bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Mutation of ADAMTS13 Gene in Congenital Thrombotic Thrombocytopenic Purpura
Authors: Bai Xia , Su Jian , Jiang Minghua , Wang Zhaoyue , Ruan Changgeng
Published: October 2019

Abstract 4674

Congenital thrombotic thrombocytopenic purura (TTP) is caused by gene mutations of von Willebrand factor-cleaving protease (a disintegrin and metalloprotease with thrombospondin type I domains 13, ADAMTS13). In this study, one novel mutation in the ADAMTS13 gene was found in a woman whose presents are first cousins. Thrombocytopenia occurred during the second trimesters in her first pregnancy, and she died of recurrent attacks after diagnosis of TTP. The ADAMTS13 activity measured using the recombinant FRET-VWF73 during her acute episode was less than 5%. ADAMTS13 inhibitor was negative measured by 9:1 mixture of patient and pooled normal plasma followed by ADAMTS13 activity assay using the VWF multimer electrophoresis. The 29 exons and exon-intron boundary sites of ADAMTS13 gene was analyzed using the human genomic DNA extracted from peripheral leukocytes of the patient. The results demonstrated she was homozygous for R498C. This novel mutant was constructed using the expression plasmid pSectag containing ADAMTS13 cDNA, and the vector was introduced by linpofectamine 2000 to Hela cells. Western Blot revealed that rADAMTS13-wide type (WT) was synthesized as a single band with molecular mass close to 190 Kda in the conditioned media, however, no detectable ADAMTS13 of this mutant existed. The lysates of cells expressing the mutant showed the same protein amounts compared to the rADAMTS13-WT. The immunofluorescence study demonstrated that mutant had the same localization pattern at Endoplasmic Reticulum(ER)and Golgi-compartments compared to the rADAMTS13-WT. The results imply that this mutant may be retained in the cellular ER and Golgi-comparments, but rapidly degraded or insufficiently secreted.

Disclosures:

No relevant conflicts of interest to declare.
Early Management of Heparin-Induced Thrombocytopenia Using a Simple Scoring System
Authors: Messmore Harry L. , Fabbrini Nancy , Bird Mary L. , Choudhury Abdul M. , Cerejo Miguel , Prechel M. Margaret , Jeske Walter , Siddiqui Arshea , Thethi Indermohan , Wehrmacher William H. , Walenga Jeanine M.
Published: October 2019

Abstract 4679

Since stopping heparin therapy lessens the probability of severe complications of heparin-induced thrombocytopenia (HIT) such as arterial and venous thromboembolism, it is important to stop heparin as soon as the diagnosis is suspected. As HIT is a complex disorder and its diagnosis is difficult, the use of a clinical scoring system would facilitate clinical decision making. One of the foremost issues in these patients is whether to discontinue heparin (to avoid HIT complications) or not (to avoid thrombotic complications). This study was performed to develop a simple scoring system to aid in the early clinical management of suspected HIT patients with regard to decisions for continued heparin therapy. The system was designed to arrive at low (0) or possible (1) probability scores based on clinical criteria without knowledge of lab test results, except platelet counts, to avoid delays. As the safest clinical approach is to discontinue heparin, intermediate and high scores were combined. Historically, laboratory tests such as the heparin-induced platelet aggregation test (PA) and 14C-serotonin release assay (SRA) and nonfunctional tests such as the enzyme-linked-immunosorbent assay (ELISA) have been used for the diagnosis of HIT. These test results are frequently available only after 12 to 36 hours which, for some patients, is a risk due to continued heparin therapy while waiting for test results. One solution to this problem is to have a clinical scoring system to guide the clinician before test results are available. This study enrolled 100 critically ill VA hospitalized patients with a >=30% fall in platelet count. Assessment of platelet aggregation (PA), 14C-SRA and GTI ELISA was also performed. In this population 53% were scored 1 and of these 43% were positive by lab testing for HIT. Emphasizing the decision to discontinue heparin, the clinical signs of HIT were paramount for the immediate determination of a diagnosis of HIT. Specifically, this study demonstrated the value of a simple clinical score to aid in the clinical management decision making to continue or discontinue heparin in patients suspected of having HIT, without dependence on a positive HIT lab test. An algorithm is provided. This scoring system does not preclude reassessment of patients in terms of continuing heparin if subsequent laboratory tests and/or thrombosis become positive.

Disclosures:

No relevant conflicts of interest to declare.
Analysis of JAK2 V617F Mutation and Its Clinical Significance in Patients with Thrombocythemia and Other Myeloproliferative Neoplasms in Chinese
Authors: Xu Yue , Yin Changxin , He Han , Shu Lingling , Wu Fuqun , Meng Fanyi , Chong Beng , Yang Mo
Published: October 2019

Abstract 4687

JAK2 mutation is commonly found in Philadelphia-negative myeloproliferative neoplasms (MPNs). In Western countries, this mutation is found in approximately 96 percent of people with polycythemia vera, half of individuals with essential thrombocythemia or primary myelofibrosis. We used the method of amplification refractory mutation PCR (ARMS-PCR) to investigate MPN patients in China. We focused our study on patients with essential thrombocythemia (ET). ARMS-PCR was used to detect JAK2 V617F mutation in the bone barrow (BM) or peripheral blood of 37 MPN patients, which consisting of 7 ET, 5 polycythemia vera (PV), 5 chronic myeloid leukemia (CML), 5 chronic idiopathic myelofibrosis (CIMF), as well as 15 suspected MPNs. 17 cases of JAK2 V617F mutation (45.9%) were found in 37 patients, including 4 ET (57.1%), 4 PV (80.0%), 3 CIMF (60.0%), 6 suspected MPNs (40.0%). We did not find JAK2 V617F in the patients with CML. Our results indicated that the frequency of JAK2 V617F mutation in bcr/abl-negative MPNs in Chinese is similar to that in MPN patients in Western countries. At the same time, ARMS-PCR can distinguish the mutation is heterozygous or homozygous. Most patients were heterozygous for JAK2 but only a few were homozygous. In conclusion, our study showed that JAK2 V617F mutation frequency in Chinese MPN patients is similar to that in patients with this disorder in the West. It is the major molecular genetic abnormality in bcr-abl negative MPN and it can be used for diagnosis of MPN in China.

Disclosures:

No relevant conflicts of interest to declare.
Naive CD4+ T Cells Exert Superior Alloreactive Capacity Than Central Memory CD4+ T Cells in Fully HLA-Matched Setting
Authors: Choufi Bachra , Cherel Mathilde , Thiant Stephanie , Despicht Damien , Dessaint jean-Paul , Labalette Myriam , Yakoub-Agha Ibrahim
Published: October 2019

Abstract 4702

Background:

Allogeneic stem cell transplantation provides donor-derived mature T cells which are involved in post-transplant immune reactions including engraftment, graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) activity, and thus play a major role in determining clinical outcome. In a prior clinical study, we showed that receiving a high percentage of CD4+CCR7+ T cells that includes naive and central memory (CM) subsets, was correlated with increased incidence and severity of acute GVHD. In a recent update of our results on 126 patients, we confirmed our previous results with a particular impact of CD4+ naive subset on acute GVHD development. The aim of the present study was to investigate in vitro the alloreactive response of CD4+CCR7+CD45RA+ naïve T cells and CD4+CCR7+CD45RAneg central memory T cells in HLA-A, -B, -C, -DRB1 and -DQB1 allele matched setting (so called 10/10 match)

Materials and methods:

The alloreactivity was investigated by mixed lymphocyte dendritic cell reaction using five HLA-identical healthy male and female sibling pairs to include at least a H-Y mismatch. Stimulators were mature dendritic cells (derived from monocytes of the male sibling) co-cultured with each one of the three highly purified CD4+ T cell subsets (naive, central memory and effector memory) from the female sibling. Alloreactive response was assessed by 3H thymidine incorporation at day 5, and functionally at day 4, 6 and 10 by IFN-gamma ELISpot and measurement of Th1/Th2/Th17 cytokines in co-culture supernatants.

Results:

Four out of five sibling pairs developed an alloreactive response to mHAs. Maximal proliferation was supported by the CCR7+CD45RA+ naive CD4+ T cells with proliferation indices from 1.75 to 3.85. This proliferation was accompanied by a functional differentiation: only naive CD4+ T cells were able to produce significant amounts of IL-6, TNF-alpha and IFN-gamma at day 6 and day 10 (120 to 174 IFN-gamma spots at day 10).

Conclusion:

This study demonstrates the superior capacity of naive CD4+ T cells to mount a primary alloreactive response as compared to central memory T cells. Their proliferative response associated with a pro-inflammatory differentiation makes naive CD4+ T cells potential acute GVHD inducers. These in vitro results confirm what we have observed in clinical studies and may also lend support to approaches of selective T cell depletion for GVHD prevention.

Disclosures:

No relevant conflicts of interest to declare.
Effectiveness of Subcutaneous Low-Dose Alemtuzumab and Rituximab Combination Therapy for Steroid-Resistant Chronic Graft-Versus-Host Disease
Authors: Gutiérrez-Aguirre Cesar H , Flores-Jiménez Juan A. , Cantú-Rodríguez Olga , Cuervo-Sierra Jorge , Ceballos-López Adrián A. , Martínez-Hernández Ramón A , Gómez-Almaguer David
Published: October 2019

Abstract 4703

Background:

Chronic graft-versus-host disease (cGVHD) is a common late complication of allogeneic hematopoietic stem cell transplantation. Corticosteroids are the standard initial treatment for cGVHD. A second-line treatment is not well defined. We prospectively evaluated the effectiveness and safety of low doses of alemtuzumab and low doses of rituximab as treatment steroid-refractory cGVHD.

Materials and Methods:

Ten men and 5 women with cGVHD refractory to steroids and CSA were included. All patients received subcutaneous Alemtuzumab 10 mg/day/3 days and intravenous Rituximab 100 mg on days +1, +7, +14 and +21.

Results:

The median age was 41 years. The main organ involved were oral mucosa (86.7%), eyes (66.7%), liver (60%), skin (53%), lungs (13.3%) and intestinal tract (6.7%). The overall response was 100% at 30-day evaluation; 10 patients (67%) had partial remission (PR), and 5 patients (33%) had complete remission (CR). At 90-day evaluation, 7 (50%) patients had PR, 4 (28%) had CR; three (21%) relapsed and 1 patient not reached evaluation. Currently, 5 patients have reached the 365-day follow-up evaluation, 2 (40%) had PR, 2 had CR and 1 showed progression. The adverse effects were mainly infectious and one patient died from pneumonia.

Conclusion:

This combination therapy appears to be an efficacious and safe as treatment for steroid-refractory cGVHD.

Disclosures:

No relevant conflicts of interest to declare.
Gene Therapy of Human Blnk Deficiency in NOD/SCID/Gc Ko Murine Model
Authors: Lagresle-Peyrou Chantal , Milili Michèle , Rouiller Julien , Picard Capucine , Cavazzana-Calvo Marina , Schiff Claudine , Andre-Schmutz Isabelle
Published: October 2019

Abstract 4717

Blnk deficiency is an autosomal recessive immune disorder characterized by the absence of B cells in periphery and the absence of any seric immunoglobulins due to an early blocage at the pro-B cell stage in the bone marrow. The very rare patients affected by blnk deficiency develop severe infections. In the murine model of the disease, a similar blocage in B cell development is described as well as susceptibility to infections and to pre-B lymphomas.

A homozygote stop mutation in blnk gene was identified in a 8-yr old boy. Bone marrow cells analysis revealed that CD34+CD10+CD24-CD19- lymphoid progenitors were present as well as CD34+CD10+CD24+CD19- early B cells and CD34+CD19+ pro B cells. However, no surface IgM or seric Ig were detected in this patient.

To demonstrate the implication of Blnk in the B-cell differentiation process, we transduced CD34+ sorted bone marrow cells from this patient with a lentiviral construct containing human wild type Blnk cDNA. The transduced cells were intravenously injected into irradiated NOD/SCID/IL2rg knock-out mice. Twelve weeks after transplantation, recipients were analysed. Human engraftment was detected in bone marrow and spleen. Among bone marrow human CD45+ cells, more than 80% were CD19+ and 6 to 8% express surface IgM. In the spleen, between 14 and 30% of CD19+ cells were detected. Eight to 42% of CD19+ cells expressed surface IgM.

This is the first demonstration that Blnk is absolutely required for the differentiation of pro-B cells toward mature B cells.

Disclosures:

No relevant conflicts of interest to declare.
Manumycin Induces Apoptosis of Leukemic Stem Cells Through Down-Regulation of Bcl2
Authors: She Miaorong , Li Jinggao , Chen Xilin , Du Xin , Lin Wei , Lu Zesheng , He Yanjie , Guo Kunyuan
Published: October 2019

Abstract 4738

Acute myeloid leukemia (AML) is a serious and often lethal hematopoietic malignant disease arising from stem cells. LSCs play the central role in the relapse and refractory of AML and highlight the critical need for the new therapeutic strategies to directly target the LSC population for ultimately curing leukemia. Unfortunately, very few agents have been shown to directly target the LSC population. The present studies demonstrate that manumycin, a farnesyltransferase inhibitor, resulted in viability decrease in a dose-dependent and soft agar clonogenic formation inhibition in LSCs isolated from KG1a cell line. Furthermore, manumycin induces LSC apoptosis in a dose-dependent. The molecular mechanism of manumycin mediated apoptosis is associated with down regulation of Bcl2 and Bcl-xL. In conclusion, manumycin triggers apoptosis in LSC and represents a potentially important new class of drugs for LSC-targeted therapy.

Disclosures:

No relevant conflicts of interest to declare.
Application of Clinical Microsystems Improves the Speed of the Initial Pain Relief in Sickle Cell Disease (SCD) Patients Admitted with Acute Vaso-Occlusive Crisis (VOC)
Authors: Aghalar Jahan , Barnett Barbara , Cohen Rubin , Tan Venessa , Marcus David , Ouchi Kei , Cascetta Krystal , Fulman Magda , Jaffrey Fatima , Roy Rajasree
Published: October 2019

Abstract 4740

Background-

The management of acute vaso-occlusive crisis continues to be a challenge in patients with sickle cell anemia. Long Island Jewish Medical Center is a 500 adult bed, tertiary care facility, with more than 500 reported admissions for acute vaso-occlusive crisis in 2009. Suboptimal pain management in the Emergency Department (ED) frequently resulted in unrelieved pain, recurrent admissions, prolonged hospitalization, and increased patient dissatisfaction.

Methods-

An interdisciplinary team which included a patient with SCD was assembled to meet weekly to address the above issues. The team was educated in Clinical Microsystem methods as described by Nelson et.al (1) over 6 months. Preliminary data was collected to assess performance with respect to pain management in adult SCD patients presenting to the ED through pain diaries. The admission process was analyzed and significant delays were noted from time of presentation to the time that sufficient sustained analgesia was achieved. The PDSA (Plan Do Study Act) cycle method to test and study change was utilized. The specific aim was to achieve a sustained 2 point reduction in subjective pain score within 2 hours of inpatient admission. Four interventions were simultaneously tested. First, given the high frequency of admission from our ED for this patient population, we tested proactive bed designation initiated when the patient presented to the ED. Second, we educated the housestaff on providing appropriately dosed narcotic administration upon admission after pharmacy verification of previous patient specific dose requirements. Third, we arranged expedited transfer to the medical floors for these patients. Lastly, we arranged for storage of PCA pumps on the medical unit designated for SCD patients, facilitating immediate initiation of pain medication via this modality. Timing of analgesic response was assessed based on patient interviews and the above mentioned diaries.

Results-

During the pre-intervention stage it took an average of 55 hours and 20 minutes to achieve a sustained reduction in pain by two points. Post intervention, the team achieved a sustained 2 point reduction in subjective pain score by an average of 9 hours and 58 minutes (see figure).

Conclusion-

The Clinical Microsystems Methodology is an effective means of engaging an interdisciplinary team in improving the initial analgesic response to treatment in sickle cell patients admitted with acute vaso-occlusive crisis.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy of Short Oral Leucovorin Rescue (SOLR) In Managing Recurrent Pralatrexate (Folotyn) Induced Mucositis (RPIM) Despite Dose Reduction
Authors: Haddad Philip A
Published: October 2019

Abstract 4745

Mucositis is a frequent complication experienced by patients receiving weekly Pralatrexate, an antifolate agent, which often leads to significant morbidity, decreased quality of life, and early discontinuation of this active therapy in aggressive and almost universally fatal Peripheral T-cell Lymphomas (PTCL). Despite its frequency, there is no standard medical management currently except omitting doses followed by one step dose reduction and subsequent discontinuation, many times despite notable responses. Since Leucovorin is approved as antidote for Pralatrexate overdose, we reviewed our clinical experience with its reactive (not pre-emptive) use in our PTCL patients with Grade 2+ RPIM that have already gone through dose omissions and dose reductions and had no other alternative but to discontinue therapy. SOLR consisted of 25 mg po Q6h for 5 days the week of RPIM to be stopped at least 48hours prior to the planned subsequent weekly dose. We reviewed our experience with such approach which amounted to 17 such interventions. All 17 SOLR regardless of grade lead to resolution of subjective symptoms by the 7th day with time to initial subjective improvement at around the 4th day from SOLR initiation. Sixteen SOLR interventions had complete resolution of RPIM on exam on the 7th day and one had almost complete resolution. As such, all cases were ready to resume weekly recommended dosing per standard PTCL regimen. None of SOLR patients discontinued Pralatrexate for RPIM. None of SOLR patients lost their response attained prior to the intervention. In fact, the patient with transformed Mycosis Fungoides continued to show notable further skin response. When compared to the PROPEL Trial which used dose omission and delay to address mucositis, SOLR further shortened the time to resolution by 3–5 days (7 days vs. median of 10 days for Grade 2 and 12 days for Grade 3- Allos unpublished data).

Although further controlled studies are recommended, our clinical observation suggests that reactive SOLR intervention in RPIM offers rapid and effective symptom management by aiding faster mucosal healing and further decreases drug holidays and discontinuation of therapy without any discernable compromise in efficacy.

Disclosures:

Haddad: Allos: Consultancy, Honoraria, Research Funding, Speakers Bureau. Off Label Use: Use of Leucovorin in treating Pralatrexate induced Mucositis.
Participation for Innovation: Survey of Motivation for Clinical Studies Evaluating New Treatments for Hemophilia
Authors: Henrard Severine , Speybroeck Niko , Lambert Catherine M , Hermans Cedric R.
Published: October 2019

Abstract 4778

Over the last 50 years, clinical research into new treatments has helped to transform the care of individuals with hemophilia. Life expectancy for severe hemophiliacs has indeed increased from 11 years in the 1960s to more than 60 years today and many patients with hemophilia are now able to lead an active and fulfilled life. All this has been possible largely through the commitment of people with hemophilia to active participation in clinical research. While much has been achieved, much remains to be accomplished such as the development and validation of longer acting clotting factor concentrates, new bypassing agents and the validation of treatment strategies aiming at preventing or reducing the risk of inhibitor development. Although many clinical trials are currently ongoing around the world, most of them are facing difficulties in recruiting candidate patients, mainly in developed countries. In this context, there is a major need to improve the patients' awareness of the importance of clinical research in hemophilia and highlight the opportunities of an active participation in clinical trials. A survey was launched to evaluate the motivation of patients with hemophilia for clinical studies and identify factors which might influence their willingness to participate. A specific questionnaire was sent to 189 patients with hemophilia (127 adults and 62 children) regularly attending the hemophilia treatment Center of the Cliniques Universitaires Saint-Luc, Brussels, Belgium. Different aspects such as socio-demographic status, familial situation, current hemophilia treatment, knowledge about principles and perceived benefits and risks of clinical studies, positive and negative factors for motivation were extensively addressed. Preliminary results show that most patients have a limited knowledge about the general principles of clinical research. Patients on prophylaxis show a higher motivation to participate than patients treated on demand. Patients are mainly concerned by possible side effects of new treatments. The time lost from school or work refrains many motivated patients from an active participation. Moreover, very few patients are ready to take part in phase 1 trial fearing unknown reactions or side-effects. This survey, which will soon be extended to several hemophilia centers in Europe, highlights the importance to raise awareness and provide better knowledge about the modalities, the risks and benefits of clinical research.

Disclosures:

No relevant conflicts of interest to declare.
Hypoxia Alters the Main Characteristics of the Hematopoietic Stem and Progenitor Cell Microenvironment in Vitro
Authors: Wobus Manja , Duohui Jing , Poitz David M , Müller Katrin , Ordemann Rainer , Ehninger Gerhard , Bornhäuser Martin
Published: October 2019

Abstract 4803

Background:

Hematopoietic stem and progenitor cells (HSPC) are located in a specialized microenvironment, called the stem cell niche, where their stem cell phenotype and differentiation are tightly regulated via interactions with the supporting mesenchymal stromal cells (MSC). These niches have been shown to be localized in regions with a lower oxygen tension which may also impact on the functional properties of MSC. For a better understanding to what extent hypoxia contributes to the establishment of an undifferentiated niche microenvironment that prevents inopportune differentiation of HSPC, we investigated MSC/HSPC co-cultures as well as MSC single cultures under low oxygen conditions.

Design and Methods:

Distribution, functional and phenotypical characteristics of CD34+ HSPC in hypoxic co-cultures (0.5% O2) were analyzed by flow cytometry. The effect of co-culture medium on the HSPC migration potential was tested in a transwell assay. The secretion of vascular endothelial growth factor A (VEGF-A), stromal-derived factor 1 (SDF-1), IL-6 and IL-8 by MSC was determined using ELISA whereas the expression of cell surface molecules was detected by flow cytometry. Moreover, the MSC proliferation as well as adipogenic and osteogenic differentiation was compared between hypoxic and normoxic culture conditions.

Results:

In the hypoxic co-culture, the adhesion of HSPC to the MSC layer was inhibited, whereas HSPC transmigration beneath the MSC layer was favoured. Increased VEGF-A secretion by MSC under hypoxic conditions, which enhanced the permeability of the MSC monolayer, was responsible for this effect. Furthermore, VEGF expression in hypoxic MSC was induced via hypoxia-inducible factor (HIF) signalling. Whereas IL-6 and IL-8 secretion were increased, SDF-1 expression by MSC was down-regulated under hypoxic conditions in a HIF-independent manner. The MSC immunophenotype which is characterized by expression of CD73, CD90, CD105, and CD166 was not significantly changed by hypoxia. Interestingly, a significant decrease of CD146 mRNA and protein expression levels was observed. The MSC proliferation was not significantly affected by lower oxygen tension. Culture of MSC in adipogenic induction medium for 14 days under hypoxia resulted in a reduced appearance of adipocyte-like cells containing lipid droplets and almost 50 % lower mRNA levels of fatty acid binding protein 2. The ALP activity as readout for osteogenic differentiation was decreased between 10% and 60% in hypoxic MSC.

Conclusions:

Low oxygen tension reduces the in vitro differentiation capacity and alters the cytokine secretion profile of primary human MSC. These functional changes may favour the homing and maintenance of quiescent HSC simulating the physiologically hypoxic niche conditions in vitro.

Disclosures:

No relevant conflicts of interest to declare.
The Changes on hematopoietic Stem Cells and mIcroenvIronment of childhood acute Lymphoblastic Leukemia at dIagnosIs and after chemotherapy
Authors: Lee Mee Jeong , Park Chan-Jeoung , Yoon Chan-Hee , Jang Seongsoo , Chi Hyun-Sook , Im Ho Joon , Kim Bo Eun , Seo Jong Jin
Published: October 2019

Abstract 4807

Background:

Hematopoietic microenvironment consists of endosteal and vascular microenvironment. Osteopontin and osteonectic are released by osteoblast which is one of the factors regulating endosteal microenvironment. CXCL12 and CXCR4 are included in the factors regulating vascular microenvironment. Markers indicating the degree of differentiation of hematopoietic stem cells (HSC) include CD133, CD34 and CD117. This study is to evaluate the expression pattern of markers of hematopoietic microenvironment and HSC in childhood acute lymphoblastic leukemia (ALL) at diagnosis and the changes of them after chemotherapy.

Methods:

Between January, 2007 and December, 2009, 32 patients were diagnosed as ALL at Asan Medical Center. Bone marrow (BM) biopsies were obtained at diagnosis, after induction, consolidation, interim maintenance and delayed intensification. There were 22 male and 10 female patients with a mean age of 7.33 years. The diagnoses were a common cell ALL in 29. By immunohistochemistry, we analyzed the expressions of CD133, CD34, CD117, osteopontin, osteonectin, CXCL12, and CXCR4 in these BM biopsy specimens.

Results:

CD133+ cells decreased at diagnosis, and recovered after consolidation. The CD34+ cells decreased after induction, and then gradually increased again. CD117+ cells were fewer at diagnosis, but increased after chemotherapy. The expression of osteopontin was depressed at diagnosis, and gradually recovered. The expression of osteonectin was also suppressed at diagnosis and recovered after delayed intensification. CXCL12 was suppressed at diagnosis, recovered after consolidation and decreased after delayed intensification. CXCR4 was also suppressed at diagnosis, but increased after therapy.

Conclusions:

This study is the first report on the changes of hematopoietic microenvironment and HSC of childhood ALL at diagnosis and after chemotherapy, evaluated by immunohistochemistry. CD133+ cells, CD34+ cells and CD117+ cells increased in the reconstructive phase. CD34 can be a marker reflecting the BM recovery. Proliferation of osteoblasts and stromal cells were active after interim maintenance. The reconstruction of HSC and BM matrix increased after consolidation. The recovery of BM microenvironment was established after consolidation and interim maintenance chemotherapy. These findings may serve as basic data for future studies dealing with the hematopoietic microenvironment and HSC of childhood ALL.

Disclosures:

No relevant conflicts of interest to declare.
The Growth Response of Erythrocyte Production to Increased Dense Condition
Authors: Choi Hye sook , Lee Eun-mi , Ajjappala Brijesh Siddaveerappa , Huh Ji Young , Yoon Hwi-Joong , Baek Eun Jung
Published: October 2019

Abstract 4812

Erythroid precursors proliferate, differentiate, and enucleate in the bone marrow niches called erythroblastic islands in contact with macrophages. However, the cell-to-cell communication between erythroblasts which are undergoing terminal maturation leading to enucleation is not well known. Moreover, the cell-to-cell interaction at a terminal erythroblast maturation stage was hardly detected in conventional suspension culture systems due to cell surface characteristics of electrostatic repulsion.

To elucidate the communication between late maturing erythroblasts, erythroid cells, which were derived from human CD34+ cells, were cultured at conventional or supra-optimal densities. Surprisingly, the final yields of red blood cells (RBCs) were significantly increased in supra-optimal culture density, even in the absence of feeder stromal cells and serum or plasma. Also observed were increases in the rates of cell survival, expansion, and enucleation. Contact between cells observed in these supra-optimal cultures was associated with increases in adhesion-related signaling proteins (150 kDa and 123 kDa), the latter of which was previously unknown to be involved in erythropoiesis. The cultured erythroid cells showed erythropoiesis specific markers such as CD71, GPA, GATA-1 and b-globin.

Therefore, we firstly elucidated that the erythroid cells which were in the final maturation step can communicate each other and affect their survival and maturation including enucleation without the help of macrophages. Also, the increased attachments between erythroid cells evoked adhesion-related signals. We anticipate that the culture of these cells at supra-optimal density may enhance in vitro RBC production for human transfusion.

Disclosures:

No relevant conflicts of interest to declare.
Ex Vivo Expanded Megakaryocyte Progenitors Generated From Human Mobilized Peripheral Blood CD34+ Stem Cells Can Be Cryopreserved and Transplanted Into NSG Mice to Generate Functional Platelets
Authors: Karsunky Holger , Tressler Robert J. , Chananukul Joy
Published: October 2019

Abstract 482

Thrombocytopenia is a common side effect of high-dose chemotherapy that can compromise cancer treatment by requiring treatment delay and/or dose reduction for the patient. Platelet transfusion is typically given to prevent severe hemorrhage. However, several factors including acquisition, banking, and associated risks of bacterial infections and alloimmunization are hampering reliance on platelet transfusion. Growth factors are also used to stimulate proliferation and differentiation of megakaryocytes to increase platelet production, but in severely myelosuppressed patients these have only had modest benefit. The limitations of these two modalities for the treatment of chemotherapy-induced thrombocytopenia indicates that additional treatment approaches are needed. We have developed a novel approach to reconstitute megakaryocytes and platelets in thrombocytopenic patients which is presented here. We have identified a scalable culture system using serum-free medium and a defined cytokine cocktail free of animal products to expand CD34+ hematopoietic stem cells from G-CSF mobilized peripheral blood donors in vitro and direct their development to the megakaryocyte lineage to yield committed human megakaryocyte progenitors (MKPs). These MKPs can be readily cryopreserved while retaining their capacity to generate CFU-MK and platelets in vitro. When infused into NSG mice, ex vivo expanded MKP generate clinically relevant platelet levels of platelets in blood within a few days with sustained platelet levels for several weeks. The platelets generated from MKP in vivo are also functional as assessed by CD62P expression in responses to ADP stimulation in vitro. Our results present a compelling approach for the development of off-the-shelf storable MKPs for the treatment of thrombocytopenia.

Disclosures:

Karsunky: Cellerant Therapeutics Inc.: Employment, Patents & Royalties. Tressler:Cellerant Therapeutics, Inc.: Employment, Equity Ownership. Chananukul:Cellerant Therapeutics Inc.: Employment, Patents & Royalties.
A Sensitive and Quantitative Polymerase Chain Reaction-Based Non-Homologous End Joining in Vitro Assay for Hematopoietic Stem Cells
Authors: Shao Lijian , Feng Wei , Zhou Daohong
Published: October 2019

Abstract 4825

Hematopoietic stem cells (HSCs) are longevity cells that are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative in vitro non-homologous end joining (NHEJ) assay using the Bgl II-digested linear pDsRed2ER plasmids and real-time polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 0.25 μg nuclear proteins or nuclear extracts from about 1000 human BM CD34+ hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34+CD38− cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34+CD38+ cells). In contrast, mouse quiescent HSCs (Pyronin-Ylow LSK+ cells) repaired the damage more efficiently than cycling HPCs (LSK− cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as ATM and ligase 4. These findings suggest that the qPCR-based plasmid NHEJ assay can be used to sensitively measure the ability of HSCs to repair DSBs. This assay can also be applied to other rare tissue stem cells as well.

Disclosures:

No relevant conflicts of interest to declare.
Increased Body Mass Index Correlates with Higher Risk of Disease Relapse and Differentiation Syndrome in Patients with Acute Promyelocytic Leukemia Treated with Aida Protocols
Authors: Breccia Massimo , Mazzarella Luca , Loglisci Giuseppina , Bagnardi Vincenzo , Disalvatore Davide , Latagliata Roberto , Testi Anna Maria , Cimino Giuseppe , Avvisati Giuseppe , Petti Maria Concetta , Pelicci Pier Guiseppe , Foá Robin , Lo Coco Francesco
Published: October 2019

Abstract 4866

The association between obesity and outcome of acute promyelocytic leukemia (APL) patients is still not defined. We investigated whether increased body mass index (BMI) might have a role in the outcome of APL patients. The study population includes 144 patients consecutively diagnosed as having APL between January 1993 and December 2005 at the “Sapienza” University of Rome and treated with AIDA or AIDA2000 schedule. Diagnosis was confirmed for all patients at molecular level. Patient demographic information, weight and height, treatments received, events and survival outcome data were collected. In particular the diagnosis of definitively present retinoic differentiation syndrome (DS) was clinically established by the presence of at least three of the following signs weight gain, respiratory distress, unexplained fever, interstitial pulmonary infiltrates, pleural or pericardial effusions, according to Frankel et al. There were 66 males and 78 females, median age 39.3 years. Seventy-five patients were aged more than 40 years at diagnosis. Twenty-four patients out of 144 were classified as having variant type according to FAB classification and 83 patients have BCR1 type of transcript. BMI was defined as the individual's body weight divided by the square of him or her height: we stratified 4 categories of patients, according to height and weight at the time of diagnosis: underweight (BMI< 18.5), normal weight (BMI 18.5-<24.9), overweight (BMI 25->29.9) and obese (BMI ≥30). Fifty-seven (39.6%) and 87 (60.4%) patients were underweight/normal (BMI<25) and overweight/obese (BMI≥25), respectively. Increased BMI was associated with older age (p=0.0002) and male gender (p=0.004). Twenty-one patients (14.5%) experienced DS during induction therapy according to reported criteria: we evaluated in multivariable logistic regression model clinico-pathological features at baseline possibly associated to an increased risk of DS and we identified as the significant factor only BMI (p=0.014, OR 7.2, 95% CI 1.5–34.9). After a median follow-up time of 6 years, the estimated cumulative incidence of relapse (CIR) at 5 years was 31.6% (95% CI: 22.7%,43.8%) in overweight/obese and 11.2% (95% CI: 5.3%,23.8%) in underweight/normal weight patients (p=0.029). Multivariable analysis showed that BMI was an independent predictor of relapse (HR = 2.45, 95% CI 1.00, 5.99, in overweight/obese vs under/normal weight patients, p=0.049). In conclusion, increased BMI at diagnosis is associated to higher risk of RAS and relapse in APL.

Disclosures:

Breccia: novartis: Honoraria; Bristol Myers Squibb: Honoraria.
HSP90 Chaperone – An Important Player in CML: Study At RNA and Protein Level
Authors: Zackova Marketa , Lopotova Tereza , Nadvornikova Sylvie , Klamova Hana , Moravcova Jana
Published: October 2019

Abstract 4878

Chronic myeloid leukemia (CML) is the myeloproliferative disease characterised by presence of BCR-ABL tyrosine kinase, which has been proved to play the basic role in CML ontogenesis. BCR-ABL as well as other kinases active during disease progression (e.g. Src kinase) belong to HSP90 client proteins. It is known, that HSP90 protein is overexpressed in cancer cells and leukemias and is related to therapy resistance in CML cells (Gorre et.al. Blood 2002;100:3041-3044).

HSP90 exists in two isoforms: alpha - inducible and beta – constitutively expressed. Taherian et.al. (Biochemistry and Cell Biology, 2008, 86:(1) 37–45) demonstrated that Hsp90α and Hsp90β exhibit similar interactions with cochaperones, but significantly different substrate specificity under stress conditions. Those results reveal both functional similarities and key functional differences between the individual members of this protein family.

In our previous study we found high protein expression of total HSP90 in leukocytes of CML patients in advanced disease states and in patients with poor responses (Zackova et.al. EHA 2011). The HSP90 seems to be an indicator of disease deterioration in patients with chronic myeloid leukemia.

In the current study, we extended our field of interest on HSP90 isoforms alpha and beta on mRNA level. We aimed to find out possible correlation between protein and mRNA expression levels, as well as between mRNA levels and response to the therapy. We wondered to know whether the monitoring of total HSP90 or its isoforms in CML can early predict the disease deterioration.

We tested HSP90 alpha and HSP90 beta mRNA expression levels in patients with various response of CML by real-time RT-PCR method. The mRNA profiles showed high similarity with the data obtained from previous western blot analyses. The analyses showed that high HSP90 levels are associated with poor response to therapy and in advanced disease phases. These levels are probably represented by HSP90-beta isoform (constitutively expressed), which is expressed in higher levels comparing to HSP90 alpha in all samples tested. Expression of HSP90 alpha isoform is much lower while its mRNA expression level highly increases only in blast crisis.

Studying both isoformes separately could distinguish various mechanisms in disease progression.

The results of this and previous studies suggest HSP90 (on protein and mRNA levels) is an important molecule for studying of prognosis in CML patients. Thus HSP90 appears to be a candidate for novel marker of CML progression.

Disclosures:

No relevant conflicts of interest to declare.
Establishment of Bioluminescence Imaging Based Leukemia In Vivo Model for Preclinical Testing
Authors: Lee Myoung Woo , Kim Hye Jin , Kim Dae Seong , Son Meong Hi , Lee Soo Hyun , Jung Hye Lim , Yoo Keon Hee , Sung Ki Woong , Koo Hong Hoe
Published: October 2019

Abstract 4879

Background.

A hematological malignant animal model is an essential tool for evaluating efficacy of anti-cancer drugs and elucidating underlying mechanism of leukemogenesis. Intraperitoneal (IP) and intravenous (IV) xenograft of acute lymphoblastic leukemia (ALL) cells have limited capacity as in vivo anti-cancer drug screening system.

Purpose.

In this study, we aimed to establish an ALL animal model using NOD/SCID mouse and evaluate efficiency and sensitivity of the model as a preclinical drug screening system.

Materials and Methods.

Firefly luciferase (fLuc)-gene introduced ALL (ALL/fLuc) cell line and patient-originated ALL cells were transplanted into a tibia of NOD/SCID mouse. We conducted a comparative analysis of intra-bone marrow (IBMT) transplanted leukemia model with IP and IV transplantation of leukemic cells.

Results.

IBMT of ALL/fLuc cells effectively established a bioluminescent leukemia NOD/SCID mouse model. Upon comparison of IBMT model with IP and IV transplantation models, infusing identical number of ALL/fLuc cells into NOD/SCID mice resulted in IBMT model with evaluable bioluminescent signal, but not in IP and IV models. In IBMT model, bioluminescent signals of ALL/fLuc cells emitted from peripheral blood, tibia and infiltrated organs indicated that leukemia model was established. The changes in these signals' strength reflected dose-dependent cytotoxic effects of vincristine, which allowed leukemia model with evaluable bioluminescent signal to be utilized as a preclinical drug screening system. IBMT leukemia model was also established using primary ALL cells that can provide additional insights for the development of leukemia therapeutics.

Conclusion.

IBMT of ALL/fLuc cells enables development of leukemia mouse model with the greater bioluminescent sensitivity than IP and IV in NOD/SCID to evaluate candidate for development of anti-cancer drug.

Disclosures:

No relevant conflicts of interest to declare.
MYL9 Is Prerequisite for the CD3 Expression in Cytoplasm Membrane of T Lymphocytes
Authors: Aoki Takumi , Miyazaki Koji , Higashihara Masaaki
Published: October 2019

Abstract 4925

[Introduction]

Normal T lymphocytes express CD3 protein, major component of T-cell receptor, in the cytoplasm membrane. However, CD3 expression is suppressed in malignant T cells. The mechanism of suppression of CD3 expression remains unknown. We previously reported the cloning of three isoforms of non-muscle myosin light chain, including MYL9 (transcript variant 1). MYL9 mRNA was expressed in monocyte-macrophage cell lines, but not in lymphoid cell lines (J. Smooth Muscle Res.44: 29–40, 2008). We report here some evidence, suggesting the prerequisite role of MYL9 (transcript variant 1) in the process of CD3 expression in cytoplasm membrane.

[Materials and Methods]

Leukemic cell lines were cultured in RPMI1640 plus 10% FCS. mRNA contents were measured with RT-PCR. CD3 expression was measured using FACScan FITC-labeled or PE-labeled second antibody. Whole CD3 contents were measured using IntraPrep™ (Beckman Coulter). Transfection of MYL9(transcript variant 1)gene into Jurkat cell (wild-type) was performed using Trans Fasc Reagent (Promega). In the process of screening the change the percentage of surface marker, we found that some clones showed the CD3 positive, and Jurkat (clone I), which expresses CD3 (85∼90%) in the cytoplasm membrane, was selected. We also used Jurkat, Clone E6-1(TIB-152)(ATCC®), strong positive in CD3 (90∼95%) in cytoplasm membrane.

[Results]

1) CD3 were negative in Jurkat cell (wild-type), and highly positive in both clone I and clone E6-1. 2) Whole CD3 (cytosole and cytoplasm membrane) were positive in these three cells, suggesting that transportation of CD3 protein from cytosole to cytoplasm membrane is suppressed in Jurkat cell (wild-type). 3) Long term culture (i.e. 30∼60 days) induced, although weakly, both MYL9 mRNA and CD3 of cytoplasm membrane in Jurkat cell (wild-type). 4) Clone E6-1 shows positive in MYL9 mRNA. 5) In Jurkat cell (wild-type), PMA induced CD3 expression in cytoplasm membrane, but not MYL9 mRNA. Pre-treatment with staurosporine showed partial inhibitory effect on PMA-induced CD3 expression. 6) Neither concanavalin A nor A23187 induced CD3 expression in both clone I and Jurkat cell (wild-type).

[Discussion]

These results suggest that MYL9 is prerequisite for the CD3 expression in cytoplasm membrane of normal T lymphocytes. However, in T-cell malignant cells, MYL9 gene is suppressed by unknown mechanism. Our data show that CD3 expression in cytoplasm membrane may be regulated by MYL9-dependent and MYL9-independent fashions.

Disclosures:

No relevant conflicts of interest to declare.
The Mortality of Vancomycin-Resistant Enterococci Bloodstream Infections (VRE BSI)
Authors: Choi Philip Young-Ill , Straube Belinda , Cook Christine , Van Der Weyden Carrie , Ramanathan Sundra , Taylor Peter , Roncolato Fernando , Chong Beng
Published: October 2019

Abstract 4928

Background

VRE are nosocomial pathogens with resistance to most commonly used antimicrobial agents. VRE BSI occurs in as few as 4% of patients colonised with VRE[i]. A review of factors contributing to the development of VRE BSI was performed to improve patient safety at St George Hospital.

Alarmingly, there were 9 new cases of VRE BSI detected amongst haematology inpatients in early 2010, as compared with only 2 in the previous 6 months. In high risk populations, the rate of VRE BSI amongst patients colonised with VRE can be as high as 29%[ii].

VRE BSI is associated with an increased length of hospital stay from 10.5 to 46 days[iii][iv]and an estimated increased cost of $27,190 per patient[v]. Annual net savings of $100,000-150,000 can be achieved by hospitals detecting 6–9 cases of VRE BSI per year by utilising enhanced infection control strategies[vi].

Methods

Admission details for haematology in-patients and their microbiology results between 1/6/2009-30/11/2010 were reviewed. A subsequent case-controlled analysis was performed matching for patient age, disease and disease stage.

Interventions to reduce the rate of VRE transmission were introduced in July 2010: improved hand hygiene education, additional staffing allocations, additional cleaning services, antimicrobial stewardship, improved patient education, increased staff awareness, monthly census screening of all patients on the 4 East Oncology/Haematology ward for VRE and contact tracing measures.

Results

471 patients were admitted a total of 943 times. VRE was isolated in 61 patients. Average length of stay was significantly longer in patients with VRE than for patients without VRE [16.3±3.0 vs 8.4±1.0 days, p=0.01].

16 patients had VRE BSI. After a median follow-up of 9.8 months, eight of these patients have died (50% mortality), mostly due to progressive disease.

45 patients had non-BSI VRE and were followed up for a median 8.4 months: 24 died (53% mortality), also mostly due to progressive disease.

Case-controlled Analysis

An age, case and stage of disease matched analysis compared 14 patients with VRE and 14 with no evidence of VRE. Median follow-up from presentation with disease was 16.3 months and from VRE detection was 8 months. Mortality rate was 64% vs 29%. Seven out of nine deaths in the VRE cohort were due to progressive disease.

Conclusions

The clinical significance of VRE BSI over other modes of detection remains uncertain for individual patients, however high rates may reflect uncontrolled VRE transmission. Case controlled analysis demonstrates an associated additional mortality risk for VRE positive patients.

Disclosures:

No relevant conflicts of interest to declare.