bluuebunny/crossref_metadata_2025 huggingface dataset:
Preliminary Results from a Phase I Study of SHC014748M in Patients with Relapsed or Refractory Indolent B-Cell Lymphomas
Authors: Xu Wei , Jin Jie , Zhou Keshu , Wang Li , Zhu Huayuan , Qian Jiejing , Huang Jianying , Zhou Fuling , Xu Xiaohong , Wang Zhiqiang , Guo Wei , Li Jianyong
Published: November 2019

Introduction: The phosphatidylinositol-3-kinase (PI3K) pathway plays an important role in multiple cellular functions, including proliferation, differentiation, and trafficking, and deregulation of PI3K is a hallmark of many malignancies. The preferential expression of the PI3Kδ isoform on leukocytes provides an attractive target for hematologic malignancies. SHC014748M is a novel, potent, and highly selective PI3Kδ inhibitor. This study is a phase I, first-in-human study to assess the safety, tolerability and pharmacokinetics of SHC014748M in patients with relapsed or refractory indolent B-cell lymphomas (NCT03588598).

Methods: Dose escalation was performed in a 3+3 design in sequential cohorts at doses of 50-250 mg QD, and then dose expansion was conducted at the selected cohorts to find recommended Phase II dose (RP2D). Eligible patients were those with relapsed or refractory indolent B-cell lymphomas, having at least 1 measurable lesion, and with ECOG performance status 0-2. Patients received a single dose on day 1, and then after 3 days washout multiple dosing once daily was initiated until disease progression or unacceptable toxicity. Primary endpoints included safety, tolerability and pharmacokinetics. Secondary endpoints included overall response rate (ORR), lymph node response (LNR), time to response (TTR), progression-free survival (PFS) and duration of response (DOR).

Results: As of Jul 16, 2019, 38 patients with relapsed or refractory indolent B-cell lymphomas were enrolled in the study, including 23 with follicular lymphoma (FL), 11 with chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL), 1 with marginal zone lymphoma (MZL) and 3 with Waldenstrom's macroglobulinemia (WM). 15 patients were treated in the dose escalation phase and 23 were treated in the dose expansion phase (12 in 150 mg cohort, 11 in 200 mg cohort). The average number of prior therapies was 2.2 (range 1-8), and 84 percent of patients had received rituximab before. No dose-limiting toxicities occurred. The most common adverse reactions were neutropenia (39.5%), fatigue (21.1%), thrombocytopenia (18.4%), diarrhea (13.2%), pneumonia (13.2%), cough (13.2%), lymphopenia (13.2%), anemia (13.2%) and rash (13.2%). Grade ≥ 3 adverse reactions were neutropenia (23.7%), pneumonia (13.2%), rash (7.9%) and diarrhea (5.3%). Among 14 FL patients who were available for efficacy evaluation, ORR was 57.1% (8 PR, 5 SD, 1 PD). Of 9 CLL/SLL evaluable patients, 5 (55.6%) had partial response, 3 (33.3%) had partial response with lymphocytosis, and 1 had stable disease. Of 3 WM patients, 1 had partial response and 2 had minor response.

Conclusion: SHC014748M was well tolerated and demonstrated promising clinical activity in patients with relapsed or refractory indolent B-cell lymphomas.

Disclosures

Wang: Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment.
Venetoclax and DLI for Patients with Early Relapse of Acute Myeloid Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation. a Historical Prospective Multicenter Trial
Authors: Amit Odelia , Bar-On Yael , Perets Galit , Atlas Lena , Shargian Liat , Yeshurun Moshe , Yachini Bruria , Karni Chen , Ram Ron
Published: November 2019

Introduction: Prognosis in patients with post allogeneic HCT-early relapse of AML is dismal and response to salvage treatment is less than 20%. Venetoclax has been shown to be safe and efficacious in frail/elderly patients with AML. Treatment results in an increase of pro-apoptotic function, thus the addition of alloreactive T cells may augment the anti-leukemic potency. We hypothesized that the combination of donor lymphocyte infusion may further enhance this response. Methods: Eligibility criteria for this protocol were - age>18 y, early (<6 months post allogeneic HCT) relapse and no concurrent GVHD. Venetoclax was administered at a daily dose of 400 mg for up to 6 cycles with DLI in escalating doses for up to 4 doses. Additional chemotherapy was allowed according to physician discretion. Primary endpoint of study included assessment Overall response (CR/CRi/CRp) rate at 3 months post treatment. Secondary endpoints included 1-year OS, rate of acute and chronic GVHD, documented infections, hematological complications, and other safety measures. Results: From January 2017 until May 2019, 19 patients were given the study protocol. Median age was 60 (43-75) years, Table. There were no cases of tumor lysis syndrome. Microbiology documented infections occurred in 5 patients (26%) and 2 patients were admitted during protocol (10%). No cases of significant bleeding were observed, and majority of patients were able to tolerate the protocol without any admissions. Acute GVHD was observed in 2 (10%) patients and chronic GVHD was observed in 4 (21%) patients. Overall response was observed in 8 (47%) patients (CR, n=3; CRi, n=2; CRp, n=3). Median time to response was 28 (18-67) days (median number of courses 2 (1-6)) and duration of response was 106 (31-395) days. Survival at 6 months was 3.5 months (95% CI 1.5-5.5). Median survival of patients achieving complete remission was 16.2 months (CI not det.), Figure. On univariate analysis there was no association between patients and disease's characteristics and overall survival, however any type of GVHD (entered to the model as a time dependent variable) was associated with a lower rate of mortality (HR=0.2, p= .03). Conclusions: These results may endorse the hypothesis that enhancing alloreactivity as a therapy may lead to a chemotherapy free approach to relapsed AML. A phase 2 study is under preparation with a larger sample size and a standardized approach to additional salvage chemotherapy added versus Venetoclax + DLI regimen only for a more robust analysis of this therapeutic approach.

Disclosures

No relevant conflicts of interest to declare.
Stratification of Responders and Non-Responders in the Reach-1 Trial Based on Serum Proteomic Analysis
Authors: Owens Sherry , Pratta Michael A. , Liu Hao , Arbushites Michael C. , Tian Chuan , Howell Michael D.
Published: November 2019

Acute graft versus host disease (aGVHD) is a serious and potentially life-threatening condition that can develop in patients receiving hematopoietic stem cell transplantation (HSCT). Acute GVHD is characterized by increased levels of inflammatory mediators and signaling through the Janus Kinases (JAKs), leading to tissue and organ damage. Ruxolitinib, an oral JAK1/JAK2 inhibitor, is the first FDA-approved treatment for steroid refractory aGVHD. The current study evaluated proteomic signatures from participants in the REACH-1 clinical trial to identify those individuals which have an increased probability for success when treated with ruxolitinib in combination with corticosteroids.

Plasma was collected for 42 participants enrolled in the REACH-1 clinical trial (NCT02953678) prior to and at designated times following treatment. Broad proteomic analysis of more than 1000 proteins was conducted using the OLINK proximity extension assay. Participants were separated into responders (complete responders, very good partial responders, partial responders) (n=36) or non-responders (mixed responders, progressive disease) (n=6) based on their clinical response at Day 28 of treatment. Baseline differences in day 28 responders versus non-responders were compared using t-tests. Pharmacodynamics changes within responders and non-responders were assessed using paired t-tests. Significance of statistical tests was conferred at p<0.05.

Broad proteomic analysis of plasma identified a total of 145 differentially expressed proteins (p<0.05) between the responders and non-responders at baseline. Thirty-five proteins were elevated and 110 proteins were lower in responders compared to non-responders. At baseline, IL-8, IL-6, and IL-24 were among the most significantly down-regulated while SCF was one of the most significantly up-regulated in responders compared to non-responders. Pharmacodynamic analyses revealed 493 significantly modulated proteins in responders and 92 in non-responders. IL2-RA was among the most significantly down-regulated from baseline to day 28 in both responders and non-responders.

This study suggests that proteomic characterization has the potential to stratify responders and non-responders to ruxolitinib and corticosteroid treatment in steroid refractory aGVHD. Additionally, this study demonstrated robust pharmacodynamic and correlative changes in responders following treatment with ruxolitinib and corticosteroids suggesting that defining a responder population could enrich for patients predisposed to positive therapeutic response; however, this should be prospectively validated in additional studies.

Disclosures

Owens: Incyte Corporation: Employment, Equity Ownership. Pratta:Incyte Research Institute: Employment. Liu:Incyte: Employment, Equity Ownership. Arbushites:Incyte: Employment. Tian:Incyte: Employment, Equity Ownership. Howell:Incyte: Employment.
Study of Seroprevalence of Hepatites "B" and "C" Markers in Blood Donors from Hemonorte, Rio Grande Do Norte/ Brazil
Authors: Medeiros Ralfo Cavalcanti , Neto Manuel Josué Batista , Paiva Aldair Sousa , Sales Valéria SF , Freitas Rodrigo Villar , Paiva Hugo Diogenes De Oliveira , Araujo Rafael Santiago , Cavalcanti Geraldo Barroso
Published: November 2019

Viral hepatitis transmissible by blood more frequent in the Brazilian population are those of type B and C. Blood banks and hemotherapy centers (Hemocenters) must carry out serological screening, in order to statistically evaluate the percentage of infected donors the virus of these hepatitis. This study aimed to characterize the blood donor population of the State of Rio Grande do Norte (RN), located in the Northeast region of Brazil, in relation to serological markers of hepatitis B and C and to compare these donors from different regions of RN. Were performed serological tests from 254.332 blood donations at the Laboratory of Serology of Hemocenters of Rio Grande do Norte (HEMONORTE) by indirect ELISA methodology. The state of RN was divided into three regions according to the presence of transfusion units or Hemocenters in: Natal city, capital of the state of RN and nearby cities and also headquarters of HEMONORTE; Western Region comprising the Hemocenter of Mossoró city and and transfusion unit of Pau dos Ferros city; and the Serido region of the hemocenter of Caicó city and the collection and transfusion unit of the Currais Novos city. The markers used for hepatitis B detection were total anti HBc (IgM and IgG) and HBsAg. The research of the virus Hepatite C virus carried out with those with third generation kits containing core proteins NS3 and NS4, in addition to NS5. The samples were considered reactive (seropositive) when optical density was obtained up to 20% below the cut-off value, which is the criterion used by most blood centers in Brazil. Among the various serological markers of hepatitis B and C, anti-HBc was the most prevalent with a percentage of 0.98%. Anti-HCV presented a prevalence of 0.77%, while HBsAg had a low prevalence (0.07%). When comparing the prevalence by region, we found that the Natal city region had the lowest rates of hepatitis B (0.93%) and hepatitis C (0.71%). The western region presented the highest percentages of reactivity for hepatitis B (1.24%) and C (1.16%). The seridó region of obtained a prevalence of hepatitis markers smaller than the western region and larger than that of the , 0.99% for hepatitis B and 0.91% for hepatitis C. This difference of percentages between regions should be investigated , to find out the reason for this prevalence variation. The strategy of clinical recruitment and screening should be reassessed and unified among hemotherapy services in Rio Grande do Norte.

Disclosures

No relevant conflicts of interest to declare.
Chronic Lymphocytic Leukemia in Kuwait
Authors: Alshemmari Salem , Pandita Ramesh , Hamadah Abdulaziz , Alhuraiji Ahmad
Published: November 2019

Background :Chronic lymphocytic leukemia (CLL) is common malignancy in Western countries. However, little known about this disease entity in our area. This study exploring the biology in out patients' population. Method:Patients with confirmed CLL under IGHV and TP53 mutational analysis at presentation or during follow up. We also integrated other clinical and biological parameter in this study. Results: A total of 137 cases were analyzed, median age 61 years (range:34-89); 30% of the cases age was<55 years at presentation. There was 108 males vs. 29 females M:F ratio 3.7. Two patients gave a family history of CLL, while 1 patient gave a history of other lymphoproliferative disorders. Binet staging system available in 134 cases, A: 109 (81.3%), B: 12 (9%), C:13 (9.7%). B2 macroglobulin elevated in 40/112 (36%) cases and 10/103 (10%) had M-spike. CD38 positivity reported in 37/112 (33%) of cases. Cytogenetics data evaluable in 85 cases: isolated del(13q): 35%, isolated trisomy 12 (16.5%), del(11q) (4.5%), del(17p)(2.4%). IGHV mutational status mutated vs unmutated: 40% vs 60%. Cases with available treatment information on 132 cases. Fifty cases required treatment due to disease progression. First line treatment Bendamustine-Rituximab (BR) 3 cases, Fludarabine Cyclophosphamide Rituximab (FCR) 30 cases and Chlorambucil with anti-CD 20 antibody 6 cases. At the time of review, 3 cases on ibrutinib (2 in 3rdline and 1 case in the 4thline). Conclusion: This is the first study to shed light on CLL in our area. There are biological differences between our patients' population and the western countries.

Disclosures

Pandita: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
The Effect of Continuous Screening of Carbapenem-Resistant Enterobacteriaceae on Theprevention and Control of Bloodstream Infections in Patients with Hematopoietic Stemcell Transplantation
Authors: Yang Tingting , Luo Xueping , Yang Qing , Chen Hongchao , Luo Yi , Zhao Yanmin , Lai Xiaoyu , Yu Jian , Tan Yamin , Wei Guoqing , Huang He , Shi Jimin
Published: November 2019

Carbapenem-resistant Enterobacteriaceae (CRE) bloodstream infections (BSIs) have become emerging cause of death in patients with hematopoietic stem cell

transplantation (HSCT). Patients underwent HSCT from September 2017 to June 2018, and from July 2018 to February 2019, were assigned as single screening group and continuous screening group; patients transplanted from January 2016 to August 2017 were assigned as control group. Continuous screening significantly improved the CRE gut detection rate compared with single screening (10% vs 1.5%, p=0.001). The CRE infection rate in the pre-intervention period, single screening period and continuous screening period were 1.6%, 2% and 0, respectively; while related mortality was were 66.7%, 50% and 0, respectively. The time from the onset of symptoms of infection to use of tigecycline in survival patients with BSIs were shorter than died patients (24 hours vs 72 hours). For 11 CRE carriers with neutropenic fever, all received tigecycline therapy-based therapy (time from detection to therapy, -3 to 17 days) and did not develop BSIs. These results suggest that continuous screening can more effectively identify high-risk groups of CRE colonization and guide targeted preemptive treatment in the presence of infection symptoms, so as to reduce the incidence of BSIs and improve the prognosis of patients.

Disclosures

No relevant conflicts of interest to declare.
ZNF802 (JAZF1), a Possible New Therapeutic Target for Treatment of β-Thalassemia
Authors: Wongborisuth Chokdee , Tangprasittipap Amornrat , Anurathapan Usanarat , Sathirapongsasuti Nuankanya , Sripichai Orapan , Songdej Duantida , Hongeng Suradej
Published: November 2019

β-Thalassemias are groups of inherited blood disorders caused by reduced or absent production of β-globin chains of adult hemoglobin (HbA; α2β2). The severely affected patients with β-thalassemia require lifelong blood transfusion and chelation therapy. Reactivation of fetal hemoglobin (HbF; α2γ2) could ameliorate the symptoms of patients via increased total hemoglobin production and reduced imbalanced α- to non-α-globin synthesis. Hemoglobin switching during human development requires activities of several transcription factor proteins, including those bind to the globin gene promoters. However, the regulation of fetal to adult hemoglobin switching is not fully understood. Previously, we have carried out the microarray analysis comparing human erythroblasts derived from therapeutic aborted fetal liver and adult peripheral blood. The results showed that ZNF802 mRNA is among the transcripts that up-regulated in adult erythroblast of greater than 2-fold change. ZNF802, also called JAZF1, is a 27 kDa protein containing two zinc finger motifs that acting as transcriptional repressor by binding to the direct repeat (DR) elements on DNA. In this study, quantitative RT-PCR was performed in order to validate the microarray data. We observed 13-fold up-regulation of ZNF802 in adult erythroid cells compared to those cells derived from fetal liver. To further elucidate the role of ZNF802 in reduction of HbF expression in adult erythroids, the lentivirus carrying ZNF802 specific shRNA and non-targeted shRNA were designed and constructed for transduction in erythroblasts. Hematopoietic CD34+ progenitor cells were isolated from β-thalassemia/HbE patients (n=9) and subjected to ex vivo two-phase liquid erythroid culture. The results showed the reduction of greater than 90% of ZNF802 transcript and nearly undetectable level of ZNF802 protein in cells transduced with the ZNF802 shRNA lentivirus. As expected, the level of γ-globin transcript and levels of HbF were increased in ZNF802 knockdown erythroid cells in β-thalassemia/HbE (5-fold changed and HbF 26.9±7.4% vs 18.5±6.8% in non-targeted shRNA (p-value = 0.04), respectively). Interestingly, we found up-regulation of the embryonic ε-globin and ζ-globin transcripts in knockdown ZNF802 cells compared to non-targeted shRNA control (15-fold and 10-fold changed (p-value = 0.01), respectively). To note, the diminishing of ZNF802 effect on the proliferation and differentiation pattern of erythriod cells was not found. Altogether, our findings suggested that ZNF802 may play a role as the transcriptional repressor of γ-globin gene in human adult erythroid cells, thus served as a possible new therapeutic target for treatment of β-thalassemia. The molecular mechanisms underlying the ZNF802 mediated HbF reduction are underway.

Disclosures

No relevant conflicts of interest to declare.
Haploidentical Hematopoietic Stem Cell Transplantation May Improve Prognosis in Non-Infant Children with t(v;11q23)/MLL-Rearranged B-Acute Lymphoblastic Leukemia
Authors: Bai Lu , Cheng Yi-Fei , Lu Ai-Dong , Suo Pan , Wang Yu , Zuo Ying-Xi , Yan Chen-Hua , Wu Jun , Jia Yue-Ping , Sun Yu-Qian , Chen Yu-Hong , Chen Huan , Liu Kai-yan , Han Wei , Xu Lan-Ping , Wang Jing-Bo , Tang Xiang-Feng , Chen Hui-Ren , Zhang Le-Ping , Huang Xiao-Jun
Published: November 2019

Background: B-acute lymphoblastic leukemia (B-ALL) with t(v;11q23)/MLL-rearrangement (MLL-r) in children (1 year or older) is rare, and its outcome and optimal treatment options remain controversial. This study aimed to analyze the clinical characteristics and outcomes of these patients, and to explore the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially haploidentical HSCT (haplo-HSCT) in the treatment of these patients.

Methods: At the time of the last follow-up (July 1, 2019), we retrospectively analyzed clinical data of 42 non-infant children with t(v;11q23)/MLL-r B-ALL. Comparison of outcomes was made between patients received allo-HSCT in the first complete remission (CR1) and chemotherapy only.

Results: The median follow-up was 41 (1-106) months. The median age at diagnosis was 4.5(1-14) years and the median leukocyte count was 56.0 (2.2-735.2)×109/L. One was excluded for death during induction. For the remaining 41 patients, the complete remission rate after induction therapy was 40/41 (97.6%), the estimated 4-year probabilities of overall survival (OS), event-free survival (EFS) and cumulative incidence of relapse (CIR) were 59.7%, 51.6% and 43.4%, respectively. 19 patients received allo-HSCT in CR1 (HSCT in CR1 cohort), notably, 18/19 cases in this cohort received haplo-HSCT, and the remaining 22 patients continued the consolidation therapy (Non-HSCT in CR1 cohort). The estimated 4-year probabilities of OS, EFS and CIR in the HSCT in CR1 cohort were 86.6%, 89.2% and 5.3%, respectively. Meanwhile, the estimated 4-year probabilities of OS, EFS and CIR in the Non-HSCT in CR1 cohort were 37.5%, 19.9% and 75.6%, respectively. They were considered to be statistically significant. Of the 17 patients who relapsed during consolidation chemotherapy, 9 patients who underwent chemotherapy only (Non-HSCT after relapse cohort) all died within 44 months. For the remaining 8 patients who chose allo-HSCT (HSCT in CR2 cohort) when they achieved the second complete remission (CR2), the estimated 4-year probability of OS was 47.6% (P=0.002). Multivariate analysis showed that HSCT in CR1 was the only independent protective factor for OS, EFS and CIR, and age at diagnosis (≥10 years) was an independent risk factor of OS.

Conclusions: Allo-HSCT (especially haplo-HSCT) in CR1 may reduce the risk of relapse and improve prognosis in non-infant children with MLL-r B-ALL. In addition, allo-HSCT also seemed to be an effective approach to improve the prognosis of relapsed patients. Thus, haplo-HSCT could be an alternative approach for non-infant children with MLL-r B-ALL.

Disclosures

No relevant conflicts of interest to declare.
Genetic Variants of ATM & Non-Homologous End Joining Pathway Genes in Acute Myeloid Leukemia: A South India Case-Control Study
Authors: Vuree Sugunakar , Cingeetham Anuradha , Nageswara Rao Dunna , Gorre Manjula , Sinha Sudha , Digumarthi Raghunadharao , Satti Vishnupriya , Gundeti Sadashivudu
Published: November 2019

Purpose of the study:

Deregulated DNA repair is one of the hallmarks of cancers including Acute Myeloid Leukemia (AML), as it results in genomic instability. ATM gene functions as a sensor, activates cascade of events leading to stimulation of multiple DNA damage- responsive signaling pathways. Principal DNA repair mechanism activated in the hematopoietic stem cells is the Non Homologous End Joining (NHEJ) pathway. However, this pathway was shown to be error prone. Functional SNPs in the genes involved in DNA repair might influence the gene expression leading to altered DNA repair which might confer the risk to AML.

Materials & Methods:

This hospital-based case-control study included 225 AML patients and 326 cancer-free controls from South Indian population. Six polymorphisms of XRCC5, XRCC6, XRCC7 and ATM were genotyped using polymerase chain reaction (PCR)-Restriction Fragment Length Polymorphism (PCR- RFLP) method. Statistical analyses were performed by using SPSS (version20v) and SNPSTAT online tool. Protein-Protein Interaction (PPI) analysis was also done to see the relationship between these genes.

Results:

We found that there was an elevated risk of AML associated with the XRCC5 VNTR 0R repeat and A allele of 2408G>A polymorphism (p-0.04 and p<0.0001 respectively), the frequencies of G allele (p-<0.0001) of XRCC6 -1310C>G and T allele (p-0.003) of ATM -5144A>T polymorphisms were also significantly increased in AML cases. Further, analyses of the variant genotypes with epidemiological and clinical variables revealed a significant association of the risk genotypes with development and progression of AML.

Conclusion: The XRCC5 0R repeat, 2408G>A, XRCC6 -1310 C>G and ATM- 5144A>T polymorphisms, but not XRCC6 -61C>G and XRCC7 6721G>T polymorphisms, play an important role in the pathogenesis of AML.

Figure

Disclosures

No relevant conflicts of interest to declare.
Revealing Homologous Clonality By Synchronous Trisomy 8 in Langerhans CELL Histiocytosis and ACUTE Myeloid Leukemia
Authors: Wang Xinkai , Wang Zhao
Published: November 2019

Purpose Langerhans cell histiocytosis (LCH) is a disorder in which excess immature Langerhans cells build up in the body. Increasing reports have revealed LCH may share a common origin with myeloid-derived malignancies. Here we report a case of a 45-year-old male, diagnosed as synchronous LCH and acute myeloid leukemia (AML). Relevant tests have been performed to investigate the clonal relationship between LCH and AML. Methods The diagnosis of LCH and AML were based on skin and bone marrow biopsy respectively in Beijing Friendship Hospital, Capital Medical University. No cytotoxic drugs were used before the two diseases were diagnosed. Immunophenotyping was run by immunochemistry or flow cytometry. Karyotype analysis was carried out with G-banding, and the findings were confirmed by fluorescence in situ hybridization (FISH). Mutational analysis of BRAF, KRAS and NRAS genes were performed by ARMS-PCR. Additional gene screening was detected by next-generation sequencing (NGS). Results The case expressed CD1a, S-100, Langerin on the Langerhans cells and CD13, CD34, CD38, CD117, MPO on the leukemic blasts. No other intermingling markers was found on the LCH and AML except CD33 and CD68. Cytogenetic studies showed a karyotype with 47, XY, +8[10], and a confirmatory test revealed the existence of trisomy 8 in both skin and bone marrow lesions by FISH. Cell rate of trisomy 8 on skin, bone marrow and oral mucosa were 9%, 49% and 1%. Mutation analysis revealed KRAS c.436G>A:p.A146T (exon4) in AML and LCH cells, but BRAF c.1799T>A:p.V600E (exon15) only in LCH. Conclusion These findings suggest that LCH cells are derived from myeloid precursors in this case, and BRAF mutation may play an important role in pathogenesis of LCH.

Disclosures

No relevant conflicts of interest to declare.
Defective Autophagy in MPN Were Predominantly Detected in Megakaryocytes in Philadelphia Negative (Ph -) Myeloproliferative Neoplasm
Authors: Wang Jen-Chin , Shi Guanfang , Josan Karan , Ramachandran Preethi , Gotlieb Vladimir , Joseph Gardith , Nasr Sherif A.
Published: November 2019

We previously reported that autophagy was defective in classical Philadelphia negative (Ph -) Myeloproliferative Neoplasm (MPN) by demonstrating increased p62, decreased Beclin-1 by RT-PCR and Western Blot (WB) methods and decreased LC3-II by WB (ASH annual meeting poster 2018) in peripheral blood mononuclear cells. Now, we further performed immunohistochemical staining of these autophagy markers on the bone marrow biopsy specimens. Methods: Formalin-fixed and paraffin-embedded bone marrow samples were stained with primary antibodies against Beclin-1 (mouse monoclonal, Millipore), LC3B (rabbit monoclonal, Cell Signaling Technology), and p62 (mouse monoclonal, Cell Signaling Technology). The tissue sections were analyzed using VENTANA BenchMark Ultra System (Ventana Medical Systems, Inc.) according to the manufacturer's protocol. Patients who were studied included 12 essential thrombocythemia (ET), 10 polycythemia (PV), and 5 myelofibrosis (MF) (including 1 post-ET MF and 1 post-PV MF). Scoring was given based on the degree of cytoplasmic staining. Score 1 included weak cytoplasmic stain, score 2 included moderate cytoplasmic stain and score 3 was given for strong cytoplasmic staining . Results: 1) As shown in Fig1, the predominant cells which stained positive including p62, Beclin-1 , or LC3 B were mostly found on the megakaryoctes, although very few of other cell types were found to have positive staining as well 2) As shown in Table 1, the immunostaining results were in general correlated to the RT-PCR , or western blot findings that a defective autophagy was demonstrated in MPN patients with combined finding of a stronger staining in p62,and weak staining in Beclin-1 and LC3B. Conclusion: We have demonstrated defective autophagy in these Ph (-) MPN, by immunostaining of the bone marrow specimens, and demonstrating positivity for defective autophagy predominantly in megakaryocytes. Since megakaryocytes are the most important cells involved in the pathogenesis of MPN, we propose that defective autophagy in megakaryocytes play an important role in the pathogenesis of these diseases.

Disclosures

Wang: Incyt: Research Funding.
Modeling of Immune Reconstitution Post CD34 Selected Stem Cell Transplantation in Pediatric Patients with Severe Combined Immune Deficiency
Authors: Diana Jean-Sebastien , Bouazza Naim , Couzin Chloé , Castelle Martin , Magnani Alessandra , Magrin Elisa , Joseph Laure , Delville Marianne , Lefrere Francois , Treluyer jean-Marc , Picard Capucine , Moshous Despina , André Isabelle , Blanche Stephane , Neven Benedicte , Cavazzana Marina
Published: November 2019

Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited disorders characterized by a profound reduction or alteration of T lymphocyte function. They arise from a variety of molecular defects which affect T lymphocytes development and function. The number of infections prior hematopietic stem cells tansplantaton (HSCT), genotype, and the type of donor are described as prognostic factors for stem cell transplants.

In this retrospective study, we included 30 pediatric patients suffering from SCID who underwent to CD34+-selected grafts between January 2008 to December 2017 in our center. Diagnosis of reticular dysgenesis, ADA deficiency, or leaky SCIDs and intra thymic deficiency were excluded. A mechanistic mathematical model of all available data was performed and provided a dynamic appreciation of immune reconstitution, while removing bias. T-cell populations were maintained through proliferation and loss model and thymic output have been integrated to the production function. This joint modeling approach aimed to predict rate and extent of T cell immune reconstitution over time (mainly CD3+ T cells, CD3+CD4+ helper T cells, and the CD3+CD4+ CD45RA+ cells).

With a median follow-up time of 97.28 months [range 0.85; 131.54], there were 345 points of T cell phenotyping concentrations in total with a median of 12 samples per patient (range, 0 -35 samples) taken post-transplantation. In this data-set, 13 % of the patients (n= 4) died from infections. Time to reach half of the maximal T cell concentrations was estimated to 3.4 months, 95%CI [2.5 - 4.6]. In covariate analysis, genetic diagnosis (p= 0.0047) and conditioning regimen (p= 0.01) were found to be significant pre transplant covariates which impacted the trajectory of T cell concentration with time.

This modeling approach appeared to be the best method to learn about the dynamic T cell reconstitution after transplant in patients suffering from SCID. In the context of new cell therapy approach for T cell depletion and in vitro thymic maturation, this mechanistic joint model can be used for the design and analysis of incoming clinical trials.

Figure

Disclosures

Cavazzana: Smartimmune: Other: Founder of Smartimmune.
PEDF Can Rescue the Inhibition of Injured Endothelial Cells on Hematopoietic Stem Cell Expansion Ex Vivo
Authors: Zeng Lingyu , Lu Wenyi , Ding Lan , Ju Wen , Qiao Jianlin , Xu Kailin
Published: November 2019

Introduction: Endothelial cells (ECs) provide a fertile niche for hematopoietic stem cell (HSC) maintenance, differentiation, and migration.Several studies have indicated that bone marrow (BM) vascular niche was impaired after HSC transplantation and severely inhibited hematopoietic reconstruction. Pigment epithelium-derived factor (PEDF) is an important potential cytoprotection and therapeutic agent for injured cells. The direct role of the injured endothelial cells on hematopoietic stem cells and whether PEDF has protective effect in this system remain unknown. This study aims to observe the influence of enjured ECs on HSCs and to explore the role of PEDF in endothelial-HSC coculture system.

Methods: Injury of Endothelial cells by two important preparative regimenconditioning radiation and Busulfan respectively was evaluated with CCK8 assay. The expression of endothelial tight junctions(TJs),adherent junctions related molecules and endothelial to Mesenchymal Transition molecules such as ZO-1, Occludin,VE-cadherin, ICAM, α-SMA, CD31 and VCAM were detected by RT-qPCR, flow cytometry, immunofluorescence and western blot. The effects of injured endothelial cells on HSC self-renewal, differentiation, cell cycle and apoptosis were evaluated by flow cytometry, photography, viable cell count and clone formation assay. Hematopoiesis regulation factors SCF, IL-6, TGF-β and TNF-α were detected by ELISA. The protective effect of PEDF was also explored.

Results: Both radiation and Busulfan could decrease cell viability of endothelial cells. The expression level of ZO-1, Occludin, VE-cadherin, ICAM, CD31 and VCAM were decreased and α-SMA was increased when EC exposed to radiation or Busulfan suggesting endothelial activation, impaired EC permeability and endothelial to Mesenchymal Transition after EC injured. Compared with normal endothelial cells and hematopoietic stem cell co-culture group, the HSC% of injured endothelial cells and hematopoietic stem cells co-cultured group were significantly decreased, the cell colony formation ability was decreased, the proportion of mature cells increased, and the damage of endothelial cells could not maintain the characteristics of HSC, weakened the self-renewal and multidirectional differentiation potential of HSC and promoted the maturation of HSC. After the administration of PEDF, endothelial to Mesenchymal Transition of EC was suppressed and the EC permeability was improved. Most importantly, the proportion of HSC was significantly increased, and the proportion of mature cells decreased in the coculture system.

Conclusion: Injured endothelial cells can inhibit proliferation of hematopoietic stem cells, self-renewal and promote HSC differentiation. PEDF could ameliorate endothelial injury and promote HSC expansion by suppressing endothelial-mesenchymal transition and protecting TJs and AJs.

Disclosures

No relevant conflicts of interest to declare.
A New Step in Understanding of Fanconi Patients Peripheral Stem Cell Harvesting, a Bridge to Gene Therapy
Authors: Diana Jean-Sebastien , Manceau Sandra , Leblanc Thierry , Couzin Chloé , Magnani Alessandra , Magrin Elisa , Bendavid Matthieux , Joseph Laure , Delville Marianne , Blanche Stephane , Soulier Jean , Cavazzana Marina , Lefrere Francois
Published: November 2019

Fanconi anemia (FA) is an inherited disorder, clinically characterized by congenital abnormalities, a fatal progressive bone marrow failure (BMF), and a predisposition to develop malignancies. Gene therapy by infusion of FA-corrected autologous hematopoietic stem cells (HSCs) may offer a potential alternative cure and to get around the problems of the Hematopoietic stem cell transplantation toxicity or the donor restriction. For gene therapy, an adequate number of HSC collected is a key point to a successful engraftment. However, the HSC collection in FA patients implies particular challenges because of their reduced BM stem cells numbers and implies a theorical risk of an inner depletion in stem cell reserve following collection.The main objective of this pilot study was to evaluate the feasibility and the safety of co-administration of G-CSF and plerixafor in patients with FA for the mobilization and collection of peripheral HSC for potential use in a GT trial.

We present the results of this open-label phase I/II trial (N°EUDRACT 2014-005264-14) from 4 selected FANCA mutated patients (FA-A) with a weight >10 Kg and an age between 2 to 18 years old. A systematic combination of G-CSF (12μg/kg twice a day) plus plerixafor (Mozobil® 0.240 mg/kg/d ) was used to maximise the CD34+ cells mobilization. CD34+ cells and white blood cells (WBC) blood counts were monitored tightly along the mobilization protocol. No short-term adverse events linked to the mobilization and the collection procedures were observed. The combination of G-CSF and Plerixafor allowed crossing the PB mobilization threshold (≥5 CD34+cells/μL) for 2 patients. Interestingly, CD34+cells were mobilized quickly but transitionally after plerixafor injection. One patient mobilization had more than 100 CD34+cells μ/L with a early peak 2h after injection. The peak disappeared 11 hours after injection. We adapted the time of collection to the C34+ cells mobilization. No CD34+ blood cell rebound was observed after the apheresis was stopped.

Our new datas suggest that mobilization of FA patients with G-CSF and plerixafor is safe. However, the age of the patient, a potential cytopenia or the lack of bone marrow progenitor cell may heavely compromise the collection. Nevertheless, the datas show a stable cytopenia despite the stimulation and collection of stem cells during the following months. This study underlines that a very cautious collection of stem cell in the Fanconi anemia to consider gene therapy is a necessity. These results also confirm that the kinetic of CD34+ cells mobilization is one of the key point to a successful stem cell harvesting for gene therapy trial.

Disclosures

Cavazzana: Smartimmune: Other: Founder of Smartimmune.
CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets
Authors: Fišer Karel , Kalina Tomas , Perez-Andres Martin , Kužílková Daniela , Engel Pablo , Cuenca Marta , Álvarez Elena Blanco , Bartol Sophinus J.W. , van Zelm Menno C.
Published: November 2019

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets and their malignant counterparts.

Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-100 (n=110) on 47 immune cell subsets from blood, thymus and tonsil using an 8-color standardized EuroFlow approach and quantification of expression.

The resulting dataset included median antibody binding capacities (ABC) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters.

The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as, to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

CD Maps project is supported with reagents from BD Biosciences, BioLegend and Exbio. TK is supported by Ministry of Health Czech Republic grant 15-26588A and LO1604. KF is supported by Ministry of Health Czech Republic grant NV18-08-00385.

Disclosures

No relevant conflicts of interest to declare.
Risk of Postoperative Bleeding Following Dental Extractions in Patients on Antithrombotic Treatment
Authors: Alsheef Mohammed Abdullah , Gray Jenny , Zaidi Abdul Rehman Z. , AlJabab AbdulSalam S
Published: November 2019

Background:

Risk of bleeding post dental extractions in patients on the anticoagulant medication is not well-established. The aim of the study is to investigate the incidence of postoperative bleeding following dental extractions in adult patients on antithrombotic medication in Saudi Arabia.

Methods:

A retrospective study of 539 patients aged 18 to 93 years of age, attended 840 visits for dental extractions from January 2012 to June 2016 at a tertiary care hospital in Saudi Arabia. All returning patients were treated as outpatients with local hemostatic measures.

Results:

Only 1.7% of the visits were associated with post-operative bleeding following dental extractions. The highest risk of bleeding occurred in patients on Warfarin (3.88%), while those on Clopidogrel had no risk associated with bleeding. Women were found to have the highest rate of bleeding associated with dental extractions especially those on new oral anticoagulant medications.

Conclusion:

Dental extractions can be safely performed on adults receiving antithrombotic treatment provided established guidelines are followed. Health care professionals must exercise caution when planning invasive dental treatment for patients on continued antithrombotic therapy.

Disclosures

No relevant conflicts of interest to declare.
Purification Platelets from Mouse Blood with Sickle Cell Disease Using Iohexol Gradient Medium
Authors: Nasimuzzaman Md
Published: November 2019

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. Since RBC and WBC contain significantly more RNA and proteins than platelets, the presence of even a small number of these cells can interfere with transcriptomic and proteomic analyses of RNA and proteins derived from platelets. Several protocols have described the isolation of platelets from human, dog, rat, and non-human primate by various methods. Some of the methods require multiple steps such as collection of platelet-rich plasma by centrifugation, filtration by separation column, negative selection of platelets with RBC- and WBC-specific antibody conjugated to magnetic beads, and so on, which are time-consuming and may degrade platelets and their contents. Moreover, mouse blood with sickle cell disease contains a significant level of fragments of RBC which should be removed from the platelet preparation.

However, the mouse yields a relatively smaller volume of blood, which makes it difficult to purify platelets. If the same small volume of gradient medium and blood samples are used, the platelet layer cannot be clearly separated from RBC-WBC layer after centrifugation. We describe a simple method for purification of platelets from mouse blood with sickle cell disease using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 °C. The platelet layer is collected and centrifuged again at 200 x g for 20 min at 20 °C to remove the residual fragments of RBC. The recovery/yield of the purified platelets were 10-17%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets were activated with thrombin indicating their viability. We confirmed that the purified platelets were sufficiently pure using flow cytometric and microscopic evaluation. Although flow cytometric analysis of purified platelet from sickle cell disease mice showed a few RBC events after staining with anti-TER119 antibody, the microscopic study did not show any intact RBC or larger fragments indicating that these are smaller fragments of RBC which do not interfere with the biochemical and functional studies. This method can be used for purification of platelets from the blood of other species and disease models as well.

Disclosures

No relevant conflicts of interest to declare.
Type I Interferon Signaling before Hematopoietic Stem Cell Transplantation Lowers Donor T Cell Activation Via Reduced Allogenicity of Recipient Cells
Authors: Thiele Orberg Erik , Fischer Julius Clemens , Göttert Sascha , Bassermann Florian , Poeck Hendrik
Published: November 2019

Background: Recent studies highlight immunoregulatory functions of type I interferons (IFN-I) during the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that selective activation of IFN-I pathways including RIG-I/MAVS and cGAS/STING prior to allo-HSCT conditioning therapy can ameliorate the course of GVHD. However, direct effects of IFN-Is on immune cells remain ill characterised.

Methods: We applied selective RIG-I agonists (3pRNA) to stimulate IFN-I production in murine models of conditioning therapy with total body irradiation (TBI) and GVHD.

Results: Using IFNAR1-deficient donor T and hematopoietic donor cells, we found that endogenous and RIG-I-induced IFN-Is do not reduce GVHD by acting on these respective cell types. However, 3pRNA applied before conditioning therapy reduced the ability of CD11c+ recipient cells to stimulate proliferation and interferon gamma expression of allogeneic T cells. Consistently, RIG-I activation before TBI reduced the proliferation of transplanted T-cells after allo-HSCT. The reduced allogenicity of CD11c+ recipient cells was dependent on IFN-I signalling. Notably, this immunosuppressive function of DCs was restricted to a scenario of genotoxic tissue damage as neither RIG-I activation and IFN-I induction in naive (non-irradiated) mice altered allogeneic T cell activation.

Conclusion: Our findings uncover a hitherto unknown IFN-I- and context dependent immunosuppressive function of dendritic cells. This needs to be considered in the development of IFN-I based therapeutic approaches to modulate donor T cell activation after allo-HSCT.

Disclosures

No relevant conflicts of interest to declare.