bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphisms of CTLA-4 Are Associated with Treatment Outcome in Multiple Myeloma Patients Receiving Bortezomib-Based Regimens
Authors: Qin Xiao-Ying , Lu Jin , Li Guo-Xuan , Wen Lei , Xu Lan-Ping , Chang Ying-Jun , Liu Kai-Yan , Jiang Zheng-Fan , Huang Xiao-Jun
Published: October 2019

Objectives: SNPs of CTLA-4 have been shown to be important risk factors associated with autoimmune disease and malignancy , the objectives of this study were to explore the association of CTLA-4 SNPs with the development of myeloma and to evaluate the outcome of patients receiving bortezomib-based regimens in relation to CTLA-4 SNPs.

Methods: Peripheral blood samples from 86 MM patients and 154 controls were obtained for the investigation of CTLA4 polymorphisms, The five SNP genotypes of CTLA-4, namely, -1772(rs733618),-1661 (rs4553808), -318 (rs5742909), CT60 (rs3087243), and +49 (rs231775), were determined by TaqMan SNP genotyping assays (Applied Biosystems) ,

Results: Some of the CTLA-4 polymorphisms display frequencies that are different among the different ethnic groups. The Kaplan-Meier analysis revealed that patients with rs733618 GG showed a significantly lower DFS (0% vs. 57.4%, P = 0.020) and OS (46.3% vs. 83.3%, P = 0.026) than those with GA+AA in MM patients after Botizomib based therapy. Multivariate analyses showed that rs733618 GG were risk factor for OS (HR= 0.025; 95% CI= 0.004-0.161;P=0.000). The incidence of nonhematologic grade 3/4 adverse events was significantly increased in the rs 4553808 GA+GG group compared to AA group(P=0.036).

Conclusion: In summary, CTLA-4 rs733618 GG reduced the progression-free survival and overall survival in MM patients after Botizomib based therapy,knowledge of the CTLA-4 polymorphism and haplotype may provide useful information for MM therapy. The exact effect of the CTLA-4 polymorphism and haplotype on MM therapy outcome should be determined in different cohorts with substantially larger number of subjects.

Correspondence: Professor X-J Huang, Peking University People's Hospital, Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Peking-Tsinghua Center for Life Sciences, 11 Xizhimen South Street, Beijing 100044, P.R. China. E-mail: huangxiaojun@bjmu.edu.cn

The first 2 authors contributed equally to this work.

Disclosures

No relevant conflicts of interest to declare.
Functional and Pain Score Improvement with Vertebroplasty in De Novo Multiple Myeloma in a Public Institution in Mexico
Authors: Torres Jorge Carlos , Espinoza Ramiro , Alarcon Silvia , Zapata Nidia , Cervera Eduardo , Sosa Alejandro , Vieyra Magnolia , Espinoza Javier , Sosa Luis , Oñate Luis , Lahuerta Juan José , Aguilar Manuel
Published: October 2019

Multiple myeloma a plasmatic cell disorder, characterized by bone marrow infiltration of clonal plasmatic cells, monoclonal immunoglobulin production and end stage organ damage. This disease has a wide range of clinical manifestations, including solitary disease or plasmacytoma. It´s the second most common hematologic disease just behind Non Hodgkin Lymphoma. More than 60% of de novo MM patients present vertebral involvement.

80% of MM patients refer pain as the first symptom of the disease, and this often leads to opioid abuse and adverse effects related to pain medication. Plasmocytoma is one of the three neoplastic lesions of the vertebra that can be susceptible of percutaneous vertebroplasty.

The preferred method of treatment is a percutaneous vertebroplasty (PV), a radiologic invasive treatment that can treat multiple pathologies, and is now widely used for treatment of osteoporosis vertebral fractures.

For the purpose of the study we aimed to assess functional status and reincorporation to daily living, we used the Karnofsky Performance Status; and the Barthel index. For pain improvement we used the Visual Analog Scale. The Likert satisfaction scale (introduced in 1932) measures patient satisfaction in an analog scale.

The main objective of the study was to assess the repercussion of the PV in pain score modification and improvement of functional scores in patients with MM at the Pain Clinic of the Instituto Nacional de Cancerología (INCan) in Mexico City.

We carried out the study between January 2004 and October 2015. We compared VP treatment to bisphosphonate (BP) and radiotherapy (RT) according to the visual analog scale; and according to Karnofsky and Barthel scales, we determined the reincorporation to daily activities.

We included 119 new cases con MM, of this only 64 were included in the study. Of this 54 were female, and 65 were male with a mean age of 59 years (28-87). 88% of patients referred pain as their main symptom, and 70% presented with a vertebral fracture. 26.9% had an ECOG of 3 or more. 96% (114) received RT and 99% (118) BP.

64 (54%) of patients were treated with VP. Of this, 67% referred a VAS of 4 or more (moderate to severe pain). And 92% of the patients achieved a VAS of 4 or less. As for the Karnofsky we assessed an improvement of 50/80 points pre VP to 80/90 post VP. The same result was assessed in the Barthel scale, with an improvement of 50/80 points pre VP to 80/100 post VP.

This data provides solid evidence of the importance of multidisciplinary approach, and that VP is a safe and effective treatment, coupled of course with BP and RT.

Disclosures

No relevant conflicts of interest to declare.
Basal CD34+ Cell Count Predicts Peripheral Blood Hematopoietic Progenitor Cells Mobilization in Healthy Donors after Administration of G-CSF
Authors: Martino Massimo , Gori Mercedes , Pitino Annalisa , Dattola Antonella , Fedele Roberta , Gallo Giuseppe Alberto , Barillà Santina , Pontari Antonella , Irrera Giuseppe , Messina Giuseppe , Moscato Tiziana , Console Giuseppe , Morabito Fortunato , Tripepi Giovanni
Published: October 2019

BACKGROUND: Peripheral blood (PB) hematopoietic progenitor cells (HPC) mobilized with G-CSF are the first-choice source for allogeneic stem cell transplantation. We carried out a prospective study on healthy donors (HDs), to identify donor characteristics that could influence the effectiveness of mobilization.

STUDY DESIGN AND METHODS: PB-HPC allogeneic donations from sibling HDs were analyzed. We tested somatic variables (sex, age, weight, height, volemia) and blood counts (WBC, platelets, hemoglobin, CD34+ cell count). Two different determinations of CD34+ cells were done in each HD: baseline (before G-CSF administration) and in PB on the morning of the fifth day (after G-CSF administration). HDs received G-CSF subcutaneously at a dose of 10 µg/kg per day.

RESULTS: 128 consecutive HDs (66 males) with a median age of 43 years were enrolled. The mean value of CD34+ on day 5 was 90.8 cells/µL, 84.2 cells/µL in females and 97.2 cells/µL in males. The median values of CD34+ on day 5 were 75.5 cells/µL for the overall sample, 74 cells/µL in females and 80 cells/µL in males. On univariate correlation analysis, donor weight r=0.19, P<0.05, height (r=0.20, P<0.05), volemia (r=0.18, P<0.05) and baseline CD34+ (r=0.38, P<0.001) were significant predictors of CD34+ on day 5. Data analysis by gender showed that CD34+ on day 5 were predicted by age (r=-0.30, P<0.05), baseline WBC (r=0.25, P<0.05), baseline CD34+ (r=0.31, P<0.05) in males and by baseline CD34+ (r=0.45, P<0.001) in females. We performed two multivariate analyses either by using ordinary regression (to predict the mean value of CD34+ on day 5) or median regression (to predict the median value of CD34+ on day 5) according to the values of CD34+ at baseline (see table) in the whole study population and separately in females. These results indicate that in the whole study population and by using ordinary regression, a baseline value of CD34+ ranging from 0.5 to 2 cells/µL predicts a mean CD34+ value of 75.7 cells/µL at 5 days whereas a baseline value of CD34+ ≥3 cells/µL predicts a mean CD34+ value of 107.5 cells/µL at 5 days. Furthermore, in the whole study population but by using the median regression, a baseline value of CD34+ ranging from 0.5 to 2 cells/µL predicts a median CD34 + value of 64.6 cells/µL on day 5 whereas a baseline value of CD34+≥3 predicts a median CD34 value of 101.1 cells/µL on day 5.

CONCLUSIONS: Baseline, HDs PB CD34+ cell counts correlated with the effectiveness of allogeneic HPC mobilization and could be useful to plan the collection.

Disclosures

No relevant conflicts of interest to declare.
SHQ1 Regulates MYC mRNA Splicing and Promotes T Cell Leukemogenesis
Authors: Su Hexiu , Hu Yufeng , Jiang Jue , Chen Zhichao , Liu Hudan
Published: October 2019

T-acute lymphoblastic leukemias (T-ALLs) are aggressive hematologic tumors resulting from the malignant transformation of T cell progenitors. In this context, constitutive activation of NOTCH1 signaling is the most prominent oncogenic pathway in T cell transformation. Yet functional role(s) of NOTCH1 in T-ALL pathogenesis and precise mechanism(s) of action remain to be fully determined. SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins which are required for spliceosomal small nuclear RNA (snRNA) maturation. We here identify SHQ1 as a NOTCH1 downstream target that plays a pivotal role in maintaining MYC splicing fidelity and promoting T-ALL cell proliferation in vitro and in vivo.

We identified SHQ1 as a NOTCH1-regulated gene from multiple human T-ALL genome-wide expression studies. To validate this finding, we analyzed SHQ1 expression in a spectrum of human T-ALL cells upon NOTCH1 pathway inhibition. NOTCH1 inactivation caused a marked downregulation of SHQ1 in all T-ALL cell lines tested. We further demonstrated NOTCH1 bound to the canonical CSL binding sites in the SHQ1 promoter and directly activated the transcription.

We next systematically analyzed the SHQ1 expression in 174 T-ALL primary samples and found that SHQ1 expression was significantly elevated in T-ALL compared with normal peripheral blood. To explore the functional role of SHQ1, we knocked it down in T-ALL using specific shRNAs. SHQ1 depletion profoundly inhibited T-ALL cell proliferation and induced massive apoptotic cell death. Consistent with these in vitro findings, SHQ1 depletion in a human T-ALL xenograft significantly delayed leukemia onset and prolonged survival.

To decipher the molecular mechanism whereby SHQ1 contributes to T cell leukemogenesis, we performed genome-wide RNA-Seq and analyzed alternative splicing across the genome. Approximately 70% of genes exhibited abnormal intron retention upon SHQ1 depletion. Among those whose expression levels and mRNA splicing were prominently altered by SHQ1, MYC gained our attention because of its vital role in T-ALL. Depletion of SHQ1 resulted in marked downregulation of mature MYC mRNA and protein. Endogenous mRNA analysis and mini-gene experiments showed aberrant accumulation of MYC pre-mRNA in SHQ1-depleted cells. Expectedly, SHQ1 knockdown had minimal effects on T-ALL cells constitutively expressing a human MYC protein.

In addition to the well-documented transcriptional control of MYC by NOTCH1, we herein report a previously unsuspected mechanism in which NOTCH1 modulates MYC splicing by activation of SHQ1. Our findings not only shed new insights in the molecular pathology of NOTCH1-induced T-ALL but also provide a new layer of regulation of oncogene MYC at the post-transcriptional level, mechanism of which may apply to other MYC involved tumors.

Disclosures

No relevant conflicts of interest to declare.
The Outcomes of Korean Patients with Primary Plasma Cell Leukemia: Analysis of Korean Multiple Myeloma Working Party (KMM160)
Authors: Jung Sung-Hoon , Lee Je-Jung , Kim Kihyun , Yoon Dok Hyun , Min Chang-Ki , Sohn Sang Kyun , Choi Chul Won , Lee Ho Sup , Kim Hyo Jung , Shin Ho-Jin , Bang Soo-Mee , Yoon Sung-Soo , Park Seong Kyu , Yhim Ho-Young , Kim Min Kyoung , Jo Jae-Cheol , Mun Yeung-Chul , Lee Jae-Hoon , Kim Jin Seok
Published: October 2019

Primary plasma cell leukemia (pPCL) is a rare and aggressive plasma cell neoplasm, with rapid clinical course. In this study, we evaluated the treatment status and survival outcomes of Korean patients with pPCL. Seventy patients were diagnosed with pPCL between February 1998 and December 2015. The median age of the patients was 63.5 years (range, 34-85). Most of patients (70.0%) had International Staging System III.Conventional karyotyping was assessed in 47 patients and FISH analysis was performed in 43 patients. Abnormal karyotype was detected in 34 patients (72.3%). The most common abnormalities were complex karyotype (65.9%). Hypodiploidy, del (13q14), del (17p13), and t(11;14) were found16 (34%), 14 (29%), 12 (25%), and 11 (23%) patients, respectively. Amongthe 70 patients, 60 patients were treated.Thirty-six patients were initially treated with novel agents and 12 received novel-containing regimens as salvage therapy. Twelve patients received conventional chemotherapies only. Twenty-two patients underwent high dose chemotherapy and autologous stem cell transplantation (HDT/ASCT), and one received the allogeneic stem cell transplantation. After a median follow-up of 16.5 months, overall survival (OS) was 16.1 months (95% CI, 11.7-20.8). Twenty patients (33.3%) died within less than one year following the diagnosis, and the early mortality rate was lower in patients who were initially treated with novel agents (22.2% vs. 50.0%, p = 0.049). The median OS of three treatment groups comprising conventional chemotherapy only, novel agents only, and novel agents + HDT/ASCT were 2.9, 12.3, and 31.1 months, respectively (P=0.001). Patients who achieved complete response (CR)after initial therapy had significantly improved OS than did others (36.4 vs. 14.1 months, P < 0.011). On multivariate analysis, achievement of complete response (CR) after induction therapy (HR 0.066, 95% CI 0.007-0.615, P = 0.017), increased lactate dehydrogenase (HR 4.803, 95% CI 1.202-19.186, P = 0.026), andserum beta2-microglobulin > 5.5 mg/dl (HR 3.218, 95% CI 1.011-10.238, P = 0.048)were significantly associated with survival outcomes. In young patients (age < 65 years), performance of HDT/ASCT (HR 0.038, 95% CI 0.004-0.381, P = 0.005) was significantly associated with improved OS. In conclusion, patients with pPCL had poor survival outcomes, and achieving CR after induction therapy associated with increased OS. In addition, performance of HDT/ASCT is important treatment modality to improve OS in young patients with pPCL.

Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures

No relevant conflicts of interest to declare.
Prevention of Chronic Gvhd By Targeting Xbp-1 Genetically or Pharmacologically in Mice
Authors: Schutt Steven D , Tang Chih-Hang Anthony , Wu Yongxia , Bastian David A , Del Valle Juan , Hu Chih-Chi Andrew , Yu Xue-Zhong
Published: October 2019

Inhibition of the endoplasmic reticulum (ER) stress response via blockade of inositol-requiring enzyme-1α (IRE-1α) is currently a promising therapeutic strategy to treat B-cell leukemia, lymphoma, and myeloma. Because B cells play an important role in the development of chronic graft-versus-host disease (cGVHD), we hypothesize that the ER stress response contributes to B-cell function and pathogenicity in cGVHD. Here, we report that the ER stress response mediated by IRE-1α and its target X-box binding protein-1 (XBP-1) plays a critical role in cGVHD pathophysiology and represents a potential therapeutic target to prevent cGVHD.

We tested the role of XBP-1 specifically in B cells by testing XBP-1 conditional knockout B cell grafts (XBP1fl/flCD19Cre+) in two mouse models of cGVHD. In the first model (B6 to BALB/c), recipients given XBP-1-deficient donor grafts showed significantly reduced cGVHD clinical scores, which were associated with reduced frequencies of donor-derived CD4 helper T cells within the lungs compared to the recipients of XBP-1fl/flCD19Cre- littermate donor grafts. XBP-1-deficient B cells produced significantly higher levels of IL-10 compared to WT control B cells after activation ex vivo. In the second model (B6 to B10.BR), the conversion of donor B cells to plasma cells (B220+CD38+CD138+) was reduced in both the spleens and lungs of recipients transplanted with XBP1fl/flCD19Cre+ grafts compared to those of the recipients given XBP1fl/flCD19Cre- grafts. Recipients given XBP1fl/flCD19Cre+ grafts also showed significantly higher total splenocytes and vastly increased splenic B-cell populations when compared with the recipients of XBP1fl/flCD19Cre- grafts.

To expand on these findings, we tested if systemic XBP-1 blockade via a novel IRE-1α inhibitor, B-I09, would attenuate cGVHD. In a cutaneous model of cGVHD (B10.D2 to BALB/c), we found that prophylactic administration of B-I09 significantly reduced clinical features of cGVHD compared to vehicle controls (Fig. 1A). Validating these findings, hematoxylin and eosin stained skin sections of B-I09-treated mice had significantly lower pathology scores compared to vehicle controls (Fig. 1B). Isolated skin lymphocytes from recipients treated with B-I09 showed significant reductions in donor derived T cells and DCs compared to those treated with vehicle controls (Fig. 1C and D).

Taken together, our findings reveal a novel role of the IRE-1α/XBP-1 pathway of the ER stress response in cGVHD pathophysiology and provide a readily translatable strategy to prevent the development of cGVHD in the clinic.

Disclosures

No relevant conflicts of interest to declare.
Pretransplant Plasmatic Citrulline and Monocyte-Derived Macrophage Reactivity : Two Useful Surrogate Markers to Predict Acute GvHD
Authors: Hueso Thomas , Magro Leonardo , Jonquel-chevalier Curt Marie , Yakoub-Agha Ibrahim , Seguy David
Published: October 2019

Acute graft versus host disease (aGvHD) represents the first complication after allogeneic stem cell transplantation (allo-HCT). During conditioning regimen (CR) intestinal damage induces microbial translocation, which primes macrophage reactivity and leads to donor-derived T cells alloreactivity. To date, little is reported on intestinal status and macrophage reactivity prior to CR. This study aimed to assess citrulline, an amino acid reflecting intestinal damage, and circulating monocytes-derived macrophage reactivity 30 days before CR.

Blood samples from consecutive patients undergoing allo-HCT for hematological malignancies and from 10 healthy donors were analyzed. Citrulline levels were assessed by high performance liquid chromatography associated to spectrometry. Monocytes were isolated and stimulated with pathogen-associated molecular patterns as LPS, PAM, flagelline, MDP, curdlan and PBS as control. In each condition, cytokines (TNF-α, IL-1β, IL-6, IL-10) releasing was evaluated by multiplex fluorescent immunoassay. Citrulline and cytokine levels were analyzed relatively to aGvHD onset within 100 days post-transplant.

Forty-seven patients underwent allo-HCT for different indications, including acute leukemia (n=26) and myelodysplastic syndrome (n=11). Twenty patients developed an aGvHD I-IV. Among the groups, differences were depicted for levels of citrulline, IL-1β, IL-6 and IL-10. Citrulline levels were lower in aGvHD group than in no-aGvHD group (p<0.05). Among all cytokines, only IL-6 and IL-10 were greater in aGvHD group than no-aGvHD group, especially after curdlan stimulation (p<0.05). Citrulline levels below the normal value (<20 μmol/L), IL-6 and IL-10 above thresholds (326 pg/ml and 78pg/ml after curdlan stimulation respectively) were associated with the aGvHD development (log rank test, p<0.05). Any patients with citrulline>20 µmol/L, IL-6<326 pg/ml and IL-10<78 pg/ml (n=10) declared aGvHD within the 100-days post-transplant.

Our results highlight the role of intestinal damage and macrophage reactivity in aGVHD development. Pretransplant plasmatic citrulline level and monocyte-derived-macrophage reactivity are promising accessible surrogate markers suitable for identifying patients likely to develop aGvHD.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Expression Pattern of CD55 and CD59 on Red Blood Cells in Sickle Cell Disease
Authors: Al Humood Salah A. , Al-Faris Lama A. , Al-Rukhayes Monera
Published: October 2019

Background: Altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins, might result in increased susceptibility of red blood cells (RBCs) to complement-mediated lysis. Limited information is available on the pattern of CD55 and CD59 expression on RBCs of sickle cell disease (SCD) patients.

Methods: Flow cytometric analysis was performed on RBCs from 71 adult SCD patients and 53 healthy controls, using the commercial REDQUANT kit.

Results: CD59 deficiency was significantly higher among SCD patients than among healthy controls. The proportions of CD55-deficient and CD59-deficient RBCs from SCD patients were significantly higher when compared with those from healthy controls (0.17 vs. 0.09 and 2.1 vs. 1.2, respectively). The MFI of CD55 and CD59 expression on RBCs in SCD was significantly reduced when compared to the expression healthy controls (5.2 vs. 6.4 and 19.4 vs 20.3, respectively). The pattern of CD55 and CD59 expression was not correlated with anemia, biomarkers of hemolysis, erythropoietin level or other pro-inflammatory markers.

Conclusions: There is an altered pattern of CD55 and CD59 expression on RBCs of SCD Patients; however, it does not seem to play a causal role in the pathophysiology of anemia, and is unlikely to be influenced by the level of erythropoietin or other inflammatory mediators.

Disclosures

No relevant conflicts of interest to declare.
Arterio-Venous Fistula Is an Applicable Vascular Access for Erythracytapheresis in Patients with Sickle Cell Disease
Authors: Delville Marianne , Manceau Sandra , Ait Abdallah Nassim , Stolba Jan , Awad Sameh , Damy Thibaud , Gellen Barnabas , Sabbah Laurent , Debbache Karima , Audard Vincent , Beaumont Jean-Louis , Arnaud Cecile , Chantalat-Auger Christelle , Driss Françoise , Lefrere François , Cavazzana Marina , Franco Gilbert , Galacteros Frédéric , Ribeil Jean-Antoine , Gellen-Dautremer Justine
Published: October 2019

Introduction Erythracytapheresis (ER) can improve outcome in patients with sickle cell disease (SCD). A good vascular access is required but can be difficult to obtain for sickle cells patients. Arterio-venous fistula (AVF) has been suggested for ER in SCD supported by limited evidence. We report the largest cohort of ER performed with AVF from three French sickle cell disease reference centers.

Methods Data of SCD patients undergoing ER with AVF in the French SCD reference center were retrospectively collected. The inclusion criteria were: SS or Sβ-Thalassemia and AVF surgery for ER. AVF for hemodialysis were excluded. SCD-related complications, transfusion history, details about AVF surgical procedure, echocardiographic data before and after AVF, AVF-related surgical and hemodynamical complications were collected.

Results Twenty-six patients (mean age 20.5 years; median follow-up 68 months [11; 279]) were included. Twenty-three patients (88.5%) required a central vascular access before AVF. Fifteen AVF (58%) were created on the forearm and 11 (42%) on the arm. Nineteen patients (73%) had stenotic, thrombotic or infectious AVF complications. The median AVF lifespan was 34 months [13; 66]. One patient with severe pulmonary hypertension worsened after AVF creation, and died.

Conclusions We report the first series of SCD patients with AVF for ER demonstrating AVF could be considered as a potential vascular access for ER. Patients with increased risk for hemodynamic intolerance of AVF must be carefully identified, in order to consider alternative vascular accesses.

Disclosures

Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding.
Characterization of B- and T-Cell Immune Repertoires By Anchored Multiplex PCR and Next-Generation Sequencing
Authors: Eberlein Jens , Harrison Thomas , McKittrick Ian , Wemmer Megan , Griffin Laura , Culver Brady P. , Johnson Laura , Kudlow Brian A.
Published: October 2019

The adaptive immune system is involved in various disease conditions including cancer, chronic infection, autoimmune disease and transplant rejection. Adaptive immunity is mediated by B and T lymphocytes, which are activated upon antigen binding to antigen receptors expressed on their surface. Therefore, the spectrum of these antigen receptors, or immune repertoire (IR), provides a means to monitor adaptive immune responses to disease, vaccination and therapeutic interventions. Next-generation sequencing (NGS) of antigen receptor genes is a valuable tool in the study of disease states and responses to various interventions. Traditional amplicon-based NGS assays use opposing primers for targeted amplification of rearranged antigen receptor genes. Thus, large primer panels are required to capture the extensive combinatorial diversity exhibited by the IR. Quantification from such assays requires a complex system of synthetic controls to account for differential amplification efficiency across segment combinations.

Here, we describe an Anchored Multiplex PCR (AMP)-based NGS assay to analyze the IR, employing a minimal set of gene-specific primers in conjunction with molecular barcodes (MBCs) to reduce amplification bias. AMP uses MBCs ligated to cDNA ends and gene-specific primers for amplification, enabling immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Furthermore, this facilitates CDR3 sequence capture from highly fragmented RNA inputs. We validated the quantitative reproducibility and sensitivity of AMP-based B- and T-cell IR assays using high-quality mRNA isolated from peripheral blood leukocytes and highly fragmented RNA isolated from formalin-fixed paraffin-embedded (FFPE) samples.

Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%. Furthermore, our data indicate that clonal diversity in sequencing data is driven by input quantity, total T-cell number, and, to a lesser degree, mRNA quality. Therefore, AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire. This enables sensitive clone tracking and measurement of lymphocyte diversity from fragmented RNA samples.

Disclosures

Eberlein: ArcherDX, Inc.: Employment. Harrison:ArcherDX, Inc.: Employment. McKittrick:ArcherDX, Inc.: Employment. Wemmer:ArcherDX, Inc.: Employment. Griffin:ArcherDX, Inc.: Employment. Culver:ArcherDX, Inc.: Employment. Johnson:ArcherDX, Inc.: Employment. Kudlow:ArcherDX, Inc.: Employment.
Circulating Histone Is Elevated in Acute Leukemia and Protects Leukemic Cell Death through Enhancement of Leukemic Cell Adhesion to Endothelium
Authors: Kim Hyunkyung
Published: October 2019

Background: Leukocytes release nuclear contents into extracellular milieu called by extracellular traps (ET) when they are stimulated by reactive oxygen species (ROS). The nuclear contents mainly compose of histone-DNA complex and neutrophil elastase. This study investigated whether the leukemic cells could release ET and the released histone could induce endothelial activation, finally resulting in leukemic progression.

Methods: The circulating ET biomarkers (histone-DNA complex, cell free DNA, neutrophil elastase) were measured in 80 patients with hematologic diseases and 40 healthy controls by ELISA. ET formation and ROS level were investigated during leukemic cell proliferation in vitro. Histone-induced endothelial adhesion molecule expression and survival were measured by flow cytometry.

Results: Acute leukemia showed high ET biomarkers, which correlated with peripheral blast count. Leukemic cells produced high ROS and release extracellular histone, which was significantly blocked by antioxidants. Histone significantly induced 3 endothelial adhesion molecules expression, promoting leukemic cell adhesion to endothelial cells, which was inhibited by histone inhibitors (heparin, polysialic acid, activated protein C), 3 neutralizing antibodies against adhesion molecules, and Toll like receptor(TLR)9 antagonist. When the leukemic cell co-cultured with endothelial cells, the adherent leukemic cells showed better survival than the non-adherent, demonstrating that histone-treated endothelial cells protected leukemic cells from both spontaneous and chemotherapy-induced death.

Conclusion: Our data for the first time supports that extracellular histone could be released from leukemic cells through ROS dependent mechanism. The released histone could promote leukemic cell adhesion through endothelial adhesion molecule induction and finally protect from leukemic cell death.

Disclosures

No relevant conflicts of interest to declare.
A Prospective Study to Evaluate the Efficacy, Safety and Response Duration of Newly Diagnosed Adult Immune Thrombocytopenia Patients Treated with Low Dose Rituximab and High Dose of Dexamethasone
Authors: Dolai Tuphan Kanti , Mukherjee Soumya , Mandal Prakas Kumar , De Rajib , Chakrabarti Prantar
Published: October 2019

Introduction

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder that involves antibody and cell mediated destruction of platelets as well as suppression of their production. Corticosteroids are initial standard therapy in adults. Initially Rituximab (dose375mg/m2) was approved as one of the important second line therapy in chronic ITP. In the present prospective study, we will evaluate the efficacy, safety and response duration of low dose Rituximab (100mg) and high dose Dexamethasone as a front line therapy in new onset adult ITP cases.

Methods

Inj. Rituximab at a fixed dose of 100mg intravenous infusion for four doses (day 1,8,15,22) and Dexamethasone tablet 40 mg/daily PO for four days in a fifteen days' interval (day 1-4 and day 15-18). Response rate evaluated according to published guidelines.

Results

Total no. of cases was 15. Male 5, female 10. Age range 18 to 62 years with median age of 36 years. Course completed in 15 patients. Median follow up of the patients was 6 months. Complete response (CR) was achieved after 1st dose of rituximab in 3 cases, 2 after 2nd dose and 4 after 4th dose of Rituximab. Partial response (PR) was achieved in 2 cases after completion of therapy and no response seen in 4 cases. Seven cases (46.66%) achieving CR maintaining sustained response after 6 months of completion of therapy. Two cases who achieved CR initially reverted to PR after 6 months with dropping of platelet count but without any evidence of bleeding symptoms. Minor side effects like GI intolerance, nausea, vomiting occurred in 3 patients, High blood sugar level during treatment seen in 2 patients and severe adverse effects like pneumonitis and intestinal obstruction seen in 2 patients who needed hospitalization. We did not observe any infusion related reaction.

Conclusions

CR and PR rate was 60% (9/15) and 14%(2/15) respectively. Six months after completion of therapy 77.8% (7/9) showed sustained response after achieving CR. Low dose Rituximab and high dose Dexamethasone is a useful treatment option in adult acute ITP patients.

Disclosures

No relevant conflicts of interest to declare.
Aptamer Mediated Inhibition of Protein S
Authors: Plautz William E. , Layzer Juliana , Sullenger Bruce A. , Majumder Rinku
Published: October 2019

Protein S (PS) is an anticoagulant that acts as a direct allosteric inhibitor of the activated form of factor IX (FIXa); FIXa is a coagulant, in both the presence and absence of its cofactor, factor VIIIa. Inhibition of FIXa by PS results in a retarded rate of activation of factor X; FX plays an important role as the initial protease common to the two pathways of blood coagulation. Factor IX (FIX) deficiencies present in the form of varying severities of hemophilia B; additionally, elevated levels of PS result in lengthened clotting times.

We intend to increase the efficacy and functional lifetime of natural and infused FIXa in hemophilia B patients by creating a high affinity aptamer that selectively inhibits PS in its interaction with FIXa. Although inhibitory aptamers that target pro-coagulant proteins have been produced in a facile manner, the hemostasis field has yet to direct its sights towards a therapeutically viable aptamer that inhibits a naturally occurring anticoagulant.

Through the use of previously selected, and experimentally successful, aptamer pools targeting either Gla-domain or plasma proteomes, we were able to use SELEX to select novel PS ligands. After two rounds of aptamer selection, using the focused pools as an initial state, we have found by nitrocellulose binding assays a strong enrichment from the focused Gla domain proteome, whereas the plasma proteome showed a weaker response.

Following development of an inhibitory PS aptamer, we will perform fluorescence binding and substrate cleavage studies to obtain kinetic constants, and ex vivo studies, including aPTT and thrombin generation assays, to demonstrate aptamer efficacy in a physiological system. Ultimately, we plan to test the aptamer in vivo, to demonstrate its use as a possible adjunct therapy to current treatments of hemophilia B.

Disclosures

No relevant conflicts of interest to declare.
Tracing the Formation of Haematopoietic Stem Cells in Mouse Embryos By Single-Cell Functional and RNA-Seq Analyses
Authors: Tang Fuchou
Published: October 2019

Haematopoietic stem cells (HSCs) are derived early from embryonic precursor cells, such as haemogenic endothelial cells and pre-HSCs. However, the identity of precursor cells remains elusive due to their rareness, transience, and inability to be isolated efficiently. Here we employed potent surface markers to capture the nascent pre-HSCs at 30% purity, as rigorously validated by single-cell-initiated serial transplantation assay. Then we applied single-cell RNA-Seq technique to analyse five populations closely related to HSC formation: endothelial cells, CD45- and CD45+ pre-HSCs in E11 aorta-gonad-mesonephros (AGM) region, and mature HSCs in E12 and E14 foetal liver. In comparison, the pre-HSCs showed unique features in transcriptional machinery, arterial signature, apoptosis, metabolism state, signalling pathway, transcription factor network, and lncRNA expression pattern. Among signalling pathways enriched in pre-HSCs, the mTOR activation was uncovered indispensable for the emergence of HSCs but not haematopoietic progenitors from endothelial cells in vivo. Transcriptome data-based functional analysis revealed de novo the remarkable heterogeneity in cell cycle status of pre-HSCs, with considerable proportion being actively proliferative. By comparing with proximal populations without HSC potential, the core molecular signature of pre-HSCs was identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating the step-wise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical application.

Disclosures

No relevant conflicts of interest to declare.
Influence of Biological Character of Acute Lymphoblastic Leukemia Cell Linet after Overexpression of C/EBPa and PU.1
Authors: Xu Linwei , Hao Xiaoxiao , Wei Yongqiang , Wei Xiaolei , Wei Qi , Feng Ru
Published: October 2019

Objective: To observe the influence of biological character of acute lymphoblastic leukemia cell line Jurkat in vitro and in vivo after overexpression of C/EBPa and PU.1.

Methods: To establish transducing plasmids, using 293T cells to package lentiviral vector and to transfect Jurkat cell line. Flow cytometry is used to analyze the expression of membrane cluster differentiation antigen, apoptosis rate, proliferation and cell cycle of cells. Cell morphology and Q-PCR was used to analyze the changes of cell morphology and molecule gene. Six to eight weeks old immunodeficiency (NSI) mice were transplanted by tail vein injected with 2x106 cells after transduction. To observe the biological behavior and survival time, using Flow cytometric, cell morphology, pathological HE staining, immunohistochemistry to analyze the invasion of leukemia cells.

Results: The expression of CD3 (mark antigen of lymphocyte) fall as low as 20% after overexpression of C/EBPa and PU.1, CD11b (mark antigen of myeloid cell) increase as high as 30%, which has significant difference with Jurkat-gfp group. The apoptosis rate has significantly increase, proliferation rate has significantly decrease after overexpression of C/EBPa, which blocked in S period. Adding myeloid cytokine (GM-CSF, M-CSF, IL-3, FLT-3L,100ng/mL) on the supernatant of transduction cells, Jurkat cells can be reprogrammed to attached cells from suspension after overexpression of C/EBPa and PU.1 in the 5th day. Attached cells can chemotaxis and consuming yeast. Cell multicore and small amount of purple particles in cytoplasm has observed in Jurkat after overexpression of C/EBPa and PU.1, high expression of myeloid gene (CD14, CD64) and low expression of lymphocyte gene (BCL-11B, GATA-3) has observed in cells after transduction. All the mice have leukemia between 1 to 2 months after injection with transfected cells. The survival time is longest in overexpression of C/EBPa group, which shows significant difference (P<0.001). Cells of peripheral blood, liver, spleen and bone marrow can partly transduce to myeloid cells after overexpression of C/EBPa and PU.1, additionally, the expression of CD11b is observed in liver and spleen cells by immunohistochemistry.

Conclusion: Jurkat cells can be partly reprogrammed to monocyte- macrophages, while with the help of myeloid cytokines, it can be reprogrammed to macrophages with chemotactic and phagocytic function. Jurkat cells can be partly reprogrammed to monocyte- macrophages in vivo and prolong the survival time after overexpression of C/EBPa and PU.1.

Disclosures

No relevant conflicts of interest to declare.
Excelent Option Therapy of BONE Marrow Failure in Fanconi Anemia Patients Withouth Full Match Donnor
Authors: Ribeiro Lisandro L , Nichele Samantha , Bitencourt marco Antonio , Petterle Ricardo , Loth Gisele , Pilonetto Daniela , Bonfim Carmem
Published: October 2019

The main cause of morbidity and mortality of FA pts is bone marrow failure (BMF), which usually arises in the first decade of life and progresses to transfusion dependence and severe neutropenia. Androgen treatment has been recommended for FA pts with BMF for whom there is no acceptable hematopoietic stem cell transplant donor. Oxymetholone and Danazol are frequently used in these pts. We retrospectively analyzed data on 67 FA pts who received oxymetholone or danazol for the treatment of their BMF. The starting dose was approximately 1mg/kg for oxy and 2-4mg/kg for danazol. The hematological parameters at the initiation of treatment were hemoglobin (Hb) < 8 g/dL and/or thrombocytes < 30.000/μl. Patients were diagnosed between 01.2005 and 01.2016. The median age was 10.5 ys (2.9 - 40ys). Gender: 39M/27F. The median duration of treatment was 18m (3m - 95m). Fifty-three patients (79%) showed hematological response and became transfusion independence at a median of 3 months after beginning oxymetholone (2-9m) and 5 months after danazol (4-7m). Two adult pts treated with danazol achieved total hematological response with 2.5mg/kg. Seven pts are stable after tapering and stopping androgen with a median follow up of 4 ys (6m-8.5ys). Fourteen pts did not respond to treatment (21%). Eleven pts received an HSCT and seven are alive and well. Three pts were not transplanted and two are alive but transfusion dependent and one pt died from CNS bleeding. All patients developed variable degree of virilization but it was more evident with oxymetholone therapy. Older age at starting therapy was related to less virilization. Conclusion: This study shows the largest number of FA pts treated with androgen up till now. Androgen is an effective and well-tolerated treatment option for FA pts who develop BMF with 79% of them showing transfusion free after 3-5 months. This response may give us time to search for better donors.

Disclosures

No relevant conflicts of interest to declare.
Set7 Is Highly Expressed in B-Lineage Acute Lymphoblastic Leukemia Cells and Overexpression of Set7 Exhibits Antileukemia Effect
Authors: Ma Xiao-Tong , Mou Ya-Kun , Zhao Yang-Yang , Wang Nan , Liu Xiao-Yan , Yin Jing , Ma Yan-Ping , Guo Dan
Published: October 2019

Set7 is a member of protein lysine methyltransferase family that is highly conserved in vertebrates. Set7 can regulate the maintenance of chromosome structure, cell cycle and apoptosis, and plays an important role in many kinds of cancer, metabolism and inflammatory process. However, studies concerning its pathogenic role in leukemia are scarce. We analyzed the expression of Set7 in different types of leukemia and healthy human bone marrow cells or peripheral blood from the Oncomine databases (http://www.oncomine.org), and showed that Set7 was highly expressed in acute lymphoid leukemia (ALL) patients, especially in patients with B-ALL. In this study, we examined the expression of Set7 in various hematopoietic tumor cell lines, bone marrow samples from patients and healthy volunteers. We found that Set7 was highly expressed in B-ALL cell lines Nalm6 and REH compared with other hematopoietic malignant cell lines. Furthermore, Set7 was overexpressed in bone marrow mononuclear cells of adult and pediatric patients with B-ALL than in healthy human bone marrow samples, and sorted CD19+ cells, as well (P<0.001). To investigate Set7 gene function, retroviral-mediated overexpression or removal experiments were performed. Gain-of-function and loss-of-function analyses in Nalm6 and REH cells demonstrated that Set7 inhibited cell growth, colony formation and enhanced the chemosensitivity of B-ALL cells to Vincristine (VCR), Arabinocytidine (Ara) and Daunorubicin (DNR). Protein sequence analysis showed that Ebf1 might be a potential substrate for Set7. The expression of Rad51 and Tnsf11, the downstream target genes of Ebf1, were changed as expected when Set7 was overexpressed in Nalm6 and REH. Our study shows for the first time that overexpression of Set7 has an inhibitory effect on B-ALL. These findings suggest that Set7 may be a promising molecular therapy target for B-ALL treatment and provide new clues for understanding the molecular mechanisms of the leukemogenesis of B-ALL.

Disclosures

No relevant conflicts of interest to declare.
Mir-130a Is Aberrantly Overexpressed in Adult Acute Myeloid Leukemia with t(8;21) and Its Suppression Induces AML Cell Death
Authors: Dai Haiping , Ding Chao , Chen Suning , MacLeod Roderick A. F. , Ehrentraut Stefan , Drexler Hans G. , Nagel Stefan , Wu Depei , Sun Aining
Published: October 2019

Emerging evidence has revealed that miRNAs can function as oncogenes or tumor suppressor genes in leukemia. By profiling miRNA expression across a cohort (n=156) with de novo acute myeloid leukemia (AML), we identified miR-130a as significantly overexpressed in AML with t(8;21). Expression of miR-130a decreased significantly once patients with t(8;21) achieved complete remission, but increased sharply at the time of relapse. In patients with t(8;21) AML, KIT mutational status was associated with miR-130a expression - with higher expression associated with KIT activating mutations. Increased miR-130a expression in t(8;21) AML was associated with worse event-free survival, however no impact on overall survival was observed. Knockdown of RUNX1/RUNX1T1 (AML1/ETO) protein in the SKNO-1 cell line resulted in decrease of expression of miR-130a. Direct binding of RUNX1/RUNX1T1 fusion protein with the promoter sequence of miR-130a was detected with luciferase reporter gene assay. Following miR-130a knockdown, SKNO-1 demonstrated increased sensitivity to etoposide. Taken together, our data suggest that miR-130a is directly activated by RUNX1/RUNX1T1, and can be used to predict leukemia burden, event-free survival and chemotherapy sensitivity in AML with t(8;21).

Disclosures

No relevant conflicts of interest to declare.
Internal Tandem Duplication (ITD) in the Tyrosine Kinase Domain of FLT3 Displays Higher Oncogenic Potential in Acute Myeloid Leukemia
Authors: Marhäll Alissa , Fischer Thomas , Heidel Florian H. , Kazi Julhash U. , Rönnstrand Lars
Published: October 2019

Up to 30% of patients with acute myeloid leukemia (AML) harbor a mutation in FMS like tyrosine kinase 3 (FLT3). This mutation is not only the most frequent, but also the most clinically challenging, because it is associated with increased risk of relapse and poor overall survival. The most common oncogenic FLT3 mutation is the internal tandem duplication (ITD) in the juxtamembrane domain (ITD-JM). Other less frequently occurring mutations include ITD mutations in the tyrosine kinase domain (ITD-TDK) and point mutations in the TKD (e. g. D835Y-TKD). Use of FLT3 inhibitors has shown initial promise, though an acquired resistance remains a problem. A need therefore remains for additional therapeutic targets, and in order to identify these we need to have a better understanding of the mechanisms by which the FLT3 mutations drive leukemogenesis. Although ITD-JM and D835Y-TKD mutations have been studied extensively, the role of the recently identified ITD-TKD remains poorly understood. In this study we compared the ITD-TKD domain mutations with other well-studied FLT3 mutations. We observed that transfection of cytokine-dependent Ba/F3 cells with ITD-TKD is sufficient to induce formation of colonies in semisolid medium in the absence of cytokines. Number and size of colonies were comparable to that of ITD-JM, while D835Y-TKD transfected cells failed to form colonies suggesting that the ITD-TKD mutations have stronger transforming potential than other TKD mutations. Similar to colony formation assays, proliferation and cell survival was significantly higher in ITD-TKD transfected cells compared to cells transfected with D835Y-TKD. ITD-TKD selectively enhanced STAT5 and AKT phosphorylation while ERK1/2 and p38 phosphorylation remained unchanged. Collectively our data suggest that ITD-TKD is a more potent oncogenic mutant compared to the TKD mutants.

Disclosures

Fischer: Novartis: Consultancy, Honoraria.
Sequential Induction of Cell Cycle Arrest and Mitotic Catastrophe Followed By Apoptosis Via Activation of PTEN By GL-V9 in Human T-Cell Malignancies
Authors: Hui Hui , Xu Jingyan
Published: October 2019

GL-V9, a newly synthetic flavonoid derivative, is an active cytotoxic component and exhibits anticancer activities. Here, we demonstrated the sequence of death reponses to different concentration of GL-V9 in human T-cell malignancies cell Jurkat and HuT78. Responses included transient accumulation of cells at the G2/M border followed by also transient rise in several mitotic markers and mitotic attempts were largely abnormal, resulting in numerous micronucleated and multinucleated cells in low drug concentration (2 μM), as shown by flow cytometre analyses and Giemsa staining. These events, indicative of mitotic catastrophe, were not associated with immediate cell death. However, MTT assay showed that high concentration of GL-V9 (4, 8, 16 μM) causes near complete cell growth inhibition at 12 h. Also, the classical manifestations of apoptosis (including phosphatidylserine externalization, mitochondrial dysfunction, and caspase activation) were marginal at 24 h, as shown by Annexin V-PI staining, JC-1, and western blot assay. The mechanism research reveals that low dose of GL-V9 induced mitotic catastrophe via decreasing EEA1, which is required for cytokinesis, and increasing the expression of PTEN. Besides, high concentration of GL-V9 induced cell apoptosis by up-regulating the expression of apoptosis-related proteins caspase 3 and Bim in lymphoid tumor cell lines and primary ALL cells. Collectively, these findings show that different concentrations of GL-V9 can induce mitotic catastrophe and ultimate cell death event of human T-cell malignancies respectively, which would be a potential therapeutical compound for treating human lymphoid tumor.

Disclosures

No relevant conflicts of interest to declare.