bluuebunny/crossref_metadata_2025 huggingface dataset:
A Tailor-Made Protein Synthesis Program Drives Erythroid Development and Disease
Authors: Forester Craig M , Shi Zhen , Barna Maria , Ruggero Davide
Published: October 2019

Erythropoiesis constitutes the largest demand on the hematopoietic system due to its extraordinary production on a daily basis. The erythroid proteome requires an integration of multiple external cues to coordinate programs of differentiation as well as maintenance of erythroid precursors. The biomedical relevance of this critical process is underscored by recent findings showing impaired ribosome function in an entire class of clinical disorders with severe impairments in erythroid differentiation, known as ribosomopathies, which remain poorly understood. One of the main signaling pathways controlling post-transcriptional gene expression during erythropoiesis is the mTOR pathway. mTOR activation downstream of SCF/Epo in erythroid progenitors controls the activity of the major cap-binding protein eIF4E. However, the functional role of eIF4E during erythropoiesis and protein synthesis control in this cell type remains unexplored. Here we show that eIF4E activity, through mTOR-dependent phosphorylation of its inhibitory protein 4EBP1, unexpectedly undergoes a dynamic switch between early erythroid precursor populations and during terminal erythrocyte maturation, where eIF4E becomes progressively silenced. Employing a unique eIF4E transgenic mouse model, we strikingly show that overexpression of eIF4E in the bone marrow compartment results in an early accumulation of erythrocyte precursors and a block in erythrocyte differentiation. Surprisingly, this new role of eIF4E in erythropoiesis is independent from control of global protein synthesis but instead may promote a specialized program of translation control that is customized for erythroid cell function. Employing state of the art unbiased proteomics, our work is uncovering distinct networks of proteins, whose expression levels are controlled by eIF4E dosage during specific phases of erythrocyte maturation. Together, our research highlights a novel molecular program linking exquisite regulation of eIF4E activity to specialized translational control underlying erythroid development, providing unprecedented insight into the etiology of erythroid dysfunction in ribosomopathies.

Disclosures

No relevant conflicts of interest to declare.
ASXL1 Is a Key Regulator for Erythroid Development and Asxl1 Loss Impairs Erythropoiesis In Vivo
Authors: Yamamoto Shohei , Shi Hui , Chen Shi , Zhang Peng , Zhou Yuan , Xu Mingjiang , Yang Feng-Chun
Published: October 2019

Objective:

Recently, ASXL1 mutations are found in a spectrum of numerous of myeloid malignancies, including myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). ASXL1 mutation is a poor prognostic markers. Recently, we reported that Asxl1+/- mice developed myelodysplastic syndrome (MDS)-like disease and MDS/MPN. Regardless of the existence of defective erythroid lineage maturation and anima in patients with MDS or MPN, the role of ASXL1 in erythropoiesis remains unveiled.

Methods:

Asxl1+/- mice were utilized for hematopoietic analysis, including peripheral blood counts, blood smear, morphology, flow cytometry and histology. To assess the erythroid phenotypes in vivo, blood smears were subjected to acridine staining for reticulocyte counts. The percentage of reticulocytes and erythroid cell differentiation in peripheral blood and bone marrow were evaluated flow cytometrically following thiazole orange and CD71/Ter119 staining. Colony-forming cells (CFU) and in vitro erythroid differentiation assays were performed with methylcellulose (EPO, SCF) and liquid (EPO, transferrin) cultures of Lin-Kit+ cells of BM, spleen and fetal liver cells. In addition, the nuclear fractions were prepared from cultured erythoblasts of WT and Asxl1+/- mice and subjected to western blotting analyses to determine the histone levels of H3K27me3. RNA-Seq was performed to survey the differentially expressed genes in LK cells of WT and Asxl1+/- mice. Real time PCR was performed to confirm the alteration of erythroid development related gene.

Results:

Adult Asxl1+/- mice were anemic. High frequencies of immature and dysplastic (tear drop) red blood cells were observed in the peripheral blood of Asxl1+/- mice. Flow cytometric analysis revealed an increased percentage of pro-erythroblasts and basophilic erythroblasts populations in peripheral blood and bone marrow cells compared to that of WT controls, suggesting a blockage of erythroid maturation in Asxl1+/- mice. In vitro methylcellulose cultures showed a dramatic decrease of BFU-E and CFU-GEMM in the bone marrow of Asxl1+/- mice compared to WT controls. Western blot analysis showed that Asxl1+/- erythroblast cells exhibited decreased global levels of H3K27me3 and qPCR of Asxl1+/- erythroblast cells revealed altered expression of genes involved in development and homeostasis of erythroid lineage, including Gata1, Ldb1, Cdc42, and Zfpm1.

Conclusion:

Our study demonstrates that loss of Asxl1 results in blockage of erythroid cell maturation, indicating that ASXL1 is a key regulator for erythroid development and Asxl1 loss impairs erythropoiesis in vivo.

Disclosures

No relevant conflicts of interest to declare.
CHD7 Deficiency Inhibits CBFB-MYH11 -Induced Leukemogenesis
Authors: Zhen Tao , Zhao Ling , Hyde R. Katherine , Alemu Lemlem , Liu Pu Paul
Published: October 2019

Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. Mechanistic studies showed that the interaction between CBFbeta-SMMHC (encoded by CBFB-MYH11) and RUNX1 plays a critical role in the leukemogenesis of this kind of leukemia. Recently, it was shown that chromodomain-helicase-DNA binding protein 7 (CHD7) interacts with RUNX1 and suppresses RUNX1-induced expansion of hematopoietic stem and progenitor cells. These results suggest that CHD7 also plays a role in CBFB-MYH11 induced leukemogenesis. To test this hypothesis, we crossed Cre-based conditional Chd7 knockout mice (Chd7f/f) with Cre-based conditional Cbfb-MYH11 knockin mice (Cbfb+/56M) to generate Chd7f/f, Mx1-Cre, Cbfb+/56M mice, which express Cbfbeta-SMMHC but not CHD7 after poly I:C treatment to induce Cre expression. Three weeks after poly I:C treatment, the Lin- and the Lin-/Sca1-/c-Kit+ (LK) progenitor cell populations were significantly lower in Chd7f/f, Mx1-Cre, Cbfb+/56M mice than that in Mx1-Cre, Cbfb+/56M mice. In addition, there were fewer BrdU+ cells in the Lin- and LK populations in the Chd7f/f, Mx1-Cre, Cbfb+/56M mice. Importantly, Chd7f/f, Mx1-Cre, Cbfb+/56M mice developed leukemia with a much longer latency than the Mx1-Cre, Cbfb+/56M mice. Secondary transplantation assay demostrated that spleen cells isolated from Chd7f/f, Mx1-Cre, Cbfb+/56M and Mx1-Cre, Cbfb+/56M leukemic mice were transplantable, but recipients that received spleen cells from Chd7f/f, Mx1-Cre, Cbfb+/56M leukemic mice had a longer latency than those who received cells from Mx1-Cre, Cbfb+/56M mice. The above results suggest that CHD7 deficiency slows leukemia initiation and development by inhibiting the proliferation ability of the Lin- and LK populations. Further mechanistic studies showed that CHD7 interacted with RUNX1 but not CBFbeta-SMMHC by co-Immunoprecipitation and immunofluorescence staining, suggesting that CHD7 is a partner of the RUNX1-CBFbeta-SMMHC transcription complex. In vitro luciferase assay showed that CHD7 enhanced RUNX1 and CBFbeta-SMMHC's transcriptional activity on M-CSFR, a RUNX1 target gene. These data indicate that CHD7 is important for Cbfb-MYH11 induced leukemogenesis by facilitating RUNX1 regulation of transcription and cellular proliferation.

Disclosures

No relevant conflicts of interest to declare.
Ten Year Single-Center Experience in Fertility Preservation of 459 Patients Suffering from Acute Leukaemia
Authors: Noetzli Jasmine , Voruz Sophie , Wunder Dorothea , Primi Marie-Pierre , Crosazzo Laura , Gavillet Mathilde , Spertini Olivier , Blum Sabine
Published: October 2019

Patients with acute leukaemias suffer from a life threatening disease that has to be treated immediately. Fertility preservation is recommended in these patients, but quite often not possible.

Methods

This study is a retrospective analysis of 459 patients suffering from acute leukaemia, 221 with acute myeloid leukaemia (AML) or high risk MDS, 238 with acute lymphoblastic leukaemia (ALL) diagnosed between 2002 and 2012 in the University clinics Lausanne, Switzerland. Patients were aged between 0 and 93 years. Of course, not all of these patients were send to the preservation laboratry because of their age or their performance status.

Results

Of 221 AML cases, 113 were male and 108 female. 46 male patients were between 14 and 60 years of age, of these, 14 were sent for cryopreservation. 12 sperm samples could be frozen, 2 were sample failures due to azoospermia. Of the frozen samples, 7 were destroyed (6 because of death of the patient, 1 according to wish of patient), 4 are still frozen without tempting procreation and 1 was used for in vitro fertilization (IVF) resulting in a healthy child in 2010. 32 female patients were between 17 and 45. No sample of ovocytes was taken, 1 sample of ovarian tissue could be frozen, that was afterwards destroyed because of death of the patient.

Of 238 ALL cases, 123 were male and 115 female. 55 male patients were between 14 and 60 years of age, of these 19 were sent for cryopreservation. 16 sperm samples could be frozen, 2 were sample failures due to azoospermia, 1 because of ejaculation failure. Of the frozen samples, 11 were destroyed (8 because of death of the patient, 2 due to missing payment and 1 according to the wish of the patient). 5 samples are still frozen, 1 was used for a IVF resulting in a healthy child in 2009. 12 female patients were between 17-45. No sample taking was tempted.

Discussion

In a 10-year period in our institution, of 459 patients suffering from acute leukaemia, 145 of these within the age range defined by our institution to qualifying to cryoconservation. Only 29 samples were taken (20%), non of these in paediatric patients. These samples were leading to 2 IVFs. Of note, one male patient whose samples were taken and resulted in one of the IVFs was suffering from ALL and had an allogenous stem cell transplantation with full conditioning. This patient fathered after the IVF two other healthy children without assisted fertilization. The other patient whose sperm sample was taken for a IVF suffered from AML, he too fathered a child spontaneously after the IVF.

The data show that only 20% of eligible acute leukaemia patients had the opportunity to freeze sperm or ovocytes samples. Certainly, this was not possible for a lot of patients due to performance status, but more effort has to be done to improve sampling in these patients.

Disclosures

No relevant conflicts of interest to declare.
First-in-Man CD123-Specific Chimeric Antigen Receptor-Modified T Cells for the Treatment of Refractory Acute Myeloid Leukemia
Authors: Luo Yi , Chang Lung-Ji , Hu Yongxian , Dong Lujia , Wei Guoqing , Huang He
Published: October 2019

Introduction: Adoptive immunotherapy using T-cells endowed with chimeric antigen receptors (CARs) has emerged as a promising new approach to treating CD19+ acute lymphoblastic leukemia (ALL). However,treatments for relapse/refractory acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and some AML patients have very poor prognosis. The interleukin-3 receptor alpha chain (CD123) has been identified as a potential immunotherapeutic target due to its overexpression in AML cells compared with normal hematopoietic stem cells. Antibodies targeting CD123 for the treatment of AML have demonstrated promising anti-leukemic activity in murine models but showed limited efficacy in clinical trials, suggesting that alternative and more potent therapies targeting CD123 are required.

Methods: We have generated a 4th generation, apoptosis-inducible lentiviral CAR targeting CD123: CD123-scFv/CD28/CD137/CD27/CD3ζ-iCasp9 (4SCAR123), and demonstrated its high AML killing and AP1903-inducible apoptosis functions in ex vivo analyses. In a pilot trial of 4SCAR123, we enrolled a 47-year-old male patient with AML-M2 (FLT3/ITD+). The patient underwent allogeneic hematopoietic stem cell transplantation and relapsed. After 3 chemotherapies combined with sorafenib, his AML cells kept at 59% in bone marrow. He received CTX 250mg/kg/day for 3 days as conditioning regimen followed by 1.8x106/kg 4SCAR123 T cell infusion. Serum cytokine levels were measured by flow cytometric bead assay.

Results: At day 1 after 4SCAR123 T infusion, the patient experienced rigorous chills and fevers, low blood pressure and hypoxemia. We detected elevated serum cytokine levels including interleukin-6 (2,500pg/ml) and tumor necrosis factor-α (33.8 pg/ml) at day 8, and the patient suffered from severe cytokine release syndrome (CRS) on day 4, which was controlled by one dose of Tocilizumab. BM examination detected a decrease of blasts from 59% to 45% 20 days after CAR-T therapy.

Conclusion: Here we report a first-in-man pilot safety study of CD123 CAR-T therapy for AML patients. The 4SCAR123 exhibited potent cytotoxicity against AML in vitro, and in this pilot trial, the patient developed a rapid response consistent with CRS and achieved partial remission within 20 days. Importantly, although CD123 is universally expressed in myeloid and endothelial cells, we did not observe overt off-target cytotoxicity from the 4SCAR123 T cells, except for a controllable CRS. Our pilot study warrants further exploration of CD123 CAR for the management of refractory AML.

Disclosures

Dong: America Yuva Biomed: Consultancy.
Induction of NK Cell Reactivity Against Myeloid Leukemia By a Novel Fc-Optimized CD133 Antibody
Authors: Rothfelder Kathrin , Koerner Samuel , Andre Maya , Leibold Julia , Kousis Philaretos , Bühring Hans-Jörg , Haen Sebastian P. , Kuebler Ayline , Kanz Lothar , Grosse-Hovest Ludger , Jung Gundram , Salih Helmut R
Published: October 2019

NK cells largely contribute to the success of monoclonal antibody (mAb) application in cancer due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), a feature considered critical for therapeutic success. Up to now, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Recently, we reported on the development of mAb targeting CD133, which is expressed on a wide variety of tumor cells (Koerner et al., Blood 2014 124:2309). Here we extend our analyses and provide further data on the preclinical characterization of an Fc-engineered CD133 mAb for the treatment of myeloid leukemia. Compared to two other anti-human CD133 mAb (clones AC133 and W6B3), which both bound to primary AML and CML cells in 15/25 and 7/10 cases, respectively, clone 293C3 recognized the leukemic cells in 22/25 AML cases and 7/10 CML cases. Based on these results, clone 293C3 was chosen to generate chimeric mAb with either a wildtype Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance affinity to the activating Fc receptor CD16 on NK cells (293C3-SDIE). Treatment with 293C3-SDIE resulted in significantly enhanced activation, degranulation and lysis of primary CD133-positive AML cells by NK cells in allogeneic and autologous experimental ex vivo settings as compared to its wildtype counterpart. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow (BM) cells. In line with the observed lower expression levels of CD133 on healthy compared to malignant hematopoietic cells no relevant toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells was observed. Moreover, 293C3-SDIE did not induce lysis of of healthy BM cells by allogeneic or autologous NK cells. In a NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) xenotransplantation model, induction of ADCC by treatment with 293C3-SDIE resulted in the elimination of patient AML cells by NK cells from a matched human donor. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound, in particular for the elimination of minimal residual disease in CD133 bearing leukemia in the context of allogenic SCT.

Disclosures

No relevant conflicts of interest to declare.
Infiltration of Effector Regulatory T Cells Is Associated with Poor Prognosis in Diffuse Large B-Cell Lymphoma, Not Otherwise Specified
Authors: Shoko Nakayama , Motomu Tsuji , Taiji Yokote , Toshikazu Akioka , Takuji Miyoshi , Nobuya Hiraoka , Yuji Hirata , Kazuki Iwaki , Ayami Takayama , Uta Nishiwaki , Yuki Masuda , Jun Hatooka , Toshiaki Hanafusa , Hiroyoshi Nishikawa
Published: October 2019

(Background) Regulatory T cells (Tregs) are the focus of interest as one of the tumor microenvironmental factors because of their critical role in the modification of immune response, such as suppression of tumor-associated antigen-reactive lymphocytes. Tregs-mediated immunosuppression contributes to the progression of tumors and poor prognosis of patients. Forkhead box P3 (FOXP3) had been regarded as a specific marker of Tregs, but recently, FOXP3-positive cells were proved to be heterogeneous in phenotype and function. FOXP3-positive cells include effector Tregs (eTregs), naïve Tregs, and non-Tregs. Of them, eTregs have a substantial immunosuppressive effect. So far, no study has investigated the relationship between infiltration of eTregs and prognosis of patients with lymphoma.

(Aim) The present study aimed to evaluate the relationship between infiltration of eTregs doubly positive for FOXP3/cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and prognosis of patients with diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS).

(Methods) Seventy-four DLBCL, NOS specimens were doubly immunostained with anti-FOXP3 and anti-CTLA-4 antibodies. The relationship between the degree of infiltration of only FOXP3-positive cells or FOXP3/CTLA-4 double-positive eTregs and the prognosis of DLBCL, NOS patients was analyzed using Cox regression analysis. (Results) Presence of numerous FOXP3-positive cells (>52x102/cm2) was significantly predictive of better prognosis than that of fewer FOXP3-positive cells (<52x102/cm2) in terms of overall survival (OS) (p =0.0481) (Figure 1). In contrast, presence of many eTregs (>18/cm2) was significantly predictive of worse prognosis than that of fewer eTregs (<18/cm2) in terms of OS and progression-free survival (p =0.0342 and p =0.0269, respectively) (Figure 2). This was independent of the international prognostic index.

(Conclusions) Higher infiltration of only FOXP3-positive cells showed better prognosis of DLBCL, NOS patients, consistent with existing reports. In contrast, higher infiltration of eTregs showed worse prognosis of DLBCL, NOS. This suggests that infiltrating eTregs play an important role in DLBCL, NOS progression. Recently, immunotherapies targeting eTregs-depletion or functional alteration of eTregs-are being developed and are being tried in some solid tumors. These immunotherapies could also become a novel strategy of treatment for DLBCL, NOS patients with higher eTregs infiltration.

Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures

Shoko: Osaka Community Foundation: Research Funding.
Palbociclib (PD-0332991), a Potent Selective CDK4/6 Inhibitor, Overcomes Therapy-Resistant and Sensitizes Diffuse Large B-Cell Lymphoma Pre-Clinical Models to Chemo-Reagents
Authors: Gu Juan J , Zhang Qunling , Mavis Cory , Czuczman Myron S. , Hernandez-Ilizaliturri Francisco J.
Published: October 2019

Background: Results from the Collaborative trial in relapsed aggressive lymphoma (CORAL) study highlighted the poor clinical outcome of relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). We found that RRCL exhibited a unique cell cycle distribution (accumulation in S-phase) and an increase in cyclin D Kinase-4 (CDk4) mRNA/protein levels. In our current work, we evaluated Palbociclib, a novel orally bioavailable CDK4/6 inhibitor recently approved by the Food and Drug Administration for the treatment of breast cancer in rituximab-sensitive and -resistant lymphoma pre-clinical models.

Methods: A panel of rituximab-sensitive cell lines (RSCL) or RRCL were exposed to escalating dosages of PD0332991 (0-80µM) for 24-72 hrs. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 values were calculated using the GraphPad Prism6 software. Subsequently RSCL or RRCL were exposed to PD0332991 or vehicle control and doxorubicin (0.5, 1, 2µM), dexamethasone (1µM), ibrutinib (1µM), bortezomib (10-20nM) or carfilzomib (10nM) for 48 hours. Changes in cell viability were determined by the Presto Blue assay. Coefficient of synergy was calculated using the CalcuSyn software.

Results: Palbociclib induced dose- and time-dependent decrease in cell viability in both RSCL and RRCL. Of interest, palbociclib was more potent in RRCL than RSCL. The IC50 values were lower in RRCL Raji-4RH and RL-4RH (12.12µM and 13.43µM) than in RSCL Raji and RL cells (14.7µM and 19.99µM). In vitro exposure of lymphoma cells to palbociclib resulted in G1 cell cycle arrested and it was more evident in RRCL than in RSCL. Synergistic activity was observed by combining palbociclib with doxorubicin, dexamethasone and proteasome inhibitors (bortezomib and carfilzomib) in vitro. Our data suggests that, palbociclib is active against in rituximab-sensitive or resistant pre-clinical models. In addition, CDK4/6 inhibition resulted in synergistic effects with several chemotherapeutic agents. A better understanding in the molecular events triggered by palbociclib is necessary to better develop optimal combination strategies using this novel agent in future clinical trials. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund).

Disclosures

Czuczman: Celgene: Employment; MorphoSys: Consultancy; Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board.
The Quality, Content, Accuracy and Readability of Information about Sickle Cell Anemia on the Internet
Authors: Breakey Vicky R. , Harris Lauren , Davis G Omar , Agarwal Arnav , Ouellette Carley , Akinnawo Elizabeth M , Stinson Jennifer N
Published: October 2019

Objective: A comprehensive website review was conducted to assess the quality, content and readability of online information for teens with sickle cell anemia (SCA).

Methods. Key words relevant to SCA were searched across the five most commonly used search engine domains. Websites that contained information about the diagnosis and management of SCA were reviewed. Quality of the information was appraised using the validated DISCERN tool. Two physicians rated website content completeness and accuracy independently. Readability of the sites was documented using SMOG scores and the Flesch Reading ease scoring system.

Results. Search results yielded more than 600 sites of which 25 websites met the criteria for DISCERN quality review. The majority of sites targeted parents and only 5/25 (20%) were specific to teens with SCA. The overall quality of the website information was "fair", with the average DISCERN quality rating score being 50.1 (± 9.3, range 31.0-67.5). Only 12/25(48%)of the websites had DISCERN scores above 50 (mean 57.37 + 4.93, range 52.17-67.50). The average completeness score of the sites was 20 out of 29 (±5; range 12-27) and accuracy was consistently rated 4/4, indicating high accuracy with moderate completeness. The average SMOG score was 12.44 (±2.01; range 10.21-16.08), and the mean Flesch Reading Ease score was 46.45 (±13.22; range 17.50-66.10) indicating that the material was written well above the acceptable level for patient education materials.

Conclusion. Given the paucity of high quality Internet health information at an appropriate reading level for teens with SCA, there is a critical need for the development of Internet programs to meet their unique self-management needs.

Disclosures

No relevant conflicts of interest to declare.
Relationship Between Myelodysplastic Syndrome and Paroxysmal Nocturnal Hemoglobinuria: Spanish Erythropathology Group and Spanish Paroxysmal Nocturnal Hemoglobinuria Working Group Experience
Authors: Lopez Rubio Montserrat , Gaya Anna , Morado Marta , Carrasco Vicente , Gonzalez Fernandez Ataulfo , Villegas Ana
Published: October 2019

INTRODUCTION

PNH clones appear frequently in patients with myelodysplastic syndrome (MDS) and aplastic anemia (AA) which may even evolve into classic PNH. It has been described isolated cases of clonal evolution from AA, through classic PNH and MDS; and at the end, acute leukemia (AL) has rarely been described. These patients show a progressive replacement of the PNH clone by a dysplastic one carrying a monosomy of chromosome 7.

OBJECTIVE

We designed a retrospective study of patients included in the database of the Spanish PNH Registry in search of patients with a MDS and a PNH clone or those with a classic PNH who evolved into MDS. Our aim was to study the characteristics of patients who showed some of these entities and AA history.

RESULTS

Our search retrieved 6 patients, of whom 3 were MDS cases with a small PNH clone (Table 1). One of the patients had had a previous classic PNH followed by a MDS. The remaining two patients had been diagnosed with AA, developing classical PNH and MDS later; of these, one (patient 2) died with an acute leukemia.

The median follow-up time was 83 (range 56-480) months, while the follow-up time from diagnosis of MDS was 68 (range 6-108) months. The patient characteristics are describe in Table 1.

There are two remarkable features, diagnosis of a RAEB-1 in two cases and the frequency of trisomies of chromosome 8 and 21 and deletion of chromosome 7. These chromosomal abnormalities appeared sequentially as MDS evolved in 3 patients. The PNH clone disappeared in those patients who developed a MDS after an AA/classic PNH and persisted in those MDS cases who showed a PNH clone at diagnosis of MDS.

Table 1. Patients Characteristics Previous diagnosis Follow-up time MDS type Karyotype Case 1 None 70 RARS + 8 Case 2 AA + PNH 56 RCMD -7 evolution to -7, + 21 Case 3 Classical PNH 83 RAEB-1 + 8 Case 4 None 83 RAEB-1 +21 evolution to +21, +8 Case 5 AA + classical PNH 480 RCMD + 8 evolution to +8, -7 Case 6 None 108 RCMD Normal

CONCLUSIONS

Patients with a diagnosis of AA, classic PNH or MDS with a PNH clone must be followed up for life, with bone marrow studies with karyotype and measurements of the PNH clone, to rule out the development of other hematological entities. Our series is a small one and only studies with a larger number of patients will allow us to establish definitive conclusions. The inclusion of these patients in the International PNH Registry is strongly advised.

Disclosures

No relevant conflicts of interest to declare.
Prevalence of Anemia and Treatment Outcomes after Bariatric Surgery-a Retrospective Korean Study
Authors: Kyoung Ha Kim , Min-Young Lee , Kim Han Jo , Kim Se Hyoung , Kim Hyun Jung , Sang-Cheol Lee , Sang Byung Bae , Chan-Kyu Kim , Kyu Taeg Lee , Sung Kyu Park , Won Jong-Ho , Hong Dae Sik , Kim Yong Jin
Published: October 2019

Background

Since the first bariatric surgery was performed in 2003, the number of bariatric surgeries performed in Korea has exponentially escalated along with the trend in Asia and recently surpassed 1,000 cases annually. Sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) are the most common types of bariatric surgery currently performed. Various types of anemia, commonly secondary to iron, folic acid, and vitamin B12 deficiencies, may complicate bariatric surgery in the long term. Thus, we evaluated the clinical course of iron deficiency anemia (IDA) in patients who underwent bariatric surgery.

Methods

A retrospective review of the prospectively established database identified all patients who underwent with RYGB for bariatric surgery between January 2008 and Feb 2014. Bariatric surgery candidates were selected according to international federation for surgery of obesity Asia Pacific Chapter Consensus statements in 2011; patients with a body mass index (BMI) ≥ 30kg/m2 with inadequately controlled obesity-related comorbidities (eg. Diabetes, obstructive sleep apnea, hypertension, or obesity related arthropathy) or with a BMI ≥ 35kg/m2. Anemia was defined as a hemoglobin level <13g/dL in men and <12 g/dL in women. Our institutional guideline of bariatric surgery is

Results

The records of 523 consecutive patients (total 796 bariatric surgeries) who underwent RYGB between January 2011 and 2015 were retrospectively reviewed. The mean preoperative body mass index was 38.0 ± 5.8kg/m2. Female was 419 (80.1%) patients and median age was 38 (range 16-67) years. Of all patients, 17.4 % patients had IDA postoperatively. Median duration from bariatric surgery to time of anemia onset was 13 (3.5-17) months. Oral iron induced 81 % overall response and 13.5% patients were required intravenous iron due to intolerance.

Conclusion

Anemia after bariatric surgery is mainly IDA, iron supplements should be administered to patients in early postoperative periods. Oral iron supplements were effective than expected.

Disclosures

No relevant conflicts of interest to declare.
Predictors of Thalassaemia Carriers in Trinidad and Tobago
Authors: Vuma Sehlule Sinqobile , Mayers Hasina , Legall George
Published: October 2019

Objective: To determine possible predictors of thalassaemia carriers among prospective blood donors in Trinidad and Tobago

Background: Thalassaemias are prevalent in India, South-East Asia, Mediterannean, Middle East and respective migrants. Trinidad ad Tobago (TT) has 1.3million people (34.2% African origin, 35.4% Indian origin, 22.8% mixed heritage, (and the rest include Caucasian, Chinese and indigenous people). DNA analysis for thalassaemia diagnosis/neonatal/population screening is not routinely available. Simple inexpensive thalassaemia predictors are needed. The main indicator is microcytosis, the commonent differential of which is Iron Deficiency Anaemia (IDA). Tiwari(2009), showed 5.4% microcytosis among Indian blood donors. (52%-IDA, 36%-Beta-thalassaemia trait, 8%-mixture of both, 4% neither). Combinations of RBC-parameters improve discriminative powers (Urechaga, 2013). No similar studies have been done in TT.

Design and Methods: 460 prospective blood donors were screened for microcytosis (MCV<83fL) by performing a CBC using a Sysmex XE-2100. Microcytic samples further had blood film analysis, iron studies, haemoglobin electrophoresis, haemoglonin F/A2 quantification, and DNA analysis for thalassaemia mutations. SPSS stastitical analysis was performed to determine predictors of thalassaemia trait. Variables tested were ethnicity, haemoglobin, MCV, MCH, MCHC, RBC (RCC), RDW-SD, and RDW-CV. Mathematical formulae were also used ie the Mentzer, Shine and Lal, Green and King, and Srivastava Indicies.

Results: 86(18.7%) subjects had microcytosis. 44 of these had DNA results. 22(50%) had MCV between 80-83fL. 31(70.5%) had thalassaemia trait: 25(80.6%) with genotype -α/αα, 4(12.9%) α-/α-(all α3.7), one IVS I-5 G/C and one IVSII-666 T/C. The MCV, RDW-SD and RDW-CV were useful in predicting thalassaemia trait. RDW-SD identified 30(96.8%) of the 31 carriers, the Green and King formula identified 27 (87.1%), and Ricerca index identified all 31.

Conclusion: MCV, RDW-SD, Green and King formula and Ricerca index may be useful predictors of thalassaemia trait in Trinidad and Tobago. A higher cut off for MCV (83fL as opposed to the 72fL or 78fL usually used for screening purposes), improves the thalassaemia-trait pick up rate.

(Acknowledgments: aMarie-D Hardy-Dessource, bChristian Saint-Martin, aMarc Romana - (aINSERM S_763, CHU de Pointe-à-Pitre, Pointe-à-Pitre, Guadeloupe: bLaboratoire de l'Unité transversale de la Drépanocytose, CHU de Pointe-à-Pitre, Pointe-à-Pitre, Guadeloupe)

Disclosures

No relevant conflicts of interest to declare.
Total Oxidant and Antioxidant Capacity in Patients with Transfusion Dependent and Nondependent Beta Thalassemia
Authors: Karakas Zeynep , Yilmaz Yasin , Celik Dolay Damla , Annayev Agageldi , Demirel Selcan , Erdem Kuruca Serap
Published: October 2019

Background: The patients with β-thalassemia are usually under oxidative stress due to iron overload as a result of ineffective erythropoiesis and repeated transfusions. The endogen (uric acid, indirect bilirubin) and exogenous (ascorbic acid and vitamin E) antioxidants protect the cells from oxidative damage. The evaluation of antioxidant defense system can be easily done by measuring serum total antioxidant capacity. The aim of this study is to investigate the total oxidant (TOC) and antioxidant capacity (TAC) of patients with transfusion dependent (TDT) and nondependent (NTDT) β -thalassemia.

Methods: Fifty eight patients (age range 3-59; median age 24 years) with β-thalassemia (16 nontransfusion dependent, 42 transfusion dependent) who were followed-up by Istanbul Medical Faculty Thalassemia Center were enrolled in this study. The sex and age matched 25 healthy subjects were used as control group. The total oxidant and antioxidant capacity were measured by Rel Assay Diagnostics, Total Oxidant Status (TOS) kit and Total Antioxidant Status (TAS) kit. The statistics of results were done by SPSS version 21.

Results: 58 patients were included consisting of β thalassemia intermedia (n=16) and β thalassemia major (n=42). The total oxidant capacity was found higher in patients than control group without significant differences. Beside this, the antioxidant capacity of patients was significantly increased (2.75 mmol/l vs 1.94 mmol/l; p=0.001). Within patients group, there was no significant differences in terms of TAC while the TOC level was high in NTDT (2.99 mmol/l vs 1.88 mmol/l; p=0.08). There was no significant relationship between TOC and sex, age (<18 vs >18 years), ferritin and splenectomy status, as the same for TAC.

Conclusions: The oxidative damage in patients with β-thalassemia can trigger the morbidity. The measurement of TOC and TAC status stands a useful, rapid, and simple method to evaluate the complex oxidative mechanism of disease. There are still controversial results about antioxidant status in thalassemia patients. The irregular consume of supportive treatment such as zinc, vitamin C, vitamin E and herbal medicine may affect the TOC and TAC status. The results of this study can guide us to use the antioxidants to decrease the oxidative stress.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Nano-Formulated Hydroxyurea Enhances Drug Delivery in K562 Cells
Authors: Mohtar Omar , Tagne Jean Bosco , Klings Elizabeth S
Published: October 2019

The vasculopathy of sickle cell disease (SCD), characterized by a chronic hemolytic anemia, endothelial dysfunction and recurrent vaso-occlusive events, underlies most of the clinical complications. Hydroxyurea (HU), the only FDA approved medication to treat patients with SCD, acts via the induction of fetal hemoglobin (HbF) production. Recent studies suggest that HU increases endothelial nitric oxide (NO) production and reduces adhesion molecule expression as another potential mechanism for the observed reduction in vaso-occlusive events and improved survival in SCD. Despite its benefits, HU is currently under-utilized and we hypothesize that improved targeted drug delivery may increase its usage. The use of nano-emulsions, a class of stable emulsions formed by a monolayer of phospholipids and/or biodegradable material, is a mechanism by which to enhance targeted cellular delivery of therapeutics. We hypothesized that a nano-formulation of HU would enhance drug delivery to the bone marrow and this may be a direct way of impacting HbF expression in SCD. Nano-formulated HU (NHU) was developed using a phospholipid approach and this compound was found to be biologically stable for > 6 months.

Chronic myelogenous leukemia bone marrow (K562) cells were treated with a NHU or HU suspension in concentrations ranging from 25µM to 250µM over a course of 4, 5 and 6 days. Cells were harvested for RNA isolation and qPCR was performed using primers for HbF and basal cell adhesion molecule (BCAM, a cellular adhesion molecule on red and white blood cells. On day 5, when HbF expression in K562 cells peaks, there was a five-fold increase in HbF expression treated with lower concentrations of NHU compared to untreated cells and a two-fold increase in expression compared with HU treated cells. With increasing concentrations of NHU and HU, HbF expression became similar. This suggests that the use of NHU can provide increased HbF expression at a lower concentration with likely less toxicity. Treatment of K562 cells with NHU and HU resulted in decreased BCAM expression on day 6 of treatment. Treatment with NHU produced a four-fold decrease in BCAM expression compared to the same concentration of a HU suspension which supports the concept that NHU can impact gene expression at lower doses than unformulated HU. Nanoformulated HU produces a molecule which is highly stable, and able to achieve higher intracellular concentrations with increased efficacy in K562 cells. This suggests a potential novel therapeutic for patients with sickle cell disease.

Disclosures

Klings: Pfizer: Consultancy; Actelion Pharmaceuticals: Research Funding.
A Meta-Analysis of Gene Expression Studies Highlights the Role of Innate Immunity in the Pathogenesis of Sickle Cell Disease and Reveals Novel Potential Regulators at the Transcriptomic Level
Authors: Hounkpe Bidossessi Wilfried , Fiusa Maiara Marx Luz , Colella Marina Pereira , da Costa Loredana Nilkenes Gomes , Benatti Rafaela Oliveira , Saad Sara Terezinha Olalla , Costa Fernando Ferreira , Santos Magnun Nueldo , De Paula Erich Vinicius
Published: October 2019

Sickle cell disease (SCD) is characterized by chronic inflammation and multisystem tissue damage. Despite the detailed characterization of the inflammatory changes observed in these patients, the precise identity of their critical triggers, as well as the hierarchical relationship between the elements involved in the pathogenesis of this complex disease are yet to be described. Meta-analysis of gene expression studies from public repositories represents a novel strategy, capable to identify key pathogenic mediators and therapeutic targets in complex diseases such as diabetes. The recent development of comprehensive bioinformatics tools further facilitated the identification of relevant genes, pathways and regulatory elements from high-throughput transcriptomic data. Here we performed a meta-analysis of recent gene expression studies with SCD patients, available at the Gene Expression Omnibus public repository. Two databases (GSE35007 and GSE53441) including samples from adults and children with SCD respectively fulfilled our inclusion criteria. We also performed additional meta-analysis comparing the gene expression pattern of these clinical samples with that of heme-stimulated endothelial cells (GSE25014). Meta-analysis was performed with the Inmex bioinformatics tool. Raw data was downloaded, annotated, preprocessed, and submitted to quality check. Differentially expressed genes were identified and ranked based on the RankProd package. Gene set analysis using more than 60 libraries was performed using EnrichR. Transcription factor, kinase enrichment, and pathway cluster analysis were performed using appropriate bioinformatics tools. Only pathways that were identified in more than one library were included. Our results demonstrate that the well-characterized association between innate immunity, hemostasis, angiogenesis and heme metabolism with SCD was also consistently observed at the transcriptomic level, across independent gene expression studies. The enrichment of genes and pathways associated with innate immunity and damage repair-associated pathways supports the model of erythroid danger-associated molecular patterns (DAMPs) as key mediators of the pathogenesis of SCD. In addition, our study generated a novel and large database of candidate genes, pathways, transcription factors and kinases not previously associated with the pathogenesis of SCD, that could be used in future and independent studies in SCD.

Disclosures

No relevant conflicts of interest to declare.
Impact of a Dedicated Sickle Cell Acute Care Observation Unit on Rate of Hospital Admission for Acute Pain Crisis
Authors: Gowhari Michel , Kavoliunaite Laura , Bonnye Johnson , Brown Stephen , Molokie Robert E. , Hsu Lewis L. , Gordeuk Victor R.
Published: October 2019

Introduction: Pain is the top concern of individuals with sickle cell disease (SCD). Acute painful vaso-occlusive episodes are the leading cause of emergency department (ED) encounters and frequent hospital admissions. There are well-documented disparities for patients with SCD, including significant delays in starting therapy and under treatment of pain in the ED. An acute care observation unit (ACOU) staffed with SCD specialists can help to address these disparities. Here we study the service impact of increasing the hours of operation of a dedicated sickle cell ACOU on utilization and hospital admissions at the University of Illinois at Chicago (UIC), a regional sickle cell resource. We hypothesized that increased hours of operations will lead to decreased ED utilization and inpatient hospitalizations.

Methods: The outcomes of individuals >16 years of age presenting with an acute painful episode to the sickle cell ACOU at UIC were assessed for the 12 months before and the 12 months after increasing the hours of operation from 9 hours/day to 15 hours/day Monday through Friday in February 2014. The outcomes of SCD patients presenting to the ED during the 12-month period following expanding hours in the ACOU were also assessed. The main outcome measures were ACOU and ED utilization and hospital admission rates.

Results: There were 344 encounters in the sickle cell ACOU in the 12 months before expansion of service hours compared to 796 in the 12 months after expanding the hours, an increase of 131%. This represents 0.15 patients treated per hour before increasing the hours versus 0.2 per hour after increasing the hours. Seventy-two percent of the patients treated at the sickle cell ACOU were discharged home in the 12 months prior to expanding hours versus 75% after. During the comparative 12-month period following expansion of hours in the ACOU, there were 1074 encounters for SCD acute painful episodes in our ED, representing 0.12 patients treated per hour of operation. Only 35% of SCD patients treated in the ED for an acute painful episode were discharged home.

Conclusion: The sickle cell ACOU at UIC more than doubled its patient volume following the expansion of operation from 9 to 15 hours/day during weekdays. Based on the hours of operation, during a comparative 12-month period the sickle cell ACOU treated twice as many SCD patients with an acute painful compared to the ED while discharging rather than hospitalizing twice as often. These observations suggest that allocating resources to a dedicated sickle cell ACOU can decrease ED utilization and subsequent inpatient hospitalizations.

Disclosures

No relevant conflicts of interest to declare.
Sickle Cell Disease in Central India: High Prevalence of Sickle/Beta Thalassemia and Severe Dsiease Phenotype
Authors: Jain Dipty , Warthe Vinit , Colah Roshan , Serjeant Graham Roger
Published: October 2019

Objectives: To assess the clinical, haematological and molecular features of sickle cell disease in central India where the disease has been reported to be more severe than the mild clinical course usually observed in the Asian haplotype of homozygous sickle cell (SS) disease.

Methods: A cross-sectional assessment of 91 consecutive patients with sickle cell disease attending clinics at the Akola Government Medical College, Akola, Maharastra State, India.

Results: Of the 91 patients, who were predominantly of the scheduled caste community, 49 had SS disease, 6 had sickle cell-HbD Punjab, and 36 had sickle cell-beta thalassaemia. Of the patients with sickle cell-beta thalassaemia, the beta thalassemia mutation was IVS1-5 G>C mutation in 25 patients (69%) while the rest had one of seven other molecular mutations identified (Table1). Contrary to commonly held beliefs, alpha thalassaemia occurred in only 9/90 (10%) of subjects but fetal haemoglobin (HbF) levels were markedly elevated with mean and median levels of 24.4%. All except 3 SS disease patients had the Xmn1(+/+) polymorphism. These patients exhibited many of the severe manifestations of sickle cell disease. Comparison of SS disease and sickle cell-beta thalassaemia showed no differences in the prevalence of dactylitis, bone pain crisis, acute chest syndrome, haemoglobin level, reticulocyte counts or hydroxyurea usage but patients with sickle cell-beta thalassaemia had significantly more hospital admissions, blood transfusions, and greater frequencies of splenomegaly and hepatomegaly.

Conclusions: Many of the patients with sickle cell disease in central India appear to have relatively severe manifestations. This appears to be due to much lower frequencies of alpha thalassaemia and more frequent sickle cell-beta thalassaemia. There is a need for assessment of the indications and policies for blood transfusion and for hydroxyurea.

Table 1. Beta Thalassemia mutations associated with HbS/Beta Thalassemia in Akola Mutation Expression Number IVS 1-5 (G>C) severe b+ 22 IVS 1-1 (G>A) bo 3 Cd 15 (-T) bo 2 Cd 30 (G>C) bo 2 Cd 15 (G>A) bo 1 Cd 39 (C>T) bo 1 Cd 41/42 (-CTTT) bo 1 619 bp deletion bo 1 Total 36

Disclosures

No relevant conflicts of interest to declare.
Coinheritance of Alpha-3.7 Deletion and Hb Questembert [131alpha-2 Tct > CCT](Ser131>Pro)] in an African American Patient Results in an Alpha Thalassemia Phenotype
Authors: Shakir Asiya , Pace Betty , Patel Niren , Kutlar Ferdane , Kutlar Abdullah , Ameri Afshin
Published: October 2019

Patients with Hemoglobin Questembert carry a Ser131Pro mutation in one of their alpha globin chains encoded by a TCT-CCT mutation of one of their alpha 2 globin gene. This mutation usually results in an unstable hemoglobin variant with mild anemia that is rarely symptomatic. Rarely these patients may show increased extravascular hemolysis during stress, due to oxidative membrane damage. Our patient is a 7 year old African American girl that came to medical attention due to severe anemia, reticulocytosis, absent haptoglobin levels, persistent jaundice and hyperbilirubinemia. Over a period of several years the patient progressed to develop marked splenomegaly with progressive anemia. Isoelectric focusing in this patient revealed Hb A, A2 and a variant "S" band which was less than 1% and thus not detectable by HPLC. We speculate that in the presence of limited alpha globin synthesis, due to a deletion of one α-2 gene and in the presence of a highly unstable alpha-2 variant, synthesis of wild-type alpha globin from the remaining alpa-1 gene is not sufficient to allow for normal alpha-globin/ beta-globin assembly. Furthermore the assembly of the alpha-globin Questembert into the hemoglobin tetramer is favored resulting in an extravascular hemolytic disorder.

Disclosures

No relevant conflicts of interest to declare.