bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel SLC1A4 Mutation (p.Y191*) Causes Spastic Tetraplegia, Thin Corpus Callosum, and Progressive Microcephaly (SPATCCM) With Seizure Disorder
Authors: Abdelrahman Hanadi A. , Al-Shamsi Aisha , John Anne , Ali Bassam R. , Al-Gazali Lihadh
Published: October 2019

Spastic tetraplegia, thin corpus callosum, and progressive microcephaly is a recently described very rare autosomal recessive neurodevelopmental disorder. This disease was first described in 2015 in several families from the Ashkenazi Jewish ancestry with a founder mutation in SLC1A4 (p.E256K) as the underlying genetic cause. SLC1A4 gene encodes for the amino acid transporter ASCT1 that is necessary for serine cellular transport to neurons. We clinically evaluated 2 Pakistani siblings with severe global developmental delay, progressive microcephaly, and seizure disorder. We performed exome sequencing, Sanger sequencing, and segregation analysis to identify the genetic cause of the phenotype followed by in silico analysis to evaluate the pathogenicity of the identified mutation. We identified a novel homozygous variant (c.573T>G) in both patients. The mutation is predicted to cause nonsense mutation (p.Y191*) in the ASCT1 protein. Here, we report the fifth disease causing mutation in SLC1A4 gene and review all previously reported cases.
CD34+ Cobblestone Area-Forming Cells Are a Valuable Indicator to Screen the Soluble Inhibitors of Hematopoiesis in the Serum of Aplastic Anemia and MDS Patients.
Authors: Kikuchi Shohei , Kobune Masayoshi , Murase Kazuyuki , Iyama Satoshi , Sato Tsutomu , Kawano Yutaka , Miyanishi Koji , Sato Yasushi , Takimoto Rishu , Kato Junji
Published: October 2019

Abstract 1439

Poster Board I-462

We have previously shown that cobblestone area (CA) could be observed underneath human stromal cells 2 weeks after coculture in serum free condition. However, it was difficult to evaluate 5 week CA-forming cells (CAFC) which is a surrogate indicator of hematopoietic stem cells because human stromal cells could not survive more than 4 weeks in a serum free media, X-VIVO10. In the present study, we optimized serum free media, combination of cytokines and chemical agents such as stem cell factor (SCF), thrombopoietin (TPO), flt3 ligand (FL) with or without delta like protein 4 (DLL4) and GSK inhibitor. In addition, we added the serum derived from aplastic anemia and MDS patients in this coculture system and evaluated the effect of serum on in vitro hematopoiesis. We found that certain serum free media could maintain the layer of human stromal cells more than 8 weeks. By using this serum free media, bone marrow (BM) CD34+ cells could be cocultured for 8 weeks without disruption of the human stromal layer. Five week CAFC could be observed in all condition of cytokine combination with or without chemical inhibitor. However, because cytoplasmic appearance of cells in some CAs is quite irregular without DLL4 or GSK inhibitor, we conducted immunohistochemical staining by using FITC-labeled anti human CD34, CD11b or CD33 antibodies. Surprisingly, we found that CD34+CAFCs were exclusively observed in the presence of DLL4 or GSK inhibitor. Otherwise, CAs was composed of CD11b+/CD45+ hematopoietic cells. Moreover, NOD/SCID repopulating cells were detected in the coculture containing CD34+CAFCs, suggesting that CD34+CAFCs were a valuable indicator of hematopoietic stem cells (HSCs). Using CD34+CAFCs as a surrogate maker of HSCs, we assessed whether the serum derived from patients of aplastic anemia and MDS could inhibit formation of CD34+CAFC. Notably, some of patient's serum could reduce the number of CD34+CAFCs. These results indicated the possibility that CD34+CAFCs underneath human stromal layer could be useful for screening certain soluble factors to inhibit hematopoiesis in aplastic anemia and MDS.

Disclosures

No relevant conflicts of interest to declare.
CXCR7 Expression Restricts CXCR4/SDF-1 Mediated Hematopoietic-Supporting Activity of Stromal Cells by Decreasing Extracellular SDF-1 Availability.
Authors: Bouamar Hakim , Zhang Yanyan , Jouni Dima , Wittner Monika , Bensidhoum Morad , Jarrier Peggy , Petite Hervé , Vainchenker William , Albagli Olivier , Louache Fawzia
Published: October 2019

Abstract 1452

Poster Board I-475

Stromal cell-derived factor 1 (SDF-1)/CXCR4 axis plays a major role in regulating the interactions between hematopoietic stem and progenitor cells (HPSC) and their stromal microenvironment within the bone marrow. A second SDF-1/CXCL12 receptor, CXCR7 binds SDF-1 with high affinity but little is known about its function in hematopoiesis. In the present study, we demonstrate that the activity of CXCR7 is crucial for proper maintenance of hematopoietic activity on stromal layers. Using quantitative reverse transcription-PCR analysis, we demonstrate that CXCR7 is highly expressed in stromal cells in contrast to hematopoietic cells showing that it functions primarily in the stromal microenvironment compartment. CXCR7 stable expression in UT7 hematopoietic cells fails to support SDF-1 induced migration and signalling but inhibits migration of CXCR4 expressing cells in paracrine manner. Overexpression of CXCR7 in MS-5 stromal cells lead to a reduction of SDF-1 concentration in the supernatants. In addition, supernatants from these cells had substantially lower efficiency in promoting integrin alpha-4 beta-1–mediated adhesion and migration of Mo7e cells to vascular cell adhesion molecule-1 (VCAM-1) and CS-1/fibronectin than their control GFP counterparts. Moreover, human cord blood CD34+ hematopoietic progenitor cells displayed SDF-1–dependent reduced responses in chemotaxis, transendothelial migration, and up-regulation of adhesion to VCAM-1 when supernatants from CXCR7 expressing MS-5 cells were used compared with supernatants from GFP expressing cells. Finally, phenotypical primitive murine cells displayed reduced hematopoietic activity when cultured on CXCR7 expressing MS-5 cells. This reduced hematopoietic activity is partly reverted when recombinant SDF-1 was added to CXCR7 overexpressing MS-5 cells. Taken together, our results indicate that CXCR7-controlled disponibility of SDF-1 expression influences BM cell migration, adhesion and hematopoietic activity and thus behaves as a decoy receptor regulating the SDF-1 level.

Disclosures

Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.
Single-Stranded DNA Binding Proteins (SSBPs) Interact with LDB1 Homodimers to Facilitate DNA Looping.
Authors: Cai Ying , Nagarajan Lalitha , Brandt Stephen J.
Published: October 2019

Abstract 1469

Poster Board I-492

The LIM domain binding protein LDB1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in hematopoiesis and other developmental programs. We have shown that LDB1 and its LIM-HD and LMO interaction partners are protected from ubiquitylation by a small family of SSBPs. Recently, we demonstrated that these SSBPs bind to LDB1 homodimers to promote formation of a ternary complex containing two molecules of LDB1 and SSBP. This was dependent on an intact LDB1 dimerization domain (DD) and produced a shift, directly or indirectly, in the equilibrium between LDB1 monomer and dimer. In the present study, we introduced a 24-glycine linker between two full-length LDB1 peptide-coding sequences and expressed this forced or tethered LDB1 dimer (TD-LDB1) in vitro and in vivo. First, both TD-LDB1, introduced into cells by transfection, and endogenous LDB1 were found to have the same turnover rate, indicating that protection from ubiquitylation was independent of dimerization status. Second, TD-LDB1 was fused to the DNA binding domain of GAL4 (GAL4DBD) and in a second construct to the activation domain of herpesvirus VP16 (VP16AD) and these constructs were expressed in cells with a GAL4 reporter plasmid. Co-expression of GAL4-LDB1 and VP16-LDB1 significantly increased reporter luciferase activity as a result of dimerization, while co-expression of GAL4-TD-LDB1 with VP16-LDB1 did not, ruling out formation of LDB1 trimers. Likewise, co-expression of GAL4-TD-LDB1 with VP16-TD-LDB1 did not significantly affect luciferase activity, indicating that LDB1 also cannot form protein tetramers. In contrast, TD-LDB1 was able to bind SSBP2 and SSBP3 in both chemical cross-linking and mammalian two-hybrid assays, consistent with the SSBP interacting with preformed LDB1 dimers. Finally, the complete 200-amino acid DD of LDB1, reported as necessary and sufficient for protein dimerization, was confirmed in cross-linking analysis to act as a dominant negative inhibitor of LDB1 dimerization. When the DD was introduced into Lhx2-, Ldb1-, and Ssbp3-expressing cells, application of a modified electrophoretic mobility assay that can detect linking of two DNA probes in solution revealed that the DD reduced formation of a ‘looped’ complex containing two DNA probes and led to the appearance of a new complex containing Lhx2, Ssbp3, and Ldb1, apparently in a monomeric form. In summary, this work elucidates a novel function of SSBPs in enhancing LDB1 dimerization and, ultimately, long-range communication between cis regulatory regions in genes. In addition, it suggests that SSBPs bind dimeric LDB1 and induce an allosteric change in the adjacent SSBP interaction domain rather than vice versa. Finally, these results lead to the prediction that an SSBP- and LDB1-containing complex could promote looping between promoter-proximal and promoter-distal LIM-HD binding elements.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Control of C/EBPa by Distant Synergic Regulatory Elements.
Authors: Ebralidze Alexander K , Ruscio Annalisa Di , Lee Sanghoon , O'Brien Karen , Tenen Daniel G.
Published: October 2019

Abstract 1470

Poster Board I-493

The transcription factor C/EBPa plays a pivotal role in hematopoietic stem cell (HSC) commitment and differentiation. Expression of the C/EBPa gene is tightly regulated during normal hematopoietic development, and dysregulation of C/EBPa expression can lead to lung cancer and leukemia. However, little is known about how the C/EBPa gene is regulated in vivo.

In this study, we demonstrate synergetic regulation of C/EBPa by two distant cis-elemets located 5' and 3' to the gene and their effect on chromatin architecture.

Previous studies have indicated that as much as 4.8 kb of 5' upstream C/EBPa regulatory sequences were unable to express significant levels of reporter gene activity in transgenic mice. Therefore, we initiated a search for important distal elements in the C/EBPa locus. We have applied a combination of 1) comparative analysis of human and mouse genomic sequences; 2) DNase I hypersensitive studies; 3) chromosome conformation capture (3C); 4) analysis of reporter constructs in stable cells lines; and 5) generation and analysis of transgenic mouse lines. This let us to identify the regulatory role of two distal conserved homology elements located at ∼38 kb 5' of the transcription start site (TSS) of murine C/EBPa (corresponding to ∼45 kb 5' of the TSS of human C/EBPa) and at ∼33 kb 3' to TSS of both murine and human C/EBPa. We show that the constructs lacking both distal elements were unable to express C/EBPa mRNA, while addition of each region resulted in detectable (by Northern blot analysis) expression in transgenic animals.

We have observed a cooperative effect of these two regions on C/EBPa expression, a construct carrying both elements expresses ∼2.5-fold level over constructs carrying either one element alone.

We have investigated the mechanism for the increased expression by these distal elements by using deletion constructs. Our results suggest that lack of these elements results in aberrant gene expression due to proximal promoter DNA hypermethylation and point to a novel mechanism in establishment of critical epigenetic marks.

Disclosures:

No relevant conflicts of interest to declare.
Clonal Analysis of the Human Hematopoietic Hierarchy Reveals An Early Lymphoid Progenitor with Extensive Monocytic Potential.
Authors: Doulatov Sergei , Notta Faiyaz , Dick John E
Published: October 2019

Abstract 1503

Poster Board I-526

The classical model of hematopoiesis posits the segregation of lymphoid and myeloid lineages as the earliest fate decision. Although the validity of this model in the mouse has recently been questioned, its status in human hematopoiesis is unclear, since little is known concerning lineage potential of human progenitors at the clonal level. We isolated and clonally mapped the developmental potential of each major progenitor class from neonatal cord blood and adult bone marrow providing the first comprehensive analysis of the human hematopoietic hierarchy. Human myeloid commitment follows the classical pattern of lineage restriction, however lymphoid development is initiated by a novel cell type, termed lympho-myeloid progenitor (LMP), which displays extensive monocytic potential. However, this myeloid capacity is lost following B- or T/NK-cell lineage commitment. The myeloid potential of LMPs is sensitive to extrinsic signals and can be directed towards differentiation into dendritic cells (DCs). Thus, human lymphoid development does not follow a rigid model of segregation of myeloid-lymphoid lineages, but proceeds through LMPs. LMPs can be massively expanded and differentiated into mature T-cells and DCs that are functionally indistinguishable from DCs derived from peripheral blood monocytes. The prospective isolation and elucidation of clonal lineage potential of human progenitors provides the basis for novel cellular therapeutics and a powerful means to uncover the cellular and molecular regulators that govern human lineage commitment.

Disclosures:

Dick: Roche: Research Funding; CSL Ltd: Research Funding.
The First Trimester Human Placenta Is An Embryonic Hematopoietic Organ with An Unexpected Function in Primitive Erythroid Maturation.
Authors: Van Handel Ben , Prashad Sacha , Huang Andy , Hassanzedeh-Kiabi Nargess , Magnusson Mattias , Atanassova Boriana , Hamalainen Eija , Chen Angela , Mikkola Hanna
Published: October 2019

Abstract 1508

Poster Board I-531

Developmental hematopoiesis satisfies the immediate needs of the embryo and provides the hematopoietic stem cells necessary for lifelong blood homeostasis. Recently, the mid-gestation mouse placenta was identified as an important definitive hematopoietic organ; data from us and others indicates that the human placenta functions analogously, harboring and potentially generating hematopoietic stem and progenitor cells. The function of the human placenta in embryonic hematopoiesis has not been addressed. We assessed the hematopoietic potential of placental tissues before the onset of feto-placental circulation and found robust de novo generation of clonogenic progenitors. Interestingly, pre-circulation progenitors were greatly enriched (69 ± 14% of total) in macrophage progenitors. Immunostaining demonstrated that these progenitors are generated in the chorionic plate. As development progresses, placental macrophages migrate to the villous stroma. Surprisingly, in the villi, placental macrophages associate closely with an unexpected population of extravascular, z-globin+ primitive erythroid cells, prompting the hypothesis that embryonic macrophages promote the maturation of primitive erythroblasts in the placenta. Concordantly, we found that human primitive erythroblasts enucleate, as has been recently demonstrated in mice. Interestingly, the first enucleated erythroid cells were found in the villous stroma; between 5-7 weeks developmental age, their relative frequency in the stroma was 2-4 fold higher than in lumens. We also observed free nuclei in the placental stroma; a similar population of ejected red cell nuclei, termed pyrenocytes, has recently been described in mouse embryos. Immunohistochemistry and FACS confirmed that these free nuclei in the placenta were pyrenocytes. Electron microscopy revealed placental macrophages containing ingested red cell nuclei. Together, this data suggests that placenta-derived macrophages promote the terminal maturation of primitive erythroblasts in the villous stroma, nominating the first trimester human placenta as a site of primitive hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
Association of G6PD −202/−376 with Lower Hemoglobin Concentration but Not Increased Hemolysis in Patients with Sickle Cell Anemia.
Authors: Nouraie Mehdi , Reading Noel S. , Campbell Andrew , Minniti Caterina , Rana Sohail R , Luchtman-Jones Lori , Gladwin Mark , Kato Gregory J , Castro Oswaldo L , Prchal Josef T. , Gordeuk Victor R.
Published: October 2019

Abstract 1511

Poster Board I-534

Background

What determines the degree of hemolysis and of anemia in patients with hemoglobin SS is not fully known. The rate of hemolysis and severity of anemia are ameliorated by the presence of alpha thalassemia and by higher hemoglobin F percentage. Mild G6PD deficiency in the form of G6PD-202/-376 may be associated with episodic hemolysis in individuals of African descent, but past studies indicated little influence of G6PD-202/-376 on the degree of hemolysis and anemia in sickle cell disease patients (1,2). In this study we examined the roles of single and double α-globin deletions and G6PD-202/-376 on the degree of hemolysis and the hemoglobin concentration in hemoglobin SS patients.

Methods

Two hundred sixty two children and adolescents with hemoglobin SS were recruited at three tertiary medical centers and studied at steady state. Principal component analysis was used to develop a hemolytic component from concentrations of lactate dehydrogenase, aspartate aminotransferase and bilirubin. PCR was used to determine the presence of α-thalassemia and G6PD-202/-376. Multivariate models were employed to determine the independent effects of these genotypes on hemoglobin concentration and degree of hemolysis.

Results

Single a-globin deletion was associated with an estimated 0.4 g/dL increase in steady-state hemoglobin concentration and double α-globin gene deletion with a 0.8 g/dL increase (P = 0.005 for trend) due to, progressively lower degrees of hemolysis (P = 0.004). G6PD-202/-376 was associated with an estimated 0.7 g/dL decrease in the hemoglobin concentration (P = 0.003) (Figure 1a), but this observation could not be explained by increased hemolysis. Rather, the reticulocyte count was an estimated 22% lower with G6PD-202/-376 (P = 0.032) (Figure 1b).

Discussion

G6PD -202/-376 may be associated with lower hemoglobin concentration in sickle cell anemia and the mechanism is probably impaired erythropoiesis rather than hemolysis. A recent study (3) indicates that G6PD is needed for definitive erythropoiesis as well as for normal survival of red blood cells in the periphery. Our present findings raise the possibility that, in the setting of the markedly increased erythropoiesis of sickle cell anemia, G6PD-202/-376 may result in impairment in erythropoiesis that is discernible in the peripheral blood hemoglobin concentration.

Disclosures

No relevant conflicts of interest to declare.
Distinct Roles for the α, β and γ Isoforms of the Catalytic Subunit of Protein Phosphatase 1 in Outside-in Integrin αIIbβ3 Signaling.
Authors: Alrehani Nawaf , Pradhan Subhashree , Vijayan K. Vinod
Published: October 2019

Abstract 154

Outside-in integrin αIIbγ3 signaling mediated by kinases and phosphatases regulate platelet adhesion and thrombus formation. The catalytic subunit of protein phosphatase 1 (PP1c), expressed as α, β and γ isoforms regulates a variety of cellular functions. We have previously demonstrated that fibrinogen-αIIbβ3 engagement during outside-in signaling dissociated integrin αIIb*β3 anchored PP1c, which was followed by PP1c activation and dephosphorylation of myosin light chain (MLC). However, it is unknown whether any functions regulated by outside-in integrin αIIbβ3 signaling are modulated by PP1c. To overcome the lack of specificity of pharmacological agents towards PP1c, we have pursued a genetic approach to inhibit PP1c in 293 cells overexpressing αIIbβ3. In this study, we demonstrated that PP1cα interacts with αIIbβ3 in platelets and 293 cells by co-immunoprecipitation and GST pull down assays. Knockdown of PP1cα by short interference RNA resulted in a 50-60% enhanced adhesion (p<0.003) of 293 cells to immobilized fibrinogen. Conversely, overexpression of PP1cα inhibited (p<0.002) αIIbβ3 adhesiveness to fibrinogen. Furthermore, the enhanced adhesiveness of PP1cα depleted 293 cells was also observed with von Willebrand factor, indicating that the differential adhesion due to the lack of PP1cα was not ligand specific. Signaling pathways implicated in the reorganization of cellular cytoskeleton, like extracellular-regulated kinase (ERK1/2) and AKT were activated in PP1cα depleted 293 cells. Such observations imply that PP1cα, knockdown of PP1cγ, which also associates with αIIbβ3, did not affect the adhesion to fibrinogen. Indeed, platelets from the PP1cγ null and wild type mice showed comparable adhesion to fibrinogen. Unexpectedly, depletion of PP1cβ isoform in 293 cells significantly (p<0.01) inhibited the ability of these cells to adhere to fibrinogen. Since MLC is a specific substrate of PP1cβ, it is likely that alterations in the MLC phosphorylation may underlie the loss of adhesiveness in PP1cβ depleted cells. Finally, depletion of all the PP1c isoforms also significantly (p<0.001) inhibited the adhesion of 293 cells to fibrinogen. These data illustrate that each isoform of PP1c contributes distinctly to the adhesive phenotype of integrin αIIbβ3 and implies that the control of outside-in integrin αIIbβ3 signaling response might be a composite of all PP1c isoforms. Perhaps, each isoforms have specific substrates that contribute to the process of adhesion, such that alterations in the phosphorylation of these substrates may underlie the distinct functional roles for PP1c.

Disclosures:

No relevant conflicts of interest to declare.
Reproducibility of International Harmonization Project (IHP)-Based Interpretations of Posttherapy PET/CT in Patients with Hodgkin's and Aggressive Non-Hodgkin's Lymphoma.
Authors: Juweid Malik E. , Thomas Damita , Berbaum Kevin , Bushnell David , Graham Michael M
Published: October 2019

Abstract 1556

Poster Board I-579

Background

IHP-based interpretation of PET/CT post-tx of lymphoma is now widely used but there is little data regarding its reproducibility and accuracy. We determined interobserver variability using IHP criteria and compared its accuracy with semiquantitative approaches using the residual mass (RM) absolute standardized uptake value (SUVmax) alone and the ratio of the RM SUVmax to mediastinal blood pool (MBP) SUVmean.

Methods

Three expert nuclear medicine physicians blinded to patient (pt) outcome independently visually interpreted post-tx PET/CT scans based on the IHP-criteria. RMs were scored on a scale of 0-4, where 3-4 was positive for RMs ≥ 2 cm whereas 1-4 was positive for RMs < 2 cm; scans without RMs were similarly scored, depending on whether there were PET positive or negative. RM SUVmax, MBP SUVmean and the RM SUVmax to MBP SUVmean ratio were determined. Since outcome data were available on all 50 pts (median follow-up of pts without progression = 49 months), the positive predictive value (PPV), negative predictive value (NPV) and accuracy for prediction of residual disease/progression were compared between the IHP-criteria and the semiquantitative approaches.

Results

A total of 50 post-tx scans in 50 pts with aggressive NHL (n=24) and HL (n=26) were interpreted by the 3 readers. On a patient-by-patient basis, the 3 readers were in complete agreement with respect to final scan interpretation (i.e., pos/neg) 90% of the time using the IHP-criteria. Percentage of PET+ scans ranged from 20%-30% and PET+ RMs from 45%-55% for the 3 readers. On a per-patient basis, visual criteria resulted in PPVs of 47%-70%; NPVs of 86%-88% and accuracies of 74%-84% for the 3 readers. In contrast, cut-off RM SUVmax of 2.5 or cut-off RM SUVmax to MBP SUVmean ratio of 1.75 resulted in PPV, NPV and accuracy of 75%, 86% and 84% with areas under the receiver operating characteristic (ROC) curve of 0.765 and 0.787, respectively.

Conclusions

Visual IHP-criteria provide fairly reproducible interpretations among nuclear medicine experts with fair to moderate diagnostic/prognostic accuracy. Compared with the IHP criteria, the semiquantitative approaches provide, at least for some readers, substantial improvement in PPV and overall accuracy. These approaches should, therefore be considered for a more consistent scan interpretations after standardization of the timing of imaging after FDG injection.

Disclosures

No relevant conflicts of interest to declare.
Impact of Routine Surveillance Imaging on the Outcome of Patients with Relapsed Hodgkin Lymphoma.
Authors: Basciano Basciano A. , Moskowitz Craig , Zelenetz Andrew D.
Published: October 2019

Abstract 1558

Poster Board I-581

Purpose

To determine the role of asymptomatic post-remission surveillance imaging in the diagnosis of first relapse and its impact on outcomes in patients with Hodgkin lymphoma (HL).

Patients and Methods

The impact of surveillance imaging on diagnosis of first relapse and outcomes was determined by analysis of a defined patient population with relapsed HL who underwent high dose therapy followed by autologous stem cell rescue (HDT/ASCR). We retrospectively identified 114 patients from the HDT/ASCR database with biopsy-confirmed, relapsed HL; 94 patients had adequate data and follow-up for inclusion in the analysis. Details of surveillance imaging were obtained including: frequency; type; indication; and results of post-remission imaging. The indication for imaging was classified as asymptomatic surveillance (AS) or clinically indicated (CI; i.e. to investigate symptoms or physical examination findings). We have previously reported a validated prognostic model for relapsed HL that found time from initial therapy, presence of B symptoms at relapse and extra-nodal disease at relapse to be adverse factors (Moskowitz et al. Blood 97:616). We determined the prognostic risk group (PRG) (low [L] 0-1 factor, intermediate [I] 2 factors, high risk [H] 3 factors) for all patients. Overall and failure-free survivals were determined using the methods of Kaplan and Meier.

Results

Patient characteristics included: median age 32 years; PRG L: 65%, I: 31%, H: 4%; AS 36 (38%), CI 58 (62%). The median follow-up for surviving patients was 7.4 years. The PRG (L, I/H) correlated to outcome, validating its applicability in this patient cohort: FFS at 5 years was 64.8% and 49.4% respectively, p=0.045. PRGs were evenly distributed between the AS and CI groups: L: 64% v 66%; I/H: 36% v 37% p=0.48. The FFS at 5 years for patients in the AS and CI groups was 58.4% and 59.3% respectively, p=0.9; similar there was no difference in 5 year OS, AS 62.4% and CI 73.3% p=0.6. Within a given risk group (L or I/H) patients in the AS group did not have a superior outcome compared to the CI group.

Conclusion

AS does not identify a group of patients with first relapse of HL with a more favorable risk profile according the MSKCC prognostic model. While nearly 40% of patients were identified with relapse as a consequence of AS imaging, this did not identify a group of patients with relapsed HL with improved outcomes compared to patients who had CI imaging. If these results are confirmed in a prospective study, AS may be safely eliminated in HL after remission to reduce cost and long-term risk of the diagnostic imaging.

Disclosures

No relevant conflicts of interest to declare.
Apolipoprotein AIV (ApoA-IV) Is a Novel Ligand of Platelet β3 Integrin That Negatively Regulates Platelet Adhesion, Aggregation, and Thrombosis.
Authors: Spring Christopher M. , Jin Wuxun , Yang Hong , Reheman Adili , Zhu Guangheng , She Yimin , Cyr Terry , Tso Patrick , Davidson Sean , Freedman John , Ni Heyu
Published: October 2019

Abstract 156

Platelet adhesion and aggregation at sites of vascular injury are key events required for haemostasis and thrombosis. It has been documented that von Willebrand factor (VWF) and fibrinogen (Fg) are required for platelet adhesion and aggregation. However, we previously showed that occlusive thrombi still form in mice deficient for both Fg and VWF (Fg/VWF−/−) via a β3 integrin-dependent pathway. Here, we have investigated novel, non-classical ligands of β3 integrin that may regulate platelet adhesion and aggregation. To identify potential ligand(s) of β3 integrin, latex beads were coated with purified human platelet β3 integrin and incubated with human plasma. Protein(s) specifically associated with β3 integrin were electrophoresed and apolipoprotein AIV (ApoA-IV) was identified by mass spectrometry. We found that ApoA-IV binds to the surface of stimulated platelets, but not to quiescent platelets or β3−/− platelets, and ApoA-IV/platelet association was blocked by the addition of a specific anti-β3 integrin monoclonal antibody. It appears that ApoA-IV binds to, but is not internalized by platelet β3 integrins. ApoA-IV-deficient (ApoA-IV−/−) mice exhibited enhanced platelet aggregation induced by ADP, Collagen, and TRAP in plasma (but not PIPES buffer) compared to wild type (WT) littermates. This enhancement was diminished when ApoA-IV−/− plasma was replaced by WT plasma, indicating that the reduction was due to plasma ApoA-IV and not an unrelated platelet effect. When platelets were incubated with FITC-Fg, ApoA-IV was able to reduce platelet/Fg association, indicating that ApoA-IV may act to displace pro-thrombotic β3 integrin ligand(s). In support of this, ApoA-IV reduced the number of adherent platelets on immobilized Fg in perfusion chamber assays and enhanced thrombus formation was observed when ApoA-IV−/− mouse blood was perfused over collagen. We found that addition of recombinant ApoA-IV inhibited platelet aggregation and thrombus formation in vitro, while the control apolipoprotein ApoA-I did not. Using intravital microscopy, we further demonstrated that early platelet deposition was increased, and the time for thrombus formation and vessel occlusion were shorter in ApoA-IV−/− mice, which can be corrected by recombinant ApoA-IV transfusion. Furthermore, recombinant ApoA-IV inhibited WT platelet aggregation, thrombus formation and enhanced thrombus dissolution both in vitro and in vivo. Our data demonstrate for the first time that ApoA-IV is a novel ligand of platelet β3 integrin that negatively regulates thrombosis. These new data are consistent with the reported association between ApoA-IV and reduced cardiovascular diseases, and establish the first link between ApoA-IV and thrombosis.

Disclosures:

No relevant conflicts of interest to declare.
Low-Level FLT3-ITD Mutations Do Not Predict for Higher Relapse Rate in AML with Standard Risk Karyotpye.
Authors: Ward Richard , Kamel-Reid Suzanne , Xu Wei , Minden Mark D. , Schimmer Aaron D. , Yee Karen WL , Schuh Andre , Gupta Vikas , Brandwein Joseph
Published: October 2019

Abstract 1591

Poster Board I-617

High-level FLT3-ITD mutations predict for a higher relapse rate and inferior survival among patients with standard risk karyotype AML. The influence of low-level FLT3-ITD mutations is less clear, although the MRC group (Gale et al, 2008) reported that low-level mutations are associated with an inferior prognosis as compared to an unmutated group. In the absence of FLT3-ITD, NPM1 mutations predict for a lower relapse rate. We analyzed outcomes of intensive chemotherapy in patients with standard risk karyotype AML (n=131) between 2004-2008. Patients received uniform induction chemotherapy consisting of cytarabine plus daunorubicin (7+3), followed by 2 consolidations consisting of high-dose cytarabine plus daunorubicin for pts under age 60 (n=96) or 7+3 followed by NOVE for pts age 60+ (n=35). Patients were divided into 3 groups based on the ratio of FLT3-ITD:FLT3-wt: There were 73 patients with negative FLT3-ITD (defined as FLT3-ITD:wt ratio < 0.01), 30 patients with low-level ITD (ratio > 0.01 – 0.37), and 28 patients with high-level ITD (ratio > 0.37). The mean presenting WBC was 32.2 ×109/L in the group with negative ITD, 49.4 ×109/L in the group with low-level ITD and 97.2 ×109/L in the group with high-level ITD (p < 0.0001). At a median follow-up of 19 months (range 2-95 months), the median OS of the entire group was 21.3 months. Patients with high-level ITD had a significantly inferior RFS (p = 0.0009) and OS (p = 0.003) as compared to the low-level and negative groups. Comparing the negative ITD and low-level ITD groups, there was no significant difference in RFS (p = 0.65) or OS (p = 0.18). The frequency of NPM1 mutations was not significantly different between these two groups (42% vs. 35%). These data were re-analyzed using a different cutoff according to % FLT-ITD positivity: negative < 1% (n=73), low-level 1 – 50% (n=42), high level > 50% (n=16). Using these ranges, the low-level FLT3-ITD group had a significantly lower CR rate (74% vs. 93%, p = 0.004) and OS (36% vs. 61%, p = 0.01) as compared to the group with negative FLT3-ITD, but there was no significant difference between these two groups with respect to RFS (2 year RFS 48% in low-level ITD group vs. 40% in negative ITD group). The high-level ITD group demonstrated significantly inferior RFS (p = 0.02) and OS (p = 0.01) as compared to the other two groups. These findings confirm the poor prognosis associated with high-level FLT3-ITD mutations due to high relapse rates. However, in contrast to previously reported data, we did not find that low level FLT3 –ITD mutations were associated with inferior RFS, although we did find, unexpectedly, that this low-level group had a lower CR rate.

Disclosures

No relevant conflicts of interest to declare.
Chemokines in Leukemic Infiltration of the Central Nervous System in Childhood Acute Lymphoblastic Leukemia.
Authors: Ramirez Manuel , Gomez Ana M , Martinez Carolina , Lassaletta Alvaro , Fuster Jose L , Esquembre Carlos , Guibelalde Mercedes , Pedro Gomez , Calvo Carlota , Molina Javier , Vivanco Jose L , de Toledo Jose Sanchez , Bureo Encarna , Madero Luis
Published: October 2019

Abstract 1627

Poster Board I-653

Background

Leukemic blasts from B-cell acute lymphoblastic leukemia (B-ALL) and T-ALL circulate through the blood stream and may infiltrate different organs. Extramedullary organs may act as sanctuaries for lymphoblasts, preventing the exposure to adequate levels of chemotherapeutic drugs. Typical extramedullary relapses are seen in testes and in central nervous system (CNS). We aimed at determining whether chemokines may play a role in the infiltration of the CNS by leukemic blasts in childhood ALL. We studied the expression of chemokine receptors in ALL blasts in marrow, as well as the levels of chemokine ligands in the cerebrospinal fluid (CSF) of children with B- or T-ALL. If chemokines had a role in CNS leukemic infiltration, the following should be confirmed: leukemic blasts should express high levels of the chemokine receptor/s for which high levels of its corresponding ligand/s were detected at the CNS.

Methods

This prospective study was approved by the local ethical committees for clinical research. Samples from 80 children in 10 Spanish pediatric oncology units were obtained. We detected the presence of leukemic blasts in CFS by flow cytometry. We defined leukemic infiltration of CFS samples as the presence of cells with the same immunophenotype as the leukemia in the marrow aspirates. We detected the expression levels of 9 CCR and 6 CXCR molecules in ALL blasts by flow cytometry in marrow aspirates. Levels of chemokine ligands were quantitated by Cytometric Bead Array or by commercial ELISA kits in CSF samples.

Results

We found that chemokine receptors expression and levels of chemokine ligands varied depending upon the lineage (T versus B), the maturation state (pre-T versus T; pro-B versus pre-B versus common-B) and the risk-status (high versus non-high) of the leukemia. T-ALL patients with high levels of CNS leukemic infiltration expressed significantly higher levels of CXCL10 compared to the same parameters of T-ALL patients with low/absent levels of CNS disease (p=0,049). Common B-ALL patients with high levels of CNS leukemic infiltration expressed higher levels of CCL22 compared to that of common B-ALL patients with low/absent levels of CNS disease (p=0,059). Among the 4 patients with a CNS relapse, we detected higher levels of CXCR3 (p=0,0038) and of its ligand, CXCL10 (p=0,0169), compared to patients who did not relapse.

Conclusions

Our study suggests that the CXCR3/CXCL10 axis may be involved in the CNS relapse of high-risk ALL in children: high expression of CXCR3 on marrow blasts plus high levels of CXCL10 in the CNS was associated with leukemic CNS relapse.

Disclosures

No relevant conflicts of interest to declare.
Heightened Susceptibility to Influenza Mortality in Immunodeficient Mice Caused by a T-Cell Specific Defect in SOD2.
Authors: Case Adam John , Mcgill Jodi L. , Sangster Stephen C. , Madsen Joshua M. , Tygrett Lorraine T. , Johns Anna G. , Takahashi Takamune , Shirasawa Takuji , Spitz Douglas R. , Meyerholz David K. , Waldschmidt Thomas J , Legge Kevin L , Domann Frederick E.
Published: October 2019

Abstract 1655

Poster Board I-681

The role of SOD2 in development and function of the immune system remains unknown. Due to the early lethality of constitutive SOD2 knock-out mice, we recently reported the creation of T-cell specific SOD2 conditional knock-out mice to identify an immunological role, if any, for MnSOD in a viable organism. Knock-out mice displayed decreases in T-cell numbers (∼2-5 fold) within all lymphoid organs assayed (i.e. thymus, spleen, lymph nodes, and peripheral blood). Quantitative morphometry on transmission electron micrographs of SOD2 knock-out thymocytes indicated defects in mitochondrial morphology compared to controls. Further analysis of T-cell subpopulations by flow cytometry revealed that áβ T-cells had the largest decrease in number (CD4-,CD8+ affected more than CD4+,CD8-), while the γd T-cell population appeared unaffected or increased. The SOD2 knock-out T-cells demonstrated a more sizable activated population (CD44+, CD62L-) than the controls (∼2 fold for both CD4-,CD8+ and CD4+,CD8-), and mRNA expression microarray analysis showed up-regulation of numerous genes involved in T-cell activation. To query immune system function in the mice with SOD2-/- T-cells, we challenged the knock-out mice with influenza A virus (IAV). Upon infection, the knockout animals suffered from increased morbidity (i.e. weight loss) and 100% succumbed to infection within 11 days, whereas all control animals made a full recovery despite initial morbidity. Characterization of T-cell response to IAV in the knock-outs revealed limited T-cell migration to the lungs (∼2-fold fewer), smaller IAV-specific tetramer-positive populations, and attenuation of interferon gamma (INFγ) production. Taken together, these results indicate that MnSOD is critical for proper immune system development and function. Future studies will examine the mechanism of the decrease in T-cell numbers as well testing pharmaceutical SOD mimetics that may demonstrate a rescue of the observed phenotype. This work was supported by NIH grants CA73612 and CA115438.

Disclosures

No relevant conflicts of interest to declare.
Targeting Bcl-2 Family Proteins in Adult T-Cell Leukemia/Lymphoma: In Vitro and In Vivo Effects of a Novel Bcl-2 Family Inhibitor ABT-737.
Authors: Ishitsuka Kenji , Ishikawa Chie , Yotsumoto Fusanori , Katsuya Hiroo , Kunami Naoko , Sawada Shigeki , Tanji Hiroe , Takeshita Morishige , Miyamoto Shingo , Mori Naoki , Tamura Kazuo
Published: October 2019

Abstract 1685

Poster Board I-711

Adult T-cell lymphoma/leukemia (ATLL) is a T-cell malignancy caused by human T-lymphotrophic virus type I (HTLV-I), and its therapeutic outcome is still remains very poor. Therefore, novel therapeutic strategies are needed to improve patient outcome. In this study, we elucidated the therapeutic potential to target anti-apoptotic Bcl-2 family proteins for the treatment of ATLL by using ABT-737 (Abbott Laboratories, Abbott Park, IL, USA), a small molecule inhibitor of Bcl-2, Bcl-XL and Bcl-w. We first validated the rationale of this study by assessing the expression of Bcl-2 family proteins among 25 lymph-node specimens derived from ATLL patients by using immunohistochemistory. Both or either of Bcl-2 and Bcl-XL proteins was highly expressed in 80% of specimens. We next examined the cytotoxicity of ABT-737 against ATLL cell lines. ABT-737 significantly inhibited growth of MT-1, MT-2 and HuT 102 cells with a concentration of 50 percent inhibition (IC50) at 72 h of 2.4, 0.23 and 0.008μM, respectively. We then elucidated the mechanism of growth inhibition induced by ABT-737 using MT-1 and MT-2 cells. ABT-737 induced apoptosis in MT-1, MT-2 cells with cleavage of caspase 9, 3 and PARP. ABT-737 also induced apoptosis in fresh tumor cells derived from patients with ATLL. We next elucidated the potential of ABT-737 to enhance the cytotoxicity induced by conventional chemotherapeutic agents. The interaction between them was evaluated using the Chou-Talalay method by determining the combination index. ABT-737 synergistically enhanced the cytotoxicity and apoptosis induced by either of doxorubicin, vincristine or etoposide, which is a current key drug to treat ATLL. Most importantly, ABT-737 significantly inhibited tumor growth of in vivo ATLL model using SCID mice inoculated by HuT 102 cells subcutaneously. The mean tumor volume, weight and serum level of soluble interleukin-2 receptor á of ABT-737 (100mg/kg/day)-treated mice were significantly lower than those of vehicle-treated mice after treatment for 21 days. Moreover, massive induction of apoptosis in tumors treated by ABT-737 was observed by immunofluorescent TUNEL assay. These results suggest that ABT-737 used either alone or in combination with conventional cytotoxic drugs, represents a promising novel targeted approach to overcome drug resistance and improve patient outcome in ATLL.

Disclosures

No relevant conflicts of interest to declare.
Adoptive Therapy for Lymphoma with CD4 Memory T Cells Depleted of CD137-Expressing Tregs.
Authors: Goldstein Matthew J , Houot Roch , Kohrt Holbrook E , Brody Joshua , Levy Ronald
Published: October 2019

Abstract 1693

Poster Board I-719

A central goal of cancer immunotherapy is an adaptive immune response against tumors. The memory T cell is a critical mediator of this response as it can give rise to effector cells as well as self-renew. Regulatory T cells (Tregs) present a barrier to effective cancer immunotherapy. Indeed, cancer patients have increased numbers of CD4+CD25+ Tregs and cancer vaccination strategies in some cases expand this cell population. Here, we demonstrate that (1) CD4+CD44hi memory T cells are effective in mediating anti-tumor immunity and (2) that expression of CD137 can be used to exclude tumor-reactive Tregs from the CD4+CD44hi population.

We have established a model for adoptive therapy of mantle cell lymphoma in which CD4 T cells mediate anti-tumor immunity. Specifically, we use a whole tumor-cell vaccine to induce anti-tumor immune cells in vivo. These cells are isolated and adoptively transferred into lethally irradiated, syngeneic, recipient mice. We show that CD4 but not CD8 T cells from vaccinated donor mice can prevent tumor growth when adoptively transferred into irradiated recipient mice.

We observed that a majority of anti-tumor T cells display a memory phenotype. 10 days after transfer, T cells from recipient mice were co-cultured with irradiated lymphoma cells for 24 hours. Only CD4 T cells produced IFN-γ in response to co-culture and greater than 95% of IFN-γ+ CD4 T cells expressed the memory marker CD44. Finally, we observed that transferred CD4+CD44hi T cells persisted for over 100 days suggesting that this CD4 subset is important for lasting anti-tumor immunity.

Contaminating Tregs may limit the effectiveness of our CD4 T cell adoptive therapy. In order to functionally deplete these Tregs from the CD4 population, we sought to identify a surface marker that could uniquely distinguish tumor-reactive Tregs from other CD4 T cells. T cells from vaccinated donor mice were co-cultured with irradiated lymphoma cells for 24 hours. We evaluated a panel of activation markers and observed that CD137 expression defined a distinct population of Tregs. Based on these results, we used flow cytometry to isolate a sub-population of CD4+CD44hiCD137- T cells from vaccinated donor mice. Adoptive transfer of less than 100,000 CD44hiCD137- but not other sub-populations of CD4 T cells provided significant protection from tumor challenge. These results suggest that CD137 defines a novel population of tumor-reactive Tregs. This marker can facilitate the enrichment of anti-tumor CD4 memory T cells within the CD44hiCD137- population.

In conclusion, these findings highlight the potential use of CD4 T cells in adoptive therapy for mantle cell lymphoma.

Disclosures

No relevant conflicts of interest to declare.
The CXCR4 Antagonist AMD3100 Has Dual Effects, Initially Enhancing and Later Inhibiting Survival and Proliferation, in Myeloid Leukemia Cells in Vitro.
Authors: Kim Ha-Yon , Hwang Ji-Young , Kim Seong-Woo , Yun Gak-Won , Yang Young-Joon , Lee Hyo-Jin , Jo Deog-Yeon
Published: October 2019

Abstract 1721

Poster Board I-747

AMD3100, a small bicyclam antagonist for chemokine receptor CXCR4, induces the peripheral mobilization of hematopoietic stem cells. It also induces the segregation of leukemia cells in the bone marrow microenvironment, which should enhance the chemosensitivity of the cells. Based on these observations, AMD3100 is being considered for clinical use. However, AMD3100 activates G-protein coupled with CXCR4 and acts as a partial CXCR4 agonist. In this study, we explored whether AMD3100 affects the proliferation and survival of myeloid leukemia cells. As demonstrated previously, both AMD3100 and T140, another CXCR4 antagonist, markedly inhibited stromal cell-derived factor-1 (SDF-1)-induced chemotaxis and induced the internalization of CXCR4 in myeloid leukemia cell lines (U937, HL-60, MO7e, KG1a, and K562 cells) and CD34+ primary human acute myeloid leukemia (AML) cells. SDF-1 alone did not stimulate the proliferation of these leukemia cells, nor did it rescue the cells from apoptosis induced by serum deprivation. By contrast, AMD3100, but not T140, stimulated the proliferation of all five leukemia cell lines and primary AML cells in a dose-dependent manner in serum-free conditions for up to 5 days (∼ 2-fold increases at a concentration of 10-5M), which was abrogated by pretreating the cells with pertussis toxin. AMD3100 binds to CXCR7, another SDF-1 receptor, and all of the cells examined in this study expressed CXCR4 on the cell surface to some extent. The proliferation-enhancing effects of AMD3100 were not changed by knocking-down CXCR7 using the siRNA technique, whereas knocking-down CXCR4 significantly delayed the enhanced proliferation induced by AMD3100. Neither AMD3100 nor T140 induced the phosphorylation of Akt, Stat3, MAPK p44/p42, or MAPK p38, which are involved in SDF-1 signaling. In extended cultures of these cells for up to 14 days, AMD3100, but not T140, induced a marked decrease in the number of cells, compared to the control, after incubation for 5-7 days. Adding SDF-1 at the beginning and middle of the incubation did not affect the early increase or later decrease in the number of cells. AMD3100 reduced the apoptosis of these cells to a modest degree over the first 5-7 days and then markedly increased it. Consistent with the proliferation assay, AMD3100 increased the number of leukemia cell colonies during the early period of the assay, while it markedly decreased the number and size of the colonies in the later period of the assay. In conclusion, AMD3100 exerts dual effects, initially enhancing and subsequently inhibiting the survival and proliferation, in myeloid leukemia cells in vitro.

Disclosures

No relevant conflicts of interest to declare.