bluuebunny/crossref_metadata_2025 huggingface dataset:
ERV-9 LTR Retrotransposon Modulates Globin Gene Switching
Authors: Tuan Dorothy
Published: October 2019

In the human beta-globin gene locus, an LTR retrotransposon derived from ERV-9 human endogenous retrovirus is located near the locus control region (LCR) far upstream of the globin genes. In transgenic mice carrying the 100 kb human globin gene locus, deleting the ERV-9 LTR by cre-loxP mediated in situ recombination inactivates transcription of beta-globin gene by ~50% and activates that of gamma-globin gene by up to 5 fold, to at least 20% the level of beta-globin mRNA in both fetal and adult erythroid cells. Chromosome-conformation-capture (3C) shows that the ERV-9 LTR preferentially loops with beta-globin gene, even in fetal erythroid cells where gamma-globin gene is predominantly transcribed. Unique in the locus, the ERV-9 LTR contains high density of CCAAT motifs that strongly bind transcription factor (TF) NF-Y critical for assembling the LTR enhancer complex but no CCNCNCCC motifs that bind master erythroid TF KLF1, while the beta-globin promoter contains two tandem CCNCNCCC motifs that bind KLF1 but a CCAAT motif that only weakly bind NF-Y. However, both the LTR and beta-globin promoter associate in vivo with high levels of NF-Y and KLF1 in ChIP assays. Protein immunoprecipitation shows that neither NF-Y nor KLF1 associates with transcription co-regulator Ldb1, which has been reported to mediate chromosome looping of the LCR with the globin genes. Thus, the Ldb1/LMO2/GATA -1, -2 complex potentially assembled by the GATA motifs distributed throughout the locus does not appear to mediate the preferential looping of ERV-9 LTR with beta-globin promoter. Through interaction of NF-Y and KLF1 mediated by the to be identified co-regulator, the ERV-9 LTR interacts with and activates beta-globin gene, and suppresses gamma-globin gene by a competitive mechanism. Deletion of the ERV-9 LTR removes the competitive advantage of beta-globin gene, thus re-activating gamma-globin gene. Our findings suggest that deleting the ERV-9 LTR from the globin locus by genome-editing could provide a potential new therapy for beta-hemoglobinopathies.

Disclosures

No relevant conflicts of interest to declare.
Prothrombin Complex Concentrate Is Safe and Effective for Achieving Hemostasis in the Setting of Urgent Surgery for Patients Treated with Direct Xa Inhibitors
Authors: Barzilai Merav , Kirgner Ilya , Steimatzky Arza , Salzer Gotler Dalit , Belnick Yulia , Avivi Irit , Varon David , Shacham Adi , Yahalom Vered , Nakav Sigal , Yussim Ethan , Bahar Gil , Rozovski Uri , Spectre Galia
Published: October 2019

Introduction: Patients treated with direct Xa inhibitors may require an urgent surgery. A reversal agent for Xa inhibitors was recently approved for major bleeding but not prior to urgent surgery. Administration of prothrombin complex concentrate (PCC) in this setting is a common practice; however, it is based on limited experience in healthy volunteers.

Objective: To characterize the population receiving PCC for apixaban/rivaroxaban reversal prior to an urgent surgery/procecdure and evaluate its efficacy and safety.

Methods: A retrospective study in two tertiary hospitals. Bleeding was evaluated by surgical reports, hemoglobin drop and use of packed red blood cells or additional PCC during 48h. Safety measures were thrombotic complications and 30-day mortality.

Results: Sixty-two patients, aged 80.7±9, received PCC prior to urgent invasive procedures ;39 (63%) received apixaban and 23( 37%) rivaroxaban. Ninety percent of them received anticoagulation due to atrial fibrillation. Most urgent procedures were abdominal surgery (61%), orthopedic surgery (13%) or transhepatic cholecystostomy insertion (10%). Mean dose of PCC was 26.6±8 U/kg. Fourteen patients (23%) received 1 gram of tranexamic acid. Bleeding was reported by surgeons in 3(5%) patients and no patient required additional PCC. Sixteen (26%) patients received packed cells (median 1 unit, range 1-5). Thirty day mortality and thrombosis were 13(21%) and 2(3%) respectively. Cause of death was related to the primary disease or sepsis. No patient died due to bleeding/thrombosis.

Conclusions: PCC is safe and effective for the reversal of Xa inhibitors prior to an urgent surgery/procedure.

Disclosures

No relevant conflicts of interest to declare.
Medium-Dose VP-16 Intensified Conditioning Regimen Is Effective in Allogeneic Stem Cell Transplantation for Acute Lymphoblastic Leukemia
Authors: Sun Feifei , Fang Xiaosheng , Yujie Jiang , Sui Xiaohui , Liu Xin , Wang Xianghua , Li Ying , Wang Na , Zhen Changqing , Qu Huiting , Fan Juan , Xu Hongzhi , Wang Xin
Published: October 2019

Objective: Retrospectively analysing the outcome of additional medium-dose etoposide (VP-16 30mg/kg) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute lymphoblastic leukemia (ALL).

Methods: From 2010.10.1 to 2018.6.30, 53 ALL patients received allo-HSCT in Shandong provincial hospital. 20 conditioning with medium-dose VP-16 (15 in CR , 5 in refractory and/or relapse disease) (VP-16 group), 33 conditioning without medium-dose VP-16 (32 in CR and 1 in refractory and/or relapse disease) (control group) . The basic conditions of the primary disease, treatment-related toxicity, leukemia-free survival (LFS), overall survival (OS), graft-versus-host disease (GVHD) and relapse-free survival (GRFS), non-relapse mortality (NRM), relapse incidence (RI), GVHD and so on were compared between the two groups.

Results: The VP-16 group included more R/R(refractory and/or relapse disease) patients and was associated with improved 3-year-LFS (70.7% vs. 56.5%, p= 0.565) and 3-year-OS (68% vs. 59.4%, p=0.825), although there was no statistical difference. Moreover, the additional of VP-16 lead to reduced incidence of aGVHD (II,III,IV) (10% vs. 30.7%, p=0.146) and cGVHD (9.1% vs 24%, p=0.25), resulting in improved GRFS (63.6% vs. 48.7%, P=0.343). RI was similar between the two group (29.3% vs 27.2%, p=0.76). Meanwhile, the addition of VP-16 enhanced the intensity of conditioning regimen, its NRM was relatively lower than control group (0% vs. 20.2%, P=0.371).

Conclusion: The intensified conditioning regimen with medium-dose VP-16 did not increase NRM and GVHD, with tendency to increase OS and LFS, may become an effective pre-treatment scheme in allo-HSCT for ALL.

Disclosures

No relevant conflicts of interest to declare.
Regulation of HACE1 Expression Enhances HDAC Inhibitor Lmk-235-Mediated Apoptosis in Diffuse Large B-Cell Lymphoma Cells
Authors: Li Xinyao , Ma Dan , Lu Tingting , Kuang Xingyi , Yu Kunling , Zhang Zhaoyuan , Wang Weili , Wang Jishi
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL). It is a group of adult lymphomas with a large clinical presentation and prognosis heterogeneity. In order to improve the cure rate of diffuse large B-cell lymphoma, it is necessary to find a new DLBCL treatment plan that can control the progression of the disease and explore its unknown potential therapeutic targets. Epigenetic modification has become a new treatment for leukemia in recent years, and histone deacetylase inhibitor (HDACi) is a novel drug for the treatment of DLBCL in recent years. However, in addition to effective clinical efficacy, Pan-HDACis has side effects. In contrast, LMK-235 is well tolerated as a novel inhibitor of HDAC4 and HDAC5. HACE1 is a potential tumor suppressor gene located on chromosome 6q21, which is involved in the regulation of tumor cell growth and apoptosis by mediating autophagy and ubiquitination of Rac1. The potential target of HACE1 as an HDAC inhibitor plays an important role in the occurrence, invasion and metastasis of human malignant tumors, but its underlying mechanism of HDACi-mediated apoptosis is still unclear. In this study, we investigated the potential mechanisms by which HACE1 regulates LMK-235-mediated apoptosis in DLBCL cells. In vitro, LMK-235 mediates apoptosis in DLBCL cells in a dose-dependent manner, and up-regulation of HACE1 by lentiviral transfection promotes LMK-235-induced apoptosis. At the same time, high levels of HACE1 can inhibit the expression of HO-1 at mRNA and protein levels. As a previous study in our laboratory, low expression of HO-1 enhanced the apoptotic rate of DLBCL cells. However, the correlation between HACE1 and HO-1 has not been reported. Therefore, we regulated HACE1 levels and found that HACE1 regulates HO-1, which affects the sensitivity of DLBCL cells to LMK-235. Our data show that HACE1 plays a key role in LMK-235-induced apoptosis in DLBCL cells. This provides new hope for the treatment of diffuse large B-cell lymphoma and is a potential new target for the treatment of diffuse large B-cell lymphoma.

Disclosures

No relevant conflicts of interest to declare.
microRNA-22 and TET-2 Mutation in Myelodysplastic Syndrome Patients Treated with Hypomethylating Agents
Authors: Park Seong Kyu , Kim Se Hyung , Lim Sung Hee , Kim Chan Kyu , Won Jong-Ho , Hong Dae-Sik
Published: October 2019

Background: MicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. Some reports suggested that aberrations in the miR-22-TET2 regulatory network are common in myelodysplastic syndrome (MDS) and leukemia, and its aberrant expression correlates with poor survival. We attempted to identify the clinical role of miR-22 and TET-2 in patients with myelodysplastic syndrome.

Materials and Methods: A total of 41 MDS patients who treated with hypomethylating agents were recruited. Real time RT-PCR was performed to assess the expression levels of miR-22 and TET-2 mutation in bone marrow samples at the time of diagnosis. And we investigated the relationship between its results and clinical outcomes.

Results: TET2 mutation frequency in the higher risk group based the IPSS and IPSS-R was lower than that of lower risk group (11.1% vs 38.1%). miR22 expression was also down-regulated in higher risk group (higher risk: 1.70 ± 0.96 vs lower risk: 3.14 ± 1.38, p=0.006). TET2 mutation seemed to be different according to the responsiveness to hypomethylating agents. TET2 mutation, IPSS, and IPSS-R were significantly associated with the risk of leukemic transformation. Patients with an decreased value in the consecutive assessment of miR-22 at the time of diagnosis and 3 months after initial treatment tended to be associated with poor survival outcome (survival rate at 3 years: 18.2% vs 35.1% for patients with an increase, p=0.168). Prognostic factors for survival included TET-2 mutation, cytogenetics, IPSS or IPSS-R, and leukemic transformation.

Conclusion: miR-22 expression and TET2 mutation had a clinical impact on outcomes in MDS patients treated with hypomethylating agents. And these biomarkers might have a potential as a prognostic factor for MDS patients.

Disclosures

No relevant conflicts of interest to declare.
Quantification of Non-Hodgkin Lymphoma Disease Burden Using FDG PET-CT Helps Predict the Severity of Cytokine Release Syndrome
Authors: Wang Jiasheng , Hu Yongxian , Zhang Yanlei , Wei Guoqing , Xu Huijun , Wu Wenjun , Liu Shaohui , Wang Kangyan , Guo Zhenxing , Chang Alex Hongsheng , Zhao Kui , Huang He
Published: October 2019

Background: CD19-targeting chimeric antigen receptor (CAR) T-cell therapy has shown great efficacy in patients with refractory/relapsed non-Hodgkin lymphoma (NHL), but was associated with serious adverse effects such as cytokine release syndrome (CRS). It has been speculated that NHL baseline disease burden might affect clinical outcome and CRS, but such assumption has not been explored in detail in previous studies. Metabolic tumor volume (MTV) and total lesion glycolysis (TLG), calculated using FDG PET-CT, are quantitative indicators of baseline tumor burden.

Methods: Utilizing FDG PET-CT, we calculated MTV and TLG at baseline and post CAR T-cell therapy in 15 patients with NHL.

Results: Among all the patients, the median MTV was 72 (range 0.02-3024.9) cm3 and the median TLG was 610.1 (range 0.011-13156.3). After a median follow-up of 6 months, the overall response rate (ORR) was 66.7% (95% CI 38.4-88.2%). The baseline MTV and TLG did not have significant difference in patients with or without response (p=0.271 and 0.95, respectively). Cox-regression analysis did not find lower baseline MTV and TLG significantly associated with better overall survival (p=0.67 and 0.45, respectively). Patients with mild and moderate CRS (defined as Grade 0-2) had significantly lower MTV and TLG than those with severe CRS (Grade 3, 4) (median MTV: 49.3 v.s. 1137.7 cm3, p=0.012; median TLG: 379.1 v.s. 9384, p=0.012, Figure A, B). The median MTV in patients received tocilizumab, an IL-6 antagonist for the treatment of severe CRS, was 963.4 cm3, which was significantly higher than the patients not receiving tocilizumab (58.1 cm3, p=0.037). The median TLG in patients received tocilizumab was 9187.1, compared with 610.1 in patients not receiving tocilizumab (p=0.053). Using FDG PET-CT, we also demonstrated that CAR T-cell therapy in NHL patients could associate with severe local complications such as local compression and local inflammation.

Conclusions: Low NHL baseline disease burden is not associated with better response rate or long-term outcome. Patients with higher baseline disease burden have more severe CRS

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
Triple-Negative Myeloproliferative Neoplasms Vs. Calr, JAK2 or MPL-Mutated Myeloproliferative Neoplasms: Distinct Molecular Characteristics
Authors: Yun Jiwon , Kim Jung-Ah , Hwang Byungjin , Park Hee Sue , Im Kyongok , Kim Sung-Min , Jeong Dajeong , Lim Kyu Min , Bang Duhee , Lee Dong Soon
Published: October 2019

Background: We compared the clinical, cytogenetic, molecular features, and telomere lengths of patients with triple negative MPN and MPN with any of CALR, JAK2 or MPL mutations.

Methods: Target capture sequencing of 87 genes and molecular cytogenetic studies were performed in 61 Korean patients with MPN. Also, we searched the newly reported mutations and novel mutations in triple negative MPN [JAK2-G335D (germline), JAK2-F556V, JAK2-G571S (germline), JAK2-V625F (germline), MPL-T119I, MPL-S204F/P, MPL-E230G, MPL-V285E (germline), MPL-R321W (germline), MPL-Y591D, MPL-S505N and MPL-W515R]. We compared clinical and molecular characteristics between two groups. Additionally, we performed telomere quantitative FISH for 48 patients' samples and measured telomere/centromere ratios of them.

Results: Among 61 patients, 13 patients showed mutations in CALR, 34 in JAK2, and 3 in MPL. All of JAK2 mutation and CALR mutation site were reported sites, but 2 among 5 mutation site of MPL were novel mutation [D128N, D261Y]. Twelve patients showed triple negative (7 of PMF 7, 2 of ET, and 3 of MPN-U) - they showed 8 different mutation sites among 6 different genes (ASXL1, DNMT3A, NPM1, POLG, SRSF2, and ZMYM3). NPM1, POLG, and ZMYM3 mutations were seen only in triple negative patients. NPM1 mutation was significantly higher in triple negative MPN (P=0.0301). In telomere length study, there was no statistical difference between triple negative group (T/C ratio mean 12.5) and CALR, JAK2 or MPL mutated group (T/C ratio mean 10.0). Although, MPN patients with telomere length shorter than normal control's lower 10% (7.04) showed poor prognosis (P=0.0045).

Conclusions: Patients with triple negative MPN are characterized by high frequency of NPM1 mutation and less number of somatic mutations. Since the mutational analysis for diagnostic purposes is limited to exons 14 of JAK2, exon 10 of MPL and exon 9 of CALR at present, search for JAK2 and MPL mutations in other sites are essential in triple negative MPNs.

Keywords: Myeloproliferative neoplasms, next-generation sequencing, triple negative MPN, chromosome, FISH, telomere

Disclosures

No relevant conflicts of interest to declare.
Hematologic Responses in Patients with Heavily Pretreated Light Chain Deposition Disease (LCDD) Receiving Daratumumab
Authors: Milani Paolo , Basset Marco , Foli Andrea , Merlini Giampaolo , Palladini Giovanni
Published: October 2019

Introduction: The monoclonal antibody daratumumab showed high response rates and a good safety profile in multiple myeloma and is being evaluated in clinical trials in AL amyloidosis. Light chain deposition disease (LCDD) is a rare monoclonal gammopathy of renal significance. Treatment directed against the underlying plasma cell clone can prevent renal progression. Bortezomib is commonly used upfront in these patients and daratumumab may represent a powerful novel option.

Methods: We report the outcome of six patients with refractory light chain deposition disease (LCDD) treated with daratumumab at the Amyloidosis Research and Treatment Center of Pavia. All patients gave written informed consent. Hematologic response was assessed according to the International Society of Amyloidosis criteria.

Results: Six patients (5 males and 1 female) received daratumumab intravenously at 16 mg/kg weekly for 8 weeks, followed by every other week infusions for 8 doses and then monthly infusions. Patients' clinical characteristics are reported in Table 1. All patients received daratumumab single agent except one who was treated with daratumumb and bortezomib combination (this patient received only 1 prior line of therapy). All patients were refractory to the last line of therapy. All patients received at least two months of therapy. All patients were previously treated with bortezomib, pomalidomide was used in 4 cases, lenalidomide, thalidomide and bendamustine in 2 cases each, and autologous stem cell transplant was performed in 4 subjects. The median time from LCDD diagnosis to daratumumab initiation was 8.3 years (range 8 - 147 months). Five of the 6 patients obtained hematologic response with at least a reduction of 50% of the dFLC value (partial response). Three patients obtained a very good partial response (dFLC <40 mg/L). The estimated glomerular filtration rate improved in one subject (from 30 to 45 mL/min per 1.73 m2) and in all the others remained stable. In 2 subjects treatment was temporarily discontinued due to pneumonia.

Conclusions: This is the first report of the use of daratumumab in LCDD. This antibody yielded rapid and significant hematologic responses in five of six heavily pretreated patients with this disease, preventing renal progression. Daratumumab represents a promising option for these patients and larger, international studies are warranted.

Disclosures

Merlini: Akcea: Consultancy; Ionis: Consultancy; Prothena: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Pfizer: Consultancy. Palladini:Celgene: Other: Travel support; Janssen: Membership on an entity's Board of Directors or advisory committees; Prothena: Honoraria.
The NUP98-HOXD13 Fusion Oncogene Induces Thymocyte Self-Renewal Via Lmo2/Lyl1
Authors: Shields Benjamin J. , Slape Chris , Jackson Jacob T. , Vo Anh , Shi Wei , Curtis David J. , McCormack Matthew P.
Published: October 2019

T-cell Acute Lymphoblastic Leukaemias (T-ALL) cases include subfamilies that overexpress the TAL1/LMO, TLX1/3 and HOXA transcription factor oncogenes. Whilst it has been shown that TAL1/LMO transcription factors induce self-renewal of thymocytes, whether this is true for other transcription factor oncogenes is unknown. To address this, we have studied NUP98-HOXD13-transgenic (NHD13-Tg) mice, which overexpress HOXA transcription factors throughout haematopoiesis and develop both myelodysplastic syndrome (MDS) progressing to acute myeloid leukemia (AML) as well as T-ALL. We find that thymocytes from preleukemic NHD13-Tg mice can serially transplant, demonstrating that they have self-renewal capacity. Transcriptome analysis shows that NHD13-Tg thymocytes exhibit a stem cell-like transcriptional program closely resembling that induced by Lmo2, including Lmo2 itself and its critical cofactor Lyl1. To determine whether Lmo2/Lyl1 are required for NHD13-induced thymocyte self-renewal, NHD13-Tg mice were crossed with Lyl1 knockout mice. This showed that Lyl1 is essential for expression of the stem cell-like gene expression program in thymocytes and self-renewal. Surprisingly however, NHD13 transgenic mice lacking Lyl1 showed accelerated T-ALL and absence of transformation to AML, associated with a loss of multipotent progenitors in the bone marrow. Thus, multiple T-cell oncogenes induce thymocyte self-renewal via Lmo2/Lyl1, however NHD13 can also promote T-ALL via an alternative pathway.

Disclosures

Curtis: MERCK: Membership on an entity's Board of Directors or advisory committees; CRC Cancer Therapeutics: Patents & Royalties, Research Funding.
Pyrimido-Indole Derivative, UM171 Expands Distinct Types of Myeloid and Lymphoid Progenitors from Human Pluripotent Stem Cells
Authors: Mesquitta Walatta-Tseyon , Kang Hyunjun , Wandsnider Matthew , Brok-Volchanskaya Vera , Suknuntha Kran , Slukvin Igor
Published: October 2019

Human pluripotent stem cell (hPSCs) have created alternative platforms for producing blood cells for transfusion, immunotherapies, and transplantation. Advancing blood cell manufacturing from hPSCs and translating hPSC-based technologies to the clinic requires improving the scalabilty of blood cell production through enhancing hematopoietic differentiation from hPSCs, and increasing expansion of lineage committed hematopoietic progenitors. The pyrimido-indole derivative UM171 has been described as one the most potent small molecules agonists for HSC expansion in vitro. However, the effect and mechanism of UM171 action on hPSC-derived hematopoietic progenitors (HPs) has not been explored. It also remains unclear whether UM171 selectively expands the most primitive multipotential HPs with lin-CD34+CD43+HSC phenotype, or affects progenitors already committed to a particular hematopoietic cell lineage. In our studies, we evaluated the effect of UM171 on the expansion and differentiation of CD34+CD43+HPs that were generated in chemically defined, serum- and feeder-free conditions from hPSCs. We revealed that culture of hPSC-derived HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3, IL3 and IL6) in the presence of UM171 selectively expanded HPs with a unique CD34+CD41loCD45+phenotype. Isolation of this population by FACS revealed that it mostly possesses myeloid potential and is highly enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4 in presence of SCF, Flt-3 and IL7, UM171 predominately expands CD34+CD7+CD41a-lymphoid progenitors with NK cell potential and increases up to 10-fold NK cell output in NK differentiation cultures. NK cells generated with UM171 possess strong cytotoxicity, express perforin and upregulate IFNg production, following stimulation with K562 or PMA. As determined by annexin V immunostaining, UM171 treatment decreased the number of apoptotic HPs in expansion cultures. In addition, UM171 expansion of HPs was associated with increased proliferation, as determined by BrdU assay and Ki67 staining. Extending these observations, cell cycle analysis revealed that UM171 predominantly increases the proportion of HPs in the early S phase of the cell cycle. These studies should improve our understanding of the effect of UM171 on de novo generated HPs and facilitate development of protocols for robust granulocyte and lymphoid cell production from hPSCs for adoptive immunotherapies.

Disclosures

Slukvin: Cynata Therapeutics Limited: Consultancy, Equity Ownership.
Nuclear Factor I-X May Regulate a Myeloid-Biased Hematopoietic Stem Cell Population during Stress Hematopoiesis
Authors: Walker Megan , Hall Trent , Brown Scott A , McKinney-Freeman Shannon
Published: October 2019

Hematopoietic stem and progenitor cells (HSPC) are routinely exploited in the clinic to treat patients with cancers or other hematologic diseases. The transcription factor nuclear factor I-X (NFIX) has a proven role regulating migration, adhesion, differentiation and quiescence in neural stem cells. Importantly, Nfix was recently shown to be upregulated in FLT3-ITD+ acute myeloid leukemia (AML) patient samples and the NFI binding motif was protected during DNase hypersensitivity screening. We recently identified Nfix as a novel regulator of HSPC repopulating potential. Using our mouse models, Nfixflox/floxRosa26-CreERT2 and Nfix+/+Rosa26-CreERT2, we induced the deletion of Nfix and performed competitive transplants; here we observe a 50% increase in chimerism 20 weeks post-transplant accompanied by a 10% increase in reconstitution of peripheral blood (PB) B cells. Primary recipient whole bone marrow (WBM) was then transplanted into secondary recipients. Beginning four weeks post-transplant, a 2.5-fold loss of PB myeloid reconstitution was observed in secondary recipients of Nfix-deficient WBM. This effect persisted beyond 20 weeks post-secondary transplant, suggesting that a self-renewing myeloid-biased HSPC is functionally perturbed by the loss of Nfix. To identify genes transcriptionally regulated by NFIX, we recently validated an anti-NFIX monoclonal antibody that is currently being used for chromatin immunoprecipitation (ChIP) followed by paired-end sequencing. Future efforts will focus on investigating Nfix-deficient myeloid-biased HSC subsets. This study will illuminate the transcriptional control of specific HSC subsets.

Disclosures

No relevant conflicts of interest to declare.
Bone Marrow T Cell Changes By Multiplex IHC after Treatment with Flotetuzumab, a CD123 x CD3 Bispecific Dart® Protein, in a Primary Refractory t-AML Patient
Authors: Godwin John , Ballesteros-Merino Carmen , Bifulco Carlo B , Lelièvre Helene , Wigginton Jon , Davidson-Moncada Jan K , Fox Bernard
Published: October 2019

Therapy-related acute myelogenous leukemia (t-AML) is associated with adverse genetic lesions, complex karyotype, and TP53 mutation; it is challenging to treat and confers a poor prognosis. We describe a 74-year-old female patient with AML that developed 9 years after receiving 6 cycles of cytotoxic (FOLFOX) chemotherapy as adjuvant treatment of colorectal carcinoma. At diagnosis, the bone marrow (BM) biopsy revealed blast count of 35%, with normal karyotype, 5q loss and AML1 (RUNX1) locus (21q22) amplification by fluorescence in situ hybridization (FISH). Initial treatment with 5 cycles of azacitidine (AZA) failed to induce a response. The patient was subsequently treated on a Phase 1 trial of flotetuzumab (MGD006/S80880) (FLZ), a novel T-cell redirecting (CD123 x CD3) DART protein [NCT02152956]. Prior to FLZ, BM blasts demonstrated clonal evolution with a complex karyotype (92,XXXX, t(14;21)(q22;q22)) and new IDH1, and TET2 mutations.

Patient received a total of 3 cycles (28 days/cycle) of FLZ. After one FLZ cycle, the patient achieved a (complete response) CR, with normal cytogenetics, and residual IDH1 and TET2 mutations. After 2 additional cycles of FLZ consolidation, the CR was maintained, with loss of the IDH1 mutation, but persistence of the TET2 mutation. CR was maintained with no additional therapy for approximately 7 months. Serial BM samples were evaluated for T cells (CD3, CD4, CD8, FOXP3 and PD-L1) using multiplex IHC (Fig 1). BM T cells were unchanged during AZA treatment. At the initial bone marrow on FLZ (CR), significant increases in CD3+, CD3+CD8-, CD3+ CD8+, and FOXP3-positive T cells and PD-L1 expression compared to baseline were noted. CD3+CD8+ T cells persisted in the BM 1 month beyond completion of the additional 2 cycles of consolidation with FLZ, while other T cells subsets and PD-L1 expression returned to baseline (Fig. 1) Three months after the last FLZ treatment, all T cell subsets had returned to baseline, with an early increase in leukemic blasts (8%), but normal cytogenetics. Five months after FLZ treatment blasts were unchanged, but a new abnormal cytogenetic clone, with additional mutations, accompanied by a rise in PD-L1 expression cells was observed. Seven months after FLZ treatment, frank leukemia with 50% blasts developed, and all T cell subsets and PD-L1 expression had returned to pre-treatment levels.

Consistent with its proposed mechanism of action, these data highlight for the first time the induction of an increase in T-cell infiltration, which persist beyond FLZ, in the bone marrow microenvironment of an AML patient that also achieved a complete and durable anti-leukemic response after treatment with FLZ.

Figure 1. Figure 1.

Disclosures

Lelièvre: Servier: Employment. Wigginton:MacroGenics: Employment. Davidson-Moncada:MacroGenics: Employment. Fox:MacroGenics: Research Funding.
Clinical Efficacy of Therapies for Transfusion-Dependent Non-Severe Aplastic Anemia: A Retrospective Cohort Study in Multiple Hospitals
Authors: Lin Shengyun , Yang Yan , Yin Hua , Huang Chang , Chen Jian , Wu Liqiang , Shen Jianping , Ye Baodong , Hu Zhiping , Shen Yiping , Zheng Zhiyin , Shen Wenyi , Lu Xingyu , Wu Xuemei , He Guangsheng , Li Jianyong
Published: October 2019

BACKGROUND: This study aims to evaluate the clinical efficacy and prognosis of currently popular treatments for patients with transfusion-dependent non-severe aplastic anemia (TD-NSAA).

METHODS: Medical records of patients with TD-NSAA were collected from three hospitals and divided into four groups based on their treatment. Treatment efficacy was evaluated at 6 months after treatment, with subgroup analysis based on patients' disease courses. Adverse events and 3-year survival status were also reviewed.

RESULTS: A total of 175 patients' records were collected in this study. The analysis of treatment efficacy indicated that the most effective treatment was allo-hematopoietic stem cell transplant (HSCT) with the response rate of 84.62%, which was followed by Anti thymocyte globulin and ciclosporin A (ATG + CsA) (68.52%), CsA + androgen (26.92%), and androgen (0%), respectively. The subgroup analysis showed that for patients with less than 6-month disease courses, ATG + CsA treatment could achieve similar response rate as allo-HSCT (84.00%). Besides, ATG + CsA showed better efficacy than CsA + androgen in all stratified arms (P<0.05). Three-year event-free survival rates were higher in patients treated with ATG + CsA (83.3%) than with CsA + androgen (64.4%) (P=0.013).

CONCLUSIONS: Therefore, we concluded that ATG+ CsA could achieve better efficacy to treat TS-NSAA than did CsA+ androgen.

Disclosures

No relevant conflicts of interest to declare.
Dynamins 2 and 3 Are Required for Human Megakaryocytes Directional Migration
Authors: Suraneni Praveen K , Corey Seth J. , Hession Michael , Ishaq Rameez , Awomolo Arinola , Hasan Shirin , Debili Najet , Crispino John D. , Eklund Elizabeth A. , Chen Yolande
Published: October 2019

Abstract:

Megakaryocytes (MKs) undergo directional migration from the proliferative osteoblastic niche within the bone marrow (BM) environment to the capillary-rich vascular niche for platelet production and release into the pulmonary circulation. This process is regulated, in part by dynamins, large GTPase proteins that regulate cellular functions such as endocytosis, vesicle transport and cell migration. Additional functions of dynamins include the formation of actin-rich structures, such as lamellipodia and dorsal membrane ruffles, invadopodia and podosomes. Previous studies have shown that mutations in Dynamin 2 (DNM2) cause thrombocytopenia in humans. To explore the function of dynamins in megakaryocyte migration and platelet production in more depth, we monitored the response of cells to chemotaxis SDF1α gradient signal by a microfluidic device-based approach. We observed an impaired directional migration by both human megakaryocytic cell lines and primary cells treated either with dynasore, a small molecule inhibitor of dynamins, or shRNA knockdown of Dynamin 2 and 3 (DNM2, DNM3). Since directional cell migration is tightly regulated by actin cytoskeletal rearrangements, we next measured actin polymerization and RhoA activity. We observed a profound decrease in the F-actin and Rho GTPase activity upon loss of DNM2 and DNM3 function. Next, since the response to chemoattractant signal is navigated by SDF1 through its receptor CXCR4, we explored the CXCR4 receptor response to ligand in dynamin defective megakaryocytes. Interestingly we observed an increase in CXCR4 expression in the dynasore treated primary human cells. Additionally, combined inhibition of DNM2 and DNM3 or over expression of dominant negative Dnm2-K44A or GTPase-defective DNM3 decreased the active β1- integrin (ITGB1) activity, which indicates a decrease in the integrin mediated endo/exocytic cycling during cell migration. Finally, to understand the role of dynamin in endosome recycling, we assayed the distribution of Rab11, a marker of recycling endosomes. We noticed an abnormal clustered staining pattern of Rab11 in dynasore-treated MKs which is indicative of a disruption in recycling pathways. This observation suggests decreased recruitment of the recycling pathway in dynasore-treated cells. Altogether, in this study we demonstrate that dynamins regulate MKs directional migration towards the SDF1α chemotaxis signal in the bone marrow and governs endocytosis and cell receptor trafficking.

Disclosures

Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.
Manufacture of Clinical-Grade Universal Antigen Presenting Cells (UAPC) for the Ex Vivo Expansion and Activation of Natural Killer (NK) Cells
Authors: Liu Enli , Ang Sonny , Tong Yijiu , Li Li , Basar Rafet , Kerbauy Lucila , Daher May , Banerjee Pinaki P , Lim Francesca Lorraine Wei Inng , Kaur Indreshpal , Champlin Richard E , Shpall Elizabeth J. , Rezvani Katy
Published: October 2019

NK cells are potent cellular immunotherapeutic agents against a wide array of human malignancies. Ex vivo expansion of NK cells to achieve clinically relevant numbers must overcome poor in vitro growth kinetics, low starting percentages within the mononuclear cell fraction (especially from autologous donors with active disease), and limited in vivo life span.

We targeted three universally critical NK signaling pathways, namely IL-21, 4-1BB, and SLAM family member 4 (SLAMF4), to increase NK cell proliferation and enhance survival. We genetically-engineered HLA-A-ve and -B-ve K562 cells to enforce expression of membrane-bound IL-21 (mbIL21), 4-1BB-L, and CD48, forming a universal antigen presenting cell (UAPC) to generate highly potent clinical-grade umbilical cord blood (CB-NK) or peripheral blood NK cells (PB-NK). While the mbIL21 and 4-1BB signaling nexuses have been utilized previously, we highlight here SLAMF-mediated immunological sculpting of NK cells for clinical applications.

SLAMF triggering of co-receptors modulate NK cell activation, in particular through high-avidity interactions between SLAMF4 (2B4/CD244) and its heterophilic, robust affinity, and physiological ligand CD48, a glycosyl-phosphatidyl-inositol (GPI)-anchored cell surface protein. Upon ligand binding and receptor phosphorylation, SLAMF4 recruits PTPN11/SHP-2 and SH2D1A/SAP for downstream signaling, including significant increases in NK cell-mediated cytotoxicity, granule exocytosis, and production of IFN-γ and IL-2. Other than a subset of low surface density expressers from aging subjects consistently associated with inefficient and impaired activating signal transduction, the majority of NK cells express SLAMF4. The functional prominence of SLAMF4, presently recognized as an activating co-receptor, is also evidenced by loss-of-function mutations associated with X-linked lymphoproliferative (XLP) disease.

We achieved log-scale expansion of NK cells with UAPC (>1000 fold in 2 weeks), with excellent purity (>99% CD56+ve/CD3−ve and < 1% CD3+ve cells), without indications of senescence/exhaustion, even after 4 weeks of culture. Surface molecular phenotypes of UAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded without SLAMF4. Synergistic signals from IL-21/STAT3, 4-1BB/4-1BBL, and SLAMF4 drive tonic NK propagation, supporting their clinical application. Our novel and clinically accessible platform technology for generation of high purity CB-NKs, a promising source of fresh and cryopreserved allogeneic NK cells, is well-suited for adoptive cancer immunotherapy.

Disclosures

Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.
FANCI-FANCD2 Binds RNA, Which Stimulates Its Monoubiquitination
Authors: Liang Zhuobin , Teng Yaqun , Liu Jingchun , Longerich Simonne , Chen Xiaoyong , Green Allison M. , Collins Natalie , Lan Li , Sung Patrick , Kupfer Gary M.
Published: October 2019

Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and a strong cancer predisposition. FA cells are hypersensitive to DNA replicative stress, and accumulate co-transcriptional R-loops. Previous work has demonstrated that BRCA2 binds to R loops, and increased R loops are noted in FA-D2 mutant cells. Additionally, it is understood that at least one FA protein, FANCA, binds RNA.

The goal of this study was to understand the relationship between FANCD2 and RNA, especially with regard to manifestation of R loops as a part of the pathophysiology of FA. First, we confirmed the increased presence of R loops in FA mutant cells using the S9.6 monoclonal antibody immunofluorescence microscopy. RNAseH overexpression removes R loop signal and increases cell survival upon mitomycin C treatment. We also showed the presence of increased R loops in an actively transcribed region of the actin gene by bisulfite DNA sequencing. We used the Damage At RNA Transcription (DART) assay, which is designed to combine oxidative DNA damage and the genomic insertion of a hyper transcription site (Fig A). Coactivation of transcription and DNA damage results in colocalization of FANCD2 and S9.6/R loop signal at the transcriptional site (Fig B and C). Consistent with the S9.6 IF, wild type RNAseH overexpression resulted in the abrogation of FANCD2 colocalization. We then asked if FANCD2 binds RNA. FANCD2 in cell lysate bound to biotinylated RNA species, preferring GC rich RNAs. Using recombinant FANCI-FANCD2 (ID2) protein (Fig D), we found that ID2 binds preferably to single stranded RNA in a more robust manner than DNA (Fig E and F). Interestingly, an ID2 complex with a known DNA binding mutation in FANCI also was defective for RNA binding. Furthermore, ID2 bound to R loops but was mediated via the single stranded DNA component of the structure. Importantly, an in vitro monoubiquitination reconstitution system using FANCL as the E3 ligase demonstrated that monoubiquitination of ID2 was stimulated to an equal or greater degree by RNA versus DNA, with greater signal in presence of GC-rich, single-stranded RNA as well as R loops (Fig G and H and data not shown).

Collectively, our results support a novel mechanism the ID2 complex suppresses the formation of pathogenic R-loops by binding RNA species, thereby activating the FA pathway (Fig I).

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
High Incidence but Very Low Mortality of Epstein Barr Virus Related Post-Transplantation Lymphoproliferative Disorder after T-Cell Replete Haploidentical Peripheral Blood Stem Cell Transplantation
Authors: Salas Gay Queralt , Al-Shaibani Zeyad , Lam Wilson , Michelis Fotios V. , Thyagu Santhosh , Kim Dennis Dong Hwan , Lipton Jeff H. , Kumar Rajat , Viswabandya Auro , Law Arjun
Published: October 2019

BACKGROUND

The use of immunosuppressive agents such as antithymocyte globulin (ATG) as graft-versus host disease (GVHD) prophylaxis may increase the risk of Epstein Barr Virus (EBV) related post-transplant lymphoproliferative disorder (PTLD). The aim of the present study is to report the incidence, risk factors, and outcome of PTLD in the setting of haploidentical stem cell transplant (haploSCT) combining PTCy and ATG as GVHD prophylaxis.

METHODS

From August 2016 to May 2018, 55 adult patients diagnosed with hematological malignancies underwent T-cell replete haploSCT. All patients received a reduced intensity conditioning regimen (RIC) with fludarabine, busulfan, and 200cGy of total body irradiation (TBI), combined with rabbit ATG, PTCy and cyclosporine (Cy).

EBV titre was monitored by quantitative PCR in plasma samples. The cut-off value for test positivity was >600 copies of EBV DNA per milliliter of plasma. Testing was performed weekly from engraftment to day 100 and beyond in case the patients were on immunosuppression.

Data was collected through retrospective chart review. Last follow up was updated in June 31, 2018. Median follow-up was 9.5 months (range 1-21).

RESULTS

Patient and donor characteristics are summarized in Table 1. Median age was 57 (22-73) years, with 23 (43%) patients ≥60 years. Overall survival of the 55 patients at 6 months was 70% (95% CI 57-83), and 51% (95% CI 46-66) at 1 year.

EBV reactivation was documented in 31 (57%) patients. Median time to EBV reactivation was 54 (20-326) days. Three (5.5%) patients developed presumed PTLD with EBV-viremia higher than 1x10E6 copies and corroborative imaging. Three (5.5%) had biopsy-proven PTLD (Table 2). Median time since EBV reactivation to presumed/proven (P/P)-PTLD was 25 (3-36) days. All cases occurred early post-transplant within the first 100 days. Four patients were on therapeutic doses of cyclosporine. All 6 (11%) patients received treatment with weekly Rituximab 375 mg/m+. Five (83%) patients achieved complete clinical responses with PCR negativity with reduction of immunosuppression and single agent rituximab. One patient died of complications of viral encephalitis and bacterial pneumonia.

CONCLUSION

ATG based conditioning can be associated with increased viral reactivations. Frequent EBV monitoring and pre-emptive treatment may lead to rapid disease control. While no guidelines exist currently, further investigation is needed to define optimal monitoring strategies.

Disclosures

Lipton: BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.
Symptomatic Hemoglobin New Mexico and a Case of Therapeutic Benefit with Acetazolamide
Authors: Ayad Ahmed , Nofal Sara , Jobson Crystal , Raizen Yuval , Alattar Mona Lisa
Published: October 2019

INTRODUCTION

Inherited disorders due to rare structural Hb variants are not included in national, neonatal screening tests and therefore likely underdiagnosed. Hemoglobin New Mexico is one such rare variant with only one case ever reported in 1985 in conjunction heterozygosity for Hb S.1 This Hb variant involves a missense mutation of the β chain substituting the hydrophobic Proline β100 with the hydrophilic Arginine.1 We present here the second case of Hb New Mexico variant ever reported to our knowledge.

CASE PRESENTATION

An 18-year-old athletic, Latin American female initially presented with episodic chest pain, palpitations, fatigue, headaches, and dyspnea on exertion to the point that she could no longer exercise for one year. Work up revealed erythrocytosis persistent for >1 year with hemoglobin level of 17.3 g/dL, and hematocrit of 47.2 % (Hb went as high as 19g/dL) with normal erythropoietin level, hemoglobin electrophoresis, and JAK2 V617 with reflex to JAK3 exon 12-15 negative. She had extensive evaluation to rule out acquired causes including cardiac, pulmonary, and neurologic evaluation which was all normal. She underwent therapeutic phlebotomies with no clinical improvement over 3 months and symptoms worsened as well as new iron deficiency from phlebotomies so this was discontinued. Evaluation with p50 oxygen dissociation curve revealed high oxygen affinity. Further gene sequencing revealed heterozygous inheritance of Hb New Mexico with HbA: 54.5%, HbA2: 3.6%, and Hb New Mexico: 41.9%. Beta Globin Gene sequencing identified the following mutation: Beta 100, CCT>CGT, Pro>Arg. Her symptoms worsened as well as an episode of syncope but no new neurologic, cardiac, or pulmonary etiologies were identified and at the time Hg 18g/dL. A trial of low dose acetazolamide 125mg daily was given to the patient and her symptoms improved by a 6 week follow up with notable reduction of headache, fatigue, dyspnea, and chest pain.

CONCLUSIONS

Given the rarity of this hemoglobin variant in a patient with severe symptoms, data is limited. Acetazolamide, a carbonic anhydrase inhibitor often used for migraines, is known to illicit mild acidosis and therefore potentially reduce hemoglobin-oxygen affinity and perhaps allow for relief of our patient's symptoms. Further therapeutic evaluation is warranted but likely challenging given rarity of the condition.

Disclosures

No relevant conflicts of interest to declare.
CAN WE Predict Risk Factors of Venous Thromboembolism Among Cancer Inpatients?
Authors: Shrotriya Shiva , Dhakal Prajwal , Sharma Mukta , Gardiner Joseph , Al-Janadi Anas , Lupi Alex , Delshad Ahmad , Rayamajhi Supratik
Published: October 2019

Introduction

Increased risk of venous thromboembolism (VTE) has been noted among cancer patients as compared to non-cancer. VTE identified as leading cause of death among those with cancer. Cancer associated thrombosis caused increased hospitalizations, increased inpatient/outpatient medical and prescription claims, and increased total health care costs per patient. Our objective was to study demographic, clinical and laboratory risk factors for venous thromboembolism (VTE) among hospitalized cancer patients and built a predictive model for VTE risk.

Methods

Ours was a retrospective cohort study focused on patients with VTE and cancer from January 2013 - September 2015. Univariate and multivariate logistic regression analysis using stepwise approach was performed. A final predictive model was derived using receiver-operating characteristics (ROC) curves and concordance indices (c-statistics).

Results

N=3948 cancer inpatients were identified which was split into a derivation cohort and a validation cohort, each with 1957. Mean age 65.9±13.8 years; 52.6% were male; 85.6% Caucasian, 7% African Americans; 15.5% were obese; common comorbidities were hypertension (46%), pulmonary disease (34.5%), diabetes (22.9%), renal disease (20.9%) and congestive heart failure (10.4%). Overall, there was 152 (3.9%) events of VTE with 77 (3.9%) in derivation and 75 (3.8%) in validation cohort. On univariate analysis, comorbidities such as infection and renal diseases, laboratory findings such as low hemoglobin and low albumin was associated with high VTE risk. The derivation set had a c-statistic or AUC of 0.668 while the validation set had an AUC of 0.65.

Conclusions

Infection, renal disease (comorbidity) and low albumin levels were associated with a higher risk of VTE. Digestive and respiratory cancers were associated with higher VTE risk. We identified three clinical and laboratory variable that was associated with increased risk of VTE in addition to the cancer group. Future research could use this analysis as a basis for forming a risk score that could be used by clinicians to identify those cancer patients at risk for VTE.

Disclosures

No relevant conflicts of interest to declare.