bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinicopathological Features and Mutational Analysis of Patients with Aplastic Anemia and Hypoplastic Myelodysplastic Syndrome
Authors: Zhang Xiaohui , List Alan F , Lancet Jeffrey E. , Jinming Song , Moscinski Lynn C. , Sokol Lubomir , Zhang Ling
Published: October 2019

Background: Pancytopenia and bone marrow aplasia/hypoplasia are caused by a heterogeneous group of disorders, most commonly aplastic anemia (AA), hypoplastic myelodysplastic syndrome (MDS), paroxysmal nocturnal hemoglobinuria (PNH), and T-cell large granular lymphocytosis (T-LGL). Clinical and morphological distinction among these entities is often challenging, particularly between AA and hypoplastic MDS. This study is to examine the clinicopathological and genetic features of a group of AA and hypoplastic MDS patients, with or without concurrent T-LGL and/or PNH, in order to better understand and differentiate the two entities.

Methods and Materials: We retrieved 45 cases with cytopenias and hypoplastic bone marrow at Moffitt Cancer Center. Peripheral blood complete blood counts, bone marrow morphological findings, flow cytometric analyses for LGL and PNH, and cytogenetics data were extracted from electronic medical records. Targeted next-generation sequencing (54 myeloid neoplasm related genes) was performed on the bone marrow.

Results: The 45 patients showed peripheral blood cytopenias and bone marrow aplasia or marked hypocellularity. There were 26 cases diagnosed with AA with no morphologic evidence of dysplasia or increased blasts in the bone marrow, and 19 cases diagnosed with hypoplastic MDS based on morphological and cytogenetic criteria. In the meantime, distinct T-LGL population was identified in 2 of 11 cases with AA (18.2%) and 3 of 11 cases with hypoplastic MDS (27.3%); PNH clones were identified in 8 of 17 cases with AA (47%) and 3 of 11 cases with hypoplastic MDS (27.3%). Clonal cytogenetic abnormalities were found in 2 of 25 cases with AA (8%) and 11 of 18 cases with hypoplastic MDS (61.1%). Twelve of 26 cases of AA (46%) showed one or more gene mutations with allele burden ranging from 7% to 52%, and most of these cases (9 of 12; 75%) involved only one gene. In contrast, 15 of 19 cases of hypoplastic MDS (78.9%) had one or more gene mutations with allele burden ranging from 19% to 53%. Seven of the 15 cases (46.7%) had two or more gene mutations. The most common mutated genes in the two groups in this study were ASXL1 and TET2.

Conclusion: Although there are overlapping clinical and morphological features between AA and hypoplastic MDS, differences are present between the two entities including presence of PNH clones, cytogenetic changes, and gene mutation frequencies. These features may help to make differential diagnosis and identify the cases with more progression potential. Clinical outcomes with different treatment and larger scale studies are needed to better characterize and define the two different entities.

Disclosures

No relevant conflicts of interest to declare.
Bortezomib-Induced Apoptosis in Myeloma Cell Via Oxidation of Peroxiredoxin III and Thioredoxin 2
Authors: Mun Yeung-Chul , Ahn Jee-Young , Kang Seung Hye , Yoo Eun-Sun , Lee Kyoung Eun , Nam Eunmi , Lee Soon Nam , Woo Hyun Ae , Rhee Sue Goo , Seong Chu-Myong
Published: October 2019

Backgrounds : Bortezomib (BTZ) is the first generation proteosome inhibitor providing excellent response in newly diagnosed multiple myeloma. BTZ treatment increases oxidative stress in myeloma cells. However the roles of antioxidant enzymes during upregulation of ROS and the mechanisms of resistance from BTZ treatment are unclear. The aims of current study are to elucidate that the changes of redox enzyme could have a important roles of anti-myeloma effect during BTZ treatment.

Methods : MM.1S, MM.1R, and RPMI8226, the human myeloma cell lines, were treated with BTZ to induce apoptosis. 2, 7-dichlrodihydro-fluorescein-diacetate (H2DCF-DA) and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS) respectively. Sulfinic acid, (SO2) form of preoxiredoxin (Prx) was studied by western blot assay using Prx SO2 form-specific antibody. Monomer/dimer assay for subtypes of Prx and thioredoxin (Trx) was performed by western blot using non-reducing gel. To evaluate the effect of down regulation on sulfiredoxin (Srx), myeloma cells were transfected with small interfering RNA (siRNA) followed by Western blot analysis.

Results : Mitochondrial over cytosolic ROS of MM cells was increased significantly after 19 hour of BTZ (2.5 nM). Apoptosis of MM cell after BTZ treatment was increased in concordance with mitochondrial ROS increment of MM cells. N-acetylcystein (NAC) reversed BTZ-induced mitochondrial ROS elevation and apoptosis of MM cells as well. Increased expressions of cleaved caspase-9 and cleaved caspase-3 were also observed during BTZ-induced MM cell apoptosis. Monomer, indicated active and reduced form, of Prx III was decreased and dimer, indicated inactive and oxidized form, of Prx III was increased in MM cells after treatment of BTZ among Prxs. Similarly, monomer of Trx 2, mitochondrial Trx was decreased in MM cells after BTZ treatment. However, increment of cysteine SO2 Prx III was not observed. Meanwhile, Srx, reducing enzyme of SO2 Prx, was induced in MM cells after BTZ treatment. Down regulation of Srx by siRNA did not promote ROS generation or apoptosis in BTZ-treated MM cells, otherwise.

Conclusions : Our results showed inactivation of Prx III by multimer formation as hyperoxidation is found during BTZ-induced mitochondrial ROS generation and apoptosis in MM cells. This Prx III oxidation was due to down regulation of reduced Trx 2 in BTZ-treated MM cells. To design of raising ROS stress and down regulation of anti-oxidants(ie, Trx), as a treatment-strategies may be worthwhile to potentiate BTZ-induced apoptosis in MM cells.

Disclosures

No relevant conflicts of interest to declare.
Reducing on-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors By Affinity Optimization
Authors: Drent Esther , Themeli Maria , Poels Renée , van de Donk Niels W.C.J. , Lokhorst Henk M. , Mutis Tuna
Published: October 2019

Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody (ab)-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells endowed with tumor-reactive CARs are highly promising tools for immunotherapy of cancer. There are however only a few truly tumor specific molecules that can be targeted by CARs, a drawback for the broad application of CAR T cell therapy. Indeed, when we recently aimed at targeting CD38high multiple myeloma (MM) with T cells transduced with high affinity CD38CARs, we observed that they not only lysed the CD38high MM cells but also CD38+ normal hematopoietic cells, pointing towards potential safety issues of such tumor-associated, but not entirely tumor-specific CARs. Therefore, using CD38 as a model for antigen we now tested whether it would be possible to reduce the on target, off-tumor effects of such CARs by optimizing their target cell affinity. To this end, we generated a new panel of CD38 abs through the "light chain exchange" method, in which heavy chains of two high affinity CD38 abs were combined with 176 different germ line light chains. This approach revealed around 100 new abs, which displayed 10- >1000 fold lower affinity to CD38 as compared to the parental abs. After categorizing them in three classes based on CD38 binding affinity, we used 8 abs from each class to generate 24 different CD38-CAR constructs. Testing the cytotoxic activity of T cells transduced with these CD38-CARs against CD38++ MM cell lines, primary MM cells and CD38+ normal hematopoietic cells in vitro and in vivo demonstrated that CD38-CAR T cells with ca. 1000 fold lower affinity to CD38 could still effectively lyse CD38++ MM cells while there was little or no cytotoxicity against CD38+ healthy hematopoietic cells. The results of this study reveal that it is possible to reduce the on-target off-tumor effects of CARs by optimizing their affinity. Thus, tailored affinity of the ab binding domain may open up new roads for CAR therapy.

Disclosures

van de Donk: Janssen: Research Funding; Celgene: Research Funding; BMS: Research Funding; Amgen: Research Funding. Lokhorst:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.
High Disease-Free and Overall Survival Rate Following Allogeneic Hematopoietic Stem Cell Transplantation for FLT3-Mutated Acute Myeloid Leukemia Even in Non-Remission Status
Authors: Nishida Aya , Yuasa Mitsuhiro , Kageyama Kosei , Ishiwata Kazuya , Takagi Shinsuke , Yamamoto Hisashi , Asano-Mori Yuki , Yamamoto Go , Uchida Naoyuki , Izutsu Koji , Wake Atsushi , Yoneyama Akiko , Makino Shigeyoshi , Taniguchi Shuichi
Published: October 2019

[Background] FMS like tyrosine kinase 3 (FLT3) mutations occur in about 30% of patients with acute myeloid leukemia (AML). Patients with FLT3-mutated AML have a poor prognosis and are referred for early allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1). So far, there is still limited data available on allo-HSCT for FLT3-mutated AML in non-remission status.

[Objective and method] To assess the clinical features and outcome of allo-HSCT for FLT3-mutated AML, we retrospectively analyzed patients underwent first allo-HSCT for FLT3-mutated AML excluding acute promyelocytic leukemia (FAB M3) from January 2011 to March 2016.

[Result] During the study period, 332 patients received first allo-HSCT for AML in our institute. One hundred and thirty-eight were tested for the presence of FLT3-mutation and 35 showed positive results and were subjected to the analysis. The median follow-up day of survivors was 602 (101-1867). The median age of the patients was 55 years (range, 21-72). Twenty-one patients had de novo AML, 12 had AML with myelodysplasia related changes, and 2 had therapy related AML. Eighteen had normal karyotype, 4 had complex, and 13 had others. Seven were in remission (5 in CR1, and 2 in CR2), and 28 were in non-remission (8 in primary induction failure, 13 in relapse 1, and 7 in chemo naïve status). Twenty-nine patients used unrelated cord blood, 2 did unrelated bone marrow, and 4 did related peripheral blood stem cell as grafts. All but 1 received myeloablative pretransplant conditionings. At 2 years after transplantation, overall survival (OS), disease free survival (DFS), relapse rate (RR), and non-relapse mortality (NRM) of whole studied population were 65.9%, 50.2%, 28.4%, and 21.4%, respectively. Among those in non-remission before transplantation, OS, DFS, RR, and NRM at 2 years post-transplant were 62.2% (Figure 1A), 49.9%(Figure 1B), 24.3%, and 25.8%, respectively. Only younger age (<55 years) was the factor associated with better OS with statistical significance in multivariate analysis.

[Conclusion] Our data indicated that allo-HSCT could overcome the poor prognosis of FLT3-mutated AML even for those in non-remission status, despite the profound chemo-resistant character of FLT3-mutated AML.

Figure 1A Figure 1A. Figure 1B Figure 1B.

Disclosures

Izutsu: Abbvie: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical K.K.: Honoraria; Eisai: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria; Mundipharma KK: Research Funding.
Precise and Efficient Crispr/Cas9 Mediated Gene Editing in Long-Term Engrafting Human Hematopoietic Stem/Progenitor Cells
Authors: Heath Jack M , Chalishazar Aditi , Lee Christina S , Selleck William , Cotta-Ramusino Cecilia , Bumcrot David , Gori Jennifer L
Published: October 2019

Transplantation of gene-modified autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for several hematologic diseases. However, a number of blood disorders may not be amenable to gene augmentation-based therapeutics. Targeted genome editing in human HSPCs could provide a therapeutic approach for these otherwise untreatable diseases. Here we demonstrate that CRISPR/Cas9 ribonucleoprotein (RNP) edits target genes in human HSPCs with high efficiency and precision. Human adult and umbilical cord blood (CB) CD34+ cells from 20 donors were electroporated with S. pyogenes or S. aureus Cas9 RNP targeting HBB, AAVS1, or CXCR4. Sequence analysis demonstrated up to 80% editing in CB CD34+ cells (mean±s.d: 61%±9%) and up to 57% in adult CD34+ cells (39%±13%). Delivery of Cas9 RNP and a single-stranded oligodeoxynucleotide donor (ssODN) led to up to 12% ssODN-mediated homology directed repair (HDR) and also led to a 20% increase in total gene editing (HDR+NHEJ)(RNP: 48%±15%; RNP+ssODN: 69%±8%). Both Cas9 RNP gene-edited CD34+ cells and donor-matched untreated control CD34+ cells reconstituted human hematopoiesis in primary and secondary recipient immunodeficient mice, with ~85% human CD45+ cell peripheral blood reconstitution 4 months after primary transplantation. Human T and B lymphoid, erythroid, and myeloid cells were detected in the spleen, thymus, and bone marrow with 20% CD34+ cell engraftment in the marrow of mice transplanted with RNP gene-edited or control CD34+ cells. The level of targeted gene editing in human erythroid, myeloid, and CD34+ cells that were recovered and enriched from the hematopoietic organs of primary recipients (~50%) was similar to the level of gene editing detected in the pre-infusion product (~60%). In summary, these results indicate that Cas9 gene-edited human HSPCs retain long-term engraftment potential and support multilineage blood reconstitution in vivo, thus supporting further investigation of CRISPR/Cas9 mediated gene-edited hematopoietic stem/progenitor cell therapies.

Disclosures

Heath: Editas Medicine: Employment. Chalishazar:Editas Medicine: Employment. Lee:Editas Medicine: Employment. Selleck:Editas Medicine: Employment. Cotta-Ramusino:Editas Medicine: Employment. Bumcrot:Editas Medicine: Employment. Gori:Editas Medicine: Employment.
Strategies for Selection of AAV Vectors for Administration to Liver: Studies in Nonhuman Primates
Authors: Wang Lili , Wilson James M , Calcedo Roberto , Bell Peter , He Zhenning , White John , Hewitt Matthew , Makaron Leah , Kattenhorn Lisa , Wadsworth Sam , Goode Tamara
Published: October 2019

Vectors based on adeno-associated virus (AAV) have demonstrated early promise in clinical trials, including published reports in patients with hemophilia B where therapeutic levels of factor IX have been achieved using AAV serotype 8, a member of the clade E family. With the success of these hemophilia B clinical trials utilizing AAV8, numerous alternative AAV capsid types that target the liver have begun to enter clinical testing across multiple disease indications. Specific properties of the various AAV capsids should be taken into account such as overall efficiency of hepatocyte gene transfer, differential gene transfer across the porto-central axis within the liver, durability of gene expression and the potential for vector re-administration. In this study, we evaluated these aspects of AAV-directed liver gene transfer in rhesus macaques across various capsid serotypes. Animals were injected with vectors expressing the secreted reporter gene rhesus bhCG and produced using different capsids [AAV5, AAV3b and two clade E vectors (AAVrh10 and AAV8)]. Nonhuman primates (NHPs) injected with clade E vectors expressing bhCG were administered 3 months later with AAV5 or AAV3b vectors expressing rhesus derived AFP. The key findings were: 1) in naïve animals, clade E vectors demonstrated the highest levels of periportal gene transfer with AAV5 vectors having the lowest levels of periportal gene transfer; 2) AAVrh10 and AAV5 elicited higher levels of neutralizing antibodies (NAb) than AAV8 and AAV3b; 3) significant animal-to-animal variation in transgene expression was noted with AAV3b in seronegative animals; and 4) within the short time frame tested NAb elicited from AAVrh10 appears to have inhibited subsequent in vivo transduction with the serologically distinct AAV3b serotype; prior exposure to AAV8 did not interfere with AAV3b transduction. These studies highlight the influence that capsids can play in efficiency and immunogenicity of AAV vectors for liver gene therapy.

Disclosures

Wilson: Dimension Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity's Board of Directors or advisory committees; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kattenhorn:Dimension Therapeutics: Employment. Wadsworth:Dimension Therapeutics: Employment, Equity Ownership.
Mutation of a GATA1 Binding Site in ALAS2 Intron 1 Arrests Murine Erythroid Development In Vivo
Authors: Zhang Yingchi , Zhang Jingliao , An Wenbin , Wan Yang , Gao Jie , Shi Lihong , Cheng Tao , Zhu Xiaofan
Published: October 2019

Congenital sideroblastic anemia (CSA) comprises a heterogeneous group of genetic disorders characterized by decreased heme synthesis and the presence of bone marrow (BM) ring sideroblasts. The most common form of CSA is X-linked sideroblastic anemia (XLSA, OMIM#300751), which is caused by mutations in the gene encoding 5-aminolevulinic acid synthase 2 (ALAS2). However, to date the experimentally confirmed ALAS2 mutations mostly occurred at the exons of ALAS2 and are missense mutations.

In our study, we identified a non-coding GATA1 binding site mutation in ALAS2 intron 1 in a large XLSA pedigree. We addressed the functions of the GATA1 binding site in ALAS2 intron 1 in vivoby generating mice lacking this GATA site and its flanking sequence (13bp) and found that this deletion led to an embryonic lethal phenotype due to severe anemia, indicating that this fragment is indispensable during erythroid development. Next we demonstrated that GATA1 activated ALAS2 transcription by forming an enhancer loop with TAL1, LDB1 and Pol II through GATA binding sites in the promoter and in introns 1 and 8. Finally, we discovered that int-1-GATA site deletion completely abolished ALAS2 transcription due to disruption of the enhancer loop and a simultaneous lack of Pol II enrichment at the intron 1 enhancer region and promoter.

Our findings first revealed the essential roles of GATA1 binding site in ALAS2 intron 1 in erythroid development in vivo and uncovered the mechanisms how GATA1 regulated the expression of ALAS2. We believe that these findings will be of wide general interest and will provide valuable information for the clinical diagnosis of XLSA patients with unknown mutations.

Disclosures

No relevant conflicts of interest to declare.
Corticosteroids Impair Granulocyte Transfusion Therapy By Targeting NET Formation and Neutrophil Antifungal Functions Via ROS/Dectin1 Pathways
Authors: Kalleda Natarajaswamy , Amich Jorge , Poreddy Spoorthi , Arslan Berkan , Friedrich Mike , Mokhtari Zeinab , Ottmüller Katja , Jordán-Garrote Ana-Laura , Einsele Hermann , Brock Matthias , G Heinze Katrin , Beilhack Andreas
Published: October 2019

Introduction:

Invasive pulmonary Aspergillus fumigatus infections cause high morbidity and mortality in neutropenic patients. Granulocyte transfusions have been tested as an alternative therapy for the management of high-risk neutropenic patients with invasive A. fumigatus infections. To increase the granulocyte yield for transfusion, donors are treated with corticosteroids. Yet, the efficacy of granulocyte transfusion and functional defense mechanisms of granulocytes collected from corticosteroid treated donors remain largely elusive. Therefore, we investigated the efficacy of granulocyte transfusion and functional defense mechanisms of corticosteroid treated granulocytes using mouse models.

Methods:

To determine the effects of corticosteroids on granulocytes to control A. fumigatus infections, we performed granulocyte adoptive cell transfers using in vivo mouse models, in vitro human and mouse granulocyte and A. fumigatus functional co-culture experiments in combination with flow cytometry, cytokine analysis, fluorescence and electron microscopy.

Results:

Transfusion of granulocytes from corticosteroid treated mice did not protect cyclophosphamide immunosuppressed mice against lethal A. fumigatus infection in contrast to granulocytes from untreated mice. Upon infection increased levels of inflammatory cytokines helped to recruit granulocytes to the lungs without any recruitment defects in corticosteroid treated and infected mice or in cyclophosphamide immunosuppressed and infected mice that had received the granulocytes from corticosteroid treated mice. However, corticosteroid treated human or mouse neutrophils failed to form neutrophil extracellular traps (NETs) under in vitro and in vivo conditions. Furthermore, corticosteroid treated granulocytes exhibited impaired ROS production against A. fumigatus. Notably, corticosteroids impaired the β-glucan receptor Dectin-1 (CLEC7A) on mouse and human granulocytes to efficiently recognize and phagocytize A. fumigatus, which markedly impaired fungal killing.

Conclusions:

We conclude that corticosteroid treatment of granulocyte donors for increasing neutrophil yields or patients with ongoing corticosteroid treatment could result in deleterious effects on granulocyte antifungal functions, thereby limiting the benefit of granulocyte transfusion therapies against invasive fungal infections.

Disclosures

Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau.
The Role of Cbx Proteins in Human Benign and Malignant Hematopoiesis
Authors: Jung Johannes , Schepers Hein , Lazare Seka , Buisman Sonja , Weersing Ellen , Dethmers Bertien , Klauke Karin , Bystrykh Lenja , De Haan Gerald
Published: October 2019

Introduction:

Hematopoietic stem cells (HSCs) need to properly balance self-renewal and differentiation to prevent malignant transformation or HSC exhaustion. We recently showed that the presence of specific Cbx proteins in the Polycomb Repressive Complex 1 (PRC1) plays a crucial role in balancing self-renewal and differentiation of murine HSCs. Whereas Cbx7 induces self-renewal, Cbx4 and Cbx8 instead induce differentiation. In this project, we investigate the role of various CBX Polycomb proteins in human normal and malignant hematopoiesis. We aim to identify genes which are controlled by CBX7 in normal and malignant human hematopoiesis.

Methods:

We overexpressed CBX2, -4, -6, -7 and -8 in human CD34+HSPCs. We assessed functional consequences by measuring cobblestone area-forming cell (CAFC), colony-forming unit (CFU) frequencies, and performed stem cell xenotransplantation studies in NSG mice. To identify genes which are controlled by CBX7 or CBX8, we performed RNA- and Chip-Seq in CD34+ HSPCs. To explore the role of CBX7 in leukemic cells, we performed short hairpin RNA-mediated knockdown of protein expression in leukemic cell lines.

Results:

Overexpression of CBX7 and CBX8 in human CD34+ HSPCs led to a significant 5-10-fold increase of week 5 CAFC (fold increase: 9,62 for CBX7; 4,92 for CBX8) and 1,5-fold increase CFU-frequencies (fold increase: 1,32 for CBX7; 1,41 for CBX8) after 14 days. In contrast, overexpression of CBX4 and CBX2 led to equal or decreased CAFC- and CFU-frequencies. Transplantation of CBX7 overexpressing human CD34+ HSPCs in NSG mice led to higher long-term engraftment in comparison to the empty vector control (p<0,05 after 18 weeks). Even after one week of in vitro culture of CBX7-overexpressing CD34+cells significant engraftment levels were obtained. Conversely, downregulation of CBX7 in various leukemic cell lines resulted in markedly decreased proliferation and strongly induced differentiation. Expression analysis showed that genes that are upregulated upon CBX7 overexpression are preferentially expressed in primitive stem cells, and are repressed in more differentiated cell types.

Conclusions:

Our study indicates that CBX7 and CBX8 regulate self-renewal of human HSPCs. Furthermore, our data show that repression of CBX7 markedly inhibits proliferation of leukemic cells and can release the differentiation block of leukemic cells in vitro. Collectively our results indicate that targeting CBX7 may be a viable strategy to induce terminal differentiation of leukemic cells.

Disclosures

No relevant conflicts of interest to declare.
A Novel Humanised ROR1 Bi-Specific T-Cell Engager Molecule for the Treatment of Chronic Lymphocytic Leukaemia
Authors: Gohil Satyen Harish , Harrasser Micaela , Paredes-Moscosso Solange R , Nathwani Amit C , Della Peruta Marco
Published: October 2019

Bispecific T-cell Engagers (BiTEs) are a promising immunotherapeutic approach, usually consisting of two single chain variable fragments (scFv) linked in tandem. One arm binds to tumour-associated antigens, whilst the other binds CD3 on T-cells, facilitating the formation of cytotoxic synapses leading to killing of tumour targets in a MHC and co-stimulatory molecule independent manner. Blinatumomab, the most advanced BiTE in clinic targets CD19 and has shown impressive results in patients with relapsed/refractory B cell acute lymphoid leukaemia but results in elimination of normal and malignant B lymphocytes.

Receptor Tyrosine Kinase Like Orphan receptor is a surface antigen expressed on a range of haematological and solid malignancies, including Chronic Lymphocytic Leukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL), and cancer stem cell-like cells (CSC). It is however expressed at low or undetectable levels in normal tissues making it an attractive target for immunotherapy approaches. We generated a panel of novel fully humanised ROR1 antibodies and selected an optimal scFv based on ROR1 binding to use in our BiTE format.

Our ROR1 BiTE (ROR1xCD3), at low concentrations, was efficient inducing T-cell mediated cytotoxicity and consequent inflammatory cytokine secretion against a panel of haematopoietic cell lines with constitutive ROR1 expression in vitro. Importantly cytotoxicity was comparable to that seen with a control CD19 BITE. Moreover, ROR1xCD3 led to significant and selective cytotoxicity of primary CLL samples. We transitioned our in vitro studies to animal models and showed that our ROR1xCD3 is able to retard growth of established SKW cells engrafted in immunocompromised mice, as detected by bioluminescence imaging. Additionally ROR1xCD3 improved survival, with the ROR1xCD3 treated cohort showing improved median survival of 50 days compared to control mice of 18 days despite only receiving a single infusion of T-cells.

In summary, we have developed a novel ROR1 BiTE able to target an array of haematopoietic malignancies. Our ROR1 BiTE induces T-cell cytotoxicity against ROR1 positive cell lines in vitro and in vivo as well as primary CLL cells and has the potential to be a beneficial therapeutic for CLL.

Disclosures

Gohil: UCL Business: Patents & Royalties: ROR1 based immunotherapies. Paredes-Moscosso:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies.
Economic Evaluation of the Direct Oral Anticoagulants Compared to Vitamin K Antagonists in the Treatment of Deep Venous Thrombosis and Pulmonary Embolism in the Outpatient Setting: Analysis from the Queen Elizabeth II Health Sciences Centre in Halifax, Nova Scotia
Authors: Al Saleh Abdullah S. , Berrigan Patrick , Anderson David , Shivakumar Sudeep
Published: October 2019

Background: To date, there have been few economic evaluations of the direct oral anticoagulants in the treatment of venous thromboembolism (VTE) conducted from a Canadian perspective. As a result, there is a lack of consensus within existing literature regarding which treatment strategy should be adopted into the clinical setting. To our knowledge, this is the first Canadian economic evaluation in patients with VTE who are treated solely on an outpatient basis.

Objectives: We conducted a cost-minimization analysis to determine the least costly intervention for the prevention of recurrent venous thrombosis in patients with acute unprovoked VTE including apixaban, rivaroxaban, low molecular weight heparin (LMWH) in combination with dabigatran, and LMWH overlapped with vitamin K antagonists (VKA). We also conducted a cost-effectiveness analysis to assess value for money in terms of cost per quality adjusted life year (QALY) gained.

Methods: We used Microsoft Excel to develop a decision model. Decision model parameters were determined using published literature, local hospital data, expert opinion, and chart review. Our cost-minimization analysis aggregated costs related to pharmaceuticals, laboratory testing, and hematologist fees. Our cost-effectiveness analysis also included costs related to health outcomes.

Results: Our cost-minimization analysis found apixaban to be the least costly intervention at three months ($609.28). At six months and one year LMWH overlapped with VKA was the least costly intervention ($896.08 and $1,193.88, respectively). With respect to cost-effectiveness, at three months apixaban was the dominant strategy. At six months, apixaban was cost-effective given a willingness-to-pay per QALY as low as $4,986.81. At one year, LMWH overlapped with VKA was cost-effective given a willingness-to-pay per QALY below $135,670.28. Probabilistic sensitivity analysis of our cost-effectiveness analysis found apixaban to be the dominant strategy compared to LMWH overlapped with VKA in 42.6% of iterations and 70.6% of iterations resulted in incremental cost-effectiveness ratio below $100,000 per QALY gained.

Conclusions: Our findings suggest apixaban is the least costly and most cost-effective strategy for short-term anticoagulant treatment for VTE but favor LMWH overlapped with VKA for long-term treatment.

Disclosures

No relevant conflicts of interest to declare.
Global Prospective Cohort Study of Factors Associated with Venous Thromboembolism in Patients with Lung Cancer Receiving Cancer Therapy
Authors: Kuderer Nicole M. , Poniewierski Marek S. , Culakova Eva , Lyman Gary H. , Khorana Alok A. , Pabinger Ingrid , Agnelli Giancarlo , Liebman Howard A. , Vicaut Eric , Meyer Guy , Shepherd Frances A.
Published: October 2019

BACKGROUND: Patients with lung cancer are known to be at increased risk for venous thromboembolism (VTE). However, there have been few studies of risk factors for VTE in lung cancer patients undergoing systemic chemotherapy.

METHODS: CANTARISK was a prospective, non-interventional, global cohort study including patients with lung cancer initiating a new chemotherapy regimen. Clinical data were collected at baseline and at 2, 4 and 6 months follow-up. The impact of patient-, disease- and treatment-related factors on the occurrence of VTE in the first 6 months was evaluated in univariable and multivariable Cox regression analyses.

RESULTS: A total of 1,980 patients with lung cancer were enrolled from 2011-12 of which 84% were diagnosed with non-small cell lung cancer (NSCLC). Median age was 63 years (range, 25-91) and 63% were male while 82% were active or former smokers. Race was white (70%), Asian (22%), or black (4%) with similar numbers from North America, Europe, and other regions including Asia. Metastatic disease was reported in 70% and ECOG PS was ≥2 in 13%. During the first six months, 121 patients developed a VTE (6.1%), of which 47.1% had pulmonary embolism (PE), 45.5% deep venous thrombosis (DVT), 3.3% catheter-associated thrombosis, and 4.1% visceral thrombosis. Among significant factors in univariable analysis, independent predictors for VTE in multivariable Cox regression analysis included female gender, US geographic region, leg immobilization, and presence of a central venous catheter (Table) with a trend toward greater risk for higher grade histology. Although predictive of early all-cause mortality in this study population (Kuderer et al ASCO 2016), the previously validated risk score for VTE in ambulatory cancer patients (Khorana et al: Blood 2008) was not significantly associated with VTE in either univariable or multivariable analysis.

CONCLUSIONS: Several demographic, geographic, and clinical factors are significantly associated with an increased risk of VTE in patients with lung cancer receiving systemic chemotherapy. Future analysis will attempt to assess how novel targeted treatment options might impact the Khorana score's predictive ability across all lung cancer patients.

Disclosures

Kuderer: Janssen Scientific Affairs, LLC: Consultancy, Honoraria. Lyman:Amgen: Research Funding. Khorana:Bayer: Consultancy, Honoraria; Leo: Consultancy, Honoraria, Research Funding; Halozyme: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Janssen Scientific Affairs, LLC: Consultancy, Honoraria, Research Funding.
Knock-Down of the NFAT2 Long and Intermediate Isoforms Leads to CLL Acceleration
Authors: Heitmann Jonas S , Märklin Melanie , Anastasia Posdorowkin , Fuchs Alexander R , Truckenmüller Felicia M , Kopp Hans-Georg , Kanz Lothar , Rao Anjana , Wirths Stefan , Mueller Martin R
Published: October 2019

NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. We and others have previously described a significant overexpression of NFAT2 in CLL cells as compared to physiological B cells. Three major isoforms of NFAT2 with different regulatory properties have been described (700aa short isoform, 800aa intermediate isoform, 900 aa long isoform). Here, we analyzed the role of different NFAT2 transcripts in CLL with respect to disease phenotype and cell proliferation.

We investigated primary samples from CLL patients (n=30) for their expression profile of different NFAT2 isoforms using RT-PCR. Applying an shRNA approach, we generated stable knock-down cells of the CLL cell line MEC-1 for the long and intermediate isoforms and for the entire NFAT2 gene resulting in the complete ablation of all isoforms. The proliferation properties of the different MEC-1 cell lines was subsequently assessed in xeno-transplant experiments into NSG mice.

While physiological B cells express comparable levels of the short and intermediate/long isoforms, we could detect a five fold overexpression of the intermediate/long isoforms in primary CLL samples. To further analyze the differential regulation of the different NFAT2 transcripts on tumor cell proliferation and cell cycle regulation, we injected NSG mice with MEC-1 cells with intact NFAT2 (n=6), MEC-1 cells with a knock-down of the intermediate and long isoforms (n=6) and MEC-1 cells with a complete NFAT2 knock-down (n=6). MEC-1 cells with selective ablation of the intermediate and long NFAT2 isoforms grew significantly faster in NSG mice than MEC-1 cells with intact NFAT2 expression or MEC-1 cells with a complete NFAT2 knock-down. MEC-1 cells selectively lacking the intermediate and long isoforms led to accelerated tumor proliferation upon subcutaneous injection. Cell cycle analysis as assessed by flow cytometry showed a significantly increased number of cells in the G1/S-Phase for the group without expression of the short isoform, while the group with complete NFAT knock-down exhibited a compromised growth pattern as compared to wild-type MEC-1 cells.

In summary, our data demonstrate that genetic loss of the intermediate and long isoforms of NFAT2 leads to CLL acceleration

Disclosures

No relevant conflicts of interest to declare.
Ongoing Phase 1a/1b Dose-Finding Study of CWP232291 (CWP291) in Relapsed or Refractory Multiple Myeloma (MM)
Authors: Yoon Sung-Soo , Min Chang-Ki , Kim Jin Seok , Manasanch Elisabet E. , Hauptschein Robert , Choi Jeongeun , Lee Kyoung-June
Published: October 2019

Background: CWP291, a novel peptidomimetic small molecule, has potent and selective inhibitory activity on a Wnt gene reporter and decreases expression of the β-catenin target genes, cyclin D1 and survivin. It has broad anti-cancer efficacy in vitro, outperforming lenalidomide and bortezomib in multiple myeloma murine xenografts and demonstrating improved survival when used in combination with lenalidomide in bone marrow engraftment models.

Methods: Subjects with relapsed or refractory MM were infused CWP291 iv over at least 30 minutes twice weekly for 3 weeks out of a 4-week cycle in a 3+3 dose-escalation, Phase 1 design.

Results: Results as of July 15, 2016 over a dose range of 198 - 446 mg/m2 in 9 subjects are reported. Median duration of treatment was 3 cycles (range 1.3 - 6.5 cycles) with 3 subjects ongoing. The most common (>20% of subjects) drug-related adverse events (AEs) were due to GI toxicities, including diarrhea (77.8%), nausea (55.6%) and constipation (33.3%). Toxicity was mostly ≤grade 2, however grade 3/4 events were frequently related to myelosuppression, including neutropenia (33.3%), and anemia and thrombocytopenia (22.2% each). Serious adverse events were recorded for 5 subjects with 8 separate events: 6 events in 4 subjects considered unrelated were hospitalizations for pneumonia, and 2 events in 1 subject each considered possibly related were a urinary tract infection and grade 3 hypoxia. Dose-limiting toxicities occurred in 2 patients at 446 mg/m2, which were hypoxia (grade 3) and thrombocytopenia (grade 4). Accrual at 335 mg/m2 has now been initiated. Stable disease (≥3 months on therapy with no increase in M protein as per the IMWG) was seen in 4 subjects. There was also documentation of a 34.6% decrease in bone marrow plasma cells in one subject for whom a bone marrow aspirate was available. PK in the first 3 subjectsreceiving198 mg/m2showed plasma levels of the active metabolite had very little inter-patient variability, which were comparable to patients with AML receiving this same dose in another study. There was less than 4% excreted in the urine, and there was no evidence of accumulation from Day 1 to Day 18. Changes in targeted genes in blood and BM, as well as additional PK, will be provided from expanded and escalated dosing cohorts. Furthermore, preliminary observations in the Phase 1b part of this study, which combines the administration of CWP291 with lenalidomide and dexamethasone, will be reported.

Conclusions: CWP291, a novel and first in class molecule with significant preclinical activity, shows single agent safety as well as early evidence of efficacy in subjects with MM. Accrual is ongoing to establish the optimal dose level for both single agent and combination therapy.

Disclosures

Hauptschein: JW Theriac Pharmaceutical: Employment. Choi:JW Pharmaceutical Corporation: Employment. Lee:JW Pharmaceutical Corporation: Employment.
Rate of Rise of EBV Viral Load By Quantitative PCR after Allogeneic Transplantation Correlates with PTLD Facilitates Timely Institution of Rituximab
Authors: Muffly Lori S. , Shiraz Parveen , Nguyen Isabelle T , Brown Janice (Wes) M.
Published: October 2019

A growing body of data supports the benefit of first-line rituximab therapy for EBV- associated post-transplant lymphoproliferative disorder (PTLD) the potential for adoptive cellular therapy, and the benefit of early diagnosis. Quantitative PCR has proven to be an important, albeit not infallible, diagnostic test; however, data regarding the utility We reviewed the records of 731 adult patients who underwent allogeneic hematopoietic cell transplantation (HCT) at Stanford University Medical Center from January 2010 through Feb 2016.

(Table 1. Demographics) With respect to EBV screening, all patients received 400 mg po acyclovir tid for one year, preemptive strategies based on plasma PCR for CMV were used with either IV ganciclovir (MA) or valganciclovir (NMA) administered. Quantitative EBV PCR was performed on whole blood (Jan 2010 through mid-2011) or plasma (mid-2011 to present) every other week in asymptomatic patients or at any time a patient developed symptoms that may be attributed to EBV.

Observations/Conclusions:

· Both the initial measureable and the rate of increase in viral load were significantly greater in patients with PTLD than those with isolated viremia.

· Promptly administered rituximab was effective in improving resolution of PTLD when compared to historically reported data.

· Conversely, patients with low grade viremia without increase in viral load may be watched expectantly.

· Although not standard practice, importance of EBV serology of donor has been demonstrated in SOT and pediatric HCT and would be anticipated to be of equal importance in adults, especially

· Early biopsy or flow cytometric analysis of EBV+ lymphocyte to determine polymorphic versus monomorphic disease can further refine our understanding the pathophysiology of disease

· The crucial limitation of our study is that determination of EBV serology was not routine; however, by limiting our analysis to patients with EBV reactivation and the use of a standardized protocol to respond to viremia has permitted the current observations to be made.

Table 2 Table 2.

Disclosures

No relevant conflicts of interest to declare.
RAD51 Modulators Induce Mitotic Catastrophe in Aid Expressing Cells through Multiple Pathways
Authors: Mills Kevin , Hasham Muneer , Lamont Kristin , Philip Vivek , Branca Jane
Published: October 2019

The B-cell specific DNA mutase/recombinase Activation Induced Cytidine Deaminase (AID) initiates DNA double strand breaks (DSB) necessary for immunoglobulin class switch recombination in activated B-cells. However, AID-induced DSBs are not restricted to immunoglobulin loci, and we previously demonstrated that the homologous recombination (HR) factor RAD51 is critical to repair widespread AID-initiated DSBs. While it is known that failure to properly repair AID-induced genomic DSBs is cytotoxic to B-cells, the pathways culminating in cell death were not fully known. Here, we demonstrate that AID-initiated DSBs can trigger mitotic catastrophe, a non-apoptotic cell death mechanism. Using genetic and chemical biology approaches we further show that AID-induced mitotic catastrophe involves multiple pathways, including mTOR. We also demonstrate that AID-induced cytotoxicity can be used to effectively and preferentially target AID-expressing lymphoid malignancies, in xenograft cancer model. This study reveals a cell death response to genomic damage by AID, providing new insight into the mechanisms that ensure cellular survival during B-cell maturation. Our findings support the idea that RAD51 modulation may be leveraged as a novel "synthetic lethal" therapeutic approach for treating AID-expressing malignancies, via the induction of mitotic catastrophe.

Disclosures

Mills: Cyteir Therapeutics, inc: Employment, Equity Ownership, Patents & Royalties. Hasham:The Jackson Laboratory: Patents & Royalties.
Anemia in Diabetic Patients without Underlying Nephropathy. a Retrospective Cohort Study
Authors: Mahjoub Ali R. , Patel Eshan , Ali Sami , Webb Kendal , Astrow Alan , Kalavar Madhumati
Published: October 2019

Background: Anemia (defined by the World Health Organization as a hemoglobin level of less than 13 mg/dl in men and less than 12 mg/dl in women) is common in diabetic patients, particularly in those with reduced renal function. Most studies attribute anemia in diabetic patients to kidney function impairment. There are no controlled systematic studies of the prevalence and predictors of anemia in patients with diabetes in the absence of overt nephropathy.

Objective: This retrospective cohort study was designed to measure the prevalence of anemia in diabetic patients with normal kidney function (estimated glomerular filtration rate (EGFR) greater than 90 ml/min/1.73 m2 and negative micro albuminuria) and compare it to a control group of non-diabetic patients. The study was designed to investigate diabetes as an independent risk for causing anemia.

Methods: We undertook a retrospective review of medical records of 400 patients (older than 18 years old) visiting the outpatient clinic in our institution between January and June 2015. 200 patients with diabetes (glycosylated hemoglobin (HgA1C) greater than 7) were compared to 200 non-diabetic patients (HgA1C level below 5.6), to identify the prevalence of anemia, with and without kidney disease and any other associated factors.

Results:The prevalence of anemia in all diabetic patients was 22% vs. 9% in non-diabetic group (OR: 2.69, 95% CI; 1.49 to 4.86, P = 0.001). Out of the 22% (n = 44) anemic patients, 18 patients had anemia with normal kidney function, out of those; 5 had iron deficiency anemia and one had autoimmune disease, while the remaining 12 patients (6%) did not have any obvious cause of anemia other than diabetes vs. 2 patients (1%) in non-diabetic patients who we did not find any explanation for the their anemia (OR: 6.6, 95% CI; 1.45 to 30, P < 0.015).

Conclusion: Most studies have highlighted an association between anemia and nephropathy in diabetic patients. This retrospective cohort study suggests a high incidence of anemia with unidentified pathology in the diabetic population. The mechanism of this anemia is not well understood, although direct glucose toxicity to erythrocyte precursors in the bone marrow or oxidative stress to mature erythrocytes are both possibilities. Prospective study of hemoglobin levels in patients with diabetes may help elucidate the precise mechanism of anemia in this group.

Disclosures

No relevant conflicts of interest to declare.