bluuebunny/crossref_metadata_2025 huggingface dataset:
Collection of Peripheral Blood Stem Cells in 118 Small Children Weighing 20 Kg or less: Experience of a Single Centre
Authors: Wen Jianyun , He Yuelin , Chen Libai , Du Jing , Peng Zhiyong , Liao Jianyun , Liu Huaying , Zhou Xiaohui , Li Chunfu , Wu Xuedong
Published: October 2019

Background: Peripheral blood stem cells (PBSC) are increasingly used as a source of stem cells for either autologous or allogeneic hematopoietic transplantation in children.Although technically similar to adult procedures, PBSC harvest may be difficult in young children, especially in the very small children.

Aim: In this study, we aimed to evaluatethe safety and efficacy of harvesting peripheral blood hematopoietic stem cells in very small children,and to provide a guideline.

Methods: Between Jan 2013 to Mar 2016, we evaluated 118 children weighing 20 kg or less, with the smallest patient weighing 11 kg. The patients had a median age of 59 months and included 72 children with thalassemia major and 46 young donors. The granulocyte-colony stimulating factor (G-CSF) analogs were used at a dose of 10 mg/kg/day administered subcutaneously once a day and receiving oral calcium for five days before harvesting. Blood was withdrawn at a mean rate of 30-40 ml/min through a temporaryfemoral vein catheter (12 or 14 guage) to ensure adequate blood flow and returned through a larger catheter in a peripheral vein.Total nucleated cells(TNC) and CD34+ cells were estimated in the peripheral blood before collection of the apheresis product.

Results: We collected sufficient products from all the children with one to three apheresis procedures. No serious complication was detected in all children and all aphereses were completed within 4 hours.The volume of blood per kilogram processed for each apheresis ranged from 55 to 160ml (median, 85ml). The median number of TNC and CD34+ cells collected were 12×108/kg and 15×106/kg per apheresis, respectively.

Conclusions:We conclude that collection of PBSC is a safe and practical procedure in children, even in very small children.

Disclosures

No relevant conflicts of interest to declare.
Transfer of Immunity Using Vaccinate and Boost Strategy with Bispecific Antibody Armed T Cells (BATs) in Metastatic Breast Cancer Patients
Authors: Thakur Archana , Uberti Joseph P. , Ratanatharathorn Voravit , Lum Lawrence G.
Published: October 2019

Background. In our recent phase I immunotherapy (IT) trial in 23 women with metastatic breast cancer (MBC), 8 infusions of activated T cells (ATC) armed with anti-CD3 x anti-HER2 bispecific antibody (HER2Bi) given in combination with interleukin-2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) induced specific anti-breast cancer immunity. This study investigated whether specific cellular and humoral anti-breast cancer immunity induced by infusions of HER2 bispecific antibody armed T cells (BATs) could be transferred after high dose chemotherapy (HDC) and stem cell transplant (SCT) in MBC patients. Methods. T cell were activated with OKT3 and expanded in IL-2. ATC were harvested, armed with HER2Bi, and cryopreserved in 8 doses for twice weekly infusions for 4 weeks along with IL-2 and GM-CSF. Seven to 14 days after the last infusion of BATs, the patient was leukapheresed again to obtain and expand immune T cells. Multiple infusions of immune ATC after the HDC and SCT were given to boost the transferred cellular and humoral immune responses which were monitored up to 24 months. Results. Six of 8 MBC patients enrolled were evaluable in the protocol, no dose-limiting toxicity, delays in engraftment, or life-threatening infections were observed. Five of 6 evaluable patients exhibited increased anti- breast cancer cytotoxicity and IFN-γ Elispots after vaccination with BATs and up to 18 months post SCT. Serum and culture supernatants from in vitro antibody synthesis assay showed gradual increases in anti-SK-BR-3 IgG levels after SCT. Serum cytokine profile showed increases in IL-12 and Th1 cytokines. One out of 6 evaluable patients who rapidly progressed showed poor immune response, had high serum levels of Th2 cytokines and no evidence of transfer of immunity. Flow cytometry analysis of Vβ repertoire pattern in PBMC collected post IT and post SCT indicate transfer of the major Vβ clones post SCT that produced IFN-γ upon stimulation with breast cancer cells (Fig. 1). A significant correlation (r=1.0; p<0.002) between immune ATC cytotoxicity directed at breast cancer cells and time to progression (TTP) suggests that more robust vaccinations with a Th1 shift in cytokine profiles can lead to clinical benefit (Fig. 2). Conclusions. Our pilot study suggests that cellular and humoral immunity was transferred and boosted using immune T cells after SCT. There was robust reconstitution of T and B cell functions early after SCT as evidenced by CTL and NK activity, IFN-γ EliSpots, in vivo/in vitro antibody synthesis, and Th1 cytokine responses.BATs induced endogenous anti- breast cancer cellular, humoral and innate immunity that could be detected after SCT and may have provided clinically meaningful anti-tumor immunity.

Disclosures

No relevant conflicts of interest to declare.
Gene Editing of the Human Pklr Gene in Human Hematopoietic Progenitors to Correct Pyruvate Kinase Deficiency
Authors: Quintana-Bustamante Oscar , Fañanas Sara , Orman Israel , Gouble Agnès , Galetto Roman , Poirot Laurent , Bueren Juan A. , Segovia Jose C.
Published: October 2019

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. This gene encodes the erythroid specific Pyruvate Kinase (RPK) enzyme, implying that this defective enzyme fails to produce normal levels of ATP and consequently, erythrocytes from PKD patients show an energetic imbalance. Hematopoietic stem cell gene therapy using gene editing will be the safest approach to treat PKD patients. However, its direct application in hematopoietic progenitor cells (HPCs) is challenging. Different gene editing approaches are being explored to correct PKD, either by Knock-In of an optimized cDNA sequence in the first introns of the gene or by site-specific correction using ssODN. While Knock-In strategy will allow the treatment of most PKD mutations, an ssODN-mediated gene editing will correct specific PKD patient-mutations. In our Knock-In system, a recombination matrix carrying an exon 3-11 partial codon optimized version of RPK cDNA and a puromycin selection cassette is inserted in the second intron of the PKLR gene assisted by TALEN® nucleases. We have previously shown that this approach was effective to correct PKD phenotype in PKDiPSC lines. Here, we have tested the feasibility of our approach in hematopoietic progenitors. Thus, the therapeutic matrix together with specific TALEN® for the second intron of PKLR was electroporated in purified cord blood CD34+ from healthy donors. Cells were then expanded and puromycin selected to enrich the population for gene edited ones. Up to 96% of the colony forming units that survived the puromycin selection showed the specific integration of the donor cassette. After transplantation into immunodeficient NSG mice, a low though detectable percentage of gene edited cells was . Additionally, CRISPR-Cas9 site-specific correction is being developed by combining guide RNAs directed against PKD patient's mutation and precise ssODN to restore RPK protein. Overall, our data show that gene editing in engraftable HPCs is feasible, although further improvements are needed to increase efficiency of this gene therapy appraoch prior to consider its therapeutic application in PKD patients.

Disclosures

Gouble: Cellectis: Employment. Galetto:Cellectis SA: Employment. Poirot:Cellectis: Employment.
Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells Via Transcription Factor Conversion of Hemogenic Endothelium
Authors: Sugimura Ryohichi , Han Areum , Jha Deepak , Lu Yi-Fen , Goettel Jeremy A , Serrao Erik , Rowe Robert Grant , Wong Irene , Sousa Patricia , Ditadi Andrea , Keller Gordon , Engelman Alan , Snapper Scott , Doulatov Sergei , Daley George Q.
Published: October 2019

A variety of tissues can be differentiated from pluripotent stem cells (PSCs) in vitro through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors (TFs). Despite considerable effort, neither approach has yielded functional human hematopoietic stem cells (HSCs). Building upon recent evidence that HSCs derive from definitive hemogenic endothelium (HE), we performed morphogen-directed differentiation of human PSCs into HE followed by screening of 26 candidate HSC-specifying TFs for the capacity to promote multi-lineage hematopoietic engraftment in irradiated immune deficient murine hosts. From genomic PCR of engrafted cells, we recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft GLY-A+ erythrocytes, CD33+ myeloid, CD15+ CD31+ neutrophils, CD19+ IgM+ B and CD3+ T cells in primary and secondary murine recipients for 12-14 weeks. Limiting dilution analysis indicated that the frequency of repopulating cells generated by this method was 1 in 4,707-15,029, lower than the frequency in CD34+ cord blood cells (1 in 1,819-5,173). Functional characterization of terminally differentiated cells demonstrated features of definitive erythropoiesis (expression of adult beta globin and enucleation). Engrafted neutrophils responded to cytokine stimuli by activation of myeloperoxidase. Human IgM and IgG could be detected in the serum of engrafted mice, and titers of ovalbumin specific antibody increased in response to protein immunization, indicating boostable immunity. T-cells responded to PMA/Ionomycin stimuli by activation of IFNγ, and sequencing of the T cell receptor revealed a broad clonotype diversity. Proviral integration analysis demonstrated derivation of myeloid and lymphoid progeny from common clones in secondary animals, indicating generation of self-renewing, multipotential HSC-like cells from PSCs. Mechanistically, the seven TFs induced HOXA target genes (LMO2, SOX4, MEIS1 and ID2); upregulated expression of homing-related genes (CXCR4, VLA5 and S1PR1); and enhanced the endothelial to hematopoietic transition (EHT), as indicated by a 2.4-fold induction of a RUNX1c-reporter. Our combined approach of morphogen-driven differentiation and TF-mediated cell fate conversion produced HSPCs from PSCs that hold promise for modeling hematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

Disclosures

No relevant conflicts of interest to declare.
Thrombin Generation: Plasma Composition Imbalance and Mortality in HIV
Authors: Brummel-Ziedins Kathleen E. , Baker Jason V , Gissel Matthew , Neuhaus Jacqueline , Neaton James D , Tracy Russell P.
Published: October 2019

Introduction: Effective treatment of HIV infection with antiretroviral therapy (ART) has prolonged survival and shifted causes of death to non-AIDS defining illnesses such as cardiovascular disease (CVD). Increased thrombin generation is evident from increased D-dimer levels in HIV; previously we have shown that inflammation and viral replication associate with imbalances in pro and anticoagulant factors resulting in increased thrombin generation profiles when mathematically modeled. We explore for the first time the hypothesis that factor compositional imbalance increases simulated thrombin generation and predicts mortality in HIV.

Methods: In a nested case-control study of the SMART and ESPRIT participants randomized to continuous ART, we evaluated cases of all-cause mortality and controls (1:3) matched on enrollment date, age (± 5yrs), study, and country. Thrombin generation in response to a 5 pM tissue factor initiator for each individual was calculated by a mathematical model incorporating levels of factors II, V, VII, VIII, IX, X, antithrombin, tissue factor pathway inhibitor and protein C. Thrombin generation metrics included time to clot, maximum rate (MaxR), maximum level (MaxL) and area under the curve (AUC). Conditional logistic regression was used to compare parameters between cases and controls, adjusted for age, gender, and race/ethnicity.

Results: Among cases (n=129) and controls (n=369), respectively, median CD4 count was 451 and 496 cells/mm3, and 65 and 77% had HIV viral suppression (<400 copies/mL). Levels of antithrombin (median: 125% vs. 128%, p=0.084) and protein C (median: 109% vs. 117%, p=0.005) were decreased and factor V (median: 112% vs 98%, p=0.014), FVIII (median: 145% vs 129%, p=0.006) and TFPI (median: 27 ng/mL vs. 26 ng/mL, p=0.073) were higher in cases versus controls. The overall effect is a more procoagulant simulated phenotype. In mortality cases versus controls, MaxR (median: 0.44 nM/s vs. 0.35 nM/s, p=0.064), MaxL (68 nM vs. 56 nM, p=0.037) and AUC (18 µM*s vs. 14 µM*s, p=0.016) were all more procoagulant, although clot time was unaffected. Among mortality cases thrombin generation (MaxL) was greater among those in the highest versus lowest quartile of IL-6 (p=0.006). In controls thrombin generation (MaxL) did not vary by IL-6 level (p=0.93).

Conclusions: Antithrombin, FV and FVIII, and PC were seen as the major contributors to the increased simulated thrombin generation among HIV+ mortality cases. Our results are consistent with the hypothesis that HIV is associated with an increase in thrombin generation via altered balance of pro and anticoagulant factors, and the resulting coagulopathy is associated with increased mortality.

Disclosures

No relevant conflicts of interest to declare.
Ribosomal Protein Rpl22 Regulates Development and Transformation Via Altering RNA Processing
Authors: Wang Minshi , Ser Zheng , Rao Shuyun , Fahl Shawn , Zhang Yong , Peri Suraj , Kentsis Alex , Wiest David L
Published: October 2019

Although it has long been reported that mutations in ribosome proteins (RP) are associated with increased cancer risk in humans, the molecular basis why which RP mutations do so remains unclear. Nevertheless, the prevailing view is that RP mutations, such as Rps19, are thought to alter transformation potential through general impairment of ribosome biogenesis or function. Importantly, recent observations are beginning to challenge this notion as too simplistic. We have determined that the RP, Rpl22, is not essential for ribosome biogenesis or global protein synthesis; however, its inactivation impairs the development of normal T lymphocytes and increases their transformation potential. Indeed, RPL22 is inactivated in human T acute lymphoblastic leukemia (T-ALL) and this is associated with reduced survival. Moreover, Rpl22-deficiency accelerates development of leukemia in a myristylated Akt2 transgenic (MyrAkt2 Tg) mouse model of T-ALL. To gain insight into how Rpl22 inactivation facilitates development of leukemia, we are performing unbiased transcriptomic and proteomic analysis on Rpl22+/+ and Rpl22-/- thymic lymphomas arising in the MyrAkt2 Tg model, and in an Rpl22-/- lymphoma reconstituted with Rpl22. Interestingly, relatively few changes in mRNA transcript read depth were observed; however, substantial differences in the proteome were observed. Pathway analysis revealed that the loss of Rpl22 altered the expression of proteins regulating RNA-processing, in particular RNA-splicing. Interestingly, interrogation of the transrciptome data for alternative splicing revealed that alterations in exon usage. The ability of Rpl22 to influence splicing appears to be conserved across species as alternative splicing was also observed in zebrafish embryos in which Rpl22 was knocked down using morpholino oligonucleotides. Consequently, we hypothesize that Rpl22 regulates biological events through its ability of binding to RNA targets, and controlling the expression of their protein products at least in part through altering mRNA splicing. How Rpl22 changes mRNA splicing pattern is currently under investigation.

Disclosures

No relevant conflicts of interest to declare.
Measurement of Quality of Life and Depression of Spouse Caregivers for Survivors after Hematopoietic Stem Cell Transplantation
Authors: Park Silvia , Cho Juhee , Jang Jun Ho , Jung Chul Won , Heo Mi Hwa
Published: October 2019

Introduction: Hematopoietic stem cell transplantation (HSCT) is a quite exhausting process, which encompasses multitude of chemotherapy before HSCT and long term care and surveillance in post transplant period. Caregivers are also exposed to this whole process, and are often the sole provider of physical and emotional support for the patients.

Methods: This was a cross-sectional, HSCT survivor-spouse caregiver matching study to determine the caregiver's quality of life (QoL) and depression. From September 2013 to March 2015, a total of 103 HSCT survivor-partner pairs were answered to a set of questionnaire for the assessment of QOL and depression, which were measured by the World Health Organization Quality of Life (WHOQOL)- BREF 26 and Patient Health Questionnaire-9 (PHQ9), respectively.

Results: Among a total of 103 pairs, the data from 97 married couples were finally used for the analysis. Mean age of the caregiver participants was 49.7 years (range 30-73) and majority of caregivers were female (n=65, 67%). More than 70% of pairs were assessed with a monthly income of

$2,000-4,000 or more, and 55.2% of caregivers responded to have co-morbidities. Mean duration since transplant was 3.2 years, and allogeneic HSCT comprised 68.7% of cases. As a result, overall QoL, psychological health, social relationships and environmental health were not significantly different between survivors and caregivers (p=0.345, 0.424, 0.415, and 0.253) whereas physical QoL was reported to be better in caregivers compared to survivors (p=0.011). Mean depression scale of depression was comparable between survivors and caregivers with mean score of 5.3 and 5.1 (p=0.812) and there was no difference in the proportion of severe depression between the two groups (15.6% in survivors, 13.7% in caregivers) (p=0.270). It was revealed that family income had significantly correlation with caregiver's overall QoL and environmental health (p=0.013, and 0.023), and female gender, co-morbidites and family income were the important factors associated with caregiver's depression (p=0.007, 0.017 and 0.049).

Conclusion: The present study identified that there was no significant difference in QoL and the level of depression between HSCT survivors and spouse caregivers. Family income, gender and co-morbidites showed significant association with caregiver's distress. We suggest that psychosocial intervention starting from a clinical attention to both survivors and caregivers is needed, and that social support to families who are financially challenged has to be considered.

Disclosures

Jang: Kyowa Hakko Kirin Co., Ltd.: Research Funding.
Wnt Signaling in Red Blood Cells
Authors: Rosin-Arbesfeld Rina , Caspi Michal , Siman-Tov Ronen , Levi Yakir , Perry Chava
Published: October 2019

Introduction : Red blood cells (RBCs) are the most common cell type in the human body.

These cells deliver oxygen to the body's tissues and are composed of a cytoplasm that is rich in hemoglobin and is surrounded by a membrane that is essential for the cells function by providing properties such as stability and deformability. The membrane is composed of a lipid bilayer, transmembrane proteins, and a filamentous meshwork of proteins such as actin and adducin that forms the cells cytoskeleton along the entire cytoplasmic surface of the membrane. RBCs lack a nucleus and other cellular organelles that enable gene expression. Thus, it has been assumed, that signaling cascades such as the Wnt signaling pathway are not active in RBCs.

The Wnt pathways are fundamental for normal development and homeostasis and regulate, among other, cell growth, motility and differentiation. Although there is no functional data connecting between Wnt signaling and RBCs, previous proteomic studies have shown that some of the non-canonical Wnt signaling components, such as specific small GTPases and kinases are present in RBCs. In this study, we show for the first time that Wnt ligands activate signaling cascades in RBCs.

Methods : RBCs were collect from healthy donors and treated with different Wnt ligands. A large number of methods were used to evaluate the RBCs morphological properties, life span, vitality, flexibility and protein expression patterns.

Results : Our results clearly show that different Wnt ligands can dramatically increase the live span of RBCs by affecting the cells membrane cytoskeleton. Some of the Wnts affects include activation of GTPases such as Rac, JNK and RhoA, which lead to actin modification. These changes in the actin cytoskeleton result in increased membrane stability. In parallel, treating RBCs with Wnts leads to activation of PKC and RhoA resulting in phosphorylation of adducin which in turn increases the cells membrane flexibility and improves the vitality of the cell.

Summary : Our novel findings indicate that the non-canonical Wnt signaling pathway in RBCs is active and can stabilize the RBCs cytoskeleton enhancing the vitality, deformability and life span of the cell.

These novel findings may help in the development of new therapeutic strategies for people suffering from different hemolytic disease that effect the RBCs cytoskeleton and provide new insights into the improvement of stored RBC units.

Disclosures

No relevant conflicts of interest to declare.
Clinical Characteristics and Factors of the Impact on the Survival in 133 De Novo FLT3-ITD Acute Myeloid Leukemia with Intermediate Chromosome Karyoptye from a Single Center of China
Authors: Qiu Shaowei , Lin Dong , Wei Hui , Zhang Guangji , Zhou Chunlin , Liu Kaiqi , Wei Shuning , Wang Ying , Liu Bingcheng , Gong Benfa , Gong Xiaoyuan , Liu Yuntao , Mi Yingchang , Wang Jianxiang
Published: October 2019

Objects: Investigate the clinical characteristics and factors of the impact on the survival of FLT-ITD mutation in the acute myeloid leukemia(AML) with intermediate chromosome karyoptye.

Methods: We retrospectively collected and analyzed information regarding clinical characteristics and survival data from 133 patients with newly diagnosed AML with FLT3-ITD mutation and intermediate chromosome karyoptye from 2001-2015.The median follow-up time was 13.02 months.

Results: 1. The median age was 37, median WBC was 50×109/L , the incidence between male and female was similar, the most common FAB subtype was M5(60.2%), the hepatosplenomegaly incidence was 8.4% .

2.The complete remission (CR) was 64.5% after first induction chemotherapy. CR could reached 74.4% or 57.1% when induction regimen included the medium dose cytarabine(100mg/m2 d1-4;1g/m2 q12h d5-7) or standard dose cytarabine(Ara-C)(100mg/m2d1-7) respectively(p=0.063).

3. Totally 131 patients received chemotherapy, and subsequently 22 patients received transplantation after chemotherapy. When the patients only received chemotherapy, 3-year OS was 39.9%, 3-year DFS was 44.8% and while in the patients who received transplantation, 3-year OS was 73.5%, 3-year DFS was 77.5%, the outcome was better (p<0.05).

4. Univariable analysis showed that OS was related with age, hepatosplenomegaly, transplantation, WBC, the remission of induction chemotherapy. DFS was related with transplantation, courses for CR. Cox regression showed that transplantation and remission of induction chemotherapy were the independent prognosis factor. When the patient only received chemotherapy, FACS-MRD persisted negative was the independent prognosis factor.

Conclusions: The FLT3-ITD de novo AML patients with intermediate chromosome karyoptye had unique characteristics. The patients who attained CR after induction chemotherapy and received transplantation would get better outcome, while in the patients who only received chemotherapy, FACS-MRD persisted negative implied better outcome. The induction regimen that contained medium Ara-C could reach higher CR , that may translated into better survival, therefore it may be the appropriate choice for FLT3-ITD AML patients.

Disclosures

No relevant conflicts of interest to declare.
Targeting Pre-Leukemic Stem Cells in T-Acute Lymphoblastic Leukemia
Authors: Gerby Bastien , Veiga Diogo F.T , Krosl Jana , Ouellette Julianne , Haman André , Lavoie Geneviève , Fares Iman , Tremblay Mathieu , Litalien Véronique , Ottoni Elizabeth , Kosic Milena , Geoffrion Dominique , Ryan Joël , Maddox Paul , Chagraoui Jalila , Marinier Anne , Hébert Josée , Sauvageau Guy , Kwok Benjamin H , Roux Philippe P , Hoang Trang
Published: October 2019

Current chemotherapy of pediatric T cell acute lymphoblastic leukemia (T-ALL) efficiently reduces the tumor mass with, however, undesirable long term consequences and remains ineffective in adolescent and adult T-ALL. Furthermore, relapse can be caused by pre-leukemic stem cells (pre-LSCs) that were spared by current protocols and evolved to malignancy. A distinctive characteristic of pre-LSCs is their critical dependence on interactions with the microenvironment for survival, which guided our strategy to target pre-LSCs using niche-based screening assays.

Using transgenic mouse models that closely reproduce the human disease, we showed that the SCL/TAL1 and LMO1 oncogenic transcription factors establish a pre-leukemic state by reprogramming normal pro-T cells into aberrantly self-renewing pre-LSCs (Gerby et al. PloS Genetics, 2014). We now provide direct evidence that pre-LSCs are much less chemosensitive than leukemic blasts to current drugs, due to a distinctive lower proliferative state as assessed by real-time imaging in a competitive assay. We therefore designed a robust protocol for high-throughput screening (HTS) of compounds targeting primary pre-LSCs that are maintained on stromal cells engineered for optimal NOTCH1 activation to mimick the thymic microenvironement. The multiparametric readout takes into account the intrinsic complexity of primary cells to specifically monitor pre-LSCs. We screened a targeted library of 1904 compounds and identified UM0119979 that disrupts both cell autonomous and non-cell autonomous pathways: UM0119979 abrogates pre-LSC viability and self-renewal activity in vivo by specifically inhibiting the translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remain functional. Moreover, in vivo administration of UM0119979 efficiently reduced the leukemia propagating activity of primary human T-ALL samples in xenografted mice. Finally, in addition to SCL-LMO-induced T-ALL, our results reveal a novel possibility of therapeutic intervention in MYC-dependent hematologic malignancies.

In summary, our screening assay, built on the genetic dependencies of pre-LSCs, revealed their vulnerabilities to compounds that inhibit both the primary oncogenes and non-cell autonomous pathways triggered by the microenvironment. The results illustrate how recapitulating tissue-like properties of primary cells in high throughput screening is a promising avenue for innovation in cancer chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
Overexpression of Notch1 Inducing T-ALL Phenotype and Promoting Angiogenesis in Zebrafish
Authors: Shen Lijing , Chen Fangyuan , Qing Kai , LI Xiang , LIU Haiyan
Published: October 2019

Aim: Abnormal activated Notch1 gene is familiar to us as a poor prognostic factor in T cell acute lymphoblastic leukemia (T-ALL). This in vivo study aimed to establish a stable line of zebrafish overexpressing the human Notch1 gene under the control of a heat stress-inducible promoter, and explore its roles in etiology of T-ALL, and in tumor-related angiogenic process.

Methods: Rag2-ICN1(intracellular domain Notch1)-RFP Plasmid was constructed and injected into the embryos Tg (fli1-EGFP) in the one cell stage to construct the double-fluorescence tracer and double transgenic zebrafish, named as Tg(ICN1:RFP/fli1:EGFP). Expression of ICN1 gene and its influence on zebrafish were assayed through fluorescence microscope, RT-PCR, western-blot, FCM, blood smear, RFQ-PCR and drug treatment.

Results: Twenty of the 867 (2.3%) mosaic F0 zebrafish embryos were identified to be the germline transgenic zebrafish, including 9 males and 11 females. FCM and peripheral blood smears suggested a significant reduction in the proportion of red blood cells and a increasing of lymphocyte ratio in transgenic zebrafish as compared to control group. RFQ-PCR shows that lymphocytic regulatory factor ikaros, rag1 and lck were significantly reduced, indicates obvious differentiation obstacle in lymphoid lineages. These results were absolutely consistent with the results of FCM and peripheral blood smear. Quercetin suppressed the proliferation activity of T-ALL cell lines and induced apoptosis in a time- and dose-dependent way, meanwhile, quercetin can inhibited Notch1, Hes1, Dll4 and VEGF gene and these proteins expression in A3 and Molt-4 cells (T-ALL cell line). Furthermore, quercetin displayed a significantly reduction in the length of ISV and SIVs in Tg(rag2-ICN1-RFP/fli1-EGFP) zebrafish).

Conclusions: The above results induced by overexpression of Notch1 gene closely resemble the main aspects of human T-ALL, suggesting that Notch1 plays a role in the etiology of AML. Some key lymphocytic regulatory factors were apparently changed, which indicates obvious differentiation obstacle in lymphoid lineages and accompanied with the increasing small lymphocytes and precursors. Quercetin inhibited the proliferation and induced apoptosis of T-ALL cell lines as well as inhibition of zebrafish angiogenesis by downregulating the expression of Dll4/Notch1 and VEGF pathways. Therefore, this Notch1 transgenic zebrafish model facilitates dissection of Notch1-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential therapeutic targets.

Disclosures

No relevant conflicts of interest to declare.
Risks and Benefits of Rituximab Maintenance in Elderly Patients with Advanced Follicular Lymphoma
Authors: Savard Marie-France , Johnson Nathalie A.
Published: October 2019

Introduction: Based on the PRIMA trial (Lancet 2010; 377: 42-51), rituximab maintenance following induction rituximab-based chemotherapy is the standard of care in patients with advanced follicular lymphoma (FL) because it extended the 3-year progression free survival (PFS) from 58% to 75%, with a low rate of infection (<5%). However, this study was conducted in young patients (median age 57 years old) and the benefits and risks of the maintenance are unknown in elderly patients.

Methods: We reviewed the clinical characteristics, outcome and complications of 62 advanced FL patients selected based on having successfully completed induction chemotherapy and initiated maintenance rituximab at the Jewish General Hospital between 2007 and 2014. The age cut off of ≥75 years old at the time of treatment was defined as "elderly". We compared characteristics of the two groups using the χ2 test and calculated PFS using the Kaplan-Meier method and log-rank test.

Results: In total, 9/62 (15%) patients were "elderly" and 53/62 (85%) were "young", with median ages of 79 (75-87) and 55 (30-74) years old, respectively. Elderly patients had more adverse prognostic features compared to younger patients: B symptoms (4/9 vs 7/53, p=0.023), elevated lactate dehydrogenase (5/9 vs 9/51, p=0.013), lower hemoglobin (5/9 vs 3/52, p<0.001) and poor performance status (3/9 vs 1/53, p<0.001). The mean PFS was lower in elderly patients (25 months vs 65 months, p=0.05). The 3-year PFS in the young group was 80%. Toxicities were higher in the elderly group: grade 3-4 neutropenia (3/9 vs 5/53, p=0.05) and infections (5/9 vs 13/52, p=0.063).

Conclusion: Elderly patients had more adverse prognostic factors and a significantly inferior PFS with rituximab maintenance compared to younger patients. The rates of infections were higher than those reported in the PRIMA trial. Maintenance rituximab may not benefit elderly patients with advanced FL.

Disclosures

No relevant conflicts of interest to declare.
Mutations in TP53 Gene Is Independent Prognostic Factor for High Grade B-Cell Lymphoma
Authors: Misyurina Anna Evgenevna , Misyurin Vsevolod Andreevich , Misyurin Andrey Vitalievich , Kovrigina Alla Mikhailovna , Kravchenko Sergey Kirillovich , Baryakh Elena Alexandrovna , Magomedova Aminat Umaraskhabovna , Pushkova Elena Nikolaevna , Parovichnikova Elena N. , Savchenko Valery Grigorievich
Published: October 2019

Background. TP53 mutations were described to have a negative impact on prognosis of patients with B-chronic lymphoid leukemia and diffuse large cell lymphoma. However, a role of TP53 mutations in high-grade B-cell lymphomas (HGL) is not well defined in context of other genetic aberrations.

Materials and methods. 23 patients (7 males and 16 females) had diagnosis of HGL were treated in National Research Center for hematology, Moscow, Russia. Median age was 49 years old (30-76). 5 patients had HGL with c-MYC (MYC-R) and BCL2 genes rearrangements and 2 - HGB with MYC-R and BCL6 rearrangements. 17 (74%) patients had MYC-R, 11/23 (48%) had double expresser lymphoma (MYC≥40%, BCL2≥50%) (DE). Median of observation time was 29,1 months (6,3-99,8). 19 (82%) of patients had IPI score 3-5 points. 16 patients underwent LM-B-04 with rituximab (Table 1), 5 - R-(DA)-EPOCH, 2 - R-CHOP-21. In 5 cases autologous stem cell transplantation was performed. Sanger sequencing was performed to identify mutations in exons 5-8 of TP53 gene using DNA extracted from formalin fixed paraffin embedded tissue («Extra-DNA» kit, «Genetechnology» LLC). Primers to TP53 gene were synthesized based on nucleotide sequences data available online on website ncbi.com by «Evrogene». To evaluate an influence of such factors as TP53 mutation (TP53mut), MYC-R, DHL, DE, gender, therapy on overall survival (OS) and time to progression (TTP) were performed multivariate dispersion analysis and Cox regression analysis (STATISTICA 10).

Results: 8 (35%) cases with TP53mut were identified: c.535C>T 45,6% p.H179Y, c.524G>C 15,6% p.R175P, c.743G>A 75,6% p.R247Q, c.487T>A 25,2% p.Y163N, c.824G>A 75% p.C275Y, c.713G>A 87,7% p.C238Y, c.745A>G 31,9% p.R249G, c.639A>G 41,8% p.R213R. 7/8 of them harbored MYC-R, 2/8 had DHL. In univariate (Picture 1, 2) and multivariate analysis pts harboring TP53mut had worse OS (median OS was 6,2 (0,7-9,5) vs 25,5 (0,7-99,8) months, p=0,004) and shorter TTP (median TTP 3,5 (0,7-9,5) vs 23,1 (0,7-99,8) months, p=0,027) than patients without TP53mut. DHL status had also an adverse effect on OS with lower significance than in pts with TP53mut (p=0,022). Adverse effect of TP53mut trends to play a role in combination with c-MYC gene rearrangement, thus5 pts TP53mut/MYC-R had shorter TTP than 5 DHL pts (3,4 (1,1-9,5) vs 7,3 (0,6-67,1) months, p=0,07).

Conclusion: High-grade lymphoma has a more powerful and independent prognostic factor than double-hit status - TP53 mutation that contribute inferior prognosis. This factor shouldn't be underestimated in routine diagnostics because of its frequency and requirement of a different therapeutic approach.

Disclosures

No relevant conflicts of interest to declare.
High Frequency of Loss of Heterozygosity and Chromosomal Imbalances in Patients with Myelofibrosis
Authors: Junior Milton Rego de Paula , Mazzeu Juliana Forte , Taylor Aline Pic , Oliveira Silviene , Nonino Alexandre , Mascarenhas Cintia Do Couto
Published: October 2019

Myelofibrosis (MF) is a clonal hematopoietic stem cell disorder belonging to the non-chronic-myeloid-leukemia myeloproliferative neoplasms. Cardinal features of myelofibrosis include extramedullary hematopoiesis, hepatosplenomegaly and cytopenias. The disease can occur de novo as primary MF or result from the progression of polycythemia vera or essential thrombocythemia with no differences in clinical or histological characteristics. Though several large chromosome abnormalities have been described in patients with myelofibrosis there are no previous studies screening for both CNVs and LOH (loss of heterozygosity) in patients with MF. We studied 11 patients using CytoScan platform (750k or HD- Affymetrix). This platform allows the identification of copy number variations as well as segments of loss of heterozygosity. Forty-five percent (5/11) presented pathogenic/possibly pathogenic CNVs and 72% (8/11) presented large segments of loss of heterozygosity (>10Mb). Two patients had a single CNV without any LOH. Two patients had a single LOH. All others have multiple LOH with (3) or without (4) CNVs. The most frequent abnormality was LOH of 9p (3/11) and deletion 13q (2/11). All other alterations were non-recurrent. One patient had a small deletion including exons 1 and 2 of NOTCH2, a gene known to be involved in hematological malignancies. The most frequent abnormality reported in the literature is 20q deletion. In our cohort only one patient had the 20q deletion and also had multiple complex chromosome rearrangements on chromosomes 7 and 12 and a large LOH on chromosome 3p. The study shows that CNV and LOH are frequent and may be involved in pathogenesis of the disease. However, the role of these changes in the pathogenesis are not yet clear.

Disclosures

No relevant conflicts of interest to declare.
Monocyte-Derived Macrophages Are Impaired in Myelodysplastic Syndrome
Authors: Han Yu , Wang Huaquan , Shao Zonghong
Published: October 2019

The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic stem cell diseases characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Macrophages are innate immune cells that ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. Tumor-associated macrophages (TAMs) play an important role in the pathophysiology of human malignancies. They support growth of cancer cells by promoting angiogenesis, inhibiting tumor cell apoptosis and anti-tumor immune reactions. In this study, we analyzed the role of macrophages from MDS patients in vitro. Macrophages were induced from peripheral blood of patients with MDS via granulocyte macrophage colony-stimulating factor (GM-CSF). Compared with that in the normal control group, the number of monocytes increased in MDS patients. However, the monocytes showed impaired ability to induce macrophages and the number of macrophages induced from MDS samples was lower. Further, we demonstrated that the ex vivo phagocytic function of macrophages from MDS patients was impaired and levels of reorganization receptors CD206 and signal regulatory protein alpha (SIRPα) were lower. Levels of inducible nitric oxide synthase(iNOS) secreted by macrophages in MDS were increased. In conclusion, monocyte-derived macrophages are impaired in myelodysplastic syndromes.

Disclosures

No relevant conflicts of interest to declare.
Frequency of Amyloid Subtyping Among Patients with Immunoglobulin Light-Chain Amyloidosis Referred for High-Dose Chemotherapy and Autologous Stem Cell Transplant
Authors: Cowan Andrew J. , Coffey David G. , Hyun Teresa S. , Becker Pamela S. , Green Damian J. , Holmberg Leona A , Gopal Ajay K , Libby Edward N.
Published: October 2019

Background:

The amyloidoses comprise a heterogeneous group of diseases characterized by misfolding of amyloidogenic proteins and subsequent deposition as amyloid fibrils. To date, over 30 proteins are known to be amyloidogenic (Sipe Amyloid 2014). Immunoglobulin light chain (AL) amyloidosis, a plasma cell dyscrasia, is the most common subtype. The standard diagnostic algorithm in AL amyloidosis is to obtain a biopsy of a clinically involve organ, and once Congo red positivity is confirmed, perform subtyping analyses with immunohistochemistry or mass spectrometry. Accurate subtyping of amyloidosis is essential to appropriate treatment, as misdiagnosis occurs in up to 10% of patients and may lead to inappropriate administration of chemotherapy (Comenzo Blood 2006; Lachmann NEJM 2002). We sought to determine the patterns of amyloid subtyping among patients with a diagnosis of AL amyloidosis referred to a tertiary referral center for HDM/SCT.

Methods:

Sequential patients with confirmed amyloidosis, age ≥ 18 years who underwent HDM/SCT between 2001 and 2014 at the Fred Hutchinson Cancer Research Center and University of Washington Medical Center were eligible. Presence of a Congo red-positive biopsy for each patient referred for transplant was confirmed and the pathology reports and medical records were reviewed to determine if subtyping was performed, and which modality was used.

Results:

Fifty-one patients with AL amyloidosis were referred for transplant; of these, 45 proceeded with HDM/SCT. The organ systems most commonly involved were renal in 34/51, and gastrointestinal in 5/51. Of the biopsies, subtyping was performed in 35 (68.6%), and no subtyping was performed in 16 patients (31.3%). Immunofluorescence was the most common modality used for subtyping in 33 biopsies (94.2%) and laser capture/mass spectrometry (LC/MS) was used in 2 patients (5.7%). All patients had evidence of a clonal plasma cell dyscrasia by bone marrow biopsy and peripheral blood testing. Of the patients without subtyping, 8 (50%) were diagnosed before 2008.

Discussion:

Misdiagnosis of amyloidosis due to a lack of appropriate subtyping is a well-described and ongoing problem for patients with amyloidosis. These data suggest that definitive subtyping is still not routinely performed in the evaluation of amyloidosis. At our center, efforts to standardize the evaluation of Congo-red positive biopsies using definitive typing are underway.

Disclosures

Gopal: Seattle Genetics: Research Funding.
The Incidence of Adrenal Insufficiency in Myeloma Patients Receiving Pulse-Dose Dexamethasone
Authors: Afrough Aimaz , Sidorski Amy , Salvatori Roberto , Borrello Ivan
Published: October 2019

Introduction: Glucocorticoids (GC's) are major drugs in the treatment of multiple myeloma (MM). Chronic administration of supra-physiological doses of GC's suppresses the hypothalamus-pituitary-adrenal (HPA) axis and is associated with secondary adrenal insufficiency (AI). In MM, GC's are usually administered in weekly high dose pulses. Due to long GC-free periods in such therapy, GC-induced AI is not usually considered to be a consequence of GC therapy in MM (Krasner, AS. JAMA, 1999. 282(7): p. 671-6). Here we report on the incidence of AI in MM patients treated at our center with pulse-dose GC.

Methods: This is a retrospective cross-sectional study of patients with MM treated with GC-based regimens. Patients were required to have at least a random serum cortisol or a standard 250 mcg ACTH stimulation test result available in their medical record during dexamethasone-based chemotherapy. Patients were excluded if they were on dexamethasone more than 1 day a week, were lost to follow-up, had prior use of synthetic progestational agents such as megestrol or had been on oral glucocorticoids for any other medical indication. Diagnosis of AI was established by a frankly low AM serum cortisol level (<3 ug/dL) or an inadequate cortisol response to ACTH stimulation test (Salvatori, R. JAMA, 2005. 294(19): p. 2481-8).

Results: A total of 45 patients were included in this study with median age of 62 (range, 49-89). Fifteen (33.3%) of patients were diagnosed with AI. The median random cortisol level in AI group was 2.3 ug/dL (range, 0.3-7.4 ug/dL) compared to 9.9 ug/dL (range, 1.0-21.2 ug/dL) in the non-AI group. The median time between the last dexamethasone dose and the serum cortisol assay was 5 days (range, 1-21). The median number of GC-based chemotherapy cycles taken before diagnosis of AI was 15 (range, 2-60). The median cumulative dexamethasone consumption was 1280 mg (range, 180-5220 mg). There was no correlation between developing AI and dose or duration of dexamethasone treatment. We observed clinical trend between cumulative doses of ≥1500 mg (P= 0.055) or use of clarithromycin (P=0.079) and developing AI, without reaching a statistically significant difference.

Conclusion: The rate of AI was 33% among patients with MM on weekly pulse-dose dexamethasone. As such, patients should be periodically evaluated for this to enable early detection and proper management.

Disclosures

Borrello: BMS: Honoraria, Research Funding; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau.
EZH2 Inhibitors Are Broadly Efficacious in Multiple Myeloma As Single Agent and in Combination with Standard of Care Therapeutics
Authors: Arora Shilpi , Williamson Kaylyn , Apte Shruti , Balachander Srividya , Busby Jennifer , Hatton Charlie , Bryant Barbara , Trojer Patrick
Published: October 2019

Post-translational modifications of the histone proteins play a key role in regulating processes that require access to DNA. Specifically, methylation of lysine 27 on histone 3 (H3K27) is intimately linked with transcriptional repression. EZH2, a histone lysine methyl transferase is the catalytic component of the PRC2 complex, which catalyzes H3K27 methylation. EZH2 dysregulation has been observed in different malignancies and inhibition of its catalytic activity has emerged as a novel therapeutic approach to treat human cancers. Potent, selective and reversible EZH2 small molecule inhibitors are currently being tested in Ph. 1 clinical trials. We and others have reported EZH2 dependencies across non-Hodgkin Lymphoma subtypes in cancer cell lines, in xenograft mouse models and in lymphoma patients.

We identified Multiple Myeloma as potential clinical application for EZH2 inhibitors. Treatment with EZH2 inhibitors such as CPI-360, CPI-169 and CPI-1205 cause apoptosis in multiple myeloma and plasmacytoma cell models and causes tumor growth inhibition in myeloma xenograft models at well tolerated doses. An EZH2-controlled transcriptional signature across various multiple myeloma was identified using integrated RNA-sequencing and ChIP-sequencing data. Combination studies testing EZH2 inhibitors with standard of care (SOC) agents across a panel of multiple myeloma cell lines showed synergistic responses with several of the SOC agents in vitro and in vivo.

Disclosures

Arora: Constellation Pharmaceuticals: Employment, Equity Ownership. Williamson:Constellation Pharmaceuticals: Employment, Equity Ownership. Apte:Constellation Pharmaceuticals: Employment, Equity Ownership. Balachander:Constellation Pharmaceuticals: Employment, Equity Ownership. Busby:Constellation Pharmaceuticals: Employment, Equity Ownership. Hatton:Constellation Pharmaceuticals: Employment, Equity Ownership. Bryant:Constellation Pharmaceuticals: Employment, Equity Ownership. Trojer:Constellation Pharmaceuticals: Employment, Equity Ownership.