bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temporal Generation and Molecular Characterization of Functional Hematopoietic Cells From Human Embryonic Stem Cells.
Authors: Hoya-Arias Ruben , Shen Ling-Bo , Mony Ullas , Peguero Elizabeth , Pera Benet , Moore Malcolm A.S. 
Published: October 2019

Abstract
Abstract 2352
Blood is one of the most highly regenerative tissues and is generated by a rare population of hematopoietic stem cells (HSCs). Generation of HSCs seems to be restricted during early embryonic development to particular fetal tissues, such as yolk sac, aorta gonad mesonephros (AGM) region and placenta. The competence of HSCs to self-renew and differentiate towards multiple lineages sustains the generation of blood cells for an entire lifespan. Human HSCs are defined by their self-renewal and multilineage differentiation in functional xenotransplantation assays. Differentiation of human embryonic stem cells (hESCs) into transplantable HSCs continues to be a major challenge in the field. We believe that generation of functional multipotent HSCs from hESC requires an approach that recapitulates early human development both temporally and spatially. Thus, we have developed an innovative in vitro system that mimics the early events of human embryonic development prior to hematopoietic generation; where 1) induction of a single ESC monolayers to mesodermal fate; 2) single cell mesoderm progenitors are placed into a fetal AGM stromal environment under conditions that favor endothelial differentiation; and 3) hematopoietic inductors are added generating single free-floating hematopoietic progenitors in a temporal fashion. We have identified both hemogenic endothelium (adherent) and hematopoietic (non-adherent) cells in the AGM/mesoderm coculture. Hemogenic endothelium defined by expression of VE-Cadherin cocultured on AFT024 fetal liver stromal cells is able to generate hematopoietic precursors. Non-adherent cells start budding up by day 18 in the AGM/mesoderm coculture. Flow cytometry analysis shows that CD31 is the prevalent marker in this floating population. Non-adherent VE-Caherin-CD31+CD34+ cells arising between day 24 till 33 have shown the highest hematopoietic activity as measure by colony forming units (CFU) cells, cobblestone area-forming cell assays (week 4–5 CAFC) and their potential to generate both B and T-cells. Molecular characterization of hemogenic endothelium the non-adherent VE-Caherin-CD31+CD34+ cells by qRT-PCR confirm that also a temporal gene expression is taken place as cells transit from endothelium to hematopoietic cells. VE-Caherin-CD31+CD34+ cells also express CRCX4, suggesting their potential for engrafting in mice models.
This novel approach provides a unique opportunity to study the early events and to identify molecular targets in the human hematopoietic commitment, lineage specification, and maturation; these hematopoietic cells may represent a promise for clinical therapies in a diverse range of blood diseases.

Disclosures:
No relevant conflicts of interest to declare.
Regulation of PCNA Mono-Ubiqutination by FANCD2 and Rad51 in Response to Hydroxyurea.
Authors: Kupfer Gary M. , Chen Xiaoyong 
Published: October 2019

Abstract
Abstract 2377
FANCD2 is a key player in FA pathway. It has been shown that FANCD2 can interact with PCNA and with Rad18, the ubiquitin ligase responsible for PCNA mono-ubiquitination. The mono-ubiquitination of PCNA is very important for its function in translesion synthesis. We found that in response to DNA damage agent hyxdroxyurea (HU) the interaction of FANCD2 with Rad18 or Rad51, and the interaction of Rad51 with Rad18 or PCNA was enhanced. FANCD2 is required for increased interaction between Rad51 and Rad18 indicating that FANCD2, Rad51 and Rad18 form a complex in response to HU. Rad18 was required for PCNA mono-ubiquitination in response to HU. FANCD2 deficient cells failed to enhance the interaction between Rad18 and Rad51. Furthermore, PCNA mono-ubiquitination was impaired in FANCD2 deficient cells in response to HU. FANCD2 mono-ubiquitination deficient mutant partially rescued PCNA mono-ubiquitination. The partial mono-ubiquitination of PCNA in response to HU in FANCA deficient mutant confirmed the role of non-ubiquitinated FANCD2 in PCNA mono-ubiquitination. The normal mono-ubiquitination of PCNA in FANCJ deficient mutant confirmed that the effect of FANCD2 in PCNA mon-ubiquitination is not due to FA pathway deficiency. Rad51 was also involved in regulating PCNA mono-ubiquitination in response to HU. Rad51 siRNA knock down cells showed decreased PCNA mono-ubiquitination in response to HU. The role of Rad51 in regulating PCNA mono-ubiquitination did not require BRCA1, indicating that this function is independent of HR. More importantly FANCD2 deficient cells were hypersensitive to HU, whereas FANCD2 mono-ubiquitination deficient mutant cells, FANCD2 corrected cells, FANCA deficient cells and FANCJ deficient cells were not hypersensitive to HU. Our data indicate that FANCD2 plays an important role in PCNA mono-ubiquitination and translesion synthesis partially in a mono-ubiquitination independent manner. Rad51 also plays an important role in PCNA mono-ubiquitination and translesion synthesis in a HR independent fashion.

Disclosures:
No relevant conflicts of interest to declare.
Transformation by Mutant IDH and (R)-2HG Is Reversible.
Authors: Losman Julie-Aurore , Looper Ryan E. , Kaelin William G. 
Published: October 2019

Abstract
Abstract 2413
Somatic mutations in IDH1 and IDH2 are common in normal karyotype AML as well as in other clonal myeloid disorders and several solid tumors. Mutant IDH overproduces the R-enantiomer of 2-hydroxyglutarate, (R)-2HG (Dang, et al Nature 462: 739, 2009), which is hypothesized to alter the epigenetic landscape of cancer cells by inhibiting the activity of α-ketoglutarate-dependent enzymes, including the TET family of 5-methylcytosine hydroxylases and the jumonji-domain-containing family of histone demethylases. There is considerable interest in developing inhibitors of mutant IDH in the hope that, by decreasing (R)-2HG production in cancer cells, their epigenetic regulation can be restored. However, there is, to date, no evidence that transformation by mutant IDH is reversible or that inhibiting production of (R)-2HG has any effect on cancers harboring IDH mutations. Herein we report that in two different myeloid transformation assays, transformation by (R)-2HG and mutant IDH1 is reversible by removal of (R)-2HG.
We previously reported that stable expression of a tumor-derived mutant IDH1 (IDH1R132H) induces growth factor independence and blocks EPO-induced differentiation in the human TF-1 erythroleukemia cell line, and that treatment of TF-1 cells with a cell-permeable form of (R)-2HG, TFMB-(R)-2HG, is sufficient to recapitulate this phenotype (Late Breaking Abstract #LBA-4, ASH 2011). We have extended these studies and found that transformation by TFMB-(R)-2HG is reversible and that this reversibility is influenced by the duration and intensity (dose) of (R)-2HG exposure. We developed a second myeloid transformation assay using a murine myeloid leukemia cell line that is transformed by expression of a HoxB8-ER fusion protein when cultured in the presence of estrogen. Upon estrogen withdrawal, the cells undergo monocytic differentiation and apoptosis. We expressed wild-type IDH1 or IDH1R132H in the cells and found that cells expressing wild-type IDH1 differentiate normally, but cells expressing IDH1R132H do not upregulate monocytic markers CD11b/Mac1 and Gr1 upon estrogen withdrawal. Furthermore, treatment of the IDH1R132H-expressing cells with an inhibitor of mutant IDH1 restores their ability to undergo monocytic differentiation upon estrogen withdrawal. Our findings suggest that continued exposure to (R)-2HG is required to maintain the cellular changes induced by mutant IDH, and further suggest that targeting (R)-2HG production may have therapeutic efficacy in the treatment of cancers harboring IDH mutations.

Disclosures:
No relevant conflicts of interest to declare.
Morphology Vs. Multiparameter Flow Cytometry in Evaluation of AML in Cerebrospinal Fluid (CSF).
Authors: Loeb Keith R. , Cherian Sindhu , Pagel John M. , Becker Pamela S , Appelbaum Frederick R. , Estey Elihu H. 
Published: October 2019

Abstract
Abstract 2499

Background and Methods:
Multiparameter flow cytometry (MFC) is increasingly used in AML diagnostics as a supplement to morphologic examination. Here we retrospectively examine the concordance between MFC and morphologic examination for the detection of AML in CSF. Between 2005 and 2012, CSF from 604 sequential patients with AML or high-risk MDS was obtained at diagnosis or in follow-up of treatment for AML involving the central nervous system. Morphologic examination was done using conventional Wright-Giemsa stained cytospin preparations. MFC was done using a standard single-tube myeloid panel (HLA-DR/CD15/CD33/CD19/CD117/ CD13/ CD38/CD34/CD71/CD45) and/or a panel targeting the prior AML immunophenotype.

Results:
Results detecting any evidence of AML in the CSF by both techniques is noted in the Table below:
The concordance between MFC and morphology (excluding atypical morphology) was high (97%; 539/558), in large part due to the predominance of uninvolved samples using both techniques. However, of the samples positive by MFC, 13% were negative by morphology and another 15% showed atypical cells insufficient for positivity by morphology. Of samples positive by morphology, only a single sample (1.4%) was negative by MFC. In addition, 65% of cases in which morphology was judged atypical were MFC negative, suggesting suboptimal specificity. Patients typically received intrathecal treatment based on positive MFC making it difficult to know whether the disease in the CSF would have expanded without treatment in patients who were MFC positive/morphology negative.

Conclusion:
Subject to the limitation that the morphology may not have been interpreted entirely independently of the MFC result, the results suggest morphology yields a false negative result in between 13% and 28% of samples and may not be sufficiently sensitive for routine screening of CSF. To follow-up our findings we are examining whether there are features that distinguish patients who were MFC positive/ morphology negative or vice versa and performing a study in which morphology and MFC are being independently assessed.

Disclosures:
No relevant conflicts of interest to declare.
Low Levels of Mir-151-5p and Mir-451 Predict Relapse in Pediatric B-Lineage Acute Lymphoblastic Leukemia.
Authors: Avigad Smadar , Verly Iedan RN , Kaspers Gertjan J.L. , Cloos Jacqueline , Ohali Anat , Hameiri-Grossman Michal , Fonkova Eva , Trka Jan , Luria Drorit , Kodman Yona , Mirsky Hadar , Gaash Dafna , Jeison Marta , Avrahami Galia , Elitzur Sarah , Gilad Gil , Stark Batia , Yaniv Isaac 
Published: October 2019

Abstract
Abstract 2507

Aim:
microRNAs (miRNAs) have been implicated in many malignancies. Our aim was to identify specific miRNAs that can predict risk of relapse in pediatric acute lymphoblastic leukemia (ALL) patients treated according to BFM protocols already at diagnosis. The current risk group stratification is based on the amount of minimal residual disease (MRD) assessed at specific time points by real time quantitative PCR (RQ-PCR).

Material and methods:
Following miRNA expression analysis, we decided to focus on miR-151-5p and miR-451 that significantly correlated with known prognostic factors in ALL. Validation was performed by measuring the expression levels of miR-151-5p and miR-451 by RQ-PCR on bone marrow samples at diagnosis of 101 B-lineage ALL patients excluding Philadelphia positive patients.

Results:
Low expression of miR-151-5p, miR-451 or of both together resulted in significantly worse relapse free survival (RFS) (43%, 58% and 38%, respectively) compared to RFS rates when either or both miRNAs were highly expressed (81%, 75% and 75%, respectively) (p=0.001, 0.044 and 0.001, respectively). Moreover, following PCR-MRD stratification, low expression of miR-451 or both miRNAs remained significant within the PCR-MRD medium risk group (p=0.00001) and within the standard group for both miRNAs (p=0.024). Multivariate Cox regression analysis identified low expression of both miRNAs as an independent prognostic marker with 11.7 fold increased risk for relapse (p=0.002). After excluding patients harboring the adverse genetic markers Ikaros deletion and P2RY8-CRLF2 rearrangement, a patient expressing low levels of both miRs had a 35 fold risk to relapse (p=0.005).
Analyzing a non-BFM treated B-lineage ALL cohort from The Netherlands, both miRNAs significantly correlated with outcome (p=0.003).

Conclusion:
Our results identify miR-151-5p and miR-451 as novel biomarkers for outcome in pediatric B-lineage ALL patients, regardless of treatment protocol. This may allow earlier and improved risk group stratification already at diagnosis, enabling exploration of early therapeutic interventions.

Disclosures:
No relevant conflicts of interest to declare.
Germline Genetic Polymorphisms Are Associated with Disease-Free Survival in Adults with Acute Myeloid Leukemia (AML): A Genomewide Association Study From the Pgrn-Riken Global Alliance.
Authors: Yee Sook Wah , Mefford Joel , Witte John , Kubo Michiaki , Matsuda Koichi , Giacomini Kathleen , Andreadis Charalambos 
Published: October 2019

Abstract
Abstract 2548

Background:
AML is a malignancy with an average five-year survival rate of 50% in adults. Great strides have been made in deciphering the genetic heterogeneity of this disease and indentifying subgroups with favorable or unfavorable outcomes based on cytogenetic and molecular factors. We undertook this study to identify patient-specific germline determinants of treatment outcome and survival in AML.

Methods:
Our cohort was comprised of 314 adult patients with AML in first or subsequent complete remission who underwent consolidation therapy with a busulfan-based autologous stem cell transplant (Bu-ASCT) at UCSF between 1986 and 2009. DNA samples were isolated from patients' peripheral blood mononuclear cell alliquots collected via apheresis following etoposide and high-dose cytarabine consolidation at the time of confirmed complete remission. Genotyping was performed using the Illumina Human OmniExpress Beadchip. To adjust for population substructure, EIGENSTRAT software was used to determine eigenvalues for the SNP correlation matrix and the corresponding eigenvectors were used as covariates. Statistical analyses were conducted with STATA and PLINK software. Association testing with DFS was based on Cox proportional hazards models. We utilized a multiplicative (log-additive) model with a genome wide-alpha set at 10−8. The subset of 168 genetically European patients (cau) was used for the primary analysis.

Results:
Overall, 9.7% of patients in our cohort had high-risk/relapsed acute promyelocytic leukemia, 12.9% core-binding factor leukemia [inv 16 or t(8;21)], 69.5% cytogenetically-normal leukemia, and 7.9% high-risk disease either by cytogenetic or molecular abnormalities. Disease-free survival in our cohort following Bu-ASCT was 59% at 5 years. As expected, baseline risk was significantly associated with DFS but treatment era was not. We identified several significant SNP clusters in our analysis. A 4-SNP cluster emerged on chr15 with a SNP ranked 2nd and one ranked 8th in the overall analysis. The SNP (rs933813) marked 2nd was upstream of the insulin-like growth factor-1 receptor (IGF1R). After adjustment for baseline risk, this SNP was significantly associated with DFS in cau (p-value= 6.9×10−8) as well as the overall population (p-value= 1.1×10−6). Other clusters are being evaluated.

Conclusions:
We identified several significant SNPs and SNP clusters that are associated with DFS in adult patients with AML undergoing Bu-ASCT. So far a SNP upstream of IGF1R has emerged as highly significant in our analysis. Ongoing studies are focusing on imputation, fine mapping and functional validation of these results.

Disclosures:
No relevant conflicts of interest to declare.
A New High Activity Anti-CD22 Chimeric Antigen Receptor (CAR) Targeting B Cell Leukemia.
Authors: Haso Waleed , Lee Daniel W. , Pastan Ira , Dimitrov Dimiter S. , Mackall Crystal L , Orentas Rimas J 
Published: October 2019

Abstract
Abstract 2611
CD22 is expressed on the surface of B cell hematologic malignancies such as acute lymphoblastic leukemia (ALL). CD22 is a Siglec family lectin present on B cells, starting at the pre-B cell stage of development, but is not expressed on plasma cells. CD22 consists of 7 extracellular Ig domains and is found in 2 isoforms, one of which is missing the second and third N-terminal Ig domains. We generated CAR modified T cells containing anti-CD22 extracellular binding motifs fused to intracellular signaling domains for T cells activation (CD3 zeta) or costimulation (CD28 or 4-1BB). The binding motifs were derived from scFvs that targeted a membrane distal epitope of CD22, Ig domain 3, (BL22 and a higher affinity HA22 motif) or that bound a more membrane proximal, Ig domains 5–7, of CD22 (m971). The CAR constructs we generated were second-generation (CD28 and CD3 zeta; or, 4-1BB and CD3 zeta) or third generation (CD28, 4-1BB and CD3 zeta signaling domains). A CH2CH3 spacer domain from IgG1 was added in some constructs to examine the impact of extending the scFv-derived binding domain away from the transduced T cell membrane. In vitro cellular cytotoxicity and cytokine release experiments with 4 B cell-ALL cell lines (REH, SEM, NALM6, KOPN8) as well as the CD22 (+)ve Daudi and Raji cell lines were performed. Our results demonstrate that addition of the CH2CH3 domain did not improve tumor lysis and that standard affinity BL22 and higher affinity HA22-derived scFv epitopes were equivalent. With regard to signaling domains, second generation constructs were better than third generation constructs both in vitro and in vivo. In comparison between second generation constructs, CD28 containing domains outperformed 4-1BB with regard to lytic activity and cytokine release. Most surprising was the activity of the m971-derived scFv binding epitope. m971-CAR had significantly higher killing activity, a far more robust cytokine release profile, and superior in vivo activity. NSG mice were injected i.v. with 0.5× 106 NALM6-GL cells (pre-B cell ALL line engineered to express luciferase). Three days later, when disease was evident, mice were treated with 1×107 CAR+ T cells, and then followed by bioluminescent imaging to measure disease burden. The m971 CAR was significantly more potent at tumor clearance than our previously developed most active construct expressing the HA22-derived scFv domain (Figure 1). Disease progressed rapidly when non-transduced T cells were used (mock). We are currently examining the activity of different signaling domains on m971 CAR efficacy in vivo and directly comparing the anti-CD22 m971 CAR to the CD19 CAR currently being evaluated in clinical trials. These studies will guide future anti-CD22 CAR-based anti-leukemia immunotherapy trials.

Disclosures:
No relevant conflicts of interest to declare.
Anti-CD44 Antibodies Inhibit Both mTORC1 and mTORC2 Activities in Acute Myeloid Leukemia.
Authors: Gadhoum Samah Z. , Merzaban Jasmeen S. 
Published: October 2019

Abstract
Abstract 2620
The role of aberrant signaling pathways such as the PI3K/Akt and the mammalian target of rapamycin (mTor) in the uncontrolled growth and survival in cancer including acute myeloid leukemia (AML) is now clearly demonstrated; and inhibitors of mTor such as Rapamycin are currently being used in clinical trials for their potent anti-neoplastic effects. While leaving normal hematopoiesis unaffected, anti-CD44 monoclonal antibodies (mAbs) have been shown to reverse the blockage of differentiation responsible for the leukemic phenotype, inhibit proliferation of the leukemic clones, and in some types of leukemia, induce the apoptosis of leukemic blasts. However, the molecular mechanisms underlying these effects are yet to be resolved.
Here, we show that anti-CD44 mAb-induced differentiation of the HL60 cell line (AML type 2), is accompanied by a marked decrease in the phosphorylation of mTor with no affect on the total expression of mTor. This decrease was strongly correlated with an inhibition of the PI3K/Akt pathway as shown by the decrease of Akt phosphorylation on Thr308. Moreover, we observed that anti-CD44 mAbs induce a decrease of phosphorylation of p70S6k (on Thr 389) as well a decrease in the phosphorylation of Akt on Ser 473, which directly reflect the activity of mTORC1 and mTORC2 respectively. This result strongly suggests that CD44 triggering inhibits the activity of both mTORC1 and mTORC2 complexes. Previous studies have shown that the activity of mTORC1 is constitutively active in most leukemic patients, while it has been suggested that the inhibition of mTORC2 activity could have an anti-leukemic effect. Our finding unveils a new role for anti-CD44 mAbs as potent mTor inhibitors, heretofore, adding a key argument to the promise of CD44 as a strong therapeutic target in AML.

Disclosures:
No relevant conflicts of interest to declare.
The Impact of Activated Akt Expression On Clinical Outcome in Diffuse Large B-Cell Lymphoma: A Clinicopathological Study of 99 Cases.
Authors: Hong Jung Yong , Choi Moon Ki , Kim Young Saing , Maeng Chi Hoon , Lee Su Jin , Chang Won Jin , Park Silvia , Kim Seok Jin , Jang Jun Ho , Kim Kihyun , Jung Chul Won , Ko Young Hyeh , Kim Won Seog 
Published: October 2019

Abstract
Abstract 2676

Purpose
Akt is a serine/threonine kinase that plays a central role in cell proliferation and growth. To define clinical impact of Akt expression in diffuse large B-cell lymphoma(DLBCL), we investigated the expression of phospho-Akt(p-Akt) in DLBCL and analyzed clinical impact of p-Akt expression on patient survival.

Methods
We evaluated the p-Akt expression in 99 DLBCL patients using tissue microarray(TMA) technology.

Results
Positive p-Akt expression was observed in 15.2% of the patients and significantly associated with elevated lactic dehydrogenase level (P = .044). Kaplan-Meier survival analysis showed that the patients with positive p-Akt expression showed substantially poorer overall survival (p-Akt+ vs p-Akt- 25.3 months [95% confidence interval(CI), 14.4–36.2 months] vs 192.6 months [95% CI, 131.3–253.9 months], P < .001) and progression-free survival (p-Akt+ vs p-Akt- 13.6 months[95% CI, 14.4–36.2 months] vs 134.5 months [95% CI, 131.3–253.9 months], P < .001), respectively. Multivariate Cox regression analysis revealed that patients with DLBCL with p-Akt positivity showed poorer overall survival with 3.2 fold (95% CI, 1.6–6.8, P = .002) risk for death compared to patients with DLBCL with p-Akt negativity.

Conclusion
Positive expression of p-Akt in DLBCL patients is associated with poorer overall and progression-free survival. Expression of p-Akt may act as an independent poor prognostic factor and might be a novel therapeutic target for DLBCL.

Disclosures:
No relevant conflicts of interest to declare.