 4 months to 15 years(median: 5y). One hundred and eight patients had acute lymphoblastic leukemia, 15 were Non-Hodgkin lymphoma and 2 were Hodgkin lymphoma.

Result

Of 126 patients, 6 developed PRES. The initial symptoms were seizure, cortical blindness and upper limb paralysis. All the patients had hypertension before developing PRES. In addition, 3 patients had tumor lysis syndrome(TLS). The median time from the initiation of treatment was 10.5 days (range: 6–35 days), Two patients developed epilepsy as a sequela. On statistical analysis, the only risk factors for PRES was the development of TLS.

Conclusion

Although various factors were reported, TLS-related PRES has never been described. Some patients developed PRES in the early period of induction treatment while the others in the late period. The difference between them remains to be seen. However, TLS may cause renal dysfunction in combination with hypertension induced by chemotherapeutic drugs such as prednisolone. Renal dysfunction and following hypertension might have induced PRES. We must pay attention to the state especially of the patients who developed TLS in the course of chemotherapy.

Disclosures:

No relevant conflicts of interest to declare.
Learn While You Wait: An Experience of a Novel, Interactive VHA e-Notebook Education Program for Multiple Myeloma, Final Results
Authors: Yacoub Abdulraheem , Mehta Paulette
Published: October 2019

Abstract 4759

We evaluated a novel e-notebook -based education program for patients with multiple myeloma. The program was developed by the Myeloma Initiative in the Veteran's Administration (MIVA) consortium to educate patients about myeloma while they wait for clinic appointments or procedures. The goals of this study were to assess the feasibility of the program in a busy clinic setting, patients ‘knowledge after the program and patients’ acceptance of the e-notebook format. Study subjects were patients with multiple myeloma being treated at the Central Arkansas Veterans Healthcare System McClellan Hospital. Eligibility criteria were diagnosis of multiple myeloma and ability to follow instructions in the program and complete a paper questionnaire. Study subjects evaluated the module while awaiting their clinic visit and then completed a survey consisting of ten multiple-choice questions designed to measure the goals of the study. The scope of the questions were: patients' primary source of medical education, patients' perceived benefit of the education program in improving their understanding of their disease (three questions), patients' preferences in regard to educational formats (three questions), and multiple myeloma knowledge (three questions).

Fifteen consecutive patients were enrolled in the study. Patient flow was not adversely affected by use of the module in the waiting room and the program was well accepted by the patients. Most patients in our study (93%) considered their primary source of medical information about their disease to be their oncologist, followed by internet resources (20%). Most patients (93%) stated that the education program helped them better understand the information provided by their oncologists and better understand multiple myeloma treatment options. Twelve patients (60%) answered that the program motivated them to learn more about myeloma. Fourteen patients (93%) recommended that this program be expanded to spouses or family members, and twelve patients (80%) advocated creating a video format of the program for repeat viewing. Nine patients (60%) stated they plan to view the content again at a later time. Of all multiple myeloma knowledge questions, 76% were answered correctly and 60% of the patients who answered correctly attributed their answers to information they learned in the education program.

We conclude that this e-notebook -based multiple myeloma education program is well accepted and can be adopted in a busy clinic. It is an effective and motivating tool for patient education in adjunct with physician efforts. It can be a productive utilization of waiting room time and has the potential for further applications.

Disclosures:

No relevant conflicts of interest to declare.
Hypoxia-Inducible Factor 1-Mediated Human GATA1 Induction Promotes Erythroid Differentiation Under Hypoxic Conditions
Authors: Zhang Jun-Wu , Zhang Feng-Lin , Shen Guo-Min , Liu Xiao-Ling , Wang Fang
Published: October 2019

Abstract 4794

The stimulation of red blood cell (RBC) production is one of the systemic adaptions to hypoxia. Hypoxia-inducible factor (HIF) promotes erythropoiesis through coordinated cell type-specific hypoxia responses. Hematopoietic transcription factor GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells (HPCs) derived from human cord blood. Enforced HIF1Á expression increased GATA1 expression, while HIF1Á knock-down by RNA interference decreased GATA1 expression in K562 cells. We searched the human GATA1 gene sequence on NCBI and identified a putative HRE in the 3'-flanking sequence of the gene. The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1Á or GATA1. Flow cytometry analysis also indicated that hypoxia or desferrioxamine or CoCl2 induced expression of erythroid surface marker CD71 and CD235a, while expression repression of HIF1Á or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 upregulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.

Disclosures:

No relevant conflicts of interest to declare.
Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis
Authors: Lee Young-Ho , Kwon Young-hee , Hwang Kyoujung , Jun Hyunju , Park Byungbae , Park Chan-Jeoung , Yoon Chan-Hee , Jang Seongsoo , Chi Hyun-Sook , Kim Dae-Young , Lee Jung-Hee , Lee Je-Hwan , Lee Kyoo-Hyung
Published: October 2019

Abstract 4808

Background:

It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC).

Methods:

The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured.

Results:

The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001).

Conclusions:

Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application.

Disclosures:

No relevant conflicts of interest to declare.