bluuebunny/crossref_metadata_2025 huggingface dataset:
Management of Perioperative Anticoagulation in Patients with VWD
Authors: Von Depka Mario , Detering Carsten , Döpke Stefanie , Ekhlasi-Hundrieser Mahnaz
Published: October 2019

Objectives: von Willebrand disease (VWD) is the most common hereditary bleeding disorder. This study reviews the management of perioperative anticoagulation in patients with VWD undergoing surgical procedures. Risk factors for VTE with von Willebrand factor (VWF) concentrate use are older age, previous VTE, obesity, surgery, hormone replacement therapy use, antifibrinolytic therapy use and high post infusion FVIII levels. Currently, there are few data from randomized clinical trials assessing efficacy and possible complications of perioperative VTE prophylaxis in VWD patients.

Methods: A total number of 116 surgeries were performed (minor: n=64 and major: n=52) in this retrospective, single-centre study. They were divided into groups of perioperative non-anticoagulation (n=54) and perioperative anticoagulation (n=62), who all received coagulation factor concentrate (CFC). Sub-analyses were done according to the type of concentrate used. Anticoagulation was performed using different low molecular weight heparins (LMWH) according to standard protocols or body-weight adapted doses in patients with either elevated body-mass-index or additional thrombosis risk factors. Blood samples had been collected pre- and post-surgery (up to 21 days) to analyse PT, aPTT, PFA, and trough levels of FVIII coagulant activity (FVIII:C), VWF activity (VWF:GPIbM) and antigen (VWF:Ag), respectively. Furthermore, the median doses of CFC/kg and the median total number of infusions were calculated. The rates of clinically overt thrombosis as well as bleeding were assessed during the post-operative phase.

Results: The majority of patients suffered from VWD type 1 (104), 9 patients with type 2A, 2 with type 2M and 1 with type 3 VWD. Humate-P (H) was used in 55 patients and 61 patients received Wilate (W). Using W, we found parallel curves for FVIII:C, VWF-antigen and VWF:GPIbM, respectively. Using H, less concordance between VWF:Ag and VWF:GPIbM was visible and FVIII:C tends to increase between D3 to D10 in spite of decreasing VWF:Ag and VWF:GPIbM. This observation was visible in minor as well as major surgical procedures. LMWH (Enoxaparin, Nadroparin and Certaparin) were used in doses between 30 and 100 mg/injection (mean 46.0 ± 18.5 mg/injection) and a mean of 32.2 ± 24.6 injections in total (range: 8-112). In one patient a significant haematoma occurred (1/116; 0.9%), also one thrombotic event was documented in a different patient (1/116; 0.9%).

Conclusion: Using standard dose LMWH in patients with no overt increased thrombosis risk as well as body-weight-adapted LMWH in high risk patients seem to be safe and effective in VWD patients receiving coagulation factor concentrate perioperatively. However, prospective randomized comparative studies are required to determine the optimal indication as well as type of anticoagulation according to the CFC treatment regimen in this setting.

Disclosures

No relevant conflicts of interest to declare.
Efficacy and Safety of Weight-Adjusted Extended Duration Tinzaparin for Prevention of Post-Operative Venous Thromboembolism after Bariatric Surgery
Authors: Siegal Deborah M. , Handa Pankaj , Kolesar Emma , Anvari Mehran , Tiboni Maria , Douketis James D. , Crowther Mark A.
Published: October 2019

Background: Patients having bariatric surgery are at moderate-to-high risk for post-operative venous thromboembolism (VTE), with pulmonary embolism representing the most common cause of post-operative death. The optimal dosing and duration of anticoagulant prophylaxis is uncertain.

Aim: To evaluate the efficacy and safety of extended-duration, weight-adjusted tinzaparin for post-operative thromboprophylaxis after bariatric surgery.

Methods: Retrospective cohort of 793 bariatric surgery patients who received routine post-operative weight-adjusted tinzaparin, 4,500-14,000 IU daily (75 IU/kg, rounded to the nearest pre-filled syringe) for 7 days after hospital discharge. The primary efficacy and safety outcomes were the frequency of VTE and major bleeding, respectively, within 30 days of surgery in patients receiving at least 1 dose of tinzaparin.

Results: 793 patients who underwent bariatric surgery from 2009 to 2012 were reviewed. There were 44 (5.5%) patients excluded from analyses: need for therapeutic-dose anticoagulation (n=16); no post-operative tinzaparin (n=27); surgery aborted (n=1). There were 646 and 645 patients included in the 30-day efficacy and safety analyses, respectively (mean age, 44.6 years [SD 9.7], median body mass index, 47.1 kg/m2 [range: 19.4-81.0]). An additional 106 patients had outcome data only during hospital admission. VTE occurred in 1/645 patients (0.3%; pulmonary embolism) after hospital discharge and in 1/752 patients (0.1%; superior mesenteric vein thrombosis) in hospital. Major bleeding occurred in 12/645 patients (1.9%). Trough anti-Xa levels measured 7-10 days post-operatively were undetectable in 143/190 (75.3%) patients. In the other 47 patients the median trough anti-Xa level was 0.12 IU (range: 0.10-0.41).

Conclusions: Weight-adjusted, extended-duration tinzaparin appears effective and safe for post-operative thromboprophylaxis after bariatric surgery. There was no drug accumulation with large doses of tinzaparin.

Disclosures

Siegal: Interactive Forums Inc.: Other: created educational slides; Daiichi Sankyo: Other: participated in an advisory board; Boerhinger Ingelheim: Other: participated in an advisory board; Portola Pharmaceuticals: Other: participated in an advisory board. Douketis:Bayer: Consultancy; Actelion: Consultancy; Biotie: Other: Advisory board; Sanofi-Aventis: Honoraria; The Medicines Company: Other: Advisory board; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Honoraria; Boehringer Ingelheim: Consultancy, Honoraria.
Plasma Levels of Human Neutrophil Peptides and Complement Activation Markers in Patients with Acquired Autoimmune Thrombotic Thrombocytopenic Purpura
Authors: Cao Wenjing , Pham Huy Phu , Williams Lawrence A. , Ping Zheng , Lorenz Robin G. , Marques Marisa B. , Zheng X. Long
Published: October 2019

Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal syndrome, resulting from autoantibodies against the metalloprotease ADAMTS13. Autoantibodies bind and inhibit plasma ADAMTS13 activity, leading to exaggerated platelet agglutination and thrombus formation in small arteries and capillaries. However, plasma ADAMTS13 deficiency or the presence of anti-ADAMTS13 autoantibodies is not sufficient to cause the disease. Infection or inflammation often precedes the acute onset of TTP. We hypothesize that neutrophil activation or complement activation vial alternative pathway may be the trigger for TTP. In this study, plasma samples were collected prior to plasma exchange from 58 adult patients with acute TTP between 2006 and 2015 at the University of Alabama at Birmingham Medical Center. All patients were diagnosed having TTP on the basis of severe thrombocytopenia, microangiopathic hemolytic anemia, and severe deficiency of plasma ADAMTS13 activity (less than 10%) and the presence of ADAMTS13 inhibitors. All patients were treated by plasma exchange. Thirty plasma samples from healthy individuals were collected for controls. Plasma levels of human neutrophil peptides (HNP-1, -2, and -3) released from activated neutrophils and alternative pathway complement activation markers (iC3b, C4d, C5b-9, and Bb) were determined by enzyme-linked immunosorbent assays (ELISAs). We showed that plasma levels of HNP-1, -2, and -3 in TTP patients were elevated on average by 10-fold when compared with those in the healthy controls (44.12 ± 39.53 ng/ml vs. 3.54 ± 2.97 ng/ml, means ± SD) (p <0.0001). Whereas plasma levels of Bb and iC3b were only modestly increased in TTP patients (2.94 ± 1.82 µg/ml vs. 1.39 ± 0.34 µg/ml for Bb, p <0.0001, and 16.21 ± 9.71 µg/ml vs. 12.13 ± 3.07 µg/ml for iC3b, p =0.028). No significant difference was detected in the plasma levels of C4d and C5b-9 between TTP patients and healthy controls (p >0.05). To differentiate HNP-1 from HNP-2 or HNP-3, we developed a LC/MS mass spectrometric assay and showed that all subtypes of HNPs in TTP patients were significantly elevated when compared with those in the healthy controls (p<0.001). In general, there was good concordance between ELISA and LC/MS MS in the measurement of all three HNPs. These results demonstrate that innate immunity (i.e. activation of neutrophils) and to a lesser degree the activation of complements via alternative pathway may play a role in pathogenesis of TTP in light of severe ADAMTS13 deficiency. Our findings provide rationales for developing novel targeted therapies for acquired TTP.

Disclosures

No relevant conflicts of interest to declare.
NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool
Authors: Kreutmair Stefanie , Istvanffy Rouzanna , Klingeberg Cathrin , Dierks Christine , Peschel Christian , Oostendorp Robert A.J. , Duyster Justus , Illert Anna Lena
Published: October 2019

Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated.

NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipako/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipako/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipako/ko vs. wt animals (11 vs. 14.5 days).

To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipako/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response.

To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population.

Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool.

Disclosures

No relevant conflicts of interest to declare.
A Human Bone Marrow-Derived Stromal Cell Population with Hemogenic Potential
Authors: Mokhtari Saloomeh , Colletti Evan Joseph , Sanada Chad , Lamar Zanetta S. , Simmons Paul J , Atala Anthony , Krause Diane S , Zanjani Esmail D. , Porada Christopher D , Almeida-Porada Graca
Published: October 2019

During ontogeny, definitive hematopoietic stem/progenitor cells (HSC) are thought to arise from vascular endothelial cells, through an endothelial-to-hematopoietic transition, a natural process that occurs in unique, specialized embryonic hemogenic endothelial cells. Developmental studies, and experiments using pluripotent stem cells in an effort to recapitulate this process and thereby gain a better understanding of the emergence of definitive hematopoiesis, have collectively led to the prevailing view that the hemogenic endothelium constitutes a transient population of cells within the embryo that rapidly disappears during development and is absent in the adult. Herein, we provide the first evidence that at early time points of gestation, prior to the establishment of hematopoiesis, a unique subpopulation of Stro-1+ cells present within the inner part of the developing human bone marrow co-expresses APLNR, a marker of angiogenic mesoderm. Moreover, these Stro-1+APLNR+ cells express multiple other markers described for hemogenic endothelium, and subsequently contribute to the vasculature, cartilage, and bone. Importantly, we also show that cells expressing these same markers of primitive mesoderm/hemogenic endothelium persist at low frequency within the adult marrow. These adult-derived cells can be extensively expanded in vitro without loss of potential, but lack hematopoietic colony-forming potential in vitro. However, upon transplantation into a fetal microenvironment, clonally-derived populations of these adult Stro1+ isolated stromal progenitors (SIPs) not only contribute to the vasculature and nascent BM niches, but also efficiently generate, at a clonal level, hematopoietic stem cells (HSC) that are capable of robust, multilineage hematopoietic reconstitution, with generation of both myeloid and lymphoid cells upon serial transplantation. In conclusion, our studies have thus uncovered the latent potential of a highly expandable population of seemingly vestigial adult human somatic cells, whose ontogenic history includes a phenotype identical to that described for hemogenic endothelium. We have also shown that, if provided with the appropriate/necessary inductive factors, these unique adult cells are capable of giving rise to hematopoietic cells that fulfill the gold standard criteria for bona fide HSC. Therefore, these cells could potentially be more amenable to reprogramming technologies, to produce HSC that could be used to treat/cure a broad variety of blood diseases.

Disclosures

No relevant conflicts of interest to declare.
The Role of Tetraspanin CD37 in B-Cell Malignancy
Authors: Beckwith Kyle A. , Williams Erich I. , Wright Mark D. , van Spriel Annemiek B. , Byrd John C. , Muthusamy Natarajan
Published: October 2019

Members of the tetraspanin superfamily of transmembrane proteins have roles in a variety of processes including cellular adhesion, migration, activation, proliferation, survival, and solid tumor metastasis. Although numerous tetraspanin proteins are expressed by immune cells, only a few have expression that is restricted to these cells. CD37 is not found in non-hematologic tissues and is highly expressed by mature B-cells, with greater than 15 times higher expression compared to other immune cells (Deckert et al., 2013). Recently there has been significant interest in CD37 as a therapeutic target in non-Hodgkin lymphoma and Chronic Lymphocytic Leukemia (CLL), as evidenced by the ongoing clinical development of four different anti-CD37 therapies. However, very little is known about the biological function of this protein. Ligation by the CD37-targeting peptide otlertuzumab leads to induction of both pro-survival Akt activation and pro-apoptotic SHP1 signaling, although cellular death is heavily favored (Lapalombella et al., 2012). Furthermore, CD37 is critical to normal plasma cell development, clustering VLA-4 (α4β1 integrin) on the surface of germinal center B-cells to allow integrin-mediated activation of Akt (van Spriel et al., 2012). In CLL, expression of the VLA-4 subunit CD49d is associated with more aggressive disease and promotes homing and adhesion to supportive cells within lymphoid tissues. This prompted us to explore the role of CD37 expression in B-cell malignancy using a CD37-deficient Eµ-TCL1 mouse model of CLL. For these studies, we monitored mice that were CD37 wild type, heterozygous knockout, or homozygous knockout (all hemizygous for the TCL1 transgene). While no significant difference was observed in the time until appearance of leukemia in peripheral blood, loss of CD37 led to decreased survival of TCL1 mice (p=0.032). In the same timeframe, non-TCL1 mice with CD37 deficiency did not exhibit impaired survival. While CD37 is predominantly seen in B-cells, the protein serves a role in other immune cells despite substantially lower expression. Our results are consistent with the recent reports on impaired dendritic cell migration in CD37 deficient mice, which is associated with decreased anti-tumor immunity in the context of a subcutaneously injected mouse cell line expressing a foreign human antigen (Gartlan et al., 2013). Our data support a potential role for CD37 in anti-tumor immune response against the syngeneic leukemia that arises spontaneously in the TCL1 model of CLL. Given the complex nature of this global knockout model, other functions of CD37 may be obscured. Ongoing studies will examine engraftment of CD37-deficient or wildtype TCL1 leukemia into healthy mice to elucidate whether the B-cell intrinsic role of CD37 is supportive of the leukemia, independent of an extrinsic role in anti-tumor immunity.

Figure 1. Figure 1.

Disclosures

Byrd: Acerta Pharma BV: Research Funding.
Tyrosin Kinase Inhibition Restores the Membrane Localization of FLT3-ITD
Authors: Reiter Katrin S. , Polzer Harald , Krupka Christina , Vick Binje , Maiser Andreas , Subklewe Marion S. , Jeremias Irmela , Leonhardt Heinrich , Spiekermann Karsten , Greif Philipp A.
Published: October 2019

The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regards to oncogenic alterations that do not only serve as prognostic markers but also as targets for therapy in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Due to sequence-dependent protein conformation changes, FLT3-ITD tends to auto-phosphorylate and displays a constitutive intracellular localization. As tyrosine kinase inhibitors (TKIs) efficiently block FLT3 auto-phosphorylation, we set out to study the effect of TKI on the subcellular localization of the FLT3 receptor and its mutants.

We visualized FLT3 WT and its mutants (N676K, D835Y, W51 (ITD), NPOS (ITD), K644R, W51-K644R) in transiently transfected U2OS cells, using anti-FLT3 immunofluorescence in combination with confocal microscopy. Treatment with 50nM of the TKI AC220 for 6 hours restored the membrane localization of both ITD (Figure 1 A) and D835Y. The TKI-induced surface expression of FLT3-ITD was confirmed by flow cytometry (mean fluorescence intensity, MFI) of BaF3 cells stably expressing FLT3 WT or mutants and in the homozygous (ITD/ITD) AML cell line (MV4-11). Similar results were obtained in cells from an AML patient derived xenograft (pdx) with FLT3-ITD and loss of heterozygosity (LOH) (Figure 1 B). Western blot analysis showed increased glycosylation of FLT3-ITD after TKI-treatment (Figure 1 C) suggesting that auto-phosphorylation prevents physiological processing of the receptor, which is required for maturation and surface expression. To monitor the subcellular localization of FLT3 and its mutants in vivo we have generated a FLT3-GFP fusion. Proliferation assays in BaF3 cells confirmed that the C-terminal GFP-tag does not alter the function of FLT3. Upon FLT3-ligand (FL) stimulation of FLT3-GFP we observed FLT3-internalization by life cell imaging in U2OS cells. Conversely, TKI treatment increased cell surface expression of FLT3-GFP.

Our findings have translational implications when considering FLT3 as a target for immunotherapy in AML, since FLT3 surface expression might facilitate antigen recognition. Especially FLT3-ITD positive patients with LOH may benefit from a combined TKI-immunotherapy approach. Therefore, we currently study the effect of AC220 on FLT3 antibody-dependent cellular cytotoxicity (ADCC), mediated by natural killer (NK) cells. Taken together, we discovered that TKI alters the subcellular localization and protein processing of specific FLT3 mutants. However, the complex interplay between the individual posttranslational modifications and their role in FLT3 traffic remains elusive.

Disclosures

Krupka: AMGEN Research (Munich): Research Funding. Subklewe:AMGEN Research (Munich): Research Funding.
Activation of Simultaneous Apoptosis and Necroptosis to Eradicate Drug Resistant Leukemia
Authors: McComb Scott , Aguadé-Gorgorió Julia , Marovca Blerim , Harder Lena , Cario Gunnar , Eckert Cornelia , Schrappe Martin , Stanulla Martin , von Stackelberg Arend , Bourquin Jean-Pierre , Bornhauser Beat
Published: October 2019

Dysregulation of apoptotic pathways provides an indiscriminate mechanism for refractory acute lymphoblastic leukemia (ALL) to escape cell death induced by many chemotherapeutic compounds. Here we have assessed the potential of SMAC mimetic (SM) compounds to short circuit cell death resistance by blocking the pro-survival cellular inhibitor of apoptosis (cIAP) proteins. By screening a large set of patient-derived precursor B-cell ALL samples in an ex vivo model of the leukemia microenvironment we detect exquisite sensitivity to two different SM compounds, Birinapant and LCL161, in about one third of ALL samples. Strong ex vivo SM activity correlated with potent in vivo anti-leukemic efficacy against de novo refractory and relapsed ALL xenografts. Intriguingly, we find that although SM-sensitivity is independent of TNF and TNFR1 levels, expression of TNFR2 is highly predictive of response to SM in two separate cohorts of ALL samples, suggesting that TNFR2 expression may represent a promising biomarker for identifying SM-sensitive cells. Downstream, we employ a novel and powerful multi-colour Lenti-CRISPR approach to show that simultaneous disruption of both apoptotic and necroptotic genes is necessary to block SM-induced death. In contrast, disruption of RIP1 alone was adequate to block SM-induced apoptosis and necroptosis. Surprisingly, RIP1 loss had no significant impact on response to standard anti-leukemic therapies, supporting a view that the RIP1-dependent death pathway is not likely to be selected against in leukemia cells that have failed to respond to front line therapy. These results provide the first evidence that SM compounds can circumvent apoptotic escape in drug-refractory ALL through parallel activation of both RIP1-dependent apoptosis and necroptosis. Furthermore, our data strongly support further development of SM as anti-leukemic agents for treatment in resistant disease.

Disclosures

No relevant conflicts of interest to declare.
Bacillus Infection Among Children with Hematologic Malignancy, a Single Institution Experience
Authors: Shulman David S , Mehrotra Preeti , Surana Neeraj K. , Blonquist Traci M. , Silverman Lewis B. , Place Andrew E.
Published: October 2019

BACKGROUND: Bacillus species, including B. cereus and other non-anthracis species, are spore-forming, gram-positive rods, found ubiquitously in the environment and cause bacteremia and CNS infection in children with cancer. Case reports have demonstrated that Bacillus spp. infections carry a high morbidity, with rates of CNS involvement of 30%, and mortality as high as 40%. These studies indicate the preponderance of cases occur in children receiving induction chemotherapy for acute lymphoblastic leukemia (ALL). There have been no large, recent studies describing Bacillus spp. infection in children with an underlying oncologic condition.

METHODS: We performed a retrospective medical record review of pediatric cancer patients who received care at the Dana-Farber/Boston Children's Cancer and Blood Disorders Center and developed Bacillus spp. infection between January 1st, 2005 and December 31st 2014. Given that the majority of these infections were found to occur in children with underlying hematologic malignancy, we provide a more detailed description of cases with these underlying conditions.

RESULTS: Twenty-six children developed Bacillus bacteremia during the study period. Of the 26 patients, 21 (81%) had acute leukemia (18 ALL; 3 AML), 3 had neuroblastoma, one had Ewing's sarcoma and one had an optic pathway glioma. The mean age of patients with hematologic malignancies at time of Bacillus spp. infection was 7.3 years [range: 1-17]. Sixteen (76%) children were neutropenic at the time of infection. Of the children with ALL, 11 (61%) developed infection during the remission induction phase of treatment, 3 during post-induction/pre-maintenance treatment phases and 3 after relapse (1 during stem cell transplant, 1 at a year post-transplant and 1 during re-induction). Of the 26 patients, 5 (19%) developed CNS complications, all during remission induction for ALL, and the overall mortality two-weeks following Bacillus infection was 19%.

DISCUSSION: Bacillus spp. continues to cause serious infections in children with cancer, especially in those with underlying hematologic malignancies. While the mortality at our center is lower than that reported in the literature, the rate of death is still higher than what is typically seen with other organisms and CNS involvement remains common. Further study in this area will include identification of risk factors for development of this infection and recommendations regarding use of prophylactic antibiotics.

Disclosures

No relevant conflicts of interest to declare.
IMGN779, a CD33-Targeted Antibody-Drug Conjugate (ADC) with a Novel DNA-Alkylating Effector Molecule, Induces DNA Damage, Cell Cycle Arrest, and Apoptosis in AML Cells
Authors: Krystal Watkins M , Walker Russell , Fishkin Nathan , Audette Charlene , Kovtun Yelena , Romanelli Angela
Published: October 2019

IMGN779 is a CD33-targeting ADC consisting of a humanized anti-CD33 antibody, Z4681A, conjugated to DGN462, a novel DNA-alkylating agent, through a cleavable disulfide linker, sulfo-SPDB. CD33 is broadly expressed on leukemic blasts of patients with AML, making it a promising target for AML therapy. DGN462 is a member of the novel IGN class of DNA-acting cytotoxic agents, that consists of an indolino-benzodiazepine dimer containing a mono-imine moiety.

Potent killing of AML tumor cells by DGN462 and IMGN779 has previously been demonstrated in vitro (K. Whiteman et. al, ASH 2014 #2321). Here we describe studies elucidating the mechanism of action of this novel payload in the AML cell lines: MV4-11, HL60, and EOL-1. The ability of DGN462 to alkylate DNA was demonstrated in an AML cell line by isolation and analysis of the genomic DNA from cells. DGN462 was found to co-purify with DNA, demonstrating that it covalently reacts with cellular DNA. A comet assay was performed confirming that DGN462 does not cross-link DNA.

H2AX is known to be phosphorylated on serine 139 in response to DNA damage. Using flow cytometry, we assessed the phosphorylation of H2AX in AML cell lines following exposure to DGN462 and IMGN779. Increases in phosphorylated H2AX levels were detected as early as 4 hours following exposure to DGN462-SMe and 12 hours following exposure to IMGN779. Additional flow cytometry analysis showed that exposure to DGN462-SMe and IMGN779 leads to S-phase accumulation, G2/M arrest, followed by induction of apoptotic markers (cleaved PARP and cleaved Caspase-3) at later time points. Ex vivo studies using AML patient samples also showed elevation of phosphorylated H2AX and an increase in apoptosis in myeloid blasts following exposure to DGN462-SMe and IMGN779.

As a demonstration of proof-of-mechanism in AML disease models, our results suggest that cell killing by IMGN779 is mediated by DNA damage, as a consequence of DNA alkylation. The DNA damage response is accompanied by cell cycle arrest, which leads to apoptosis. This relationship will be further explored in additional AML preclinical models to support the use of phosphorylated H2AX as a pharmacodynamic biomarker for IMGN779 activity in future clinical studies.

Disclosures

Krystal: ImmunoGen, Inc.: Employment, Equity Ownership. Walker:ImmunoGen, Inc.: Employment, Equity Ownership. Fishkin:ImmunoGen, Inc.: Employment, Equity Ownership. Audette:ImmunoGen, Inc.: Employment, Equity Ownership. Kovtun:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.
Bortezomib, Dexamethasone, Thalidomide and Melphalan (VDT-Mel) Preparative Regimen in Autologous Stem Cell Transplant (ASCT) Results in a Very High Stringent CR (sCR) Rate in Multiple Myeloma (MM)
Authors: Nadiminti Kalyan , Abbi Kamal Kant Singh , Tricot Annick , Schultz Allyson , Dozeman Lindsay , Mott Sarah L , Zhan Fenghuang , Tricot Guido J
Published: October 2019

Background: Melphalan 200mg/m2 is the standard preparative regimen in MM and addition of other cytotoxic drugs has not been found to result in superior activity. The novel agents have improved outcome in MM significantly, but data on their role in preparative regimens are scarce. The purpose of this study was to understand the toxicity and efficacy of triple therapy with VDT in combination with high-dose melphalan.

Methods: An IRB approved retrospective analysis was performed on all patients who received an ASCT with the VDT-Mel during 2012-2014. Mel: 100 mg/m2 was given on days -4 and -1; V: 1 mg/m2 on days -4, -1, +2 and +5; T: 100 mg daily from -5 to +5; and D: 20 mg/day from -4 to -1 and +2 to +5. End points were treatment-related toxicity during the first 100 days and quality of response at 6 months post-transplant; 98 patients had follow-up ≥ 6 months. Patients in sCR were also minimal residual disease negative (MRD-) by 10-color flow cytometry with a sensitivity of 10-4.

Results: 100 patients received 153 transplants; 47 patients underwent single and 53 had tandem transplants (TT); 64 patients received early (≤ 12 months of induction therapy) and 36 salvage transplantation. Median age was 61 y; median followup was 16.2 months. Only 1patient had achieved a sCR and 11 a CR prior to transplantation. Best responses at 6 months were 53% sCR (and MRD-), 24% CR, and 9% VGPR. The sCR rate after single transplant was 47% (overall) and 54% (early transplant) vs 59% and 60% after TT. Grade 3-5 non-hematologic toxicities were almost entirely related to infections (38% and 53% in single and TT, respectively); the 100-day mortality rate was 2.6% (4/153), 1.8% for early transplants and 4.5% for salvage transplants. Median time to ANC recovery > 500/µL was 12 days in both early and salvage transplantation.

Conclusion: VDT-Mel is well-tolerated and resulted in minimal additional toxicity and a similar mortality rate when compared to historic data of MEL alone. Importantly, the sCR rate with MRD- by flow cytometry at 6 months in our study was very high compared to published reports. The ultimate sCR rate will be higher as at this time an additional 13 patients attained a sCR during further follow up past 6 months for a total of 66% sCR. Since both sCR and MRD- are proven early surrogate markers for progression-free and overall survival, it appears highly likely that this regimen will be superior to Mel alone and should become the new standard for ASCT in myeloma.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Disease Risk Index Is the Major Predictor of Outcome Following Myeloablative Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT) and Post-Transplant Cyclophosphamide (PT/Cy)
Authors: Solomon Scott R. , Sizemore Connie , Zhang Xu , Solh Melhem , Morris Lawrence E. , Holland H. Kent , Bashey Asad
Published: October 2019

Disease Risk Index is the Major Predictor of Outcome Following Myeloablative Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT) and Post-Transplant Cyclophosphamide (PT/Cy)

Although non-myeloablative (NMA) haplo-HSCT utilizing PT/Cy results in low rates of GVHD, infection, and non-relapse mortality (NRM), relapse remains the predominant cause of treatment failure, occurring in up to 50% of patients. To reduce the risk of relapse often associated with the use of NMA preparative regimens, we have developed a myeloablative haplo-HSCT utilizing PT/Cy. Sixty-four patients have been transplanted following either Busulfan-based (n=20) or TBI-based (n=44) myeloablative conditioning, T-cell replete PBSC infusion, PT/Cy, and tacrolimus/mycophenolate mofetil. Median age was 43 (range 21-60). Patient characteristics included a high/very high disease risk by the Dana-Farber/CIBMTR disease risk index (DRI) in 32 patients (50%), KPS<90 in 69%, and comorbidity index (CMI) of ≥2 in 58% of patients. The most common indications for transplant were AML, ALL, and advanced-phase CML in 55%, 20% and 12% of patients respectively. Median follow-up for surviving patients was 24 months. All patients have engrafted with no late graft failure. Grade II-IV, III-IV acute GVHD and moderate-severe chronic GVHD occurred in 46%, 23%, and 30% respectively. One-year NRM was 10%. Predicted three-year overall survival (OS), disease-free survival (DFS), and relapse are 53%, 53%, and 26% respectively. In multivariate analysis, high/very high DRI was the most significant negative predictor of OS (HR 13.26, p<0.001), followed by CMI≥2 (HR 3.54, p=0.01) and age (HR 1.26, p=0.038, per each 5 year increase in age). DRI was also significantly associated with DFS (HR 10.84, p<0.001), NRM (HR 15.0, p=0.004), and relapse (HR 8.85, p=0.004). In the 32 patients with standard risk disease (low/intermediate DRI), outcomes were significantly improved with one-year NRM of 0% and predicted 3-year OS, DFS, and relapse of 79%, 74% and 9% respectively. Conditioning regimen (TBI vs. Busulfan) had no significant impact on outcome. This analysis confirms that DRI is a strong predictor of outcome following myeloablative haplo-HSCT and PT/Cy and adds to a growing body of literature suggesting that haplo-HSCT is a safe and effective transplant option for patients lacking a matched sibling donor.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
The Role of Mitochondrial Metabolism and Redox Signaling in Iron Deficiency Anemia
Authors: Bullock Grant C , Richardson Chanté L , Schrott Valerie , Gunawardena Naomi D , Cole Teague Nolan , Corey Catherine G , Shiva Sruti S
Published: October 2019

Several clinical observations illustrate the link between iron and erythropoietin (EPO)-mediated signaling in committed erythroid progenitor cells. In iron deficiency anemia (IDA), erythropoiesis is blocked despite increased serum EPO concentrations. Intravenous iron improves the effectiveness of exogenous EPO in patients with EPO-refractory anemia of chronic disease. These clinical observations suggest that iron dominantly regulates EPO-receptor signaling. However, the mechanism of this iron-mediated signaling remains unclear. We recently demonstrated that 1) the aconitases, multifunctional iron-sulfur cluster proteins that convert citrate into isocitrate are essential in the iron- EPO-signaling pathway in erythroid progenitor cells, and that 2) isocitrate, the product of aconitase, can enhance the effectiveness of EPO during iron deficiency in vitro and in mice with IDA and in rats with the anemia of chronic inflammation. These observations suggest that isocitrate, or its derivatives that synergize with erythropoiesis stimulating agents, have important therapeutic application in the treatment of anemia. New data shows that cellular iron restriction regulates mitochondrial oxygen consumption rates (OCR) differentially over time during erythropoiesis, suggesting a novel link between mitochondrial function and erythropoeisis. It is unknown how iron deficiency induced inhibition of mitochondrial aconitase (ACO2) regulates mitochondrial metabolism during RBC production. Pilot data show that ACO2 inhibition by cellular iron deprivation or pharmacological inhibition of ACO2 decreases mitochondrial respiratory rates (RRs) and alters reactive oxygen species (ROS) production. Further, isocitrate normalizes mitochondrial RRs and ROS and restores RBC production. Importantly, disruption of mitochondrial ROS generation with a mitochondrial-specific anti-oxidant blocks RBC production and a subset of oxidant generators promote erythropoiesis. Targeted reduction of ACO2 protein expression and enzyme activity in iron replete stably transduced K562 cells decreases OCRs. This confirms the link between ACO2 and mitochondrial metabolism in human erythroid cells. These data inform our overarching hypothesis that iron-restriction inhibits ACO2, thereby inhibiting mitochondrial metabolism, resulting in the loss of a mitochondrial ROS signal that is required for erythropoiesis. The loss of this critical mitochondrial ROS signal inhibits the EPO signaling that is required for RBC production. These data also suggest that ACO2 is an iron-sensing regulator of mitochondrial metabolism and ROS signaling.

Disclosures

No relevant conflicts of interest to declare.
Decrease in the Severity of Painful Sickle Cell Crises with Oral Pglg
Authors: Niihara Yutaka , Tran Lan , Razon Rafael , Stark Charles , Macan Henry
Published: October 2019

Background: Our earlier work suggested an enhancement of nicotinamide adenine dinucleotide (NAD) in sickled Red Blood Cells (sRBC) by administering a precursor of NAD, L-glutamine. We hypothesized that L-glutamine would decrease the incidence of painful sickle cell crises (PSCC). Small proof of concept studies led to the development of a multi-center Phase 2 placebo-controlled trial where oral pharmaceutical grade L-glutamine (PGLG) suggested benefit when measuring PSCC incidence at 24 weeks. A large multi-center Phase 3 trial further demonstrated that there was a reduction in the incidence of PSCC by administering PGLG compared to placebo. Additional analyses on the data from this trial were conducted and we are reporting our evaluation on the severity of crises between treatment arms.

Methods: A randomized (2:1), double-blind, placebo-controlled, parallel-group Phase 3 study was conducted at 31 sites in the United States. Subjects were stratified by hydroxyurea usage. This study included subjects 5 years of age and older that had at least 2 PSCC during the year prior to enrollment. The primary endpoint for the Phase 3 trial was the number of PSCC between treatment arms. Since PSCC events were adjudicated by a blinded third party committee and data were collected as an adverse event ranked as "None"(0), "Mild" (1), "Moderate" (2) and "Severe" (3), the difference in "Severity" of PSCC between treatment arms using Cochran Mantel Haenszel (CMH) Ranked Scores statistics was evaluated post-hoc. Each patient was counted only once at the highest level of severity.

Results; A total of 230 patients were enrolled; ages 5-58; 53.9 % female; 152 were assigned to PGLG and 78 to placebo; and the groups were well balanced for baseline clinical characteristics. The comparison of Severity for PGLG vs. Placebo indicated that at (0) was 24% vs. 10% respectively; at (1) was 6% vs. 8%; at (2) was 36% vs. 35%; and at (3) was 34% vs. 47%. Summary statistics for the cohort taken as a whole indicated a treatment difference benefit for the PGLG treatment (p = 0.0167). Other adverse events in the PGLG arm were similar to those observed in the placebo arm.

Conclusion: PGLG therapy may be providing clinical benefit over placebo in adult and pediatric patients with a history of two or more crises per year by reducing the "Severity" of painful crises. Difference appeared to be driven by (0) or "None" PSCC events and by a decrease in (3) "Severe" PSCC events. PGLG was administered without difficulty and did not require special monitoring.

Disclosures

Niihara: Emmaus Medical, Inc: Employment, Equity Ownership. Tran:Emmaus Medical, Inc: Employment, Equity Ownership. Razon:Emmaus Medical, Inc: Employment. Stark:Emmaus Medical, Inc: Employment, Equity Ownership. Macan:Emmaus Medical, Inc: Employment.
Thymic-Derived Tolerizing Dendritic Cells Are up-Regulated upon Treatment with Intravenous Immunoglobulin or Splenectomy in a Murine Model of Immune Thrombocytopenia
Authors: Kapur Rick , Aslam Rukhsana , Speck Edwin R. , Kim Michael , Zufferey Anne , Guo Li , Segel George B. , Ni Heyu , Semple John W.
Published: October 2019

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet counts. ITP has a complex pathogenesis, in which both anti-platelet antibodies as well as T cells have been shown to be important. Initial management of newly diagnosed ITP may be either watchful waiting or pharmacologic intervention, such as glucocorticoids or Intravenous Immunoglobulin (IVIg), a blood product consisting of polyclonal immunoglobulin G (IgG) derived from thousands of donors. Second-line therapy may include dexamethasone, high-dose methylprednisolone, rituximab, thrombopoietin (TPO)-receptor agonists, or splenectomy. The working mechanism of IVIg is actively under investigation and is still a matter of debate, as various different working mechanisms have been suggested. One of them is that IVIg may shift the balance from a pro- to anti-inflammatory state through immunomodulating the activity of dendritic cells (DCs). To gain more insights into the role of DCs in ITP, upon IVIg treatment or splenectomy, we analyzed DC subsets in a murine model of ITP, which features both the antibody and T cell mediated thrombocytopenia. Severe combined immunodeficient (SCID) mice were administrated 4x104 splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 (or naïve control splenocytes) and the mice were treated with or without 1 g/kg IVIg twice a week. Also the same type of splenocytes were transferred into splenectomized SCID mice. Weekly platelet counts were assessed and after 4 weeks the mice were sacrificed and spleen and thymuses were harvested. Splenocytes and thymocytes were isolated and examined by flow cytometry for cross-presenting (XCR1+) and non-presenting tolerizing (SIRP alpha+) DCs. Without IVIg or splenectomy, both splenic DC subset numbers correlated positively with platelet counts and both the thymic DC subset numbers correlated negatively with platelet counts, indicating thymic retention of DC in a setting of thrombocytopenia. Interestingly, splenectomized SCID mice, apart from increased platelet counts, demonstrated a complete reversal of the DC pattern in the thymus, as thymic DC subsets correlated positively with platelet counts in splenectomized mice. Upon IVIg treatment, apart from a general increase in platelet counts, the splenic tolerizing DCs significantly increased in numbers. Moreover, the thymic retention of tolerizing DCs and thus the negative correlation with platelet counts (R2: 0.46, p<0.05) was fully abrogated upon IVIg treatment (R2: 0.02, NS). Overall, our results indicate that both splenectomy as well as IVIg treatment can immunomodulate thymic tolerizing DCs significantly, in a murine model of ITP.

Disclosures

No relevant conflicts of interest to declare.
Survival in Essential Thrombocythemia and Prefibrotic Primary Myelofibrosis - Correlation of Clinical Phenotype with Histomorphological Diagnosis
Authors: Jeryczynski Georg , Woelfler Albert , Gisslinger Bettina , Schalling Martin , Burgstaller Sonja , Buxhofer-Ausch Veronika , Krauth Maria Theresa , Geissler Klaus , Schlögl Ernst , Sliwa Thamer , Schiefer Ana-Iris , Muellauer Leonhard , Simonitsch-Klupp Ingrid , Beham-Schmid Christine , Thiele Jürgen , Gisslinger Heinz
Published: October 2019

Introduction

The correct diagnosis of myeloproliferative neoplasm (MPN), especially the correct classification of early or prodromal stages, remains a matter of debate. While the 2008 WHOcriteriarecognize the existence of an early, prefibrotic primary myelofibrosis, other diagnostic systems make no such distinction between ET and prePMF. According to WHO criteria the accurate diagnosis is based mainly onhistomorphologicassessment of bone marrow (BM) biopsy samples. However, there is an ongoing debate if ET, prePMF, PV and overt PMF are, dependent from their molecular genetic phenotype, a continuum or rather independent entities.

Methods

From a multicenter database, we selected 251 patients with ET and 185 patients with prePMF, in which the diagnosis had been established by BM biopsy and in which clinical data at time of diagnosis and for follow up were available.

We compared overall survival in patients from both entities in which common clinical features of prePMF were present at time of bone marrow biopsy. These were anemia (male <13 g/dL, female <12 g/dL),leukocyte counts ³ 11 G/L, elevated lactate dehydrogenase levels (LDH) as well as splenomegaly.

Results

Survival was substantially impaired in WHO-defined prePMF patients when compared to the ET cohort (Fig. 1). Median overall survival was significantly worse in patients with leukocytosis (9.3 vs. 14.9 years, 95% CI 6.9-11.6 vs. 9.1-20.7, p<0.001), elevated LDH levels (10.6 vs. 21.7 years, 95% CI 7.7-13.4 vs. 11.5-31.9, p<0.001) and splenomegaly (6.8 years vs. median not reached, 95% CI 6.8-10.8 vs.n.a.,p<0.001). Significance was narrowly missed in anemic patients (7.2 vs. 13.1 years, 95% CI 4.7-9.8 vs. 9.2-16.9, p=0.089).

Discussion

Our data illustrate, that the distinction of prePMF and ET patients based onhistomorphologicalcriteria by the WHO 2008 diagnostic criteria translates into different overall survival patterns in patients that may share some similar clinical features at diagnosis. This suggests that the underlying diseases are two different entities, with different underlying biology, that can both present with the same adverse clinical parameters. Further, these two entities can only be accurately differentiated by bone marrow histology.

Therefore the concept that ET, WHO-defined prePMF and overt PMF form a continuum of the same disease, with prePMF just being an advanced stage of ET, may need revision in face of this study.

Figure 1 Figure 1.

Disclosures

Burgstaller: Novartis: Consultancy, Honoraria. Geissler:Novartis: Honoraria. Gisslinger:AOP Orphan: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Baxalta: Consultancy, Honoraria.