bluuebunny/crossref_metadata_2025 huggingface dataset:
MRx102, a Novel Triptolide Analogue, Has Low Nanomolar Activity Against AML Stem/Progenitor Cells in Vitro and in a Murine Model of AML
Authors: Carter Bing Z , Mak Duncan H , Shi Yuxie , Fidler John M. , Chen Rong , Ling Xiaoyang , Plunkett William , Andreeff Michael
Published: October 2019

Abstract 1547

Current chemotherapeutic regimens are known to be ineffective against AML stem/progenitor cells that is responsible for disease relapses due to both intrinsic resistance of these cells and extrinsic environmental factors. Triptolide, isolated from the herb Tripterygium wilfordii, has been shown to potently inhibit growth and induce apoptosis in various malignant cells by inhibiting RNA synthesis and NF-κB activity. Previously, we showed that triptolide promotes apoptosis in AML cells via the mitochondria-mediated pathway, in part by decreasing levels of the anti-apoptotic proteins XIAP and Mcl-1. MRx102 is a novel triptolide derivative, acting by converting to triptolide. We investigated whether MRx102 effectively targets primary human AML cells, particularly AML stem/progenitor cells co-cultured with bone marrow (BM) derived mesenchymal stromal cells (MSCs), and examined the effects of MRx102 in NOD/SCID mice injected with Ba/F3-ITD cells. We show here that MRx102 potently promoted apoptosis in AML cell lines, with EC50 values of low nanomolars for OCI-AML3 and MV4-11 cells. Like triptolide, MRx102 decreased XIAP and Mcl-1 protein levels and inhibited RNA synthesis in OCI-AML3 cells. Interestingly, we also found that MRx102 and triptolide decreased β-catenin protein level in OCI-AML3 cells. MRx102 effectively induced apoptosis in all primary AML samples tested, regardless of the cytogenetics or clinical responses to various therapies of the patients at the time of sampling (ED50 = 132.1 ± 7.5 nM, n=12) and was less toxic to normal BM cells (ED50 = 209.9 ± 56.8 nM, n=3). In addition to bulk AML cells, MRx102 also induced apoptosis in CD34+ progenitors, and more importantly in CD34+CD38− stem/progenitor cells from AML patients, even when they were protected by co-culturing with BM-derived MSCs. The effectiveness of MRx102 on AML stem/progenitor cells may be in part due to its ability to decrease β-catenin and triptolide's ability to inhibit NF-κB activity as both Wnt/β-catenin and NF-κB are highly active in AML stem/progenitor cells. Furthermore, in vivo, MRx102 greatly decreased leukemic burden, reduced infiltration of leukemic cells to various organs, and increased survival of NOD/SCID mice harboring Ba/F3-ITD cells (median survival 43 days with MRx102 at 3.0 mg/kg/day compared with 36 days for the untreated mice, P = 0.027).

Collectively, we demonstrated that MRx102 has strong antileukemic activity both in vitro and in vivo in a murine model of AML, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation.

Disclosures:

Fidler: MyeloRx LLC: Employment.
DA-EPOCH Chemotherapy Is Highly Effective in ALK-Positive and ALK-Negative ALCL: Results of a Prospective Study of PTCL Subtypes in Adults
Authors: Dunleavy Kieron , Shovlin Margaret , Pittaluga Stefania , Grant Cliona , Steinberg Seth M. , Jaffe Elaine S. , Wilson Wyndham H
Published: October 2019

Abstract 1618

Background:

Anaplastic large cell lymphoma (ALCL) is a distinct type of peripheral T-cell lymphoma (PTCL) characterized histologically, by large anaplastic lymphoid cells that are CD30 positive. There is biologic heterogeneity within ALCL: ALK (anaplastic lymphoma kinase) positive cases harbor a translocation usually involving chromosomes 2 and 5 resulting in over-expression of ALK (variant translocations also occur) whereas ALK negative cases do not. ALK positive ALCL is also clinically distinctive, with a younger age of onset and better treatment outcomes. In the recent International PTCL and Natural Killer (NK) T-Cell Lymphoma Study (Savage et al. Blood.2008: 111 (12)), the outcome for patients with ALK positive compared to ALK negative ALCL was significantly better (5-year failure-free survival (FFS) 60% versus 36%: p=0.15). Methods: We prospectively investigated the efficacy of 6 cycles of DA-EPOCH chemotherapy in newly diagnosed patients with both ALK positive and ALK negative ALCL and compared their outcome to other subtypes of PTCL. Results: Clinical characteristics of 38 enrolled patients are shown below. 22 patients had a diagnosis of ALCL and other diagnoses were PTCL-NOS (10), hepatosplenic gamma delta T-cell lymphoma (4), enteropathy associated T-cell lymphoma (1) and angioimmunoblastic T-cell lymphoma (1). Patients with ALK positive and ALK negative ALCL had similar characteristics. With a median potential follow-up time of 10.5 years, the 5 year progression-free survival (PFS) probability for ALK positive versus negative patients was 80% versus 71% (p=0.82); 5 year overall survival (OS) was 86% in both groups (p=0.95). For patients with other subtypes of PTCL, PFS and OS were 32% and 50% respectively, at 5-years. Conclusions: In contrast to other reports, patients with ALK negative ALCL had a very favorable outcome following DA-EPOCH chemotherapy that was no different from that of ALK positive cases. This regimen is highly effective in ALCL, independent of ALK expression. Accrual continues.

Disclosures:

No relevant conflicts of interest to declare.
Increasing Grades of Follicular Lymphoma Correlate with Better Prognosis in Patients Treated with Rituximab
Authors: Wahlin Björn Engelbrekt , Sundström Christer , Holte Harald , Sander Birgitta , Østenstad Bjørn , de Nully Brown Peter , Smeland Erlend B. , Delabie Jan , Christensson Birger , Kimby Eva
Published: October 2019

Abstract 1641

Background:

The World Health Organization (WHO) classification allocates indolent follicular lymphoma to grades 1, 2 or 3A according to the proportions of small centrocytes and large centroblasts in the neoplastic follicles. The prognostic value of the WHO's grading system in indolent follicular lymphoma has not been investigated in patients given the anti-CD20 monoclonal antibody rituximab without chemotherapy.

Methods:

We prospectively reviewed the follicular lymphoma grades in 276 grade 1–3A patients who received upfront rituximab without chemotherapy in two randomized trials. Flow cytometry analyses of malignant and nonmalignant lymphocyte subsets in lymph nodes and blood were also assessed.

Results:

In these patients given upfront rituximab-containing therapy, increasing grades of 1, 2, and 3A correlated with longer time to treatment-failure (P =0.002) and overall survival (P =0.045), independently of clinical factors (including the follicular lymphoma international prognostic index). The grades' impact was also independent of the levels of CD3+, CD4+, and CD8+ T cells in lymph nodes and in blood.

Conclusion:

Increasing grades of indolent follicular lymphoma correlate with better outcome in patients treated upfront with rituximab without chemotherapy, independently of clinical and immunologic factors. This suggests that treatment with rituximab acts differentially in tumors depending on the centrocyte/centroblast ratio.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Cerebrospinal Fluid Levels in Patients with Primary Central Nervous System Lymphoma Treated with Intravenous High Dose Rituximab
Authors: Larouche Jean-Francois , Bergeron Marc , Hampson Grace , Illidge Tim , Delage Robert
Published: October 2019

Abstract 1644

INTRODUCTION:

Rituximab penetration in central nervous system is largely unknown in human. Experiments in monkeys showed that 0,1% of serum rituximab concentration was achieved in cerebrospinal fluid. Reports in patients are limited (Rubenstein JL, Blood 2003 and Petereit HF, Multiple Sclerosis 2009). They demonstrated similar low levels of cerebrospinal fluid penetration with standard dose (375 mg /m2) rituximab in patients with central nervous system (CNS) lymphoma or multiple sclerosis.

METHOD:

We conducted a phase 2 trial in patients with primary CNS lymphoma. Patients were treated with an intravenous combination of high dose methotrexate (8 g/m2), high dose cytarabine (2 g/m2) and high dose rituximab (750 mg/m2 every 1–2 weeks × 13 infusions). We obtained from four patients paired cerebrospinal fluid and serum samples and rituximab concentration were determined in each. Samples were collected at different time points just before rituximab infusion and represent trough levels.

RESULTS:

11 cerebrospinal fluid samples were available and their 11 paired serum samples. Mean cerebrospinal fluid and serum levels were 2,04 ug/mL (0,49–4,08) and 297,09 ug/mL (211,26–504,47) respectively. Mean cerebrospinal fluid levels were 0,71% (0,18–1,5%) of serum levels. No relationship was made between cerebrospinal fluid level and the number of rituximab dose administered.

CONCLUSION:

In patients with primary CNS lymphoma receiving high dose 1–2 weekly rituximab. cerebrospinal fluid concentration achieve is low compared to serum levels. However, administration of higher dose of intravenous rituximab can increase penetration in central nervous system but its clinical impact is unknown as cerebrospinal fluid levels are still low.

Disclosures:

No relevant conflicts of interest to declare.
Doxycycline Reduces Fibril Formation in a Transgenic Mouse Model of AL Amyloidosis
Authors: Seldin David C , Ward Jennifer , Ren Ruiyi , Toraldo Gianluca , SooHoo Pamela T , Guan Jian , O'Hara Carl , Jasuja Ravi , Trinkaus-Randall Vickery , Liao Ronglih , Connors Lawreen H
Published: October 2019

Abstract 1837

AL amyloidosis results from the production and aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) by a clonal bone marrow plasma cell dyscrasia. Currently, there are no genetically reproducible animal models of AL amyloidosis. We engineered transgenic mice to express an amyloidogenic human λ6 LC. We used the cytomegalovirus (CMV) promoter to express the LC in multiple tissues and organs, circumventing the disruption of B cell development by premature expression of recombined LC in the B lineage alone. LC were detected in a number of organs including epithelial tissues of the gastrointestinal and genitourinary tracts, and in the serum. The CMV-λ6 transgenic mice developed neurologic dysfunction. By 2 years of age, >80% of the transgenic mice developed Congo-red staining human LC fibrils in the stomach. Transgenic mice were treated with the antibiotic doxycycline orally for 7 months. Of treated mice, 23% had Congophilic deposits, compared with 69% of controls drinking water for the same time period. Treatment of recombinant LC in vitro with doxycycline reduced fibril formation and the drug also caused to disruption of pre-formed fibrils, producing disordered aggregates. Thus, this transgenic model replicates the process of AL amyloidosis in vivo and is useful for testing the anti-fibril potential of orally available agents. Antibiotics such as doxycycline could prove useful in AL as well as in other amyloidoses.

Disclosures:

No relevant conflicts of interest to declare.
Targeting Leukemias by CD123 Specific Chimeric Antigen Receptor
Authors: Thokala Radhika , Singh Harjeet , Olivares Simon , Champlin Richard , Cooper Laurence J N
Published: October 2019

Abstract 1908

Chimeric antigen receptors (CARs) are employed to genetically modify T cells to redirect their specificity to target antigens on tumor cells. Typically a second generation CAR is derived by fusing an extracellular domain derived from the scFv of monoclonal antibody (CAR) specific to targeted antigen with CD3 zeta, and CD28 endodomains. CD123 (IL3RA) is expressed on 45% to 95%of acute myelogenous leukemia (AML) and B-cell lineage acute lymphoblastic leukemia (B-ALL). Expression of CD123 is high in the leukemic stem cell (LSC) population, but not in normal hematopoietic stem cells. Thus, CD123 appears to be potential target for immunotherapy in leukemias through chimeric antigen receptor (CAR). We hypothesized that the generation of CD123 specific CAR can redirect the specificity of T cells to CD123 and this was tested by cloning the scFv of CD123 mAb in our CAR construct. The sleeping beauty system was used to express the CAR and DNA plasmids were electroporated into peripheral blood mononuclear cells and cells were numerically expanded on artificial antigen presenting cells genetically modified to express co stimulatory molecules CD86, 4-1BBL, membrane-bound IL-15, and CD123 antigen in presence of IL-21 and 1L–2. CAR+ T numerically expanded to clinically relevant numbers and showed antigen specific cytotoxicity in leukemic celllines. CAR+ T cells expressed both effector and memory markers showing the potential for in vivo persistence after T cell infusion. The bonemarrow homing receptor CXCR4 was expressed by CAR T cells shows the potential to target LSC that reside in BM niches. The preliminary data suggests that mirroring an approach we are using to manufacture clinical grade CD19 specific CAR+ T cells.Figure 1:(A) CAR expression on day 35. (B) Cytotoxicity of CD123CAR in leukemic cell lines.Figure 1:. (A) CAR expression on day 35. (B) Cytotoxicity of CD123CAR in leukemic cell lines.CD3CD3

Disclosures:

No relevant conflicts of interest to declare.
High Efficiency Isolation of Alloantigen-Reactive Foxp3+ Natural Regulatory T Cells According to Specific Activation Marker Signatures
Authors: Schoenbrunn Anne , Keye Jacqueline , Kohler Siegfried , Frentsch Marco , Moewes Beate , Scheffold Alexander , Romagnani Chiara , Thiel Andreas
Published: October 2019

Abstract 1915

Transplant tolerance induction and avoidance of exposure to destructive immunosuppressive drugs are still major objectives in transplantation medicine. Many preclinical animal models have proven the adoptive transfer of polyclonal CD4+CD25+Foxp3+ regulatory T (Treg) cells to be an important and effective tool for the prevention of graft rejection or graft-versus-host-disease. However, polyclonal Treg may also modulate immunity to foreign or tumor antigens. It is unclear yet, whether and how their application might lead to an unwanted general immunosuppression. Therefore the selective transfer of alloantigen-reactive Treg represents a very attractive therapeutic option. However, so far this strategy has been hampered so far by the lack of appropriate marker to assess and isolate antigen-reactive Treg cells with high efficiency.

In order to get access to alloantigen-reactive Treg, we established cytometric methodologies allowing a clear dissection between (allo)antigen-specific Treg and Teff based on expression of 4-1BB and the lack of expression of CD40L. After allogeneic stimulation CD4+ T cells expressing 4-1BB but lacking CD40L expression were highly enriched in Foxp3+ T cells. Further molecular analysis of the Foxp3 gene locus revealed that only 4-1BB+CD40L− T cells were characterised by a completely demethylated TSDR (Treg specific demethylated region). 4-1BB+CD40L− T cells highly expressed the transcription factor HELIOS recently demonstrated to specify thymic-derived Treg. Alloantigen-reactive Treg isolated according to 4-1BB and CD40L were superior with respect to their alloantigen-specific in vitro suppression as compared their polyclonal Treg counterparts. Finally 4-1BB+CD40L− alloantigen-reactive Treg could be easily expanded in vitro up to 300 fold during 3 weeks culture, maintaining alloantigen-specific suppression. Our results offer the possibility to improve current approaches for adoptive cell-therapy with alloantigen-specific Treg to achieve transplantation tolerance aiming at a specific inhibition of pathology.

Disclosures:

Scheffold: Miltenyi Biotec GmbH: Employment.
β-1,4-Galactosyltransferase 1 Expression Is Required for Platelet Production in Vitro and In Vivo
Authors: Giannini Silvia , Grozovsky Renata , Begonja Antonija Jurak , Hoffmeister Karin M.
Published: October 2019

Abstract 192

Platelet turnover requires correct glycan presentation and expression. Deficiency in β-1,4-galactosyltransferase 1 (β4GalT1), the major enzyme that transfers galactose (Gal) from UDP-Gal to terminal N-acetylglucosamine (GlcNAc) to form type-2 lactosaminyl structures, is lethal in embryonic mice. However, E14.5 β4GalT1−/− embryos are grossly indistinguishable from wild type embryos. Here we sought to determine whether platelet formation and survival require β4GalT1. Hepatic stem cells were obtained from embryonic livers at E14.5 and megakaryoctes were differentiated in vitro in the presence of thrombopoietin. β4GalT1−/−megakaryocytes mature and differentiate normally in vitro as judged by their number, morphology and expression of main surface glycoproteins specific for the megakaryocyte lineage (GPIbα/β, GPIX, αIIb and β3). However, following differentiation, β4GalT1−/−megakaryocytes were unable to produce proplatelets and platelets in vitro. Addition of exogenous β4GalT1 and the donor substrate UDP-Galactose did not improve in vitro proplatelet and platelet formation. Importantly, platelet numbers were decreased by ∼ 75% in β4GalT1−/− E14.5 embryos when compared to wild type embryos. Platelet size was increased by ∼ 50% in β4GalT1−/− embryos, indicating that β4GalT1−/− mice have increased platelet clearance and/or that larger platelets are produced in vivo. Our data strongly support the notion that glycosylation mediated by β4GalT1 activity is crucial for platelet production in vitro and in vivo. Our findings demonstrate for the first time a role for posttranslational glycan modification in platelet production.

Disclosures:

No relevant conflicts of interest to declare.
Donor Dual TCR T Cells Preferentially Expand and Mediate Pathologic Alloreactivity in Acute Graft Versus Host Disease
Authors: Morris Gerald P. , Uy Geoffrey L , Donermeyer David L , Allen Paul M , DiPersio John F.
Published: October 2019

Abstract 1972

The nature of the T cell repertoire mediating pathologic in vivo alloreactivity is an important question for understanding the development of acute graft-versus-host disease (aGvHD) following clinical allogeneic transplantation. We have previously demonstrated that the small proportion of T cells that naturally express 2 T cell receptors (TCR) as a consequence of incomplete TCRa allelic exclusion during thymic development contribute disproportionately to the alloreactive T cell repertoire, both in vitro and in vivo in a mouse model of graft versus host disease (GvHD) (J. Immunol., 182:6639, 2009). Here, we extend these findings to human biology, examining dual TCR T cells from healthy volunteer donors (n = 12) and patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) (n = 19). Peripheral blood was collected at day 30 post-HSCT or at the time of presentation with symptomatic acute GvHD. Dual TCR T cells were measured in peripheral blood by pair-wise staining with 3 commercially-available and 2 novel TCRa mAbs. Dual TCR T cells were consistently and significantly expanded in patients with symptomatic aGvHD, representing 5.3±3.8 % of peripheral T cells, compared to 1.7±0.8 % of T cells in healthy controls (p < 0.005) (Figure 1). There was no correlation between dual TCR T cell frequency and GvHD severity. Furthermore, sequential analysis of peripheral blood in 2 patients demonstrated expansion of dual TCR T cells concurrent with the development of aGvHD (Figure 2). Dual TCR T cells from patients with symptomatic aGvHD demonstrated increased expression of CD69 as compared to T cells expressing a single TCR, indicative of preferential activation of dual TCR T cells during aGvHD. Similarly, dual TCR T cells isolated from patients with symptomatic aGvHD demonstrate increased production of IFN-g ex vivo, indicative of the ability to mediate pathogenic alloreactive responses. Dual TCR T cell clones isolated from healthy donors and patients post-HSCT by single cell FACS sorting demonstrate alloreactive responses against a range of allogeneic cell lines in vitro. We propose that the increased alloreactivity of dual TCR T cells results from the less stringent thymic selection for secondary TCR, and thus provides a link between thymic selection, the TCR repertoire, and alloreactivity. These findings may lead to simple ways of phenotypically identifying specific T cells predisposed to inducing aGvHD for subsequent examination of T cell repertoires and functional studies. Furthermore, these data suggest that dual TCR T cells represent a potential predictive biomarker for aGvHD and a potential target for selective T cell depletion in HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Longitudinal Analysis of Body Mass and Composition in Survivors of Pediatric Hematological Malignancies After Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Inaba Hiroto , Yang Jie , Kaste Sue , Hartford Christine , Motosue Megan S , Srinivasan Ashok , Triplett Brandon M. , Shook David R , Pui Ching-Hon , Leung Wing
Published: October 2019

Abstract 1991

Background:

Body mass and composition have been well studied in survivors of childhood cancers, but little is known about these factors in those who have undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Methods:

We prospectively and longitudinally evaluated changes in body mass index (BMI), changes in the proportion of fat and lean body mass, and associated clinical factors in 179 survivors of pediatric hematologic malignancies after allo-HSCT. BMI was analyzed as an ordinal variable (four percentile categories: underweight, healthy weight, overweight, and obese) and as a continuous variable (Z-score); dual-energy X-ray absorptiometry data were used to derive Z-scores for percent total body fat and lean mass/height2 ratio.

Results:

With a median follow-up of 6.6 years (range, 1.0–17.7 years), BMI decreased significantly over time (mean Z-score, 0.32 pre-HSCT and −0.60 at 10 years post-HSCT) (Figure; p<0.001). These longitudinal changes differed among pre-HSCT BMI categories (p<0.001). BMI Z-scores of patients who were underweight before HSCT increased over the first 4 years after HSCT and then declined, while those of patients in the other three pre-HSCT BMI categories continuously declined. Although the mean Z-scores for percent fat mass were higher or similar to those of the general population during follow-up (0.305 at 1 year, −0.272 at 10 years), the mean Z-scores of lean mass/height2 ratio remained below that of healthy controls and diminished significantly over time (-0.296 at 1 year, −1.256 at 10 years post-HSCT) (p=0.002). Pre-HSCT BMI categories were strongly associated with post-HSCT BMI values (p<0.001) and with Z-scores of percent total body fat and lean mass/height2 ratio (both p<0.001). Patients who had extensive chronic graft-versus-host disease were significantly more likely to be in the underweight BMI category (p=0.021) and to have a low Z-scores for lean mass/height2 ratio (p<0.001). Younger patients (less than the median age, 11.3 years, at HSCT) had a higher BMI after HSCT than older patients (p<0.001), and female survivors had higher Z-scores of percent total body fat (p=0.005) and lower Z-scores of lean mass/height2 ratio (p=0.025) than male survivors.

Conclusions:

The finding of a significant decline of BMI and severe loss of lean mass after HSCT indicates a critical need for nutritional education and early intervention for these survivors.

Disclosures:

No relevant conflicts of interest to declare.
Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis: Long-Term Clinical Outcome in China
Authors: Chen Bing , Ouyang Jian , Zhou Min , Shao Xiao-Yan , Xu Yong , Zhou Rong-Fu
Published: October 2019

Abstract 2020

Background

Autologous haematopoietic stem cell transplantation (AHSCT) is a promising treatment of multiple sclerosis (MS) who had not adequately responded to conventional therapies. We retrospectively evaluated the safety and long-term clinical outcome of AHSCT in MS patients in China.

Methods

Twenty-five patients with various types of MS were treated with AHSCT. Peripheral blood stem cells were obtained by leukapheresis after mobilization with granulocyte colony stimulating factor. Then CD34+ cell selection of the graft was performed and anti-thymocyte globulin was given for T-cell depletion. Conditioning regimen was BEAM. Early and late toxicities were also recorded. Long-term outcomes were evaluated by the expanded disability status scale, progression free survival and gadolinium-enhancing magnatic resonance imaging (MRI) scans.

Results

10, 7 and 8 patients experienced neurological improvement, stabilization and progression, respectively. The median EDSS scores at every follow-up year after transplantation (5.5-7.0) were consistently lower than the baseline (8.0). The progression-free survival rate was 74%, 65% and 48% at 3, 6 and 9 years post-transplant. 58% cases (7/12) had active lesions at baseline and all turned to inactive status in the follow-up years. 25% cases (3/12) experienced progression after transplantation but had no active lesions in MRI in the whole follow-up period. 17% cases (2/12) without active lesions at baseline progressed and appeared active lesions in MRI.

Conclusion

AHSCT is a feasible treatment for severe MS and its long-term efficacy is favorable. However, more random and controlled clinical trials are required to fully assess long-term efficacy of AHSCT for MS.

Disclosures:

No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Stem Cell Transplantation Overcomes the Adverse Prognostic Impact of Minimal Residual Disease in Acute Myeloid Leukemia
Authors: Oran Betul , Singleton Timothy , Ramirez Pablo A. , Shanley Ryan , Brunstein Claudio , Ustun Celalettin , Warlick Erica D , Weisdorf Daniel J.
Published: October 2019

Abstract 2027

BACKGROUND:

The identification of minimal residual disease (MRD) can predict impending relapse in acute myeloid leukemia (AML) patients. Little data exists evaluating the prognostic impact of MRD, as determined by multiparametric flow cytometry (MFC), at the time of allogeneic (allo-HCT). Although disease outcomes may be worse for MRD positive (MRD+) patients, MRD is often associated with other adverse risk factors leaving it unclear whether MRD is an independent risk factor for relapse or a surrogate marker for underlying poor risk disease features.

METHODS:

We retrospectively analyzed 97 consecutive AML patients in complete morphological remission (CR) who underwent allo-HCT with a matched related donor (MRD, n=30, 31%), matched unrelated donor (MUD, n=4, 4%) or umbilical cord blood (UCB, n=63, 65%) at the University of Minnesota between January 2005 and June 2009. Presence of MRD at allo-HCT was determined by MFC. Analyses were done separately for myeloablative (MAC) and reduced intensity conditioning (RIC) patients, testing the impact of MRD along with conditioning intensity, age, donor type and disease status on allo-HCT outcomes.

RESULTS:

Of 97 patients, 41 (42%) had MAC; 57 (58%) had RIC. Sixty-six were in first CR (CR1) with the rest in CR2 or later remission (CR2+). MRD at allo-HCT was detected in 7 patients after MAC (17%) and 7 after RIC (12.5%); and this frequency was similar in patients in CR1 and CR2+ (13% vs. 16%, p=0.7). MRD+ and MRD- patients had similar median age (40 vs. 44 yrs), gender, donor source (MSD or MUD vs. UCB), CMV serostatus and diagnostic cytogenetic risk group. Six of 40 (15%) intermediate and 5 of 39 (13%) high risk cytogenetics patients had MRD+ (p=0.8). Only 3 of 53 patients with a cytogenetic abnormality at diagnosis had it detected prior allo-HCT and 1 of 3 had MRD.

The median follow-up of survivors was 25 months. Two-year probabilities for MAC and RIC patients were similar: Overall survival (OS), 48% and 47 % and leukemia free survival (LFS) 43% and 41% respectively. When disease outcomes were analyzed separately by MRD status (table), OS and LFS were markedly worse in MRD+ patients receiving RIC, but this difference was not statistically significant. In multivariate analysis, MRD+ was not an independent prognostic factor for OS and LFS. Although we identified no adverse prognostic factors for MAC patients, patient with RIC in CR2+ had worse OS and LFS vs. CR1 (HR 2.6, p=0.04 and HR 2.7, p=0.04 respectively).

CONCLUSION:

The negative prognostic impact of MRD was overcome by allo-HCT with MAC, but outcomes with MRD+ were suggestively inferior after RIC. However due to limited sample size, MRD in patients with RIC should be further investigated.

Disclosures:

Weisdorf: Genzyme: Consultancy, Research Funding.
A Randomized Trial of Dabigatran Versus Warfarin in the Treatment of Acute Venous Thromboembolism (RE-COVER II)
Authors: Schulman Sam , Kakkar Ajay K , Schellong Sebastian M , Goldhaber Samuel Z. , Henry Eriksson , Mismetti Patrick , Christiansen Anita Vedel , Schnee Janet , Kearon Clive
Published: October 2019

Abstract 205

Background:

Dabigatran has been compared with warfarin for treatment of acute venous thromboembolism in one previous trial (RE-COVER). Based on the low rate of the primary outcome as the RE-COVER study was running, we undertook this replica study to confirm the results of RE-COVER, and to allow for more rigorous sub-group analyses.

Methods:

In a randomized, double-blind, double-dummy trial of 2568 patients with acute VTE, treated with low molecular weight or unfractionated heparin for 5 to 11 days, we compared dabigatran, 150 mg twice daily, with warfarin, dose-adjusted to an International Normalized Ratio of 2.0 and 3.0, each given for 6 months. Primary outcome was recurrent symptomatic, objectively confirmed venous thromboembolism and deaths related to venous thromboembolism during 6 months. Safety endpoints included bleeding events, acute coronary syndrome, elevated liver function tests, and adverse events.

Results:

Of 1279 patients randomized to dabigatran, 30 (2.4%) had recurrent VTE compared with 28 (2.2%) of 1289 patients randomized to warfarin; risk difference 0.2% (95% confidence interval [CI], −1.0 to 1.5); p<0.0001 for the pre-specified non-inferiority margin. The hazard ratio for dabigatran was 1.08 (95% CI, 0.64 to 1.80). Major bleeding occurred in 15 patients treated with dabigatran and 22 patients treated with warfarin (hazard ratio 0.69; 95% CI, 0.36 to 1.32) and any bleeding occurred in 200 versus 285 patients, respectively (hazard ratio 0.67, 95% CI, 0.56 to 0.81). The frequency of reported ACS events was less than 1% in the trial, with more cases in the dabigatran treatment group than those treated with warfarin. There were 25 deaths during each treatment, and serious adverse events were similar in the two groups. Analysis of outcomes based on demographic characteristics showed consistency of effects for both safety and efficacy. This included the analysis based on Asian race, which had been limited in RE-COVER. There were 537 Asian patients in RE-COVER II compared to only 65 in RE-COVER. The event rates of recurrent VTE and of any bleeding were similar in Asians and non-Asians.

Conclusion:

The study confirms that the efficacy of dabigatran is non-inferior to warfarin in the treatment of acute VTE and with a lower risk for bleeding. The safety of dabigatran is similar in the Asian population compared with non-Asians.

Disclosures:

Off Label Use: dabigatran for treatment of venous thromboembolism. Christiansen:Boehringer Ingelheim: Employment. Schnee:Boehringer Ingelheim: Employment.
Evaluating Trends in Diagnostic and Prognostic Testing for Multiple Myeloma
Authors: Fidler C. J. , Hussein Ahmed Kamel Abou , Gandhi Neel , Karur Vinit , Sharma Manish , Klumpp Thomas R , Kropf Patricia , Martin Mary Ellen , Emmons Robert V. , Mangan Kenneth F.
Published: October 2019

Abstract 2067

Introduction:

A battery of diagnostic and prognostic testing is recommended by both the NCCN and the International Myeloma Working Group (IMWG) at the time of diagnosis of myeloma. It has been our observation that many patients referred for Autologous Stem Cell Transplant (ASCT) to our center have not had a complete myeloma workup. In this report, we attempt to quantify the adherence to recommended guidelines for the workup of myeloma.

Methods:

We reviewed the records of all patients with a diagnosis of myeloma referred to our center for ASCT between 2006 and 2011. We looked for the following tests at the time of diagnosis: bone marrow biopsy, SPEP, 24hr urine for Bence-Jones proteinuria, serum β2 microglobulin, skeletal survey, serum free light chains, serum albumin and myeloma FISH panel. We used descriptive statistics to evaluate our results.

Results:

There were a total of 64 patients who underwent an ASCT in our center between 2006 and 2011. Of these 64 patients, 9 patients were excluded from this review due to incomplete records. At the time of initial diagnosis, the most commonly obtained tests included an albumin level (55/55, 100%), SPEP (54/55, 98%), bone marrow biopsy (52/55, 95%), skeletal survey (52/55, 95%), standard cytogenetics (47/55, 85%), and β2-microglobulin (46/55, 84%). The most commonly omitted studies included the myeloma FISH panel (26/55, 47%), 24-hour urine for Bence-Jones protein (30/55, 55%), and serum free light chains (37/55, 67%). Compliance with the use of serum free light chain assay improved over time, while the frequency of obtaining a myeloma FISH panel remains relatively low and the frequency of obtaining a 24-hour urine for Bence-Jones protein decreased (Tables 1 and 2).

Conclusions:

The most commonly omitted studies were the serum free light chains, 24hr urine and myeloma FISH panel. When assessed over time, compliance increased with the ordering of the SFLC assay, stable for the myeloma FISH panel but decreased for the 24hr urine for Bence-Jones protein. This trend reflects the perception that the serum free light chain assay is replacing the 24hr urine for Bence-Jones proteinuria. Referring community physicians need further education on the ordering of initial diagnostic and prognostic tests for multiple myeloma.

Disclosures:

No relevant conflicts of interest to declare.
Role of hSET1 Complex in Epigenetic Controls of HoxB4 Expression and Development of Hematopoietic Stem Cells
Authors: Deng Changwang , Li Ying , Liang Shermi , Salz Tal , Qiu Yi , Roeder Robert , Zhao Keji , Bungert Jorg , Huang Suming
Published: October 2019

Abstract 212

Hox genes play an important role in embryonic development and are sequentially activated in a temporal and spatial fashion. Among them, HoxB4 regulates the self-renewal ability of adult and embryonic hematopoietic stem cells. It was shown that USF proteins positively regulate the transcription of HoxB4 gene. However, the epigenetic mechanism specifically controlling HoxB4 transcription during HSC formation remains unknown. In this report, we found that USF1 interacts with histone H3K4 methylatransferase SET1 complex, but not MLL proteins. The interaction is mediated by SET1/MLL core component, ASH2L. Both USF1 and hSET1 are colocalized at the HoxB4 promoter in hematopoietic progenitors, which is accompanied by H3K4 trimethylation and transcriptional activation. siRNA-mediated knockdown of both SET1 and HCF1 specifically impaired H3K4 trimetylation and the HoxB4 promoter-driven reporter gene activity. Overexpression of AUSF1, a dominant negative mutant of USF1 in which the basic DNA recognition domain is deleted and thereby, inhibits endogenous USF DNA binding activity, leads to dramatically decrease in HoxB4 transcription, disruption of embryonic body formation and inhibition of hematopoietic stem cell population in mouse ES (mES) cells and the AUSF1 transgenic mice. The suppression of USF activity drastically reduces SET1 recruitment, H3K4 trimethylation, and pol II binding at the HoxB4 promoter. Furthermore, knock-down of SET1, an enzymatic subunit of the SET1 complex, also resulted in reduction of HoxB4 transcription and impairment of hematopoietic differentiation capacity of mES cells that are accompanied by reducing the HoxB4 promoter H3K4 methylation and recruitment of RNA PolII machinery. Thus, our data reveal that recruitment of hSET1 complex and its H3K4 methyltransferase activity by USF1 is essential for HoxB4 transcription and expansion of hematopoietic stem cells.

Disclosures:

No relevant conflicts of interest to declare.
NK Cells in Hodgkin Lymphoma Are Impaired but Can Be Activated
Authors: Kessler Joerg , Reiners Katrin S. , Sauer Maike , Engert Andreas , von Strandmann Elke Pogge
Published: October 2019

Abstract 2182

Introduction:

NK cells represent the key component of the innate immune system to recognize and eliminate cancer cells. Defects in NK cell function including impaired cytotoxicity/cytokine secretion, aberrant receptor expression profile, NK cell number and NK cell anergy are reported in non Hodgkin lymphoma and correlate with a bad prognosis. So far, nothing is known about the phenotype of peripheral NK cells and serum levels of ligands for NK cell receptors in Hodgkin Lymphoma (HL) patients. Here, cytotoxicity, expression pattern of activating NK cell receptors and the serum levels of several ligands for the key cytotoxic receptors NKG2D and NKp30 are determined.

Methods:

The cytotoxicity of NK cells isolated from HL patients was analysed by europium release assay using the HL cell line L428 as target cells. The serum level of the NKp30-ligand BAT3 and ligands for NKG2D (MICA, MICB and ULBP1,2,3) was estimated in sera of 117 HL patients and 40 healthy donors by ELISA. The expression pattern of NKp30, NKp44, NKp46, CD16 and the activation markers CD25, CD69 and CD71 was determined by 4-colour FACS analysis of peripheral blood lymphocytes.

Results:

The cytotoxicity assays reveal a significantly reduced killing efficacy of NK cells from HL patients against the Hodgkin cell line L428 in comparison to NK cells from healthy donors. Correlating with the impaired NK cell function, we observed that the serum level for BAT3 and MICA was significantly elevated in HL patients, whereas other ligands (MICB and ULBP1,2,3) remained unchanged. NKG2D showed a significantly decreased expression on NK cells of HL patients. No significant difference was observed for all other receptors and activation markers tested.

Conclusion:

Our results suggest that soluble BAT3 and MICA, ligands for NKp30 and NKG2D, contribute to the NK cell inhibition in HL patients. Since soluble ligands for NK cell receptors are known to inhibit NK cell-cytotoxicity, the release of these ligands might represent an immune escape mechanism of HL tumors to avoid detection and killing by the innate immune system. To overcome NK cell inhibition in HL patients we design, express and purify bispecific proteins (immunoligands) that target NKG2D and a HL-specific tumorantigen. Work to activate HL-derived NK cells with immunoligands ex vivo will be discussed.

Disclosures:

Engert: Affimed Therapeutics AG: Honoraria, Research Funding.
The Neuronal Protein GAP-43 Associates with Microtubules and Lipid Rafts in Platelets and Megakaryocytes and Is Involved in Platelet Genesis
Authors: Begonja Antonija Jurak , Nguyen Lilly , Italiano Joseph E. , Falet Herve , Meiri Karina F. , Hartwig John H.
Published: October 2019

Abstract 2201

GAP-43 (neuromodulin) is a membrane-associated protein involved in neurite outgrowth, pathfinding and branching during development where it regulates neurotransmitter release and endocytosis, and plays a role in cytoskeletal signal transduction at the nerve ending. We have found GAP-43 to be expressed in platelets and megakaryocytes (MK) as evidenced by immunoblot and flow cytometry analysis. Immunofluorescence shows that GAP-43 localizes intracellularly as discrete punctuate structures mostly near the marginal microtubule (MT) coil of platelets and along the MT bundles in MKs and proplatelets. In contrast to neuronal cells where GAP-43 is associated with their detergent insoluble F-actin cytoskeleton, it is mostly soluble in platelets unless the platelets are first treated with taxol, a drug that stabilizes MTs. MT disruption with nocodazole or by chilling decreases the amount of GAP-43 in the detergent-insoluble cytoskeleton and disturbs its localization in proplatelets. GAP-43 colocalizes in platelets and MKs with the glycosylphosphatidylinositol-linked lipid raft marker Thy-1 and Cherry-PH PLC-delta1 (pleckstrin homology domain of phospholipase C-delta1) that stains membrane rafts. Since GAP-43 overexpression in MKs diminishes proplatelet formation, it appears to play a negative role in thrombopoiesis. Together, the data shows that GAP-43 is associated with MT and lipid rafts and suggests a role in signaling reactions between these two components to modulate proplatelet elaboration.

Disclosures:

No relevant conflicts of interest to declare.
Activated Protein C Controls p66shc: A Novel Defense Mechanism in Diabetic Nephropathy
Authors: Bock Fabian M , Shahzad Khurrum , Madhusudhan Thati , Wang Hongjie , Nawroth Peter P , Isermann Berend H.
Published: October 2019

Abstract 2204

Introduction:

Previously it has been shown that activated protein C (APC) protects against diabetic nephropathy (DN). APC's cytoprotective effect in DN may be related to its in vitro ability to downregulate reactive oxygen species (ROS) and maintain mitochondrial membrane potential in glucose stressed cells. Cellular targets, through which APC, an extracellular serine protease, regulates mitochondrial function, are unknown. Since mice lacking the ROS-inducing mitochondrial protein p66shc are protected from DN we hypothesized that APC's cytoprotective effect may be mechanistically linked to p66shc.

Material & methods:

Immortalized mouse podocytes and murine endothelial cells were stressed in vitro with glucose (30 mM). In mice persistent hyperglycaemia was induced by streptozotocin injections. Markers of DN were analysed in wild-type mice, mice expressing a thrombomodulin (TM) mutant lacking cofactor activity towards protein C activation (TMPro/Pro), mice expressing high levels of APC (hPChigh), and p66−/− x TMPro/Pro double mutant mice. Immunoblotting, RT-PCR, cellular subfractionation, immunohistochemistry and immunofluorescensce were employed for analyses.

Results:

8-OH-Desoxyguanosine, a marker for ROS, is induced in diabetic mice. Nitrotyrosine, a marker for ROS-dependent damage, localizes mainly to podocytes. This is paralleled by p66shc-upregulation in glomerular podocytes. These effects are aggravated in diabetic TMPro/Pro, ameliorated in aPChigh, and reversed in p66shc x TMPro/Pro mice. In vitro APC counteracts glucose dependent upregulation of p66shc and inhibits mitochondrial translocation of p66shc in mouse podocytes, but not in endothelial cells in vitro.

Conclusion:

This study identifies a novel mechanism through which APC mediates cytoprotection. In glucose stressed podocytes, but not endothelial cells, APC prevents mitochondrial translocation of the ROS-inducing redox-protein p66shc, which is required for protection against DN in vivo.

Disclosures:

No relevant conflicts of interest to declare.
HLADRB3*0101 Combined with DRB4*0101 Are Potential Predictor of Neonatal Alloimmune Thrombocytopenia (NAIT) Development
Authors: Salomon Ophira , Loewenthal Ron , Kalt Rivka , Rosenberg Nurit , Dardik Rima , Yahalom Vered , Avishai Ofelia , Frenkel Orit , Gazit Ephraim , Lipitz Shlomon
Published: October 2019

Abstract 2221

Neonatal alloimmune thrombocytopenia (NAIT) is a life-threatening bleeding disorder in the fetus/ neonate caused by maternal alloantibodies directed against fetal platelet antigens inherited from the father. Of the 21 allonatigens described up to date http://www.ebi.ac.uk/ipd/hpa/table2.html, the human platelet antigen (HPA) −1 defined as a leucine /proline polymorphism at residue 33 in b3 integrin is the immune-dominant antigen leading to severe NAIT. Immunization to HPA-1a occurs only in 10% of HPA-1bb pregnant women, 90% of them carrying histocompatibility leukocyte antigen (HLA) DRB3*0101. The aims of the present study were: 1. to prospectively study whether in addition to HLA DR B3*0101 there other potential HLA DR antigens involved in the immune process. 2. To analyse whether the response to IVIgG treatment correlates with maternal HLA DR genotype 3. to determine whether, anti HLA antibodies in addition to HPA1a antibodies, resulted in a more severe thrombocytopenia. We enrolled 21 pregnant women who had previously given birth to newborns with NAIT due to HPA1 incompatibility and had antibodies directed to their spouses HPA1 phenotype (leucine or proline polymorphism ) at the Sheba Medical Center from May 2001 to January 2011. The control group consisted of 21 women who delivered at term newborns with thrombocytopenia and a diagnosis of NAIT was ruled out. Twelve women of 21 (57%) in the study group carried both HLADRB3*0101 and DRB4*0101phenotype. But in none of the women in the control group such combination was detected. Interestingly is the observation that of 20 women who gave birth once more following IVIgG treatment, four did not respond to IVIgG and 3 (75%) of them harbor both DRB3*0101 and DRB4*0101. When lymphocytotoxic cross match analysis was performed in eighteen couples of the study group seven (39%) of them had positive lymphocytotoxic cross match. However the presence of both HLA and HPA1 antibodies against their partner phenotype did not affect platelets' counts in the newborn. Taken together we postulate that the presence of both HLADRB3*0101 and HLADRB4*0101 in the mother increases the risk of NAIT development when HPA1 incompatibility between the couples is found and can serve as a diagnostic tool. The presence of HLA antibodies against the spouse did not alter response to IgG treatment.

Disclosures:

No relevant conflicts of interest to declare.
Ticlopidine-, Clopidogrel-, and Prasugrel-Associated Thrombotic Thrombocytopenic Purpura: A Twenty Year Review
Authors: Qureshi Zaina Parvez , Armstrong John , Bennett Charles
Published: October 2019

Abstract 2226

Objective:

Thrombotic thrombocytopenic purpura (TTP) is a rare syndrome, with our group having identified the thienopyridines ticlopidine and clopidogrel as the most common drugs associated with TTP in Food and Drug Administration (FDA) databases. We review clinical, epidemiologic, laboratory, and drug safety findings for thienopyridine-associated TTP, including the first reported cases with prasugrel, a thienopyridine approved in 2009.

Methods:

Data sources included FDA's Adverse Event Reports database, pharmaceutical manufacturers, package inserts, physician surveys, phase III reports, insurance databases, and an epidemiologic study (1989–2011). Causality was assessed with the World Health Organization scale.

Results:

Since 2002, FDA received reports of 10 ticlopidine-, 140 clopidogrel-, and nine prasugrel-associated TTP cases, including four, 11, and 9 cases respectively, in the first year of marketing of each agent. Surveys of hematologists by our group identified 32 ticlopidine- and ten clopidogrel-associated TTP cases. Thienopyridines were administered for > two weeks for 90% of 93 ticlopidine-associated cases, 26% of 35 cases clopidogrel-associated cases, and none of nine prasugrel-associated cases. In the Phase III setting, one of 2,932 ticlopidine-, none of 27,961 clopidogrel-, and none of 1,769 prasugrel-treated patients developed TTP. Insurance databases identified three clopidogrel-associated TTP case among 15.3 million individuals. Ticlopidine- (n=30) versus clopidogrel-associated TTP patients (n=8) presented with severe ADAMTS13-deficiency (80% versus 0%) and neutralizing auto-antibodies to ADAMTS13 (100% versus 0%) and had higher survival rates following therapeutic plasma exchange (87% versus 50%) (p<0.05). Greater than 95% of ticlopidine-associated TTP cases were assessed as having a probable causal relationship versus none of the clopidogrel- or prasugrel-associated TTP cases. TTP is described in a Black Box warning for ticlopidine (1998, incidence of 1 in 2,000) and as a warning for clopidogrel (2000; 12 per million) and prasugrel (2010; no incidence reported)

Conclusion:

TTP is associated with all thienopyridines, although causal relationships remain under active investigations.

Disclosures:

Armstrong: LeadHorse Technologies Inc.: Employment.