bluuebunny/crossref_metadata_2025_split huggingface dataset:
Germinal Center Specific Activation of K-Ras, Common In Multiple Myeloma, Is Selected Against and Is Not Sufficient to Initiate Plasma Cell Transformation In Mice
Authors: Mullins Chelsea D. , Tomasson Michael , Lu Lan , Su Mack Y. , Hutchagowder Vishwanathan , Vij Ravi , Kulkarni Shashikant , Chu Liang 
Published: October 2019

Abstract
Abstract 137
Activating mutations in Ras (N- and K-) are the most common mutations known in multiple myeloma (MM), and are associated with advanced clinical stage and poor outcomes. We re-sequenced coding regions of highly expressed cytokine signaling candidate genes from MM patient samples and found rare novel mutations. Still, the most common recurrent somatic mutations we found affected N and K-Ras. Previous studies of MM patient samples suggest that Ras mutations may be late/progression events, but the role of Ras activation in MM pathogenesis has not been tested directly. Toward this end, we generated double-transgenic mice by crossing Lox-stop-lox(LSL)- K-ras G12D knock-in mice (K-ras+/G12D) with Cγ1-Cre knock-in mice (Cγ1+/Cre) expressing the Cre recominase specifically in germinal center (GC) B-cells. K-ras+/+/Cγ1+/Cre mice served as negative controls. We found 58% of unstimulated K-ras+/G12D/Cγ1+/Cre mice developed fatal double-positive CD4/8 T-cell lymphomas (n=12) and 42% developed lung adenocarcinomas (n=12). The median survival of these mice with lymphomas was 19 weeks, compared to the 300 day end point of unstimulated K-ras+/+/Cγ1+/Cre control mice. Tumor cells from these mice demonstrated successful recomination of the K-Ras locus. In contrast, no evidence of multiple myeloma or monoclonal gammopathy was found by serum ELISA, SPEP, flow cytometry or histologic examination. We isolated B-cell subsets from K-ras+/G12D/Cγ1+/Cre mice and documented successful recombination in splenic germinal center cells (B220+, GL-7+) but not in bone marrow plasma cells. Together, these data suggest that Ras activation by “leaky” off-target Cre expression was sufficient to initiate tumorigenesis in two non-B-cell compartments, but that activation of the K-rasG12D allele in germinal center B-cells was associated instead with clearance of recombined cells. We conclude that activating Ras mutations are disease progression events in MM and that earlier mutations are required before primary plasma cells are transformed. This novel mouse model will be useful for identifying and testing early genetic events that drive MM development in cooperation with Ras.

Disclosures:
No relevant conflicts of interest to declare.
Type 3 Von Willebrand Disease with Alloantibodies and Its Challenging Management During Episode of Bleeding
Authors: Verma Anupam , Yaish Hassan , Rodgers George M. 
Published: October 2019

Abstract
Abstract 1405
Type 3 Von Willebrand Disease (VWD) is the most severe and least common of the three VWD types. The incidence varies by ethnicity ranging from 0.1–5.3 cases/million. 8.5% of patients develop alloantibodies to Von Willebrand Factor (VWF), becoming non-responsive to treatment or prone to life threatening reactions. Predisposing factors include extent of previous exposure to VWF and mutations causing a null-allele gene. Variability of the clinical course is attributed to different antigenic specificities and potency of the antibodies. Management of these patients during events of bleeding is challenging. We report 2 cases of girls with type 3 VWD. The first was diagnosed at birth and had her initial VWF transfusion as a neonate. The second was diagnosed at 4 years of age. Both were frequently treated with VWF and developed alloantibodies. The clinical course, severity and circumstances leading to the diagnosis were different in both cases. Life threatening anaphylactic reactions like hypotension, bronchospasm and tongue edema was noticed in the first. The second showed poor factor recovery and response to treatment with urticaria. She continued to tolerate therapy but failed tolerance induction. Peak antibody level was 84 BU compared to 3 BU in the first. Novel VWF gene mutations were detected in both. Recently the first patient was managed for profuse bleeding post extensive dental procedure. Recombinant Factor VIII infusion was administered every 3 hours with monitoring of aPTT and Factor VIII levels. Based on peak and post 3 hour levels of Factor VIII, we concluded the half life of Factor VIII could be as short as 2 hours in patients with type 3 VWD. Post infusion peak level might approach 200% in some instances. Due to persistent bleeding despite adequate correction of Factor VIII and aPTT, platelet transfusion was cautiously administered which proved to be not only effective in controlling bleed but was also safe and not associated with any anaphylactic reaction. In conclusion, patients with type 3 VWD and alloantibodies can be successfully managed with aggressive frequent therapy with recombinant Factor VIII infusions. Platelet transfusions during episodes of severe bleeding can also be safe and effective.Administration of Factor VIII by continuous infusion could be an option if high post infusion peaks are to be avoided.

Disclosures:
No relevant conflicts of interest to declare.
Tacrolimus Induced Refractory Immune Thrombocytopenia.
Authors: Alkhateeb Hassan , Manchanda Naveen , Mehta Rakesh P 
Published: October 2019

Abstract
Abstract 1426
Tacrolimus, a calcineurin inhibitor, has become a standard immunosuppressive therapy in solid organ transplantation. It is known to cause a thrombotic microangiopathy, and has been implicated to cause immune thrombocytopenia (IT) in children. Our institution has recognized a delayed onset IT in adult liver transplant patients that appears associated with chronic therapy with Tacrolimus. Between 2007–2009, 4 patients (3 females and one male) between the ages of 28–71 years who were status post orthotopic liver transplant and were on Tacrolimus developed a refractory IT. The onset of the IT was 3–12 years after starting on the Tacrolimus. One of the patients had a history of an autoimmune hemolytic anemia, which occurred one month after transplantation and resolved with IVIG therapy. Two of the patients were diagnosed with Hepatitis C reactivation after transplantation, but were not on therapy before the onset of IT. At presentation, the platelet counts were between 2,000 – 4,000/μL. IT was diagnosed after ruling out other causes of thrombocytopenia. Bone marrow biopsies were done in all four patients, and were consistent with increased platelet destruction. The thrombocytopenia was refractory to conventional IT treatment with steroids and IVIG. Other therapies included Rituximab (3 patients), Vincristine (2 patients), and splenectomy (1 patient), but none of these significantly improved the platelet count. However, switching the Tacrolimus to Cyclosporine in all the patients led to a rise in platelet count within 6 days to two months. These patients have been observed for over 1 year without a recurrence of their IT. One patient requires chronic therapy with Romiplostim, while being treated with Ribavirin and Interferon for his Hepatitis C reactivation. His platelet count has not fallen below 100k/μL. CONCLUSION: Tacrolimus may cause a refractory IT that occurs several years after its initiation as an immunosuppressant. When this situation occurs, discontinuing the Tacrolimus and replacing the immunosuppression with Cyclosporine is the most effective therapeutic approach.

Disclosures:
No relevant conflicts of interest to declare.
Incidence of Isolated Heparin-Induced Thrombocytopenia and the Risk of Developing Subsequent Thrombosis Using the New IgG Specific Anti-PF4/Heparin ELISA
Authors: Radaideh Sofyan M. , Frenkel Eugene P. , Sarode Ravindra , Shen Yu-Min 
Published: October 2019

Abstract
Abstract 1448

BACKGROUND:
The new IgG specific ELISA for anti-platelet factor 4 (PF4)/heparin antibody is recently introduced for the diagnosis of heparin-induced thrombocytopenia (HIT). It is as sensitive but more specific than the poly-specific IgG, M, A ELISA. About 50% of patients testing positive for the anti-PF4/heparin antibody have thrombocytopenia alone as initial manifestation of HIT without thrombosis (isolated HIT). These patients are thought to be at high risk (≂f30%) for developing thrombosis within next 28 days. The incidence of isolated HIT using IgG-specific ELISA is unknown.

OBJECTIVES:
Evaluate the incidence of isolated HIT using the IgG specific anti-PF4/heparin ELISA and the risk of developing subsequent thrombosis in these patients.

PATIENTS AND METHODS:
We performed a retrospective observational study of patients being tested for the anti-PF4/heparin antibody at our institution from December 2008 to May 2010 using the IgG-specific anti-PF4/heparin ELISA with a two-step assay using high dose heparin in the second step to demonstrate heparin specificity. A positive test was defined as optical density (OD) >0.4 with >50% inhibition.

RESULTS:
319 patients were tested during the study period. 23 (7.2%) patients were diagnosed with HIT based on clinical findings (4T score) and ELISA. 16/23 (70%) had thrombosis at diagnosis whereas 7/ 23 (30%) had isolated HIT. All 7 patients with isolated HIT had follow up data for at least 3 months after diagnosis. Only 2/7 were treated with direct thrombin inhibitors (DTI) and 4/7 were treated with warfarin for at least 1 month. While only 3 of the 7 isolated HIT patients had compression ultrasonography to rule out occult lower extremity thrombus, none of these 7 patients (including 4 with an OD of >1.0) developed symptoms or signs of thrombosis in the subsequent 3 months after the diagnosis of isolated HIT.

CONCLUSION:
The incidence of isolate HIT in this study (7.2%) is significantly lower than previously reported by others and our own historical patients (57.4%, Altuntus et al, Euro J of Haematology 2008) using the IgG, M, A poly -specific anti-PF4/heparin ELISA (z value 2.092, p=0.036). It is possible that many patients previously thought to have HIT by the poly-specific anti-PF4/heparin ELISA assay were false positive. Using the IgG-specific anti-PF4/heparin ELISA not only improved the specificity of the ELISA assay, it also reduced the number of patients with isolated HIT significantly who also seem to have lower risk of developing subsequent thrombosis.

Disclosures:
No relevant conflicts of interest to declare.
Blockade of T Cell Anergy by Tyrosine Kinase Inhibitor, Sorafenib May Induce Exacerbation of Gvhd In Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Yokoyama Hisayuki , Harigae Hideo , Childs Richard 
Published: October 2019

Abstract
Abstract 1458

Combining molecular targeted drugs with allogeneic hematopoietic stem cell transplantation could be an attractive approach to treat diseases associated with a high risk of relapse after allogeneic transplantation. However, influences of molecular targeted drugs on immune function during the period around allogeneic transplantation remain obscure. In our experiment, tyrosine kinase inhibitor, sorafenib exacerbates Graft-versus-Host disease in mouse model when given following allogeneic hematopoietc stem cell transplantation (Blood in press). To explore the mechanisms of this phenomenon, we examined in vitro effects of sorafenib on proliferation of T cells. 3H uptake study showed that the addition of sorafenib did not alter mixed lymphocyte reaction, but when stimulated with anti-CD3 antibody (OKT3) alone, T cell proliferation was enhanced significantly in the presence of sorafenib (median Stimulation index: 4.6, range). This effect was disappeared when stimulated with OKT3 and anti-CD28 antibody. We hypothesize that this stimulatory effect of sorafenib would result from inhibition of T cell anergy. To test this hypothesis, expression of anergy related genes (DGKa, Itch, Egr2, GRAIL) in T cell was examined by RQ-PCR. After stimulation of OKT3 alone, expressions of these genes were increased but in the presence of sorafenib, GRAIL expression was decreased, while expression of DGKa, Itch and Egr2 was not changed. This result may indicate that sorafenib exacerbates GVHD presumably through inhibition of T cell anergy.

Disclosures:
No relevant conflicts of interest to declare.
Memory T Cells Masquerading as Naïve Cells: Implications on Adoptive T Cell Immunotherapy.
Authors: Cieri Nicoletta , Provasi Elena , Camisa Barbara , Magnani Zulma , Bondanza Attilio , Bonini Chiara 
Published: October 2019

Abstract
Abstract 1471
Differentiation stage of adoptively transferred T cells has a critical impact on the success of immune-gene therapy. Ex vivo T cell manipulation often induces their terminal differentiation, resulting in poor persistence and activity of transferred cells. We previously showed that costimulation and culture with γ-chain cytokines generates gene modified T cells with a functional central memory (TCM) phenotype superior to effector/effector memory (TEM) counterparts for expansion potential and antitumor activity. Here we investigated the consequence of initial targeting of selected T cell subpopulations. We activated and efficiently transduced FACS-sorted T Naïve (TN), TCM and TEM cells by viral vectors. In contrast to TCM and TEM, TN had a greater expansion potential and more sustained expression of IL7-Rα. Strikingly, manipulation of TN resulted in a predominant population of post-mitotic lymphocytes expressing the CD45RA+CD62L+CCR7+ naïve phenotype. These cells expressed markers common to early differentiated cells (CD27, CD28, CD127 and Bcl2) and markers proper of memory lymphocytes (CD45RO, CD122 and CXCR3). Post-mitotic TN produced lower levels of IFNg and Granzyme A, expressed higher levels of c-Kit and CXCR4, and lower levels of HLA-DR, CCR5 and PD1 than memory counterparts. To verify their self-renewal and differentiation potential upon antigen stimulations, TN, TCM and TEM were transduced to express a WT1-specific TCR. Upon multiple stimulations TN expanded at higher numbers and were unique in the ability to generate a mixed population of CD127+/CD127- lymphocytes. When infused in immunodeficient mice, transduced TN proved higher engraftment and persistence potential than memory counterparts, reconstituted a mixed CD45RA±CD62L± phenotype and were significantly higher xenoreactive. These results suggest that gene transfer into TN lymphocytes might increase the efficacy of cancer immunotherapy.

Disclosures:
Bonini: MolMed: Consultancy.
Investigating the Experiences and Educational Needs of Adolescents with Hemophilia Prior to Transition of Care: A Website Development Project.
Authors: Sterling Lynn , Nyhof-Young Joyce , Blanchette Victor S. , Breakey Vicky R. 
Published: October 2019

Abstract
Abstract 1526

Background:
Adolescents with hemophilia (AWH) have heightened educational needs as they become more independent and transition from pediatric to adult care. Poor compliance during adolescence may lead to suboptimal management and its long-term sequelae. An innovative website is being developed to meet these educational needs and foster the acquisition of self-management skills.

Purpose:
To examine the educational needs of AWH treated at The Hospital for Sick Children and explore how a web-based transition program can best support them.

Methods:
Qualitative, semi-structured interviews with a purposive sample of AWH addressed subjects’ experiences with hemophilia, views about transition, internet use, and thoughts about an educational website. The interviews were audio-recorded and transcribed verbatim. Themes were identified through constant comparative analysis by three independent reviewers.

Results:
Eleven of twelve subjects that were approached consented to be interviewed and data saturation was achieved in the areas of interest. Important themes surrounding the hemophilia experience included personal identity and perception of their condition, challenges, raising awareness, and sources of support. Some participants had concerns about transition to adult care but many also recognized its benefits. There was a broad spectrum of perceived preparedness for transition. All participants felt comfortable using the Internet and overall, felt a website would help them to learn about haemophilia and explain it to others. Social networking with others with haemophilia was deemed desirable by many.

Conclusions:
AWH are interested in a hemophilia website and have identified a variety of features which they believe may help to support them during transition of care and beyond.

Disclosures:
Breakey: Baxter Bioscience Canada: Fellowship salary provided through unrestricted educational grant.
Changing Age Distribution of Deep Venous Thrombosis In the United States
Authors: Viswanathan Prabhu , Krishnamurti Lakshmanan 
Published: October 2019

Abstract
Abstract 1539

Background:
Deep vein thrombosis (DVT) and pulmonary embolism (PE) are the major cardiovascular killers in the hospital setting. While there is increasing understanding of the public health importance of DVT there are limited data on the trends in the distribution of DVT in different age groups.

Aim:
The aim of this study was to study national trends in age distribution of DVT in patients over the age of 45 years in the last decade.

Methods:
We used the National Inpatient Sample (NIS) to analyze the epidemiology of hospitalizations in various age groups from 1997 – 2007. The NIS is a part of the Healthcare Cost and Utilization Project (HCUP) sponsored by the Agency for Health Care Research and Quality (AHRQ) and contains information from a 20% stratified sample of hospitals extrapolated to show the entire national utilization in United States. For our study we selected the following ICD-9 codes to represent DVT (ICD-9 codes 451.11, 451.19, 451.2, 451.81, 451.9, 453.40, 453.41, 453.42, 453.8, 453.9). For each hospitalization, NIS allows a total of up to 15 diagnostic entries. The entry in the diagnostic (DX) field 1 is called the Principal Diagnosis (PD) is the principal reason for admission and the entries between DX fields 1 and DX field 15 are called all-listed diagnoses and include the principal diagnosis plus additional conditions that coexist at the time of admission, or that develop during the stay. We derived at Secondary diagnoses (SD) by excluding PD of DVT from the list of AD. U.S. Population Census Bureau was used to calculate the change in population. Predictive Analytics Software, IBM PASW Statistics 18.0 data analysis tool were used for data mining and statistical analysis and least square regression analysis was used to calculate ‘p’ value.

Result:
From 1997 – 2007,

Conclusion:
These data describe for the first time, trends in the relative contribution of different age groups to the burden of DVT among hospitalized patients in the United States. The population between age group 45–64 years is growing symmetrically as a proportion of the total population, total hospitalized population and total hospitalized population with DVT. On the other hand the population 65–84 years of age is decreasing as a proportion of the total hospitalized population with DVT to an even greater degree than as a proportion of the total population and the total hospitalized population. The group over 85 years remains a disproportionately large contributor to the population with DVT. These data suggest the need for greater study of the distribution of DVT, the underlying causes and potential strategies to prevent DVT in different age groups

Disclosures:
No relevant conflicts of interest to declare.
Lack of the BH3-Only Proteins Bim, Bmf and Puma In Haematopoietic Stem and Progenitor Cells Facilitates Early Reconstitution and Long Term Haematopoiesis.
Authors: Erlacher Miriam , Bertele Daniela , Woess Claudia , Niemeyer Charlotte Marie , Villunger Andreas , Labi Verena 
Published: October 2019

Abstract
Abstract 1542
During haematopoietic stem cell transplantation (HSCT) the transplanted haematopoietic stem and progenitor cells (HSPC) suffer a transient deprivation of survival signals (i.e. cytokines or adhesion molecules) normally provided by the stem cell niche. Therefore, cell loss may occur prior to successful engraftment and thus restrict haematopoietic reconstitution.
Apoptosis induced in response to lack of cytokines or contact to the extracellular matrix is regulated by members of the Bcl-2 family. Bcl-2 family members can be divided into the pro-apoptotic ‘BH3-only’ proteins and anti-apoptotic Bcl-2 like proteins, the two subclasses antagonizing each other in their function. Using different gene-modified mouse models lacking the ‘BH3-only’ proteins Bim, Bmf or Puma, all implicated in leukocyte homeostasis, we aimed to delineate which one of these Bcl-2 family proteins is critically involved in limiting successful reconstitution of the haematopoietic system.
Our results demonstrate that HSPC lacking Bim show accelerated reconstitution of lethally irradiated recipient mice. Moreover, competitive reconstitution experiments reveal that wild type haematopoiesis is completely displaced in wt:bim-/- and strongly suppressed in wt:bmf-/- and wt:puma-/- bone marrow chimeras. The effects of Bim are comparable to those observed upon induced overexpression of Bcl-2 and cannot be enhanced further by additional loss of Puma or Bmf, identifying Bim as the major BH3-only protein limiting cell survival during haematopoiesis. Since both lymphopoiesis and myelopoiesis are similarly affected, a direct role of Bim on early progenitor or even stem cells can be assumed. However, wild type lymphocytes are additionally outcompeted by bim-/- and bcl-2 tg lymphocytes during differentiation, since the balance between Bim and Bcl-2 is critically involved in many selection processes occurring during a lymphocyte's life (i.e. negative selection in the thymus).
In summary, inhibition of apoptosis in HSPC by transiently interfering with Bim-function may be a promising strategy to increase the efficacy of HSCT and reduce transplantation-related morbidity.

Disclosures:
No relevant conflicts of interest to declare.