bluuebunny/crossref_metadata_2025 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 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 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 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 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 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 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 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 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 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.
PF4/H Complexes Induce Germinal Centers in Vivo
Authors: Joglekar Manali , Qi Rui , Arepally Gowthami M.
Published: October 2019

Abstract 269

Mice injected with ultra-large complexes (ULCs) of platelet factor-4 (PF4) and heparin (PF4/H) exhibit a strong immune response which mimics that seen in patients developing Heparin induced thrombocytopenia (HIT)(Suvarna, Blood 2005). Previous studies with this murine immunization model have shown that murine (m) PF4/H ULCs potently activate dendritic cells (DCs) (Chudasama, Blood 2010) and initiate a T-cell dependent immune response (Suvarna, Blood 2005). To examine cellular/anatomic basis of PF4/H antibody formation, we examined the role of the spleen and germinal center formation in mice injected with mPF4/H complexes. To evaluate the role of the spleen, we immunized splenectomized mice (SPL) (n=15) and wild type (WT) mice (n=15) retro-orbitally with 100 mg/mL mPF4 and 5 U/mL Hep for 5 days. Antibodies to mPF4/H complexes were detected by an ELISA developed in our laboratory (Suvarna, Blood 2005; Suvarana, Blood 2007). As shown in Figure 1, SPL animals showed a marked reduction in Ab formation compared to WT animals (mean A450nm ± SD; SPL 0.206 ± 0.253 vs. WT 0.76 ± 0.788, p<0.003). We next asked if antibody responses to PF4/H were associated with germinal center (GC) formation. The GC is a hallmark of T-cell dependent (TD) immune responses and is considered vital for development of high-affinity and isotype-switched antibodies. For these studies, mice were injected with a high dose of PF4/H (400 ug/mL PF4: 10 U/mL heparin) (n=2), standard protocol dose of PF4/H (100 ug/mL: 5 U/mL) and or buffer (Hank's Balanced Salt Solution, n=3) daily for 5 days via retro-orbital injection. As a positive control for GC formation, we injected two mice with a conventional antigen, nitrophenyl conjugated chicken gamma-globulin (NP-CGG). Mice expressing high antibody titers on Day 16 (mean A450nm± SD; high dose- 1.112 ± 0.5767; standard dose- 1.186 ± 0.7853) were sacrificed, spleens harvested and frozen in an embedding medium. Splenic sections (5m, longitudinal) were obtained as frozen sections and stained with immunohistochemical markers for GCs (GL-7 FITC; 1:400, counter-stain- FITC Alexa Fluor 488; 1:200), T-cells (TCR-β PE; 1:200) and B-cell follicles (B220 Biotin; 1:400, counter-stain- Streptavidin Alexa Fluor 350; 1:200). Images were obtained using Carl Zeiss AxioVert 200M microscope and analyzed with AxioVision Rel 4.6 software. Fluorescent images were processed with Adobe photoshop CS5.1. As shown in Figure 2, we noted that mice injected with NP-CGG, had ∼ 22 GC's, mice injected with buffer had 2 GC's and mice injected with high and standard dose of PF4/H, had ∼20–22 and 15–17 GC's respectively. These studies show that mPF4/H complexes elicit GC formation in association with secretion of mPF4/H Abs and that GC formation is comparable to that seen with conventional antigen.

Disclosures:

No relevant conflicts of interest to declare.
Long Term Safety and Efficacy Of Low Fixed Dose Hydroxyurea In Pediatric Patients With Sickle Cell Anemia: A Single Center Study From Central India
Authors: Jain Dipty L. , Krishnamurti Lakshmanan , Sarathi Vijaya , Desai Saumil , Gokhale Amruta
Published: October 2019

Introduction

Hydroxyurea (HU) therapy ameliorates the morbidity associated with Sickle cell anemia (SCA). HU is typically escalated to maximum tolerated doses (MTD) for treatment of SCA patients. Monitoring HU for toxicity poses potential challenges in low resource settings. We have previously reported the safety and efficacy of fixed low dose HU at 10mg/kg body weight ( Jain et al 2013). We studied the long term safety and efficacy of low dose HU in pediatric patients with SCA.

Methods

Long term observational follow up of 40 young Indian SCA patients was carried out for a period of 10 years in a single tertiary care center in central India. The number of clinical events including vasocclusive crises, acute chest syndrome, stroke, sequestration crises, avascular bone necrosis in the entire cohort was assessed prior to starting HU and subsequently every 3 months. Laboratory parameters including hemoglobin levels, Hb F levels, mean corpuscular volume, mean corpuscular hemoglobin concentration were also followed up over the same time period.

Results

The total number of patients in this study was 40 (17 females & 23 males). Mean age was 12.95 years ± 9.62. All patients had HbSS. There was an increase in mean hemoglobin, HbF, MCV and MCH over time in the patient population ( Table 1). There was a decrease in the mean number of hospitalizations, vasocclusive crises, acute chest syndromes, stroke and severe anemia ( Table 2). HU was well tolerated with minimal hematological or other toxicity (Table3). Patients who experienced transient hematological, liver or renal toxicity were able to resume HU at the same dose. HU was permanently discontinued in 2 patients who had manifestations of HIV/AIDS. HU was temporarily interrupted in one patient who was receiving treatment for tuberculosis and in one patient for the duration of a normal pregnancy. There was one death attributable to severe septicemia without evidence of neutropenia after 2 years follow up. There were 3 subjects who were lost to follow up, one after 2 years follow up and remaining 2 after 5 years of follow up. Of note, patients had a high HbF level at baseline. High HbF levels at baseline, the variable relationship of HbF to disease severity and amelioration of disease in Indian patients with high HbF has been previously reported ( Jain et al 2012, Patel et al 2012, Italia et al 2009).

Conclusions

Low fixed dose HU is well tolerated and is efficacious in reducing incidence of VOC, hospitalizations, severe anemia, stroke and acute chest syndrome. Low fixed dose HU may be advantageous in low resource settings because of lower toxicity and consequently less need for monitoring. There is a need for multi-center randomized comparisons of low fixed dose HU with conventional escalation of HU dose to MTD.

Disclosures:

Krishnamurti: GlycoMimetics, Inc.: Research Funding.
Hyperfiltration Is a Risk Factor For The Development Of Microalbuminuria In Sickle Cell Anemia
Authors: Vazquez Benjamin , Saraf Santosh L. , Molokie Robert E. , Gowhari Michel , Hassan Johara , Lash James P. , Gordeuk Victor R.
Published: October 2019

Hyperfiltration, which has been implicated in the early stages of development of chronic kidney disease (CKD) in diabetes mellitus, is a common finding in sickle cell anemia (SCA) patients with normal kidney function. We hypothesized that it may be a risk factor for the development of microalbuminuria, an early indicator of glomerular injury that is observed in 40-60% of adult patients with sickle cell anemia (SCA).

In the cross-sectional analysis, 233 adult SCA patients treated at the University of Illinois at Chicago Sickle Cell Center with baseline estimated glomerular filtration rates (eGFR) determined by the MDRD equation and albuminuria concentrations were screened. Data was collected from steady-state visits defined as four weeks from a vaso-occlusive pain episode or red blood cell transfusion. Eight-two patients with normoalbuminuria (urine albumin concentration < 30mg/g creatinine) and an eGFR ≥ 90mL/min/1.73m2 were evaluated of which 38 (46%) had normal eGFR (defined as eGFR 90-150mL/min/1.73m2) and 44 (54%) had hyperfiltration (defined as eGFR >150mL/min/1.73 m2). On univariate analysis, the median age of patients with hyperfiltration was 23 years compared to 34 years in patients with a normal eGFR (p<0.0001). Additionally, patients with hyperfiltration had lower hemoglobin F concentrations (3.9 vs. 7.3%, p=0.01) and higher absolute reticulocyte counts (390 vs. 288 x103/µL, p=0.03) than patients with normal eGFR, respectively. On multivariate analysis, hyperfiltration was independently associated with age (OR 0.9, 95% CI: 0.8-0.9; p=0.0001) and the natural log of hemoglobin F concentration (OR 0.5, 95% CI: 0.2-1.0; p=0.04).

Forty-six patients with normoalbuminuria and eGFR ≥ 90mL/min/1.73m2 were followed longitudinally for progression to microalbuminuria. At a median follow up of 30 months (range 1-81 months), 35% (16/46) of patients developed microalbuminuria with a mean time to progression of 49 months (range 11-57 months). The average time to develop microalbuminuria in patients with hyperfiltration was 33 months (42%, 11/26) compared to 62 months in patients with normal eGFR (25%, 5/20) (age-adjusted HR 4.0, 95% CI 1.5 – 10.4; log-rank p=0.016) (Figure 1).

In summary, hyperfiltration in SCA patients with normoalbuminuria was associated with younger age and lower hemoglobin F concentration on cross sectional analysis and with higher rates of progression to microalbuminuria on longitudinal analysis. Future research to better understand the development of hyperfiltration and its contribution to kidney damage may help guide future therapies for sickle cell nephropathy.

Disclosures:

No relevant conflicts of interest to declare.
Intermittent Use Of Sufadoxine-Pyrimethamine To Prevent Malaria In Patients With SCD In Malaria Endemic Regions: Findings From The Sickle Cell Disease Research and Control Center Of Bamako, Mali
Authors: Diallo Dapa Aly , Guindo Aldiouma , Touré Boubacari Ali , Sarro Yaya , Fané Baba , Dorie Alain
Published: October 2019

Sickle cell disease (SCD) is a chronic disorder affecting erythrocytes and is especially prevalent throughout Sub-Saharan Africa where malaria is thought to be a significant cause of morbidity and mortality in affected individuals. In the absence of effective malaria vaccine, one of the affordable alternatives to prevent malaria at present is chemoprophylaxis. In order to evaluate the effectiveness of a monthly intermittent prophylaxis treatment (IPT) with sulfadoxine-pyrimethamine (SP) during high malaria transmission season in patients with sickle cell disease, two groups of SCD patients from two different sites were compared. The first group constituted of patients followed at the Sickle Cell Disease Research and Control Center of Bamako where IPT is routinely administered while the second group consisted of individuals enrolled in the health district in the same locality but no malaria prophylaxis. In this area, the incidence of resistance of P.falciparum to SP is estimated < 10%. SP combination was administrated as follows: sulfadoxine 25mg/kg and pyrimethamine 1.25mg/kg. For both groups, diagnosis of malaria was performed by using the rapid diagnosis test for the presence of P.falciparum. From 2011 to 2012, 687 SCD patients (457 from the Sickle Cell Disease Research and Control Center and 115 from the health district) were enrolled. The observed prevalence of malaria infection in the group receiving a monthly IPT by SP (1.5%) was much lower than the group (6%) for which IPT was not offered (P=0.01). When data was stratified by hemoglobin genotypes, malaria was found to occur entirely in SS and SC patients and no malaria cases were observed in S/β-thalassemia patients. SP was well tolerated since no patient in SP arm reported pruritus and no serious adverse events including death were recorded during the study. In malaria endemic areas where the incidence of resistance to SP is low, anti-malarial prophylaxis with this combination therapy significantly reduced the incidence of malaria in SCD patients with good safety and a lower cost.

Disclosures:

No relevant conflicts of interest to declare.
Assessing B Lymphocyte Clonal Diversity, Expansion, and Convergent Evolution By High-Throughput Sequencing Of Rearranged IGH Segments From Naïve and Memory Repertoires
Authors: DeWitt William S , Lindau Paul , Emerson Ryan O , Sherwood Anna , Williamson David , Rieder Mark J , Chung Moon , Carlson Christopher S , Robins Harlan
Published: October 2019

Diversity in B lymphocyte antigen specificity, necessary for effective adaptive immunity, arises from structural diversity of the B cell receptor generated by random somatic rearrangement of the immunoglobin heavy (IgH) and light chain loci during lymphocyte development. Antigen-experienced (memory) B cells are stimulated to proliferate upon successful binding of antigen, whereas the extent to which unexperienced (naïve) cells proliferate, if it all, is unclear. During memory clonal expansion, daughter cells undergo somatic hypermutation, which introduces random single nucleotide variants to the rearranged gene segments, selectively optimizing antigen affinity. Among memory B cells that express identical receptor proteins, the extent to which the cells are identical by descent, or rather have convergently evolved from different naïve clones to the same specificity, is also unknown.

We performed high-throughput sequencing of rearranged IgH gene segments for B cells sorted into memory (CD19+, CD27+) and naïve (CD19+, CD27-,IgM+, IgD+) samples (∼5 million cells each) from each of three adults. Each sample was split among 188 libraries for multiplex PCR amplification of IgH sequences at ∼10x coverage, allowing us to estimate clonal abundance by measuring the number of libraries occupied by each sequence.

Using a maximum entropy inverse model, all three memory samples evince three distinct subpopulations, corresponding to memory cell types of differing abundance. Data from naïve samples suggest extreme diversity with more than 98% of clones occupying one library (i.e., almost surely present in only one cell in the starting material). A measure-theoretic upper bound on mean clone abundance in the naive repertoire was computed based on Monte Carlo simulation, indicating that naïve B cells typically undergo little, if any, expansion. Additionally, somatic hypermutation in the memory samples was investigated to detect convergent evolution of distinct naïve B cell clones toward common amino acid sequence (hence common antigen specificity) in the memory B cell compartment.

Disclosures:

DeWitt: Adaptive Biotechnologies: Employment, Equity Ownership. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Rieder:Adaptive Biotechnologies: Employment, Equity Ownership. Chung:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.
Patients With End Stage Congestive Heart Failure Have Decreased High Molecular Weight Von Willebrand Factor Multimers and Higher Plasma Levels Of Total TGF-β1: Implantation Of a Left Ventricular Assist Devices (LVAD) Exacerbates Both Abnormalities
Authors: Ahamed Jasimuddin , Monteagudo Juan , Kinkhabwala Mona , Suarez-Farinas Mayte , Garan Arthur Reshad , Coller Barry S. , Mancini Donna
Published: October 2019

Left ventricular assist devices (LVADs) have improved survival of patients with end stage congestive heart failure (HF), but patients are at high risk of both hemorrhage and thrombosis. Acquired von Willebrand (vW) disease is observed in most patients after continuous flow LVAD insertion, presumably due to shear-dependent vW factor (vWf) cleavage, which may contribute to hemorrhage, but there are no validated thrombosis biomarkers. Since shear can also activate platelets, and platelets contain high concentration of transforming growth factor β1 (TGF-β1), we analyzed plasma TGF-β1 levels in patients before and after LVAD implantation and correlated the data with the loss of high molecular weight (HMW) vWf multimers.

Blood samples were collected from 14 HF patients enrolled either as a destination therapy (n=2) or as a bridge to transplantation (n=12) at the Columbia University before and after LVAD implantation. Plasma was prepared by centrifuging blood at 12,000 rpm for 5 min at 4°C within 5 min of blood drawing. Total TGF-β1 levels were measured after acidification of samples using a 2-antibody ELISA (R&D Systems). The platelet α-granule proteins thrombospondin 1 (TSP-1) and platelet factor 4 (PF4) were detected by immunoblotting. vWf multimers were analyzed by electrophoresis of plasma after running in 1.2% discontinuous agarose gel followed by in-gel immunoreactivity band quantification using the LI-COR imaging system. HMW-vWf multimers were defined as the percentage of total vWf antigen contributed by multimers above the 11th identifiable band, starting from the cathodal position.

Plasma total TGF-ß1 levels were higher in HF patients before LVAD implantation than in 16 healthy controls (3.76 ± 1.55 vs. 1.0 ± 0.60 ng/ml; p <0.001). After LVAD insertion, the levels in HF patients increased further to 5.20 ± 2.30 ng/ml (p=0.014). Plasma TGF-ß1 levels in HF patients before and after LVAD implantation were positively correlated with TSP-1 levels (r=0.86; p<0.0001), but PF4 was not detectable, suggesting in vivo release of TGF-β1 from platelets. A reduced percentage of HMW-vWf multimers was observed in HF patients before LVAD insertion compared to healthy controls (18.0 ± 10.0 vs. 29.5 ± 2.5 %; p=0.018), and the percentage was further reduced after LVAD implantation (11.0 ± 9.0 %; p=0.027). There was a weak negative correlation between the percentage of HMW-vWf multimers and TGF-ß1 after LVAD implantation (r= - 0.40; p=0.05).

We conclude that HF itself, even before LVAD insertion, is associated with both loss of HMW-vWf multimers and elevation of plasma TGF-ß1 levels and both abnormalities are exacerbated by LVAD implantation.

Disclosures:

No relevant conflicts of interest to declare.
Platelet Sequestered Proteins Mediate Pre-Metastatic Communication Between Tumors and Bone
Authors: Kerr Bethany A. , Byzova Tatiana V.
Published: October 2019

Tumors can be considered parasites that exploit the host’s resources in order to promote their own growth. Tumors secrete growth factors and cytokines (tumor secretome) which recruit platelets, blood vessels, and bone marrow-derived cells (BMDCs) to promote angiogenesis and tumor progression. We have recently demonstrated that distant tumor growth can stimulate bone formation and BMDC mobilization prior to metastasis. However, the mechanism of communication between the primary tumor and the bone microenvironment remains unknown. Signals from the tumor to the bone microenvironment are likely transmitted through the circulation; nevertheless, these signals need to be protected from degradation or utilization prior to reaching their target. We hypothesized that platelets could function as regulated storage compartments for the tumor secretome. Using a murine platelet depletion model during tumor growth, we assessed the role of platelets in BMDC homing and bone remodeling. We demonstrate that platelet depletion prevents BMDC mobilization and recruitment into tumors. Further, platelet depletion inhibited tumor-induced bone formation. To determine which tumor-derived proteins in platelets were responsible for changes in BMDC mobilization and bone remodeling, we analyzed the tumor secretome components and found that 77% of proteins are enriched within platelets over plasma, and that these proteins regulate BMDC mobilization and bone remodeling. In addition, we discovered that tumor-derived proteins, specifically transforming growth factor (TGF)-β1 and matrix metalloproteinases, sequestered within platelets could stimulate osteoblast differentiation and mineralization in vitro. We also found that thrombospondin (TSP)-1 levels are increased in the platelets of tumor-bearing mice. Finally, we assessed the role of the TSP-1/TGF-β1 signaling axis in tumor-induced bone formation and BMDC mobilization. Tumors in TSP-1 null mice were larger, however tumor-induced bone remodeling decreased. Interestingly, in tumor-bearing mice, high levels of TGF-β1 were being retained in the platelets due to a lack of TSP-1 to activate and release TGF-β1. To examine the role of platelet TGF-β1 in the process of tumor-induced bone remodeling, tumors were implanted in platelet-specific TGF-β1 conditional knockout mice and the changes in tumor size and the bone microenvironment were assessed. In summary, our data show that platelet sequestration and secretion of tumor-derived proteins represent a cellular mechanism mediating communication between the bone microenvironment and the primary tumor.

Disclosures:

No relevant conflicts of interest to declare.
Adenosine Inhibits Pro-Inflammatory Thrombin Signaling In Endothelial Cells
Authors: Hassanian Seyed Mahdi , Rezaie Alireza R.
Published: October 2019

Adenosine is an important regulatory metabolite which attenuates inflammation when it binds and activates the adenosine A2A receptor subtype on immune cells. However, the effect of adenosine on inflammatory responses in endothelial cells is mostly unknown. Thrombin as a known pro-inflammatory protease is involved in a variety of pathophysiological processes associated with inflammation in stimulated endothelial cells. The present study investigated the effect of adenosine on thrombin-mediated modulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs). Adenosine, in a concentration-dependent manner (1-100µM), inhibited the barrier disruptive effect of thrombin (20nM) on endothelial monolayer. The expression level of adenosine receptors, A1, A2A, A2B and A3 was examined in HUVECs and it was found that A2A and A2B are the highest expressing receptors among the four subtypes (A2B> A2A>A1>A3) in endothelial cells. Further studies revealed that the barrier protective effect of adenosine on thrombin-induced hyperpermeability in HUVECs was abrogated by the A2A receptor specific siRNA or the A2A receptor specific antagonist, ZM-241385, but not by siRNAs targeting the other adenosine receptor subtypes. To further determine the molecular mechanisms of the barrier protective effect of adenosine, its effect on thrombin-induced RhoA activation was assessed. Pretreatment of endothelial cells with adenosine prevented both thrombin-induced RhoA activation (Rho-GTP) and its membrane translocation as evidenced by cell fractionation and Pull-down assays, respectively. Moreover, preincubation of endothelial cells with adenosine down-regulated the expression of cell surface adhesion molecules (VCAM-1 and ICAM-1) and thrombin-mediated activation of nuclear factor-kappaB (NF-kB) pathway. Adenosine also inhibited secretion of the early chemokine, MCP-1, and the late-acting pro-inflammatory cytokine, HMGB-1, by thrombin-stimulated endothelial cells. Taken together, these results suggest that adenosine can inhibit pro-inflammatory thrombin signaling responses in endothelial cells by specifically activating the A2A receptor subtype, thereby protecting endothelium during the activation of coagulation and inflammatory pathways.

Disclosures:

No relevant conflicts of interest to declare.