bluuebunny/crossref_metadata_2025 huggingface dataset:
ADAMTS13 Expressed In Glioblastoma and Astrocytoma Cells Can Modulate Endothelial Cell Migration
Authors: Lee Manfai , Hoyt Courtney , Rodgers George M
Published: October 2019

ADAMTS13, the von Willebrand factor (vWF) cleaving protease, regulates platelet aggregation and microthrombi formation by cleaving high-molecular weight vWF multimers. It is expressed primarily in hepatic stellate cells, but is also found in endothelial cells. Recently, ADAMTS13 was reported to be expressed and regulated in astrocytes, microglial, neuroblastoma, and adult human brain endothelial cells. Previous in vitro studies by our group with human umbilical vein endothelial cells (HUVEC) showed that ADAMTS13 can promote angiogenesis via upregulating the secretion of VEGF and phosphorylation of VEGFR2, suggesting that ADAMTS13 may also be involved in physiological processes unrelated to vWF cleavage (Lee, M., et al. Microvasc Res. 2012, 84, 109-115). Herein, we report an additional possible role of ADAMTS13 secreted by brain tumor cells to modulate tumor cell angiogenesis.

Using a human ADAMTS13 immunoassay, we detected ADAMTS13 in U-87 and LN-229 glioblastoma cell lysates, SW-1088 astrocytoma cell lysates, as well as in the supernatants of all cell lines (> 2.0 ng/mL). Co-incubation of U-87, LN-229, and SW-1088 tumor cell conditioned media with recombinant vWF indicated that brain tumor-secreted ADAMTS13 is biologically active in cleaving vWF multimers (measured by ELISA). Secretion of VEGF was upregulated in LN-229 and SW-1088 cell lines by ADAMTS13. 939 pg/mL and 674 pg/mL of VEGF were measured in LN-229 and SW-1088 cell lysates, respectively, after incubation with 100 ng/mL ADAMTS13. Incubation of LN-229 glioblastoma cells with 10 – 500 ng/mL rh-ADAMTS13 or 50 ng/mL VEGF165 did not affect tumor cell proliferation. No change in tumor cell proliferation was observed when LN-229 cells were incubated with a polyclonal antibody against ADAMTS13 in serum free media supplemented with 10 ng/mL ADAMTS13 or media supplemented with 5% FBS, suggesting that ADAMTS13 secreted by brain tumor cells may be involved in extracellular signaling of endothelial cells.

Brain tumor cell secreted-ADAMTS13 induced HUVEC migration in a Matrigel invasion assay. Using a modified Boyden chamber fitted with a Matrigel-coated polycarbonate membrane, LN-229 glioblastoma cells increased HUVEC migration by 83%. LN-229 cells supplemented with 10 ng/mL ADAMTS13 further increased HUVEC migration by 190%, suggesting that tumor cell-secreted ADAMTS13 may modulate EC migration (Fig. 1). Combined with our previous findings suggesting that recombinant ADAMTS13 modulates EC angiogenesis, brain tumor-secreted ADAMTS13 may also be a regulator for tumor vasculature angiogenesis.

Disclosures:

No relevant conflicts of interest to declare.
Tacrolimus-Induced Refractory Immune Thrombocytopenia In Solid Organ Transplant Patients
Authors: Mathew Aju , Woytowitz Donald , Smith Roy E. , Mehta Rakesh P
Published: October 2019

Background

Immune thrombocytopenia (IT) is an uncommon complication in patients who have undergone solid organ transplantation. A few case reports have suggested a role for tacrolimus in inducing IT in such patients.

Methods

A retrospective chart review was conducted in two major academic institutions with a high volume of transplant surgeries. We identified cases of refractory IT in solid organ transplant patients who were on tacrolimus as their graft-rejection immunosuppressant, in whom platelet counts recovered after discontinuation of the medication. Retrospective chart review was performed. All other etiologies for thrombocytopenia were excluded. Finally, descriptive statistical analysis was performed.

Results

Ten patients were identified of which five each were renal and liver transplantations. Median age was 48 years. The median time from transplantation to onset of IT was 5.9 years (range 2.1-20 years) (table 1). In all but one case, the nadir platelet count was less than 5000/μL. Multiple therapeutic strategies including intravenous immunoglobulin, steroids, chemotherapy, rituximab, splenectomy, and romiplostim were attempted without any response. Bone marrow biopsy was consistent with IT in 9 of these patients; one patient did not undergo the procedure. Two patients were treated for H.pylori IgG positivity without any platelet recovery response. After tacrolimus was discontinued, median time to platelet recovery to greater than 50,000/μL was 8.5 days (range 5-81; n=8) (table 2). Median time to platelet count recovery to greater than 100,000/μL was 14 days (range 5-88 days; n=10). Median follow-up duration after platelet recovery of greater than 100,000/μL was 30 months (range 2-60; n=10). One patient, who recovered counts after nearly 3 months of discontinuation of tacrolimus, was on romiplostim for 2 years. If this patient is excluded from the analysis, the median time to platelet count recovery of greater than 100,000/μL was 13 days (range 5-35; n=9). Of note, 6 patients were on a steroid taper at the time of discontinuation of the medication. All except one patient was transitioned to immunosuppression using cyclosporine A; one patient was safely taken off immunosuppression.

Conclusion

Tacrolimus appears to occasionally cause a refractory IT in solid organ transplant recipients, which only appears to resolve with cessation of the drug. The onset of the IT usually occurs after at least two years of taking the medication. A trial of tacrolimus discontinuation for at least a 3 month period, substituted by another immunosuppressant such as cyclosporine A, should be attempted when seeing these patients, especially if there is consideration to perform a more invasive procedure such as a splenectomy or splenic artery embolization.

Disclosures:

No relevant conflicts of interest to declare.
Induction Of Tolerance To FVIII Using Nanoparticles In a Murine Model Of Hemophilia A
Authors: Allan Zhang Ai-Hong , Rossi Robert J. , Griset Aaron , Maldonado Roberto , Kishimoto Takeshi Kei , Scott David William
Published: October 2019

Anti-FVIII inhibitor development is currently considered the most serious side effect in the treatment of hemophilia A patients. In this study, we sought to address this problem by determining the efficacy of a novel tolerogenic protocol utilizing nanoparticle formulations provided by Selecta Biosciences. Thus, hemophilia A mice (FVIII KO, n = 6-8) were treated weekly for five weeks through intravenous route with 1 µg recombinant human FVIII (rFVIII), plus immune modulating agent coupled nanoparticles or control nanoparticles (coded F1 or F2 or F3). An additional control group was included, in which the mice were treated on the same schedule with high dose IVIG + rFVIII. As expected, all the mice in the control group (later revealed to be F3) developed high levels anti-FVIII IgG after the five treatments, with the average levels at 21.2 ± 4.4 µg/ml. Both the F1 and F2 nanoparticle treatments effectively prevented the anti-FVIII inhibitory antibody development throughout the course of the 5-week treatment. One week after the final treatment, the majority (5/7) of both F1 and F2 groups remained undetectable for anti-FVIII IgG antibodies. Consistent with this pattern, the anti-FVIII inhibitor titers for the F1, F2, F3 and IVIG groups were 42.3 ± 9.9, 14.3 ± 7.2, 92.9 ± 2.5, and 30.3 ± 5.6 Bethesda Units (BU)/ml, respectively after another 7 days (p < 0.01 for F1 and F2 vs. F3). To determine the duration of the FVIII-tolerance, the mice were challenged twice with rFVIII (1µg) 29 and 53 days after the final nanoparticle treatment. After the first FVIII challenge, the inhibitor titers for the F1, F2, F3 and IVIG groups were 81.3 ± 10.8, 33.1 ± 13.2, 84.1 ± 2.2 (p = 0.41 for F1 vs. F3; and p < 0.01 for F2 vs. F3), and 50.6 ± 9.7 BU/ml, respectively. Remarkably, the FVIII-tolerance in F2 group was sustained even after additional FVIII challenges ∼ 2 months later, when the effect in IVIG group was completely lost. A therapeutic protocol using mice pre-immunized with FVIII also led to substantial reductions in anti-FVIII titers. In conclusion, the concomitant Selecta F2 nanoparticle treatments not only significantly prevented the anti-FVIII inhibitory antibody development during the treatments, but also resulted in long-term FVIII-tolerance, suggesting a potential clinical application. (Supported in part by a grant-in-aid from Selecta Bioscience and by HL061883)

Disclosures:

Griset: Selecta Biosciences: Employment, Equity Ownership. Maldonado:Selecta Biosciences: Employment, Equity Ownership. Kishimoto:Selecta Biosciences: Employment, Equity Ownership. Scott:Selecta Biosciences: Research Funding.
Natural Course Of Serum Ferritin In Childhood Cancer Survivors: Need For Iron Removal Therapy?
Authors: Maeba Hideaki , Kuroda Rie , Fujiki Toshihiro , Mase Shintaro , Araki Raita , Ikawa Yasuhiro , Yachie Akihiro , Nishimura Ryosei
Published: October 2019

Background

Iron overload has been reported in adult survivors of leukemia after chemotherapy with or without allogeneic hematopoietic stem cell transplantation (allo-HSCT). Approximately 10-15% of adult survivors suffer from liver dysfunction, endocrine disorders, and/or cardiac dysfunction due to iron overload, in which free radicals produced by iron could damage tissues. Therefore phlebotomy and iron chelation therapy in adult survivors have been used prophylactically, however iron overload has not been studied extensively in childhood survivors, so that it would be a problem how to manage the childhood patients in whom serum ferritin level was high at the completion of chemotherapy. In this study, we retrospectively analyzed the serum ferritin level over time after the completion of therapy and also referred to whether iron chelation therapy and/or phlebotomy would be needed or not in childhood survivors.

Patients and methods

We retrospectively analyzed the level of serum ferritin overtime in 48 childhood cancer survivors (ALL 19, AML 13, Lymphoma 5, Pediatric solid tumor 11) except allo-HSCT, who were transfused concentrated red cells in our hospital. All the patients did not receive any phlebotomy and iron chelation therapy throughout the course.

Results

The total mean concentrated blood transfusion volume was 114 ml/kg (114±16, ranges 7-672). At the completion of chemotherapy, the median serum ferritin level was 867 ng/ml (867±216, ranges 7-6558). Three years after chemotherapy, the median serum ferritin levels decreased to 281 ng/ml (281±77, ranges 7-1285). All patients did not show any symptoms related to iron overload such as liver dysfunction and glucose intolerance. Twelve out of 48 patients (25%) exceeded 1000 ng/ml of the serum ferritin at the time of completion of chemotherapy, which has been considered as the initiation of iron chelation therapy in adult patients. However all patients except one decreased the serum ferritin level below 1000 ng/ml in 3 years after chemotherapy without any iron removal therapy. Although serum ferritin level in the exceptional case was extraordinary high (6558 ng/ml) compared to other cases at the completion of chemotherapy, it declined to 1285 ng/ml spontaneously in 3 years, which was much better than expected.

Conclusions

Although 25 percent of our childhood cancer survivors showed high level of serum ferritin more than 1000 ng/ml at the time of completion of chemotherapy, almost all the cases eventually declined thereafter without any iron removal therapy probably due to the iron consumption with growth. Further study would be needed to make specialized criteria for initiating iron removal therapy for childhood cancer survivors.

Disclosures:

No relevant conflicts of interest to declare.
AK2 Deficiency In Zebrafish Recapitulates Human Reticular Dysgenesis, An Autosomal Recessive Form Of Severe Combined Immunodeficiency
Authors: Rissone Alberto , Jagadeesh Jaya , Simon Karen , Bishop Kevin , Sood Raman B. , Candotti Fabio
Published: October 2019

The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP+AMP to 2ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues. Mutations in the AK2 gene are responsible for reticular dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID). RD is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation and it represents less than 2% of total SCID. Affected children succumb to overwhelming infections early in life unless their immune system is successfully restored with allogeneic hematopoietic stem cells transplant (HSCT). The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has never been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). We used the zebrafish model to perform a comprehensive study of the effects of AK2 deficiency using Morpholino oligomers injections and two different kinds of AK2 mutants (a ENU-induced T371C/L124P missense mutant and two null mutant lines generated using zinc-finger nuclease technology). In situ hybridization analyses of AK2-deficient embryos indicated that only erythroid development was affected during primitive hematopoiesis. Conversely, during definitive hematopoiesis, the loss of function of AK2 resulted in abnormalities distributed along all hematopoietic lineages suggesting an impairment of hematopoietic stem cell (HSC) development. Moreover, we observed that the AK2 deficiency induced oxidative stress and consequent apoptosis in both primitive erythroid cells and definitive HSCs. Importantly, antioxidant treatment of AK2 mutant embryos rescued the hematopoietic phenotypes as indicated by the recovered expression of HSC and lymphoid markers (such as c-myb and rag1). Overall, our data indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD and testing of new therapeutic interventions. To date, our mutant lines remain the only animal model of this rare and lethal human disease.

Disclosures:

No relevant conflicts of interest to declare.
PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs
Authors: Yang Zhong-Fa , Junling Wang , Rosmarin Alan G.
Published: October 2019

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Integration Of HoxB4 and STAT3 For Self-Renewal Of Hematopoietic Stem Cells: A Model Of Molecular Convergence For Stemness
Authors: Oh Il-Hoan , Tae-Min Kim , Shim Jae-Seung
Published: October 2019

Multiple transcription factors (TFs) that regulate the self-renewal/stem cell state of hematopoietic stem cells (HSCs) have been identified, but understanding the molecular interplay of these TFs for their functional coordination remains a challenging issue.

In this study, we investigated the functional integration and transcriptional coordination of STAT3 and HoxB4, which are TFs known to have similar effects on the self-renewal of HSCs. We found that while STAT3 (STAT3-C) or HoxB4 similarly enhanced the in vitro self-renewal and in vivo repopulating activities of HSCs, simultaneous transduction of both STAT3-C and HoxB4 did not have any additive enhancing effects. In contrast, the overexpression of HoxB4 caused a ligand-independent Tyr-phosphorylation in STAT3, and the inhibition of the STAT3 activity in HoxB4-overexpressing bone marrow cells significantly abrogated the enhancing effects of HoxB4 on both the bone marrow repopulation and maintenance of the undifferentiated state, revealing a molecular integration of these two TFs for HSC self-renewal. Expression microarray analysis revealed a significant overlap of the transcriptomes regulated by STAT3 and HoxB4 in undifferentiated hematopoietic cells. Moreover, a gene set enrichment analysis (GSEA) for TFs that can recapitulate the transcriptional changes induced by HoxB4 or STAT3 showed significant overlap in the candidate TFs. Interestingly, among these identified TFs were the puripotency-related genes, Oct-4 and Nanog. These results indicate the functional integration of tissue-specific TFs for HSC self-renewal and provide insights into the functional convergence of various TFs towards a conserved transcription program for the stem cell state.

Disclosures:

No relevant conflicts of interest to declare.
B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Specific Copy Number Alterations Are Unique For Progressive Pediatric Chronic Myeloid Leukemia (CML): A Large Cohort Study
Authors: van der Sligte Naomi E , Krumbholz Manuela , Pastorczak Agata , Scheijen Blanca , Tauer Josephine T. , Nowasz Christina , Sonneveld Edwin , de Bock Truuske H. , Meeuwsen - de Boer Tiny G. , van Reijmersdal Simon V. , Kuiper Roland P. , Bradtke Jutta , Metzler Markus , Suttorp Meinolf , De Bont Evelina S. , van Leeuwen Frank N.
Published: October 2019

Chronic myeloid leukemia (CML) is a rare malignancy in children and is mostly diagnosed in the chronic phase (CP). In adults, the five-year overall survival rate is 89% for patients on Imatinib and disease progression occurs in 1-3% per year (Druker 2006). Once a blast crisis (BC) has occurred, treatment options are limited with a median survival of only a few months (Cortes 2008). Therefore, early recognition of patients at risk for developing a BC is desirable. Besides the translocation t(9;22)(q34;q11), IKZF1, PAX5, and CDKN2A deletions have been reported in CML lymphoid blast crisis (LyBC) of both adult and pediatric patients (Mullighan 2008, Alpár 2012).

The aim of this study was to investigate the presence of IKZF1 deletions and other copy number alterations (CNAs) by MLPA analysis in a large cohort of pediatric CML patients at time of diagnosis in order to determine whether CNAs commonly found in pediatric ALL might predict disease progression and / or treatment response.

Between October 1991 and October 2012 a total of 86 children with newly diagnosed CML were included. The median follow up was 31 months.

Among the 86 patients, 82 patients were diagnosed in CP, 2 patients in accelerated phase (AP), and 2 patients in LyBC. Six patients experienced progression to a BC respectively a myeloid blast crisis (MyBC) (N=2) and LyBC (N=4).

At time of diagnosis, an IKZF1 deletion was detected in one patient diagnosed with CML-AP (Table A, patient no 58). IKZF1 and EBF1 deletions were detected in one patient diagnosed with CML-LyBC (Table A, patient no 22). No CNAs were detected in the 82 patients diagnosed with CML-CP.

At time of disease progression, new CNAs were detected at time of the LyBC (Table A, patient no 62, 64, and 67). Due to the absence of material no CNAs could be detected in both patients experiencing a MyBC.

In conclusion, we were able to detect CNAs in progressive CML disease (CML-AP and CML-LyBC) and not in the samples at the time of chronic phase in this large pediatric cohort of CML patients. Therefore, the investigated CNAs could not be used to predict disease progression at time of diagnosis. The CNAs detected in patients with progressive CML were similar to specific CNAs detected in pediatric B-cell precursor ALL, indicating a similar disease development (Kuiper 2010).

Additionally, our results are in accordance with existing literature, suggesting that mechanisms of disease progression in pediatric and adult CML might be similar (Brazma, 2007).

Disclosures:

No relevant conflicts of interest to declare.
Bone Marrow Beta-Catenin Expression In Chronic Myelogenous Leukemia Patients Treated With Imatinib
Authors: Zhang Xiaohui , Sweet Kendra L , Moscinski Lynn , Hazlehurst Lori A , Pinilla-Ibarz Javier , Zhang Ling
Published: October 2019

Background

Imatinib is the standard of care for treating chronic myelogeneous leukemia (CML). However, it is rarely curative and patients often show resistance to the drug. ABL-kinase domain mutations are one mechanism of resistance in CML. Regulation of hematopoietic stem cell survival, as well as disease progression in CML has been linked to the Wnt/beta-catenin pathway. Our study investigated the expression of beta-catenin in the bone marrow (BM) of CML patients treated with imatinib as well as its possible application as a prognostic marker for response to therapy.
Piggybac Mediated Gene Transfer To Correct Hemophilia A
Authors: Staber Janice M , Pollpeter Molly , Arensdorf Angela , Sinn Patrick L , Rutkowski Thomas D , McCray Paul B
Published: October 2019

Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder, affecting about 1 out of 5,000 males; those affected suffer disabling joint and muscle hemorrhages. Hemophilia A is an attractive gene therapy candidate, because even small increases in FVIII levels (5-10%) will alter the phenotype. Non-viral vector systems are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this novel nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize that a PB transposon vector carrying a codon-optimized human FVIII cDNA along with a hyperactive transposase (iPB7) will confer persistent gene expression and correction of the hemophilia A bleeding phenotype. We engineered PB transposon to carry a codon-optimized human FVIII B-domain deleted cDNA (coFVIII-BDD). We evaluated the in vivo gene transfer efficiency in hemophilia A mice by hydrodynamic tail-vein injection using PB coFVIII-BDD driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Factor VIII null mice received 25 micrograms each of the PB coFVIII-BDD transposon and iPB7 to determine long term expression and phenotypic correction. FVIII activity and antigen levels were measured prior to injection and then every 4 weeks for 24 weeks. Results revealed therapeutic levels (50-225%) of factor VIII activity and antigen post gene transfer with stable expression for 24 weeks in most mice. A goal of gene transfer based therapies is to develop the most efficacious expression vectors with the least toxicity. To assess endoplasmic reticulum stress in the livers of treated and untreated mice, we evaluated BiP, CHOP, and EDEM levels via q-PCR. All experimental mice, null mice, and transposon treated mice without the coFVIII-BDD cassette revealed no evidence of cell stress. These data indicate codon-optimized FVIII and the piggyBac transposon vector system may provide a safe long term gene transfer strategy. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. More than 75% of mice receiving PB coFVIII-BDD transposon and iPB7 demonstrated functional correction via tail clip. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression and phenotypic correction.

Disclosures:

No relevant conflicts of interest to declare.
Notch Signaling In B-ALL Reveals PLK1 As A Potential Therapeutic Target In B-ALL: PLK1-Selective Inhibitor Poloxin Modulates Cyclin B and P53 Pathways, Leading To Growth Arrest and Apoptosis
Authors: Kannan Sankaranarayanan , Hall Mandy A , Golfman Leonard S , Zweidler-McKay Patrick A
Published: October 2019

Background

Notch is a well-known oncogene in T-ALL, yet appears to have tumor suppressor effects in B-ALL. These cell type-specific effects of Notch signaling mirror consequences seen in early lymphocyte development and raises the question of how Notch leads to such divergent consequences in closely related cell types. In exploring these Notch mechanisms we discovered a B-ALL specific Notch-mediated reduction in the cell cycle regulator Polo-like kinase-1 (PLK1), revealing a novel targetable kinase in B-ALL.

Approach

To explore the consequences of Notch-mediated down regulation of cell cycle regulator kinase PLK1, we targeted PLK1 kinase function with the novel PLK1-selective inhibitor poloxin in human B-ALL lines.

Results

PLK1 is highly expressed in B-ALL verses normal tissues (panel A), correlates with cyclin B expression, is expressed >2-fold higher in B-ALL with t(1;19) than other B-ALL samples, and may predict response of ALL to methotrexate. In our panel of human B-ALL cell lines poloxin induced G2/M growth arrest and decreased cell number by >80% (panel B), and decreased survival in B-ALL cells (>75% AnnexinV+, panel C). PLK1 inhibition led to tumor suppressor p53 stabilization, revealing >5-fold increase in p53 protein levels following poloxin treatment in B-ALL (panel D). Mechanistically, PLK1 inhibition leads to both cytoplasmic re-localization of cyclin B, disrupting the CDC2-cyclinB complex, as well as phosphorylation of p53 at Ser20, which destabilizes p53-MDM2 interaction and thus accumulation of p53.

Conclusions

While exploring the mechanisms of cell type-specific effects of Notch signaling in ALL, we have found a novel therapeutic target, the cell cycle regulator PLK1. Our findings reveal a novel therapeutic approach whereby PLK1-selective inhibition via poloxin induces growth arrest and apoptosis in human B-ALL via consequences on cyclin B and p53 pathways.

Disclosures:

No relevant conflicts of interest to declare.
Bleeding Incidence Among Patients With Venous Thromboembolism: A Large US Insurance Database Analysis
Authors: Liu Xianchen , Xie Lin , Phatak Hemant , Mardekian Jack , Tan Wilson , Baser Onur , Ramacciotti Eduardo
Published: October 2019

Introduction

It is crucial to assess clinical benefit and bleeding risk when selecting an anticoagulant for patients with venous thromboembolism (VTE). This study examined the incidence of major bleeding and non-major bleeding and clinical predictors of major bleeding among patients with VTE in the usual clinical practice setting.

Methods

VTE patients aged ≥18 years were identified from the Truven Health MarketScan commercial or Medicare supplemental insurance database. Index date was defined as the first VTE diagnosis between 07/01/2006 – 12/31/2011. Patients were required to have ≥2 VTE outpatient diagnoses within a 3-week window or have 1 VTE diagnosis in an inpatient setting, have a continuous health plan enrollment for 6 months prior to the index date (baseline), and have no VTE diagnosis in the baseline period. Major bleeding was defined as any bleeding that resulted in hospitalization or blood transfusion; all other bleeds were non-major bleeding. Patients were followed until major bleeding/non-major bleeding, death, disenrollment, or end of study. Multivariate Cox regression was used to examine factors associated with major bleeding.

Results

Of 267,655 eligible patients, 182,972 patients were with deep vein thrombosis (DVT) only (68.4%), 69,169 with pulmonary embolism (PE) only (25.8%); and 15,514 with both (5.8%). Mean age was 61.5 years, and 53.1% were female. Mean follow-up period was 17.3 months (median=12.1). The annual incidence of major bleeding and non-major bleeding were 3.9% and 20.1%, respectively. The median time to major bleeding from index VTE diagnosis was 2.2 months. Major risk factors at baseline for major bleeding (Risk increase>20%) were history of major bleeding (HR=10.78, 95%CI=10.30-11.28) and non-major bleeding (HR=1.61, 95%CI=1.54-1.68), chemotherapy (HR=1.27, 95%CI=1.20-1.35), age ≥65 vs.≤40 years (HR=1.27, 95%CI=1.18-1.37), alcohol abuse (HR=1.26, 95%CI=1.16-1.38), cancer (HR=1.25, 95%CI=1.19-1.31), and heart disease (HR=1.23, 95%CI=1.18-1.28) (Table). Baseline hormone therapy (HR=0.74, 95%CI=0.68-0.80) and pregnancy (HR=0.59, 95%CI=0.46-0.74) were associated with reduced risk for major bleeding.

Conclusions

Major and non-major bleeding were common in patients with VTE. Multiple factors, especially history of bleeding, age≥65 years, alcohol abuse, cancer and heart disease, were associated with increased risk for major bleeding. Further research needs to examine bleeding risk associated with anticoagulant therapy.

Disclosures:

Liu: Pfizer: Employment, Equity Ownership. Xie:StatInMed: Employment, Research Funding. Phatak:BMS: Employment, Equity Ownership. Mardekian:Pfizer: Employment, Equity Ownership. Tan:Pfizer: Employment, Equity Ownership. Baser:StatInMed: Employment, Research Funding. Ramacciotti:BMS: Employment, Equity Ownership.
Randomized Controlled Trial Of The Efficacy Of a Best Practice Exercise Program Post-Treatment On Physiological and Psychosocial Outcomes In Haematological Cancer Patients
Authors: Joske David J L , Furzer Bonnie , Ackland Tim , Wallman Karen , Petterson Anna , Wright Kemi , Gordon Sandy
Published: October 2019

Purpose

Following cancer treatment haematological patients exhibit significant physical deconditioning and psychological distress resulting from the treatment and the disease. Exercise has been shown as a safe and effective method of rehabilitation in cancer patients, with the potential to reverse the deleterious effects following diagnosis. Our aim was to investigate the efficacy of an immediate or delayed exercise program on various patient outcomes post treatment.

Patients and Methods

Between July 2010 and December 2011, haematological cancer patients from Western Australia, who had completed chemotherapy treatment, were randomly assigned to immediate exercise (IEG; n=18) or a 12-week delayed exercise program (DEG; n=19). The exercise intervention incorporated aerobic and resistance training performed three times per week for 12-weeks. Patient rated outcomes (PRO) included cancer related fatigue (CRF), quality of life (QoL), psychological distress, exercise habit and behaviour. Physiological outcomes included cardiovascular fitness, muscle strength, physical function and body composition.

Results

Patient adherence to the exercise program was 85%, with patients completing, on average, 74 min per week of aerobic exercise and strength training intensity that ranged from 51-87% of maximum weight. A significant time by group interaction (p<0.05) was seen for PRO and physiological outcomes including CRF (p=0.01), QoL (p≤0.001), aerobic fitness (p≤0.001) and muscle strength (p≤0.001). Follow-up t-tests showed a significant difference in change between the IEG and DEG from weeks 0-12 (p≤0.001 to p=0.046), and significant differences between groups from weeks 12-24 (p≤0.001 to p=0.050), with no significant differences between groups at baseline or final assessment on any variables.

Conclusion

A 12-week exercise program, whether commenced immediately post treatment or delayed by 12-weeks, resulted in significant statistical (p≤0.05) and clinical improvements in PRO including QoL and CRF, and physiological outcomes, including aerobic fitness, in haematological cancer patients without any exacerbation of symptoms.

Disclosures:

No relevant conflicts of interest to declare.
Yin Yang 1 (YY1) Regulates The Transcription Of The Gene Krüppel-Like Factor 4 (KLF4) In Pediatric Burkitt Lymphoma: Clinical Implications
Authors: Vega Mario I , Valencia-Hipolito Alberto , Hernandez-Atenogenes Miriam , Vega Gabriel G , Mayani Hector , Huerta-Yepez Sara , Bonavida Benjamin , Martinez-Maza Otoniel
Published: October 2019

Krüppel-like factor 4 (KLF4) is a member of the KLF zinc-finger containing transcription factor family. Supporting evidence has established that KLF4 is either an oncogene or a tumor suppressor. Reported studies have indicated the involvement of KLF4 in the regulation of apoptosis, proliferation, and differentiation of B cells and B-cell malignancies. In contrast to adult lymphomas and solid tumors, recently, we have shown in a TMA the overexpression of KLF4 in pediatric NHL tumor tissues. The KLF4 overexpresion predicted unresponsiveness to CHOP treatment. In addition, we have also reported that the transcription factor Yin Yang 1 (YY1) is overexpressed in B-NHL and is a prognostic factor. We hypothesized that the coexpression of KLF4 and YY1 may result from the transcriptional regulation of KLF4 by YY1. This hypothesis was tested in various experimental designs both in cell lines and tumor tissues derived from patients. Analysis of the B-NHL cell line Ramos revealed that both KLF4 and YY1 are overexpressed compared to normal B cells. The transfection of Ramos with siRNA YY1 showed significant inhibition of KLF4. In silico analyses of the KLF4 promoter identified the presence of four putative binding sites for YY1. We confirmed that the -126 site as the binding site for YY1 by CHIP analysis. We also used a reporter system of the KLF4 promoter and mutated the putative binding site for YY1 (-126) and confirmed it as an important site for the regulation of KLF4. The co-expressions of KLF4 and YY1 were examined in TMA of pediatric lymphomas and showed by IHC that all of the tumor tissues exhibited a positive correlation of the expressions of KLF4 and YY1 and the correlation was markedly significant in the Burkitt subtype. These correlations were consistent with the bioinformatics analyses due by ONCOMINE in several data base. KLF4 acts as a transcriptional activator of epithelial genes and as a repressor of mesenchymal genes. In addition, KLF4 suppresses the extrinsic apoptotic pathway by inhibiting the activation and cleavage of caspases (7, 9, and 3). Thus, the overexpression of KLF4 in lymphoma may be responsible, in part, in the pathogenesis, malignancy, and drug resistance. The putative function of KLF4 was examined by the use of chemical inhibitors for KLF4 (Kenpaullone) and inhibition of KLF4 resulted in the inhibition of cell proliferation and the spontaneous induction of apoptosis. The present findings suggest that both KLF4 and YY1 are prognostic biomarkers for pediatric lymphoma and are also potential therapeutic targets.

Disclosures:

No relevant conflicts of interest to declare.
Ofatumumab As Initial Therapy For Indolent B Cell Lymphomas: A Phase II Trial
Authors: Barnes Jeffrey A. , Feng Yang , Hochberg Ephraim P , Takvorian Tak , Weitzman James , Fisher David C. , Jacobsen Eric D. , Joyce Robin , Neuberg Donna S. , Abramson Jeremy S.
Published: October 2019

Background

Single agent monoclonal antibodies (mAb) such as rituximab are an attractive treatment option for patients with low grade B-cell lymphoma given proven efficacy and tolerability. Ofatumumab offers potential advantages over rituximab including increased affinity for the CD20 antigen, increased complement dependent cytotoxicity, and is fully humanized which may predict a lower incidence of infusion reactions. We therefore investigated ofatumumab\as initial therapy for indolent B-cell lymphomas.

Methods

We conducted a phase II trial of ofatmumab monotherapy. Eligible patients were adults with previously untreated indolent B-cell lymphomas including follicular lymphoma grade 1-2, marginal zone lymphoma and small lymphocytic lymphoma. Subjects had to have measurable disease >2cm and not be candidates for potentially curative radiotherapy for localized disease. Ofatumumab was administered as 8 weekly infusions at a flat dose of 1000 mg. The primary end point was complete response rate (CRR) as defined by the International Workshop Response Criteria (1999). Restaging CT scans were performed 4 weeks after the final dose of study drug. Secondary endpoints included overall response rate (ORR), progression-free survival (PFS), and toxicity. The study was designed with 90% power to show a 50% CRR, with a 30% CRR considered unworthy of further study.

Results

Between March 2011 and December 2012, 42 eligible subjects were enrolled (28 follicular lymphomas, 4 marginal zone lymphomas, and 10 small lymphocytic lymphomas). With 41 of 42 subjects completing all 8 infusions, the CRR was 9.5% with a PRR of 42.9% (ORR of 52%). The median PFS has not been reached at a median follow-up of 9 months. The 12 month PFS was 86% (95% CI [72%, 99%]). One patient died due to progressive disease. There were 22 (52%) grade 1-2 and 7 (17%) grade 3 infusion reactions despite standard pre-medications including acetaminophen, hydrocortisone, and diphenhydramine. There were no cases of grade 3-4 neutropenia.

Conclusion

Ofatumumab did not appear to improve the CRR compared to what would be expected from rituximab in patients with previously untreated indolent B-cell lymphomas. Incidence of infusion reactions was significant with this fully humanized mAb.

Disclosures:

Off Label Use: Ofatumumab is an anti-CD20 monoclonal antibody approved for the treatment of relapsed chronic lymphomcytic leukemia that is refractory to fludarabine and alemtuzumab.
The Expression Of CD200 As a Prognostic Factor In Newly Diagnosed Multiple Myeloma
Authors: Li Yi , Wu Wenjun , He Jingsong , Han Xiaoyan , Zheng Gaofeng , Zi Fuming , Yang Li , Wu Cai , He Donghua , Zhao Yi , Yang Yang , Wang Lijuan , Ye Xiujin , Huang He , Yi Qing , Cai Zhen
Published: October 2019

Introduction

multiple myeloma (MM) is currently an incurable hematological malignancy. Discovering molecular therapeutic targets is a new approach to improve the outcome in the treatment of the malignant disease. As CD200 is a type Ⅰmembrane glycoprotein expressed on myeloma cells, we asked if the expression of CD200 could serve as a prognostic marker for MM patients. Our data indicated that the expression level of CD200 is indeed correlated with the prognosis of the MM patients.

Methods

bone marrow samples from 96 newly diagnosed MM patients from April 2011 to July 2013 were evaluated by flow cytometry, using PE-conjugated anti-CD200 mAb, FITC-conjugated anti-CD138 mAb, and PE-Cy7-conjugated anti-CD45 mAb. PE-or FITC-conjugated normal mouse IgG was used as isotype-matched controls.

Results

96 MM patients were investigated in the present study, including 60 men and 36 women, with a median age of 63 years (range 34–86 years). 81/96(84%) MM patients were CD200 positive with a median Mean Fluorescence Intensity (MFI) of 127 as analyzed by flow cytometry, which was consistent with the previous studies. While in 15 of 96 patients, CD200 expression was undetectable. Among the CD200 positive ones 7.40% patients were classified as stage Ⅰ, 12.35% were stage Ⅱ, and 80.25% were stage Ⅲ according to Durie–Salmon staging criteria. 40.74% patients were stageⅠ, 22.22% were stage Ⅱ, and 37.04% were stage Ⅲ, according to the International Staging System (ISS). Analysis of the CD200 positive patients revealed the MFI<127 group had a better progression free survival (PFS) (p=0.046) (Fig 1A) and overall survival (OS) (p=0.069) compared to those with MFI≥127. In the patients with age ≥65 years old, PFS (p=0.023) (Fig 1B) and OS (p=0.044) (Fig 1C) were much shorter in the MFI≥127 group, compared to the MFI<127 ones.

Conclusions

Our study demonstrated that the expression and MFI of CD200 on primary multiple myeloma cells is correlated with the prognosis of the MM patients. The better PFS and OS were observed in the MFI<127 group compared to the patients with MFI≥127, especially in the patients with age≥65 years old. Improved PFS in CD200 positive ones is likely due to the immune suppression mediated by CD200. Our study suggests that targeting therapy against CD200 may become a new approach to the treatment of MM in clinical practice.

Disclosures:

No relevant conflicts of interest to declare.
Neutrophils Of Multiple Myeloma Are Dysfunctional and Immunosuppressive
Authors: Parrinello Nunziatina , Romano Alessandra , Conticello Concetta , Cavalli Maide , La Fauci Alessia , Rizzo Giuseppina , La Cava Piera , Chiarenza Annalisa , Tibullo Daniele , Giallongo Cesarina , Palumbo Giuseppe A , Di Raimondo Francesco
Published: October 2019

Introduction

Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. However the role and function of neutrophils in MM and monoclonal gammopathy of undetermined significance (MGUS) has been poorly investigated.

Methods

on neutrophils (N) of 65 MM at diagnosis, 74 MGUS and 30 healthy subjects we evaluated, by flow cytometer, phagocytic activity and surface expression of CD64, CD16, CD62L and CD11b, markers of neutrophil activation. We tested also the immunosuppressive properties of N isolated from MGUS or MM patients, through functional assays, based on in vitro co-culture of N isolated from patients and T-lymphocytes from healthy subjects and we evaluated the expression of the immunosuppressive molecule arginase-1 (Arg-1) by RT-PCR.

Results

Despite no differences in the absolute number of N between MM, MGUS and healthy subjects, we found a functional impairment in MM not evident in MGUS patients.

The phagocytic activity of MM-N was significantly reduced compared to healthy subjects-N (p<0.001) and MGUS-N (p<0.0001) and restored after induction chemotherapy (p=0.02).

Expression of CD64 was significantly elevated in MM-N compared to MGUS-N or healthy subjects-N (p=0.01 and p= 0.007 respectively) and was inversely correlated with phagocytic activity (p=0,01). No differences were observed among MM, MGUS and healthy subjects for the other surface markers evaluated.

MM-N exhibit an increased expression of ARG-1 compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity of ARG-1, positively correlated with advanced disease. After PHA-P stimulation,T-lymphocytes isolated from healthy subjects missed the expression of activation markers such CD71, CD69, CD25, CD3ζ in the presence of MM-N for 72 hours, and in a less extensive way in the presence of MGUS-N.

Conclusion

Compared to controls, neutrophils obtained from MM patients have a reduced phagocytic activity, a greater expression of Arg-1 and exhibit an immunosuppressive function on T lymphocytes. Taken together, these findings indicated that neutrophils may contribute to impairment of immune function that characterizes MM patients.

Disclosures:

No relevant conflicts of interest to declare.
Both Host-Type and Donor-Type Mast Cells Protect Against Acute Graft-Versus-Host Disease
Authors: Araki Raita , Maeba Hideaki , Kuroda Rie , Mase Shintaro , Fujiki Toshihiro , Ikawa Yasuhiro , Koizumi Shoichi , Yachie Akihiro , Nishimura Ryosei
Published: October 2019

Originally mast cells have been known as effector cells in the IgE-mediated allergic responses. In addition, recent studies demonstrated that mast cells exerted pro-inflammatory or anti-inflammatory effects in complicated immune response depending on the situation. In allogeneic hematopoietic stem cell transplantation (HSCT), exact role of mast cells in graft-versus-host disease (GVHD) remains unclear. First we investigated whether host mast cells protect acute GVHD or not in a murine HSCT model using mast cell deficient mice. When lethally irradiated wild type (WT) B6 or mast cell deficient mice were transplanted with bone marrow cells and spleen cells from WT Balb/c mice, mast cell deficient B6 host mice showed significantly lower survival rate than WT B6 mice (p<0.01) due to acute GVHD. Moreover when irradiated mast cell deficient host mice (B6 derived) were given BM cells and splenocytes from mast cell deficient Balb/c mice, these mice died much faster compared to the mice receiving WT BM plus splenocytes (p<0.01) as shown below. Next, we demonstrated that bone marrow derived cultured mast cells (BMCMCs) inhibited mixed lymphocyte reaction (MLR) in a dose-dependent manner, when added to the coculture (Stimulator: dendritic cells (DCs) from B6, Responder: spleen cells of Balb/c, BMCMCs from B6). In addition when mast cells generated from the same strain of responder cells (Stimulator: DCs from B6, Responder: spleen cells from Balb/c, BMCMCs from Balb/c) were added to the MLR mixture, MLR was also suppressed even in this condition. Taken together, we have clearly demonstrated that both host-type and donor-type mast cells suppressed alloreaction in vivo and in vitro. However when we used anti-CD3 and anti-CD28 monoclonal antibodies to stimulate T-cells instead of DCs, BMCMCs could not suppress the lymphoproliferation any more. It suggested that mast cells could suppress MLR via the regulation of DCs directly, not T-cells. Next we have shown that BMCMCs dominantly regulated the alloreaction in a cell contact-dependent manner by using transwell system, though mast cells contain many kinds of substances to regulate immune reactions such as IL-10. DCs highly expressed costimulatory molecules such as CD80, CD86, CD40, I-A, however expression level of these molecules was not changed during coculture of BMCMCs and DCs. Finally, we compared the MLR suppression rate between naïve and activated BMCMCs using IgE and specific antigens, however no difference was observed between them. In conclusion, both host and donor-type mast cells have a protective role against acute GVHD.

Disclosures:

No relevant conflicts of interest to declare.