bluuebunny/crossref_metadata_2025 huggingface dataset:
HoxA3 Controls Notch Pathway to Repress Blood Development
Authors: Sanghez Valentina , Kee Dustin , Beuder Steven , Rux Danielle , Osawa Mitsujiro , Markson Joe , Elowitz Michael , Kyba Michael , Iacovino Michelina
Published: October 2019

Hematopoietic stem cells (HSC) are generated from a specialized subset of endothelial cells, hemogenic endothelium. Previous studies performed by our group showed that HoxA3 restrains the cell at the hemogenic endothelium stage, inhibiting further differentiation toward blood by direct repression of Runx1. Building on our previous work, we show here that overexpression of HoxA3 affects the Notch pathway. Upon HoxA3 upregulation in endothelial cells, Jag1 is induced, Mfng (Manic) and Lfng (Lunatic) fringes are downregulated, and there is a trend towards Notch target gene repression. These data suggest that in the presence of HoxA3, endothelial cells are blocked from receiving Notch signal through ligand cis-inhibition with resulting blood inhibition. In order to test this hypothesis, we evaluated the effect of activation or inhibition of the Notch pathway during blood development. We show here that the number of blood progenitors originating from the hemogenic endothelium is decreased when the Notch pathway is inhibited. Conversely, induction of the pathway by upregulation of NICD (Notch1 Intra Cellular Domain) promotes an increase in the number of blood progenitors originating from hemogenic endothelium. Furthermore, inhibition of the pathway when HoxA3 is upregulated has little or no effect in blood while induction of the pathway in HoxA3 presence in part promotes blood development. Taken together, these results demonstrate that Notch pathway is both sufficient and essential for blood development. Specifically HoxA3 inhibits Notch signal reception in two ways: 1) HoxA3 increases Jag1 ligand expression that acts in cis-inhibition; 2) represses Manic and Lunatic fringes both necessary to increase the affinity of Notch receptors for the Delta ligands. When this blockage is bypassed by NICD upregulation, blood is formed, demonstrating that HoxA3-dependent Notch inhibition results in blood suppression.

Disclosures

No relevant conflicts of interest to declare.
The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine Suppresses the Plasma Membrane Translocation and Neoplastic Transforming Activity of NRAS and KRAS4A
Authors: Wu Min , Jiao Bo , Ren Ruibao
Published: October 2019

The RAS family includes three RAS genes, which encode four highly homologous proteins: H-, N-, and KRAS4A and 4B, the latter two being alternatively spliced forms differing only at the carboxyl terminus. RAS oncogenic mutations, mostly KRAS and NRAS, are common in human cancers. Since the enzymatic activity of RAS is used to turn itself off and its GTP binding affinity is very high, RAS proteins have been difficult to target. Identification of alternative means to block the RAS oncogenic signaling is critical for developing therapies against RAS-driven cancer. We took a chemical biology approach to discover novel therapeutic strategies for RAS-related cancer. Since the biological activity of RAS proteins relies upon lipid modifications and RAS regulates lipid metabolisms in cancer cells, we used a RAS specific cell viability assay to screen a bioactive lipid library for potential therapeutics against RAS-driven cancer. We found that endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) could effectively kill RAS-transformed cells. The structure-activity analysis showed that the anti-RAS activity of NADA relied on both the dopamine and arachidonoyl moieties in the same molecule and was independent of its ability to engage cannabinoid or vanilloid receptors. Further analysis showed that NADA was more active in killing cells transformed by oncogenic NRAS or KRAS4A than that of KRAS4B. Consistently, NADA was more active in inhibiting the phosphorylation of ERK in NRAS-transformed cells than that in KRAS4B-transformed cells. Interestingly, NADA blocked the plasma membrane translocation of NRAS and KRAS4A, but not that of KRAS4B. Significant effect of NADA on palmitoylated NRAS levels was not detected, suggesting that NADA inhibits NRAS and KRAS4A plasma membrane translocation through a novel mechanism. These results demonstrate that NADA possesses a novel property of inhibiting the plasma membrane translocation and neoplastic transforming activity of NRAS and KRAS4A. Since NRAS mutations are common in hematological malignancies as well as in solid tumors such as melanoma and colorectal carcinoma, and KRAS4A is required for carcinogen-induced lung cancer, the new findings would help to develop novel therapies for a broad range of human cancers.

Disclosures

No relevant conflicts of interest to declare.
Platelet-Derived Growth Factor Has a Potent Effect on MK Proliferation, F-Actin Reorganization and Proplatelet Formation Via PDGFR and p-Akt or p-ERK1/2 Pathways
Authors: Yang Mo , Cheng Yuan-shan , Li Su-yi , Meng Fan-yi , Lian Qi-zhou
Published: October 2019

Platelet-derived growth factor (PDGF) may intimate the existence of an autocrine and/or paracrine loop constructed by megakaryocytes (MKs) and their granular constituents in the regulation of megakaryopoiesis and thrombopoiesis. Our study demonstrated that PDGF has a promoting effect on thrombopoiesis in an irradiated-mice model (Ye et al, Haematologica, 2010). PDGF may enhance megakaryopoiesis and platelet production via PDGFR and Erk/Akt pathways. PDGF receptors were identified on platelets and megakaryocytic cells (MKs) by ELISA, flow cytometry and immunocytochemical staining. MK proliferation and platelet formation were studied by CFU-MK and pro-platelet assay. Both PDGFR subtype were identified on platelets and MKs; Levels of PDGF beta-receptors exceeded that of alpha-receptors. PDGF-BB significantly stimulated MKs proliferation; this mitogenic effect was partially neutralized by antibodies directed against the PDGF beta-receptors. Western blot also determind that these receptors mediate PDGF-BB-inducible expression of c-fos. In the MKs apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on MKs. We demonstrated that PDGF activated the Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression, suggesting that the PDGF-initiated protective effect is likely to be mediated via PDGF receptors with subsequent activation of the Akt pathway. PDGF also promoted proplatelet formation in MKs and this effect was reduced by PDGFR antibodies. In PDGF-BB (50ng/ml) and TPO (50 ng/ml) groups had more proplatelet bearing MKs; in 200 MKs, proplatelet bearing MKs in PDGF-BB treatment group was (15 % ± 3.2), and in TPO group was (18 % ± 2.5), which were significantly more than the control group (7 % ± 2.6) (n=5). Whether PDGF has effects on cytoskeleton reorganization of MKs, and whether the effect can be reduced by Erk1/2 inhibitor PD98059, MKs were staining with F-actin specific binder rhodamine-phalloidin. We observed that polymerized actin level is lower in control group refer to PDGF-BB group and distributed throughout the cytoplasm with occasionally few aggregation. In contrast, polymerization actin level was higher in PDGF-BB group. Adding PD98059, the fluorescence intensity was reduced (n=5). Our data demonstrated that treated with PDGF-BB for 30 minutes Erk1/2 was activated in MKs. This study suggests that PDGF has a potent effect on megakarpoiesis and platelet formation. This effect is likely mediated via PDGF beta-receptors with subsequent activation of p-ERK1/2 or p-Akt, which leads to MKs proliferation, F-actin reorganization and proplatelet formation.

Disclosures

No relevant conflicts of interest to declare.
Identification of Secreted Factors with a Role in Hematopoietic Stem and Progenitor Cell Engraftment in the Developing Zebrafish
Authors: Blaser Bradley Wayne , Li Brian , Tamplin Owen J. , Binder Vera , Prober David , Schier Alexander , Vidal Marc , Zon Leonard I.
Published: October 2019

The hematopoietic microenvironment regulates the behavior of hematopoietic stem and progenitor cells (HSPC) throughout vertebrate development. We sought to identify secreted factors that may play a role in HSPC engraftment in the caudal hematopoietic territory (CHT), an endothelial cell-rich vascular plexus that serves as the primary site of hematopoiesis in the developing zebrafish from 3-6 days post fertilization (dpf). We hypothesized that such factors would be highly expressed in endothelial cells relative to hematopoietic stem cells (HSCs). To identify these factors, endothelial cells and HSCs were purified from 3 dpf Flk1:mcherry; Runx1:GFP double transgenic zebrafish embryos and gene expression profiling was performed by microarray analysis. Gene set enrichment analysis of these data showed that zebrafish chemokines, cytokines, TGF-β, TNF, Notch and non-canonical WNT family members were enriched in the endothelial cell fraction with a nominal P ≤ 0.2. Genes from the leading edge of these gene sets were then used as candidates for gain-of-function testing. Coding sequences from candidate genes were cloned downstream of the zebrafish HSP70l promoter and microinjected into wildtype zebrafish embryos at the single-cell stage. Gene expression was induced in F0 transgenic animals by heat shock at 36 and 48 hours post fertilization. HSPC numbers were assayed by performing whole-mount in situ hybridization to identify runx1- and cmyb-expressing cells at 3 dpf. WNT5A was found to enhance HSPC numbers in this assay (P = 0.00046). We conclude non-canonical WNT family members, in particular WNT5A, regulate HSPC engraftment in the developing zebrafish.

Disclosures

Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Downregulation of Dicer1 Promotes Cellular Senescence and Decreases the Differentiation and Stem Cell-Supporting Capacities of MSCs in Patients with Myelodysplastic Syndrome
Authors: Zhao Youshan , Wu Dong , Fei Chengming , Guo Juan , Gu Shucheng , Xu Feng , Zhang Zheng , Chang Chunkang , Li Xiao
Published: October 2019

Myelodysplastic syndrome (MDS) is characterized by decreased blood counts caused by ineffective hematopoiesis. Although the insufficient hematopoietic support capabilities of mesenchymal stromal cells (MSCs) have been reported in patients with MDS, the underlying mechanisms remain elusive. In this study, we found that MSCs from patients with MDS displayed a significant increase in senescence, as evidenced by their decreased proliferative capacity, flattened morphology and increased expression of SA-β-gal and p21.Senescent MDS-MSCs exhibited decreased osteoblastic and adipogenic differentiation potential and decreased stem cell support capacity. Next, we found that Dicer1 expression was downregulated in MSCs from patients with lower-risk (LR)-MDS and higher-risk (HR)-MDS. Gene knockdown of Dicer1 induced senescence in the MSCs. The differentiation of MSCs into osteoblasts and adipocytes was significantly inhibited by Dicer1 knockdown (Dicer1-KD). Moreover, Dicer1-KD MSCs exhibited less vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF) expression than normal MSCs. Long-term culture assays showed that layers of MSCs with the Dicer1 gene knocked down are inefficient at supporting hematopoiesis. Further, we observed that the key senescence mediators, p21,but not p16, was elevated in Dicer1-KD MSCs. We also identified reduced expression in the miR-17 family (miR-17-5p, miR-20a/b, miR-106a/b and miR-93) as a potential factor responsible for increased p21 expression in Dicer1-KD MSCs. A verification experiment showed that miR-93 and miR-20a expression levels were significantly reduced in MSCs from patients with MDS. Taken together, our study shows that MSCs from MDS patients are prone to senescence and that Dicer1downregulation promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of MSCs in MDS patients. Dicer1 downregulation seems to contribute to the insufficient hematopoietic support capacities of MSCs in patients with MDS.

Disclosures

No relevant conflicts of interest to declare.
a Proposal for a New Staging System of Extranodal Natural Killer T-cell Lymphoma, Nasal-Type: a Multicenter Study of Chinese Southwest Oncology Group (CSWOG)
Authors: Hong Huangming , Du Xin , Zhang Mingzhi , Wang Jinghua , Zhu Jun , Huang He , Liu Ting , Meng Fanyi , Wu Gang , Guo Ye , Zhao Weili , Jin Jie , Li Juan , Deng Yanming , Gu Kangsheng , Wu Xiangyuan , Ke Xiaoyan , Xie Derong , Lin Daren , Lin Tongyu
Published: October 2019

Purpose: Extranodal NK/T-cell lymphoma, nasal type (ENKTL), is a rare and highly aggressive disease. The Ann Arbor staging system had been unsuitable to proper staging of ENKTL. This study was conducted to establish a new staging system specified for ENKTL, which can identify poor prognostic patients.

Patients and Methods: Patients with untreated, centrally reviewed diagnosis of ENKTL were included in our study. The new staging system was established based on the study of single center consecutive patients treated with CHOP-like regimens with or without involved field radiotherapy (IFRT), then we initinated a multicenter confirmation study and conducted a multicenter prospective study to validate the new staging system.

Results: From Jan 1997 to June 2006, 134 consecutive patients treated in the cancer center of Sun Yat-sen University were analyzed. The following was a summary of the classification system developed: stage I: lesions confined within nasal cavity or nasopharynx without local invasiveness (paranasal sinuses or bony or skin invasion); stage II: localized disease with local invasiveness; stage III: localized disease with regional lymph node involvement (cervical lymph nodes); and stage IV: disseminated disease (lymph nodes on both sides of diaphragm, multiple extranodal site). The distribution of stage I to IV using the new system and Ann Arbor system were 39.6%, 23.9%, 23.1%, 13.4% and 63.4%, 23.1%, 5.2%, 8.2%, respectively. The 5-year OS rate of stage I to IV using the new system were 29.5%, 23.4%, 21.3% and 0.07% compared with 23.8%, 21.3%, 0.0% and 0.0% using the Ann Arbor system. In the multicenter confirmation study conducted in 18 centers in China, 722 patients were analyzed. The results showed that the distribution of the new system compared with Ann Arbor system from stage I to IV were 24.1%, 34.9%, 18.3%, 22.7% vs 59.1%, 19.0%, 6.9%, 15.0%, respectively, and the 5-year OS rate of stage I to IV were 56.0%, 48.3%, 33.8%, 26.1% vs 50.7%, 39.1%, 10.8%, 28.0%, respectively. For the multicenter prospective study, 233 newly diagnosed ENKTL patients treated with non-CHOP-like regimens were enrolled and showed a balanced distribution of 17.2%, 39.9%, 19.3%, 23.6% vs 53.6%, 25.3%, 6.9%, 14.2% from stage I to IV and superior 5-year OS rate: 82%, 73%, 67%, 54% vs 75.2%, 65.6%, 46.9%, 73.8% from stage I to IV using the new system in compared with the Ann Arbor system.

Conclusions: The new staging system with a more balanced distribution and a superior prognostic discrimination as compared with Ann Arbor staging system no matter for CHOP-like or non-CHOP-like regimens, is more suitable for ENKTL and can be recommended used in the future.

Disclosures

No relevant conflicts of interest to declare.
Pharmacokinetics and Tolerability of Etoposide in Newly Diagnosed Lymphoma Patients with Hepatic Impairment
Authors: Lai Catherine , Cole Diane , Lucas Nicole , Steinberg Seth M. , Widemann Brigitte C , Dunleavy Kieron , Wilson Wyndham H
Published: October 2019

Background: The addition of etoposide to chemotherapy regimens (e.g.- CHOEP and EPOCH) may be beneficial for the treatment of aggressive lymphomas. Previous studies have shown decreased clearance of etoposide in hepatic impairment leading to a dose reduction or removal of drug. Total clearance of etoposide does not change significantly in patients with elevated bilirubin. However, free (or unbound) etoposide levels are a more accurate measurement of drug clearance (Stewart el al. Changes in the Clearance of Total and Unbound Etoposide in Patients with Liver Dysfunction. J Clin Oncol. 1990 Nov;8(11):1874-9.). No studies have analyzed etoposide pharmacokinetics and clinical toxicity in lymphoma patients with hepatic impairment.

Methods: Patients with newly diagnosed aggressive lymphoma received 1 to 8 cycles of DA-EPOCH +/-R at the NCI. Of 56 pts studied, 48 had normal and 8 had elevated bilirubin (median 3.4mg/dL, range 1.4-22.5mg/dL). All patients received full dose etoposide at 200mg/m2 over 96-hours on cycle 1. Blood samples were collected at 0, 22 and 96 hours after infusion began.

Results: There was no significant difference between free etoposide clearance (Cl) and the free etoposide concentration (Css) in patients with elevated versus normal bilirubin (see table below). In all patients, irrespective of bilirubin, there was no significant correlation between free etoposide Cl, free etoposide Css and creatinine clearance (CrCl) or age. Neutropenia and thrombocytopenia was higher in patients with elevated bilirubin. Complete response (CR) was achieved in 75% (6/8) versus 85% (41/48) and PR in 0% (0/8) vs 4% (2/48) of patients with elevated vs normal bilirubin.

Conclusions: Etoposide pharmacokinetics were not altered in patients with abnormal versus normal hepatic function. Although there was an increase in hematologic toxicities, there was no difference in Css or Cl of etoposide. Importantly, there were no significant differences in febrile neutropenia or grade 3/4 toxicities. The toxicity difference is likely attributed to other drugs. Response rates were similar between the 2 groups. Our results do not support the empiric dose reduction of etoposide in patients with aggressive lymphomas receiving potentially curative treatment.

Table Results Elevated Bilirubin Normal Bilirubin p-value Median age (yrs) 39 (17-59) 54 (20-75) 0.15 IPI (int hi/hi risk) 75% (6/8) 23% (11/48) 0.0070 ANC nadir < 500/mm3 (C1) 100% (8/8) 54% (26/48) 0.017 PLT nadir < 25x103/mm3 (C1) 38% (3/8) 4% (2/48) 0.017 Febrile Neutropenia (C1) 13% (1/8) 13% (6/48) 1.00 G3/4 GI and neuro tox (C1) 38% (3/8) 13% (6/48) 0.11 Median free etoposide clearance on C1 (ml/min/m2) 724 (438-1413) 663 (199-1148) 0.40 Median free etoposide concentration on C1 (μM) 0.082 (0.044-0.135) 0.089 (0.052-0.298) 0.44

Disclosures

No relevant conflicts of interest to declare.
Intratumoral Inhibition of C5a Using Anti-C5a Monoclonal Antibody Inhibits Lymphoma Progression
Authors: Veeramani Suresh , Weiner George J.
Published: October 2019

Background : The complement system can have both pro-inflammatory and immunosuppressive effects. Our preliminary studies demonstrated that C5a can limit antigen-specific responses, in part, by enhancing the induction of immunosuppressive T regulatory (Treg) cells by primary human antigen-presenting cells and malignant B cells. The current study explored the effect of intratumoral C5a on B lymphoma progression and intratumoral T cells using the A20 murine lymphoma mouse model.

Methods :

(A) Co-injection approach: Female Balb/C mice were subcutaneously injected with A20 B lymphoma cells mixed with either 20 μg of rat anti-mouse C5a neutralizing monoclonal antibody (mAb) or matching isotype control mAb. Tumor growth was followed and the phenotype of tumor-infiltrating lymphocytes analyzed by flow cytometry.

(B) Therapeutic approach: Established subcutaneous A20 tumors (7-9 mm diameter) were treated intratumorally with 20 µg of anti-mouse C5a neutralizing mAb (considered as day 1). A second dose (20 µg) of mAb was given intratumorally on day 5. Control mice were treated similarly with matching isotype control mAb. Tumor growth was followed.

Data was analyzed using standard t-test (for tumor growth and immune responses) and Log-rank (Mantel-Cox) test (for comparing survival).

Results:

(A) Co-injection approach: Co-injection of anti-C5a mAb at the time of tumor cell inoculation significantly delayed A20 tumor growth (Fig. 1; p<0.05) and led to prolonged survival of anti-C5a-treated tumor-bearing mice when compared to the control mice (p=0.001).Tumors from mice that were co-injected with anti-C5a mAb showed significantly higher infiltration of activated CD8+ T cells producing IFNg than the control mice (Fig. 2; p=0.034). Anti-C5a-treated mice also had a lower percentage of circulating Tregs (p=0.023) and splenic Tregs (p=0.0001) compared to control mice.

(B) Therapeutic approach: Treatment of pre-established subcutaneous A20 tumors with anti-mouse C5a mAb slowed tumor growth significantly when compared to the control mice (Fig. 3; **p<0.01; *p<0.05). Tumor-bearing mice treated intratumorally with anti-C5a also showed significant improvement in the survival when compared to tumor-bearing mice treated with isotype control Ab (p=0.008).

Conclusions:

Intratumoral anti-C5a mAb therapy inhibits progression of B lymphoma and improves survival, likely by reducing immunosuppression. We are currently exploring the mechanisms behind C5a-promoted tumor immunosuppression.

Disclosures

No relevant conflicts of interest to declare.
Characterization of CD34+ Hematopoietic Progenitor Cells in JAK2V617F and Calr-mutated Myeloproliferative Neoplasms
Authors: Angona Anna , Alvarez Alberto , Longarón Raquel , Camacho Laura , Fernández Concepción , Pairet Silvia , Martinez Luz , Ancochea Agueda , Senin Alicia , Bellosillo Beatriz , Besses Carlos
Published: October 2019

Introduction: The dominance of the JAK2V617F-positive clone at the CD34+ compartment is an important modifier of the disease phenotype in myeloproliferative neoplasms (MPN). Recently, mutations in the calreticulin gene (CALR) have been described in around 40-70% of JAK2V617F and MPL wild-type essential thrombocythemia (ET) and myelofibrosis (MF) patients. However, there is limited information regarding the role of CALR mutant clone in hematopoietic progenitor cells.

Objective: To study the mutant allele burden at progenitor level in JAK2V617F-positive and CALR-mutated MPN.

Methods: Sixty-five patients with MPN including 36 with polycythemia vera (PV) all JAK2V617F-positive, 13 with ET (7 JAK2V617F-positive and 6 CALR-mutated) and 16 with MF (9 JAK2V617F-positive post-PV MF, 4 CALR-mutated primary MF and 3 CALR-mutated post-ET MF) were included in the study. Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+CD38-) and progenitors (CD34+CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F and CALR allele burden was measured by quantitative PCR and PCR followed by fragment analysis, respectively, in stem cells, progenitor cells and granulocytes. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki.

Results: CALR-mutated ET patients harbored a higher mutant load in CD34+CD38- than JAK2V617F-positive ET patients (30.6 vs 6.3%, p=0.01), whereas no significant differences were observed in CD34+CD38+ and in granulocytes allele burdens. Moreover, CALR-mutated ET patients showed a higher mutational load in CD34+CD38- than JAK2V617F-positive PV (30.6% vs 15.7%, p=0.04) but the mutant load in granulocytes was lower (29.6% vs 63.3%, p<0.001).

The mutant allele burden in granulocytes and CD34+ cells was higher in patients with JAK2V617F-positive MF than in those with CALR-mutated MF (CD34+CD38-: 71% vs 47.2% p=0.05, CD34+CD38+: 68.4% vs 40.6% p=0.018, granulocytes: 76.9% vs 53.7% p=0.05).

Finally, we could demonstrate that the mutant load was lower in CALR-mutated ET patients than in CALR-mutated MF at progenitor level and in granulocytes (CD34+CD38-: 30.6% vs 47.1% p=0.08, CD34+CD38+: 17.8% vs 40.6% p=0.03, granulocytes: 29.6% vs 53.7% p=0.004).

Conclusion: CALR-mutated ET patients have a higher mutant load in CD34+CD38- than JAK2V617F-positive ET and PV patients, whereas the JAK2V617F-positive hematopoietic progenitor cells have more differentiation potential than those CALR-mutated. Moreover, in the MF phase of MPN, the expansion of the mutated clone at the progenitor level is greater in JAK2V617F-positive than in CALR-mutated patients.

Disclosures

No relevant conflicts of interest to declare.
Hydroxyurea for β-Thalassemia Major: A Meta-Analysis
Authors: Algiraigri Ali H. , Wright Nicola A. , Kassam Aliya
Published: October 2019

Background

β-thalassemia major (β-TM) is one of the most common inherited diseases worldwide, characterized by a reduced ability to produce hemoglobin resulting in life-long transfusion-dependent anemia. Chronic transfusions carry significant risks such as infection, and result in iron overload that can cause significant multisystem organ damage. Hydroxyurea, an oral chemotherapeutic drug, is anticipated to decrease the need for transfusions, either completely or partially by raising hemoglobin levels and thus decreasing the short and long term complications of chronic transfusions.

Objectives

To evaluate the clinical efficacy and safety of hydroxyurea in β-thalassemia major (β-TM) patients of any age.

Search strategy

We searched MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials (CENTRAL), ongoing trials registers, and major preceding conferences. Hand searches were also conducted using reference lists from primary studies. All searches were updated to June 5, 2014.

Selection criteria

Randomized controlled trials (RCTs) and observational studies (sample size ≥ 5) assessing the clinical efficacy of hydroxyurea alone for three months or longer, for the treatment of patients with β-TM were included.

Data collection and analysis

Two authors acted as reviewers and independently assessed study quality and extracted data from the included studies. Authors of included studies were contacted if further information was required. β-TM includes the classical β-TM as well as severe hemoglobin E/β thalassemia, both of which are characterized by lifelong transfusion needs. The effect size was estimated as a proportion (those showing response to treatment over the total number treated) and reported as overall response rate (ORR) or complete response rate (CRR). ORR was defined as ≥ 50% reduction in transfusion need and CRR was defined as complete cessation of regular transfusion. All data was analyzed using Stata, Version 13.0.

Results

A total of 10 observational studies involving 620 patients were included. Hydroxyurea was associated with a statistically significant decrease in transfusion need with CRR of 36% (95% CI, 23-50%) and ORR of 66% (95% CI, 52-79%).

All of the studies had several limitations, such as small sample size, lack of comparison group, under-reporting of data and methods, and being observational studies.

Adverse events (AEs) were transient and improved with temporary cessation of the drug and/or adjustment of the dose. No long-term AEs, including cancer or end organ damage were reported.

Authors’ conclusion

Hydroxyurea appears to be effective in the management of β-TM by decreasing the need for chronic blood transfusions completely or partially in a significant number of patients. It appears to be well tolerated and associated with mild and transient AEs. Patients with β-TM may benefit from a trial of hydroxyurea, though large RCTs assessing efficacy should be done to confirm the findings of this meta-analysis.

Disclosures

Off Label Use: Hydroxyurea for β-Thalassemia.
Liver Enzymes Changes and Safety Profile of Deferasirox Iron Chelator in Omani Children with Thalassemia Major
Authors: Mevada Surekha Tony , AlDhuli Abdaluziz Sulieman , Al-Rawas A.Hakim , Al-Khabori Murtadha K. , Nazir Hanan , Zachariah Mathew , Wali Prof. Yasser
Published: October 2019

Background: Iron overload is a leading cause of morbidity and mortality in beta thalassemia and chelation therapy remains the mainstay in reducing iron burden. For effective chelation, optimal drug dosage with close monitoring of side effects is crucial. The safety and efficacy of deferasirox has been extensively studied in adult population. We aimed at evaluating the liver enzyme changes and safety profile of deferasirox in pediatric patients with thalassemia major.

Materials and Methods: A retrospective study of 30 patients (range 2-15 years) with thalassemia major on regular follow up at the Pediatric Day care Center, Sultan Qaboos University Hospital was performed to evaluate the side effects of deferasirox over a mean of 15 month follow-up period. Data from electronic patients' records was collected for age, gender, serum ferritin, alanine transaminase, (ALT), aspartate transaminase (AST), dose and side effects of deferasirox. Data were analyzed using SPSS software version 19.

Results: Thirteen (44.8 %) patients had either ALT or AST elevation above 2 times upper limit of normal (ULN). Except for two (6.8%) patients with enzyme elevations more than 5 times ULN, the majority of patients had mild transaminitis. None of the patients had liver enzyme elevation above 10 times ULN. The other side effects included fever (17.24%), nausea and vomiting (10.34% each), diarrhoea (13.79%), skin rash (21.4%), elevated serum creatinine with either 2 consecutive readings more than 33% of baseline level and/or single reading above 60% baseline (17.24%). The mean serum ferritin dropped from initial baseline level of 1236.21 ± 354 ng/ml (Range 534 -2821 ng/ml) to a level of 950 ± 320 ng/ml (Range 550-1900 ng/ml) at the end of the study period; with a mean deferasirox dose of 32.72 ± 4.79 mg/kg/day.

Conclusion: Majority of our patients had mild transaminitis not requiring dose modification or interruption of chelation. Patients with significant elevations of liver enzymes more than 5 times ULN showed prompt recovery of transaminitis within 4-5 weeks of dose reduction of deferasirox by 5 mg/kg/day and future dose increments were well tolerated. Except for significant elevations in serum creatinine requiring dose reduction or short interruption, the other adverse events were well tolerated and did not warrant dose modification. Deferasirox was effective in reducing serum ferritin levels and was well tolerated in our young patients with thalassemia major.

Table 1 PatientCharacteristics Numberof patients, n 30 Meanage of patients, years Range,years 7.24± 3.4 2-15 Meanserum ferritin, ng/ml Range,ng/ml 1236.21± 354 534-2821 Meandose of deferasirox (mg/kg/day) 32.72± 4.79

Figure 1 Figure 1.

Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
No Association Between Platelet Function and Hemophilia B Phenotype
Authors: Schutgens Roger E.G. , Bladel Esther R. van , Fischer Kathelijn , Roest Mark , Urbanus Rolf
Published: October 2019

Introduction

In hemophilia, unravelling the mechanisms behind interindividual variability in phenotype remains a big challenge.

Objectives

To study platelet activation and platelet responsiveness in relation to hemostatic phenotypes in hemophilia B by 1) comparing hemophilia B patients to healthy controls and 2) relating these to the clinical phenotype of the individual patients with severe hemophilia B.

Methods

Platelet reactivity was determined in whole blood measuring expression of P-selectin and binding of fibrinogen with FACS analysis after stimulating or inhibiting platelets with concentration series of several (ant-)agonists, including adenosine diphosphate (ADP), convulxin (CVX) and thrombin receptor associated protein (TRAP)and iloprost. Concentration of soluble platelet activation markers (soluble P-selectin, β-Thromboglobulin and RANTES) were measured in platelet poor plasma by ELISA.

Markers of platelet activation and responsiveness were compared between healthy controls (n=12), patients with mild/moderate (n=12) or severe hemophilia B (n=19).

In severe hemophilia patients, annual FIX consumption/kg was calculated over the last 6 years. Data were analysed with linear regression using SPSS 20.

Results

Levels of basal platelet activation (both P-selectin and fibrinogen) were equal between controls, mild/moderate and severe hemophilia B (Figure 1). For basal P-selectin expression, the mean area under the curves for were 28.5 (95% CI 23.3-33.6), 33.0(95% CI 24.4-41.6) and 28.4(95% CI 23.9-32.9) respectively (p=0.4). After stimulation with ADP, CVX and TRAP (with and without iloprost), platelet responsiveness was similar in all groups.

There was no relation between annual FIX consumption and any marker of platelet activity. For basal P-selectin expression, the beta coefficient was -0.212 (p=0.38)(Figure 2).

Conclusion

In hemophilia B, platelet activity and responsiveness was similar to healthy controls. In severe hemophilia patients, there was no relation between FIX consumption and platelet activity.

Figure 1. Basal P-selectin activity between healthy controls and hemophilia patients Figure 1. Basal P-selectin activity between healthy controls and hemophilia patients

Figure 2. Basal P-selectin activity according to FIX consumption in severe hemophilia B Figure 2. Basal P-selectin activity according to FIX consumption in severe hemophilia B

Disclosures

Schutgens: Pfizer: Research Funding.
Low dose Rituximab for Preemptive Management of Thrombotic Thrombocytopenic Purpura ( TTP )
Authors: Nelson John C.
Published: October 2019

Low dose rituximab has been successful in treating some patients with autoimmune cytopenias. Recently reported data indicate preemptive rituximab may postpone hematologic relapses of TTP in patients with chronic severe ADAMTS13 deficiency.

A now 22-year-old woman had multi-relapsing TTP with five episodes over the previous 10 years, three of which were successfully treated with four doses of rituximab 375 mg/m2. The most recent course in 2009 was given preemptively because intense ADAMTS13 monitoring had shown that previous hematologic relapses followed shortly after developing severe ADAMTS13 deficiency. Asymptomatic chronic IgG deficiency less than 300 mg% had followed this most recent rituximab course given in 2009.

In 2014, continued intense ADAMTS13 monitering disclosed a new progressive decrease in ADAMTS13 levels from normal to severely deficient levels with a weak inhibitor, but no hematologic or clinical changes. Just two 100 mg rituximab doses have been followed by a progressive rise of ADAMTS13 levels to solidly normal values with significant improvement by day 13 post initiation and normalization by day 34.

Low dose rituximab could be a viable alternative for preemptively managing TTP patients with persistently severly low ADAMTS13 levels.

Disclosures

Off Label Use: Off label use of rituximab to treat TTP.
Characteristics of Intracranial Haemorrhage in Haemophilic Children-Report in Single Center in China
Authors: Ding Wenjiao , Feng Xiaoqin , Sun Jing
Published: October 2019

Objectives: Intracranial haemorrhage (ICH) is the most severe complication for patients with haemophilia. Our study objectives were to analyze the characteristics of Intracranial haemorrhage in haemophilia children.

Methods: Intracranial haemorrhage cases in haemophilic children were retrospective analyzed from Jan. 2008 to Dec. 2013. Patients with sever trauma inhospitalized in the surgical department were excluded.

Results: Of 439 severe or intermediate haemophilic children 29(6.6%) experienced 32 events of ICH. The 29 children included 20 haemophilia A boys and 9 heamophilia B boys. The age of onset of ICH from 1 month to 17 years old (mediate age 3 years old). Head trauma histories was found in 79.3% (23/29) children 1 day to 1 week before ICH. Of 29 patients 15 (51.7%) had the presentation of convulsion or coma. The haemorrhage site included 2 events in extradural, 2 events in subdural, 6 events in brain parenchyma, 1 event in subarachnoid space, 21 events had multiple sites ICH. The ICH mortality was 6.9% (2/29). Only two children had done prophylaxis treatment with low dose (10-15u/kg, 2/w) before ICH. Inhibitor were only tested in 8 children who were all positive when ICH occurred.

Conclusion: The most significant risk factors for ICH included the presence of an inhibitor and reported head trauma. Our patients with ICH had low mortality maybe because most patients had no inhibitor who could have good response to factor treatment, and these patients had excluded patients with sever trauma in the surgical department.

Disclosures

No relevant conflicts of interest to declare.
Clinical Significance of Absolute Monocyte Count in Venous Thromboembolism Patients with Lung Cancer
Authors: Go Se-Il , Song Haa-Na , Lee Gyeong-Won
Published: October 2019

Purpose: To investigate the role of the absolute monocyte count (AMC) as a predictor of response to anticoagulation and survival in lung cancer patients with venous thromboembolism (VTE).

Methods: We retrospectively collected the clinical data of 134 patients with lung cancer who were diagnosed with VTE and treated with anticoagulation between July 2008 and May 2014 in a single institution. Baseline characteristics, laboratory findings, response to anticoagulation, and survival since the diagnosis of VTE were analyzed in low (n = 67) and high (n = 67) AMC groups according to median AMC (640/μL) at the time of VTE diagnosis.

Results: The majority of patients had non-small cell lung cancer (n = 110, 82.1%), stage IV (n = 86, 64.2%), and pulmonary thromboembolism (n = 102, 76.1%) and were incidentally diagnosed with VTE without any sign of thrombosis (n = 103, 76.9%). Patients’ characteristics and laboratory values were not significantly different between low and high AMC groups. Among 90 patients available for evaluation of response to anticoagulation, high AMC group (n = 10/46, 21.7%) was more refractory to anticoagulation than low AMC group (n = 3/44, 6.8%) (p = 0.044). In addition, high AMC group showed worse overall survival than low AMC group (median 9.6 vs. 5.9 months, p = 0.038). In multivariate analysis, high AMC and low albumin were independent poor prognostic factors.

Conclusion: High AMC is associated with refractoriness to anticoagulation and poor prognosis in lung cancer patients with VTE. Further evaluation is warranted to confirm these findings.

Disclosures

No relevant conflicts of interest to declare.
Mesenteric Vein Thrombosis Associated to Elevated Factor VIII in a Patient Treated with Adjuvant Tamoxifen
Authors: Herrada Juan , Reiter Ethan A , Gupta Anuradha , Arango Carmen , Landeros Mark M , Mercedes Lorenzo-Medina , Gomez Jesus A , Vargas-Sanz Sacramento , Campos-Gines Angel F , Madrid Melissa , Briceno Jeannette
Published: October 2019

Introduction: Venous thrombosis is a well documented adverse effect in about one percent of patients treated with tamoxifen. Although the precise role of elevated factor VIII in defining venous thrombotic risk is not well understood, current data suggest that factor VIII is an independent risk factor for venous thrombosis.

Methods: Case report.

Results: In May 2011, a 76-year-old male underwent right modified radical mastectomy in May 2011. Tumor was a 2.5 cm well-differentiated, invasive ductal carcinoma, with lymphatic invasion present and widely negative surgical margins. The patient (pt) had one ipsilateral axillary lymph node (out of 26 sampled) with two microscopic foci (<1 mm) of metastatic ductal carcinoma. Additional evaluation on the mastectomy specimen showed that estrogen and progesterone receptors were strongly positive, Ki-67 was 25% and HER-2/neu showed negative (0 expression) by immunochemistry. The patient had no family history of venous thromboembolic events, and he had never smoked. His past medical history was significant for hypothyroidism and hypertension, both under good control. In June 2011 pt was started on adjuvant Tamoxifen 20 mg daily. In July of 2013 pt started with diffuse, ill-localized abdominal pain. No associated abdominal cramping, fever or nausea. No evidence of melena or rectorrahgia. An abdominal magnetic resonance imaging showed the presence of a superior mesenteric vein thrombosis. Tamoxifen was at that time discontinued and pt was started on enoxaparin 1 mg/Kg twice a day, with complete resolution of his symptoms within 7 days. Pt remained anticoagulated with enoxaparin for six months. After discontinuation of anticoagulation, radiologic and laboratory evaluation showed no evidence of underlying hypercoagulable state except for an elevation of serum factor VIII (> 150 IU/dl) that has persisted during his follow-up.

Conclusions: Our case supports the notion that high levels of factor VIII are a risk factor for venous thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Reversal of Radiation Damage to Marrow Stem Cells By Mesenchymal Stem Cell Derived Vesicles
Authors: Quesenberry Peter J , Wen Sicheng , Dooner Mark S , Carpanetto Andrea
Published: October 2019

Extracellular vesicles (exosomes and microvesicles) have been found to deliver both mRNA and transcriptional modulators to target cells and affect their phenotype. Marrow mesenchymal stem cell derived vesicles (MSC-DV) have also been shown to reverse renal and hepatic injury. We have studied the capacity of MSC-DV to reverse radiation injury to marrow stem cells.

B6.SJL mice were exposed to 100 centigrade whole body radiation (WBI), and 4, 24, and 168 hours later marrow is harvested and established in culture for 24-48 hours with either human or murine marrow-derived mesenchymal stem cells (MSV-DV) or vehicle. These cells were then transplanted into 950 cGy exposed C57/BL mice and engraftment evaluated at 3 weeks to 8 months. Alternatively cells were engrafted without competition into 200 centigrade exposed mice. Significant (p<0.05) restoration of engraftment was seen in each setting; restoration of secondary engraftment was also seen. Mice were also subjected to 500 cGy WBI and injected at 6, 24 and 72 hours with human MSC-DV and peripheral blood counts determined. Granulocyte levels were restored to 86% of control at 3 weeks post irradiation and significant restoration seen at multiple other time points. Restoration was seen was seen in both peripheral blood and marrow. Further studies on the murine hematopoietic cell line, FDC-P1 exposed to 500 centigrade showed dramatic recovery on exposure to murine or human MSC-DV. Additional work, employing differential ultracentrifugation as a separative technique for vesicles, showed that a preparation with both exosomes and microvesicles was superior to either microvesicles or exosomes alone. Studies with irradiated FDC-P1 cells indicated that exposure to vesicles decreased apoptosis. These studies indicate that vesicles from marrow-derived mesenchymal stem cells from different sources have the capacity to reverse radiation damage to bone marrow stem cells. Thus administration of MSC-DV to patients exposed to radiation could represent an important new strategy in radiation mitigation.

Disclosures

No relevant conflicts of interest to declare.
Human Mesenchymal Stem Cells Promote Resistance to Death Signals By Potentiating FGF Signaling in Mantle Cell Lymphoma
Authors: Sehgal Lalit , Mathur Rohit , Braun Frank K , Berkova Zuzana , Neelapu Sattva , Samaniego Felipe
Published: October 2019

Introduction

Mantle cell lymphoma (MCL) represents an aggressive, incurable form of non-Hodgkin’s lymphoma (NHL). The health complications associated with advanced age of MCL patients further restrict treatment with intense chemotherapy. Translocation t(11;14), responsible for overexpression of cyclin-D1 is the hallmark of MCL. An intense analysis of primary MCL had been delayed until development of a tissue culture system, using human mesenchymal stromal cells (hMSC), suitable for propagation of primary MCL cells. We hypothesized that tumor-initiating cells are responsible for MCL relapse and chemoresistance and thus, identification of survival signals responsible for survival and maintenance of MCL-initiating cells (MCL-ICs) is essential for design of successful treatment strategies.

Methods

Isolates of primary MCL cells (n=24) were co-cultured with hMSC and content of MCL-ICs was analyzed by flow-cytometry based on marker expression profile; CD34-CD3-CD45+CD19-. Cytokine array was used to identify the soluble factors enriched in the cocultures and the expression of these factors was confirmed by RT-PCR analysis. The signaling pathways employed by the newly-identified factors were blocked in 3 MCL cell lines (JVM2, Mino, Z138) and cell survival was monitored.

Results

Co-cultures of primary MCL isolates with hMSCs supported the growth of MCL cells for over 4 weeks with continued presence of MCL-ICs (CD34-CD3-CD45+CD19-) representing about 1% of MCL cells. We found IL-6 triggered a FGF/FGFR autocrine loop in primary MCL in cocultures. Patients with MCL showing high FGFR levels in MCL tissues compared to lymphocytes, and the highest FGFR expressing cohort of patients showed a lower overall survival rated compared to those with low FGFR levels. Blockage of FGF/FGFR rendered MCL cells dead and also sensitized the cells to the killing effects of death receptor ligands.

Conclusion

We establish that primary MCL use an FGFR autocrine loop for propagation in cocultures with hMSCs. We defined the factors supporting MCL and MCL-ICs survival; such knowledge is essential for targeting MCL and MCL-ICs.

Disclosures

No relevant conflicts of interest to declare.
Evidence for Vasculogenic Potential and Endothelial Differentiation of Bone-Marrow-Derived Very Small Embryonic-like Stem Cells
Authors: Guerin Coralie L , Loyer Xavier , Vilar Jose , Cras Audrey , Mirault Tristan , Gaussem Pascale , Silvestre Jean Sebastien , Smadja David M
Published: October 2019

Objective: Very small embryonic-like stem cells (VSELs) are multipotent stem cells localized in adult bone marrow (BM) that may be mobilized into peripheral blood (PB) in response to tissue injury. We aimed to quantify VSELs in BM and PB of patients with critical limb ischemia (CLI) and to test their angiogenic potential in vitro as well as their therapeutic capacity in mouse model of CLI.

Approach and Results: We isolated BM VSELs from patients with CLI and studied their potential to differentiate into vascular lineages. Flow and imaging cytometry showed that VSEL counts were lower in BM (p<0.001) and higher (p<0.001) in PB from CLI patients compared to healthy controls, suggesting that ischemia may trigger VSELs mobilization in this patient population. Sorted BM-VSELs cultured in angiogenic media acquired a mesenchymal phenotype (CD90+, Thy-1 gene positive expression). VSEL-derived cells had a pattern of secretion similar to that of endothelial progenitor cells, as they released low levels of VEGF-A and inflammatory cytokines. Noteworthy, VSELs triggered post-ischemic revascularization in immunodeficient mice (p<0.05 vs PBS treatment), and acquired an endothelial phenotype either in vitro when cultured in the presence of VEGF-B (Cdh-5 gene positive expression), or in vivo in Matrigel implants (human CD31+ staining in neo-vessels from plug sections).

Conclusions: VSELs are a potential new source of therapeutic cells that may give rise to cells of the endothelial lineage in humans.

Disclosures

No relevant conflicts of interest to declare.