bluuebunny/crossref_metadata_2025 huggingface dataset:
RPS19 Deficiency Leads to GATA1 Downregulation through TNF-Mediated p38 MAPK Activation
Authors: Youn Minyoung , Bibikova Elena , Danilova Nadia , Ono-Uruga Yukako , Konto-Ghiorghi Yoan , Ochoa Rachel , Narla Anupama , Glader Bertil , Lin Shuo , Sakamoto Kathleen
Published: November 2019

Diamond-Blackfan Anemia (DBA) is a rare inherited bone marrow failure disorder, characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. While several studies have found that the tumor suppressor protein p53 contributes to DBA pathogenesis, and that certain GATA1 mutations can give rise to DBA, the link between ribosomal protein mutations and erythroid defects is not well understood.

To investigate the molecular pathways downstream of RPS19 deficiency, we infected human cord blood CD34+ cells with RPS19 shRNA lentivirus and observed that RPS19 knockdown resulted in decreased GATA1 mRNA and protein expression, resulting in defective erythropoiesis. We also detected increased TNF-α levels in RPS19 knockdown cells, and in zebrafish that have been treated with RPS19 morpholino. TNF-α was primarily upregulated in the non-erythroid population of RPS19 deficient cells, while its receptor, TNFR1, showed increased expression on the surface of erythroid cells.

To understand the relationship between GATA1 and TNF-α, we treated primary hematopoietic CD34+ cells with TNF-α or its inhibitor etanercept. Our results demonstrated that TNF-α treatment reduced GATA1 expression, and that this effect could be rescued by the addition of etanercept. Treatment of RPS19 deficient cells with etanercept improved their erythroid differentiation, and increased GATA1 expression. Additionally, etanercept treatment successfully reversed the anemia phenotype observed in RPS19 deficient zebrafish.

To study pathways downstream of TNF-α, we examined phosphorylation of signaling pathways such as p38 MAPK, NFκB, ERK, STAT1, and STAT5 in RPS19 deficient erythroid cells using phospho-flow cytometry. Among these pathways, we found a significant increase in phosphorylation of p38 MAPK, but not ERK, NFκB, STAT1, or STAT5, suggesting that p38 MAPK activation by TNF-α contributes to decreased GATA1 expression in RPS19 cells. We suggest a novel mechanism for the erythroid defects observed in DBA, in which RPS19 deficiency leads to increased TNF-α production in non-erythroid cells, and activation of p38 MAPK, followed by decreased GATA1 expression, in erythroid cells. Our data also suggest that TNF-α inhibitors, such as etanercept, may be beneficial in treating patients with DBA.

Disclosures

No relevant conflicts of interest to declare.
High Efficiency and Tolerance of Temozolomide in Relapse/Refractory Primary Intra-Ocular Lymphoma (R/R PIOL). a Retrospective Multicentric Study from the LOC Network
Authors: Baron Marine , Gyan Emmanuel , Merle-Beral Hélène , Leblond Veronique , Cassoux Nathalie , Touitou Valerie , Bodaghi Bahram , Darugar Adil , Choquet Sylvain
Published: November 2019

Background: PIOL, now named “primary vitreo-retinal lymphoma”, is a very rare subset of non Hodgkin lymphoma, usually arising in elderly patients and characterized by a high level of relapse, mainly in the first year, with more than 20% of cases relapsing in brain, and a short survival. There is no consensus on treatment procedures, even in first line, and prospective comparative studies do not exist. Classical attitudes are systemic chemotherapy (SC), often high dose methotrexate, radiotherapy or intraocular injection of methotrexate but publication on R/R PIOL are exceptional. New treatments are necessary, especially with a good tolerance profile. As Temozolomide (Te) hassome efficiency on primary-central-nervous-system lymphoma (PCNSL) we used this drug in R/R PIOL in our center.

Methods: Inclusion criteria were R/R PIOL and/or PIOL not eligible for IV chemotherapy. Diagnosis was established on cytological and molecular analysis after vitrectomy, and the absence of brain or meningeal localization verified by MRI and lumbar puncture. Treatment consisted in Te at 150mg/m2 orally 5 days per month, without corticosteroid use, in absence of any response, dosage was increased to 200mg/m². A complete response was defined as a normalization of eye exam and intraocular interleukins 10 and 6 levels.

Results: Sixteen patients were analyzed, 3 males and 13 females, mean age 75 years [35-90]. All but two received systemic chemotherapy with at least high dose methotrexate and high dose cytarabin before Te, 8 were in second line, 4 in third, with 1 patient who relapsed after autologous stem-cell transplantation (ASCT) conditioned by thiotepa, cyclophosphamide and busulfan, and 2 in forth. The 2 patients treated in first line where more than 80 years old. Median duration of treatment was 5 months. The median follow-up (fu) is 16 months. Overall response rate is 75%, with 10 CR (63%) and 2 PR (13%). At the last fu, 7 patients are still in CR, with a median DFS of 13 months. The patient treated after ASCT relapse is still in CR at 62 months. The two old patients treated in first line are in CR at last fu. Two patients where treated a second time by Te after relapse, obtaining 2 new CR, one of 4 months, one persistent at 14 months. Median OS is not reached. Only 3 patients experienced hematological grade 3/4 toxicity.

Conclusion: This work represents the biggest study with an homogeneous treatment in R/R PIOL. Temozolomide appears as a safe and efficient treatment of R/R PIOL or in first line in patients in bad condition, even after high dose chemotherapy and/or in elderly patients. Longer follow-up, prospective and larger studies are necessary to confirm these data

Disclosures

Leblond: Roche: Honoraria, Speakers Bureau.
JAK2 Is a Direct BCL6 Target Gene: Implications for Therapy in Diffuse Large B-Cell Lymphoma
Authors: Barber Ruth , Zobel Jenny , Beck Daniel , Evans Sian , Elliott Richard , Lord Christopher J , Ashworth Alan , Porter Andrew G.C. , Wagner Simon D
Published: November 2019

Increased STAT3 signalling is a factor in driving ~50% of diffuse large B-cell lymphoma. In some cases increased cytokine production by the lymphoma is responsible for activation of JAK2 and STAT3 but the regulation of signalling through this pathway is not clear. We constructed a conditional BCL6 deficient cell line through disruption of the endogenous BCL6 loci of a genetically tractable human B-cell lymphoma by homologous recombination, and insertion of a tetracycline regulatable BCL6 transgene. On induction of BCL6 deficiency growth of the cell line slowed by 3 to 4-fold. A synthetic lethal screen employing a library of small molecule inhibitors in genetically BCL6 deficient lymphoma cells showed that lestaurtinib, a JAK2 inhibitor, enhanced loss of viability. We investigated the hypothesis that JAK2 is a direct BCL6 target gene. JAK2 mRNA and protein expression were induced by BCL6 deficiency. Inspection of the JAK2 proximal promoter region demonstrated a potential BCL6 binding site. BCL6 bound to this sequence in vitro and mutagenesis of the binding site relieved BCL6 mediated transcriptional repression in luciferase reporter assays. Analysis of ChIP-seq data showed a binding peak in the JAK2 proximal promoter region confirming in vivo BCL6 binding. Data from a large and publicly available gene expression dataset confirmed an inverse correlation between BCL6 and JAK2 mRNA whilst there was a positive correlation between JAK2 and STAT3 mRNA. STAT3 is a known target of BCL6 transcriptional repression. We suggest that BCL6 represses both JAK2 and STAT3 and that relatively high BCL6 levels will tend to reduce JAK2-STAT3 signalling whereas lower BCL6 levels will amplify this pathway. Mouse xenografts utilising our conditional BCL6 deficient cell line showed that lestaurtinib alone caused minimum reduction to tumour size but the combination of BCL6 deficiency and JAK2 inhibitor caused growth reduction with central necrosis. In summary we identify JAK2 as a BCL6 target gene and propose that BCL6 has an important role in regulation of JAK2-STAT3 signalling in DLBCL. BCL6 mRNA expression may be a biomarker to enable the rational use of JAK2 inhibitors in DLBCL.

Disclosures

No relevant conflicts of interest to declare.
The Experience of Gruppo Laziale for the Study of Ph-, Chronic Myeloproliferative Syndromes in the Treatment of Myeloproliferative Neoplasms in Blastic Phase with Azacytidine
Authors: Andriani Alessandro , Montanaro Marco , Voso Maria Teresa , Villivà Nicoletta , Ciccone Fabrizio , Andrizzi Cristina , De Gregoris Cinzia , Buccisano Francesco , Alimena Giuliana , Latagliata Roberto
Published: November 2019

Prognosis of patients with myeloproliferative neoplasms developing blast phase (MPN-BP) is very poor with a median survival of about 2-3 months; nowadays, no treatment can induce a durable complete response (CR) in patients who are not candidate to BMT. Very recently, a possible role of azacytidine (AZA) was reported in these patients, but there is still a lack of data as to its efficacy; to address this issue, we evaluated retrospectively 19 patients (M/F 15/4 F, median age 71.3 years, interquartile range (IQR) 64.5 – 77.7] with MPN-BP treated with AZA and collected in the database of our cooperative group. The primary MPN diagnosis was Essential Thrombocythemia in 6 cases (31,6%), Policythemia Vera in 3 (15,8%), Primary Myelofibrosis in 7 (36,8%) and MDS/MPN in 3 (15,8%); the JAK-2 V617F mutation was present in 11/17 patients tested (64.7%). All but 1 patient received previous chemotherapies during chronic phase. Median time from diagnosis to BP evolution was 52.6 months (IQR 11.2 – 181.8). All patients were treated with AZA at the standard dosage of 75 mg/m2: 16 patients received a 7-day schedule and 3 a 5-day schedule every 28 days. Two patients died early after 5-AZA initiation for pulmonary fungal infection and respiratory failure respectively, 4 patients had a disease progression, 4 patients maintained stable disease, 3 patients had a hematological improvement, 1 patient achieved partial response and 5 patients (26.3%) a CR after 4, 4, 4, 5, and 12 months. The median cumulative survival from BP evolution was 9.9 months (95%CI 6.7 – 13.1).A comparison with the historical cohort of 72 patients of our database presenting MPN-BP and treated with approaches other than AZA (median cumulative survival 3.1 months, 95%CI 1.1 – 5.1) showed a significant advantage for patients treated with AZA (p= 0.02). Our data confirm the relative good efficacy and safety of AZA in this group of patients with otherwise dismal prognosis, underlining the possible achievement of long-lasting responses in a sizeable portion of them.

Disclosures

No relevant conflicts of interest to declare.
Mir-148a and Mir-25 Target TULA-2 and Regulate Human Platelet FcγRIIA Signaling
Authors: Zhou Yuhang , Abraham Shaji , Andre Pierrette , Edelstein Leonard C. , Shaw Chad , Tsygankov Alexander , Kunapuli Satya P. , Bray Paul F , McKenzie Steven E.
Published: November 2019

FcγRIIA-mediated platelet activation is important in the pathogenesis of heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. FcgRIIa is also the major transmembrane signaling adapter for the human platelet fibrinogen receptor, integrin aIIbb3. In HIT, antibody to PF4/heparin is necessary but not sufficient for the disease. There is considerable inter-individual variation in platelet FcγRIIA activation; the reasons remain unclear. To identify potential regulators in the FcγRIIA pathway, we determined the platelet RNA expression profile in 154 healthy human donors who were phenotyped for platelet aggregation via FcgRIIA. 87 mRNAs and 4 DE miRNAs were identified as differentially expressed (DE) between hyper- and hypo-responsive donors by microarray and Nanostring analysis, respectively. Among them, hyper-responders show significantly lower T-cell ubiquitin ligand-2 (TULA-2) mRNA level and higher miR-148a-3p and miR-25-3p levels than hyporesponders. TULA-2, also known as UBASH3B, is a known protein tyrosine phosphatase for Syk, and thus a biologically plausible regulator of platelet FcgRIIA.

Both miR-148a-3p and miR-25-3p are predicted to target the 3’UTR region on TULA-2 mRNA (Fig., A). Overexpression of miR-148a-3p or miR-25-3p results in down-regulation of the TULA-2 protein level in HCT116 cells and HEL cells (Fig., B). Inhibition of miR-148a-3p by anti-miR (LNA) leads to elevated TULA-2 expression. A luciferase construct with the 3’UTR miR binding sites was generated. A decrease in luciferase activity was observed in assays with co-transfection of the construct and a miR, supporting the binding between miR-148a-3p and miR-25-3p with TULA-2 3’UTR (Fig., C). Finally, we show that knockdown of TULA-2 caused increased Syk-pY (Y323) level in HEL megakaryocytic cell line in response to FcγRIIA crosslinking by IV.3 (mAb against FcγRIIA) + GAM (Goat anti-mouse F(ab)’2)(Fig., D). These results for the first time show that TULA-2 serves as a negative regulator of the FcγRIIA pathway and that miR-25 and miR-148a may contribute to HIT susceptibility. Studies are in progress for the translation of these findings in vivo.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Comparison of Safety Reports to the FDA from a Pilot Introduction of Peginesatide, a Third Generation Erythropoiesis Stimulating Agent Versus Those from the Usual Care Setting: Manufacturers of Biosimilars and the FDA Should Consider Pilot Introductions of These Agents
Authors: Bennett Charles L. , Jacob Sony , Georgantopoulos Peter , Nichols Judy , Macdougall Iain C
Published: November 2019

Background: Thirty eight anaphylaxis cases were reported to the FDA during the first year of peginesatide marketing in 2012. Marketing was discontinued as a result. We compare 28 reports from a pilot peginesatide introduction conducted by the largest dialysis organization (LDO) in North America versus the 10 reports of anaphylaxis from usual care settings. The pilot introduction was conducted at 10 LDO centers, a nurse was assigned to each center, and staff were educated on peginesatide dosing, safety, pharmacokinetics, and handling.

Methods: Reports in the FDA of anaphylaxis occurring within 30 minutes of peginesatide administration were reviewed for information on administration site, patient characteristics, time to report to FDA, and patient outcome.

Findings: In comparison to 28 anaphylaxis events reported to the FDA from the pilot introduction (as described in Bennett CL et al NEJM 2014), 10 anaphylaxis reports to the FDA from the usual care settings were less often reported as fatal (0% versus 22%) or grade IV severity (10% versus 22%) associated with hypotension (20% versus 57%), or cardiorespiratory arrest (0% versus 29%), and were reported to the FDA later (median of 81 days (range, 14- 172) versus 46 days (range, 4 – 136 days). They were more often associated with clinical findings of diaphoresis (40% versus 18%), syncope (30% versus 18%), or angioedema (20% versus 11%). Onset was a median of four to five minutes after peginesatide infusion in either setting. Among 25,000 peginesatide-treated patients (19,430 in the pilot introduction), anaphylaxis rates were 1.4 per 1,000 persons in either setting.

Conclusion: Clinical characteristics of anaphylaxis events were more serious and FDA reporting more timely for peginesatide-associated anaphylaxis reported to the FDA from the pilot introduction versus usual care settings. With the anticipation of biosimiars in the United States, consideration should be given to requiring pilot introductions of these agents to facilitate safety assessment.

Disclosures

No relevant conflicts of interest to declare.
Selective Inhibition of EZH2 and EZH1 Enzymatic Activity By a Small Molecule Suppresses MLL rearrangement-Induced Leukemia
Authors: Wang Gang Greg
Published: November 2019

Enhancer of Zeste Homolog-2 (EZH2) and Related EZH1 Control Gene Expression and Promote Oncogenesis Via Methylating Histone H3 Lysine 27 (H3K27). These Methyltransferases Are Proposed to be Therapeutic Targets Due to Their Hyperactive Mutations and Overexpression Found in Cancer Including Various Hematological Malignancies. Here, We Characterized a Set of Small Molecules That Allow Pharmacological Manipulation of EZH2 and EZH1, Which Include UNC1999, a Highly Selective Inhibitor of Both Enzymes, and Its Di-Methylated Analog UNC2400, an Inactive Compound Useful to Assess Off-Target Effects. We Show That UNC1999 Suppresses H3K27 Tri- and Di-Methylation Globally and Inhibits Growth of MLL-Rearranged Leukemia Lines. Mechanistically, UNC1999 Preferentially Affects the Regulatory Elements Such As Enhancers and Distal Promoters, Leading to De-Repression of Many Known Polycomb Target Genes. Gene De-Repression Correlates with Decrease in H3K27me3 and Concurrent Gain in H3K27 Acetylation. UNC2400 Does Not Induce These Effects, Demonstrating UNC1999’s on-Target Inhibition. Oral Administration of UNC1999 Significantly Prolongs the Survival of a Well-Defined Murine Leukemia Model Generated By MLL-AF9. Thus, Our Studies Provide Detailed Profiling of a Set of Compounds for Studying EZH2 and EZH1 in Cancer and Establish Specific Inhibition of EZH2 and EZH1 Enzymatic Activities By Small Molecules As a Promising Therapeutics for MLL-Rearranged Leukemia.

Disclosures

No relevant conflicts of interest to declare.
Serologic Response to Meningococcal Vaccination in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) for the Chronic Treatment with the Terminal Complement Inhibitor Eculizumab
Authors: Roeth Alexander , Alashkar Ferras , Herich-Terhuerne Doerte , Elias Johannes , Vogel Ulrich , Duehrsen Ulrich
Published: November 2019

BACKGROUND: Eculizumab is indicated for the therapy of patients with symptomatic paroxysmal nocturnal hemoglobinuria (PNH). Due to inhibition of terminal complement cascade, patients on eculizumab are susceptible to N. meningitids infections (0.48 cases per 100-patient years). The two mainstays to reduce the risk of infection are vaccination at least two weeks prior to treatment and antibiotic prophylaxis.

PATIENTS/METHODS: In this retrospective study serologic response was analyzed after vaccination with a meningococcal vaccine in PNH patients (n=32) by measuring serum bactericidal antibody titers with external rabbit complement (rSBA) against meningococci serogroups A, C, W and Y. 78.1% of the patients (25/32) received a tetravalent conjugate vaccine, while 6.3% (2/32) received an unconjugated polysaccharide vaccine. In 9.4% (n=3) the type of vaccine administrated was not available. Serologic response was defined by an rSBA titer ≥1:8. In 23 patients the corresponding rSBA titers were available. 39.1% (9/23) had been vaccinated more than once due to chronic eculizumab treatment.

RESULTS: Overall serologic responses independent of the time of observation for the meningococcal serogroups were: A: 78.3%, C: 86.9%, W: 47.8%, Y: 69.6%. rSBA titers varied significantly for the different time points [≤2 months (n=1): A: 100%, C: 100%, W: 100%, Y: 100%; ≤4-<6 months (n=4): A: 75%, C: 100%, W: 75%, Y: 75%; ≥6-<12 months (n=5): A: 60%, C: 40%, W: 0%, Y: 40%; ≥ 12-36 months (n=13): A: 84.6%, C: 100%, W: 53.8%, Y: 76.9%]. In 5 patients (15.6%) rSBA titers against 3 serogroups were <1:8, while 5 other patients (15.6%) had rSBA titers <1:8 against 2 serogroups at the same time. Serologic response after vaccination with an unconjugated vaccine even for the third time (n=1) prior to observation due to chronic therapy was identical to patients who had been vaccinated only once, supporting a non-immunological memory post vaccination with an unconjugated vaccine. No meningococcal infections have been observed in the analyzed cohort of eculizumab treated PNH patients so far.

SUMMARY/CONCLUSIONS: Efficiency of meningococcal vaccines for the different serotypes varies significantly and serological response analyses are very useful. Revaccination with the tetravalent conjugate vaccine (Menveo®) every 3 years is recommended or should be based on the individual response. However, physicians and patients must be vigilant for meningococcal infections at all times. Furthermore, stand-by therapy with ciprofloxacin 750 mg p.o. in case of signs of meningococcal infection and immediate medical evaluation and treatment is recommended. The availability of a novel vaccine covering the B-strain (Bexsero®) may further reduce the risk of an infection.

Disclosures

Roeth: Alexion: Consultancy, Honoraria, Research Funding. Duehrsen:Alexion: Honoraria.
Value of Procalcitonin Arise Markedly after Use of ATG in Conditioning of HSCT without Value in Differentiating Bacterial and Non-Bacteria Inflammation
Authors: Ge Hui , An Shengli , Li Chunfu
Published: November 2019

Background It is difficult how to differentiate bacterial infections from viral infections and to differentiate bacterial infections from other non-infective systemic inflammation. Especially in the patients with lack of neutrophil bacteria infection is usually life-threaten. The prognosis for those patients is high dependent on early diagnosis and treatment. We still feel confusing in spite of monitoring serum procalcitonin (PCT) and C-reactive protein (CRP) levels with temperature.

Aim To find out whether PCT and CRP are helpful to differentiate bacterial infection and non-bacterial inflammation after use of anti-human T-lymphocyte globulin (ATG) in conditioning of hematopoietic stem cell transplantation (HSCT).

Method Total 60 patients were involved in current study, who underwent HSCT and received conditioning included Cyclophosphamide, Fludarabine, ivBusufan, Thiotepa and ATG (at total dose of 5mg/kg for thymoglobulin, and 15-30mg/kg for ATG-Fresenius, respectively, on day-3 to -1). Blood samples were obtained in the morning from day-3 to 0 for testing PCT and CRP associated by daily recording temperature. Patients were grouped into Group 1 (G1, with antibiotics, n=30), Group 2 (G2, without antibiotics, n=30), Group 3 (G3, bacteria culture positive, n=7) and Group 4 (G4, bacteria culture negative, n=47)

Results The value of PCT was not significant difference when G1 vs. G2 (p=0.061), and G3 vs. G4 (p=0.891), but CRP value and temperature were of significant difference between G1 and G2 (p=0.001 and 0.000, respectively). When comparing G3 with G4, CRP was significant (p=0.021) but temperature was no (0.377). However, the deference of daily CRP value was proved not to be enough to do a differential diagnosis by ROC (AUROC=0.685, 0.665, 0.643 and 0.643 on day-3 to 0, respectively).

Conclusion The current study shows the value of PCT is insignificant to differentiate bacterial infection and non-bacterial inflammation after use of ATG in conditioning of HSCT.

Disclosures

No relevant conflicts of interest to declare.
Direct Contact with Stromal Cells in Association with SDF-1 Is Important for B-Lineage Differentiation Toward CD19+ ProB Cells from Human Hematopoietic Precursors, but Dispensable for Generation of CD7+CD45RA+ Multipotent Lymphoid Precursors
Authors: Minami Hirohito , Ohishi Kohshi , Nakamori Yoshiki , Masuya Masahiro , Katayama Naoyuki
Published: November 2019

The regulatory mechanism of human early B- and T- lymphoid differentiation has not been well studied. Coculture on telomerized human bone marrow stromal cells supported the generation of CD7+CD45RA+ multipotent precursors with differentiation potential for T-, B-, NK-lineage, and monocytic cells, CD10+CD19-CD45RA+ B-biased precursors, and CD10+CD19+CD45RA+ proB cells from human CD34+lin- hematopoietic progenitors. CD7+CD45RA+ and CD10+CD19- lymphoid precursors can be developed from hematopoietic progenitors by soluble factors produced from stromal cells, but the generation of CD19+ proB cells was reduced. Replating analysis showed that direct contact with stromal cells promoted B-lineage differentiation toward CD19+ proB cells from CD34+lin- cell-derived CD7+CD45RA+ and CD10+CD19- lymphoid precursors.

SDF-1 is shown to be critical for B-lineage differentiation from studies of mice. SDF-1 was produced from the human telomerized stromal cells. Inhibition of binding to SDF-1 with neutralizing antibody against CXCR4 suppressed B-lineage differentiation to CD19+ proB cells from hematopoietic precursors, while the generation of CD7+CD45RA+ cells was not significantly affected. By replating analysis, anti-CXCR4 Ab inhibited the differentiation to CD19+ proB cells from CD7+CD45RA+ and CD10+CD19- lymphoid precursors on stromal cells.

These data indicate that direct contact with stromal cells in association with SDF-1 produced from stromal cells is important for B-lineage differentiation toward CD19+ proB cells from CD7+CD45RA+ and CD10+CD19- lymphoid precursors but dispensable for differentiation toward CD7+CD45RA+ multipotent lymphoid precursors.

Disclosures

No relevant conflicts of interest to declare.
Assessing the Mechanism of Cytarabine-Induced Killing in Acute Leukemia
Authors: Vincelette Nicole Dionne , Yun Seongseok
Published: November 2019

The nucleoside analogue cytarabine is part of standard therapy for acute myelogenous leukemia (AML) and is also widely used in various acute lymphocytic leukemia (ALL) regimens. During a 7-day infusion at 100 mg/m2/d, which is part of the “7 + 3” AML induction regimen, steady-state cytarabine concentrations in the 100-200 nM range are achieved. Previous literature suggesting the mechanism of cytarabine-induced killing in AML examined cytarabine concentrations in the micromolar range, which might be less pertinent to routinely achieved drug exposure. To better understand the mechanism of cytarabine-induced killing, AML and ALL cell lines were exposed to varying cytarabine concentrations (25-200 nM) for 24-48 hours and assayed for cell death using a variety of assays. Dose- and time-dependent induction of apoptosis was observed in both AML and ALL cell lines, as detected by phosphatidylserine externalization (Annexin V staining), DNA fragmentation (propidium iodide staining for subdiploid cells), nuclear morphological changes, and cleavage of caspase substrates. Jurkat T cell ALL variants lacking critical components of the death receptor pathway (FADD or procaspase 8) underwent apoptosis with kinetics indistinguishable from parental cells, arguing against involvement of the death receptor pathway. Immunoblotting for Bcl-2 family members in AML and ALL cell lines revealed upregulation of the BH3-only proteins Bim, Puma and Noxa across multiple lines. In further experiments, small interfering RNA (siRNA)-mediated knockdown of Bim, Puma, and Noxa or the mitochondrial proteins Bax and Bak markedly diminished the apoptotic response to cytarabine. These results suggest that cytarabine at nanomolar concentrations kills cells through the mitochondrial apoptotic pathway but, in contrast to more targeted agents, does so by upregulating multiple BH3-only Bcl-2 family members simultaneously. Additional studies to determine the mechanisms of Bim, Puma, and Noxa upregulation are underway. (Supported in part by R01 CA166741 and predoctoral fellowships from the Mayo Foundation for Education and Research.)

Disclosures

No relevant conflicts of interest to declare.
The Clinical Significance of Minimal Residual Disease Detected By Multiparametric Flow Cytometry in Acute Leukemia
Authors: Huang Fen , Jing Hui , Yi Zhengshan , Wei Xiaolei , Zheng Zhongxin , Xie Muchen , Feng Ru
Published: November 2019

Purpose

Minimal residual disease (MRD) detected by multiparametric flow cytometry is an important method for predicting relapse. According to the dynamic changes of MRD, through the first clinical treatment to prevent relapse in patients with elevated MRD levels.

Method

We retrospectively analyze dynamic level of MRD of 75 patients with acute leukemia in our department of hematology from January 2011 to June 2013. According to the retrospective data, to analyze the expression of LAIP and discuss the relationship between changes of MRD and prognosis.

Result

In this study there were 75 patients, including 20 patients with acute lymphocytic leukemia (ALL) and 55 patients with acute myeloid leukemia (AML). The specific LAIP were detected in 49 patients with AML, and the cross lineage and non-synchronous expression were majority. In cross lineage expression, detected frequency of CD45/CD34/CD33/CD13/CD56 was highest, accounted for 67.3% (33/49). In non-synchronous expression, CD45/CD34/CD117/CD33/CD64 accounted for 57.1% (28/49). In 20 patients with ALL, there were 16 patients with B-ALL and 4 patients with T-ALL. The specific LAIP were detected in all of the patients. In patients with B-ALL, detected frequency of CD45/CD19/CD22/CD10/CD13 was highest, accounting for 43.8% (7/16). In abnormal quantity of antigens, CD45/CD19/CD22/CD10/CD38 was the most common type, accounting for 50%. The abnormal expression of CD7/TDT was detected in four patients with T-ALL. In monitoring period 7 patients relapsed. We analyzed the relationship between the clinical data and replase. The results showed that level of peripheral hemoglobin at diagnosis and the times of remission induction were significantly associated with replase (P=0.021 and P=0.017, respectively). To analyze the relationship between change of MRD and prognosis, the results showed that 0.05% as the threshold was significantly related with recurrence. 20 patients of persistent MRD≥0.05% had significant differences with 28 patients of persistent MRD<0.05% in replase free survival (P=0.005).

Conclusion

It has important significance to predict relapse in patients with acute leukemia by detecting minimal residual disease. Patients of MRD ≥ 0.05% should receive early intensive therapy or hematopoietic stem cell transplantation. Even patients without relapse, it should be closely monitor dynamic levels of MRD and highly vigilant extramedullary relapse.

Disclosures

No relevant conflicts of interest to declare.
Novel Role for Rpl22 in Controlling Lymphoma Progression and Dissemination
Authors: Rao Shuyun , Cai Kathy , Stadanlick Jason , Lee Sang-Yun , Greenberg-Kushnir Noa , Solanki-Patel Nehal
Published: November 2019

Mutations in ribosomal proteins often cause bone marrow failure syndromes associated with increased risk for cancer development; however, in most cases, the basis by which they do so remains unclear. We have shown that Rpl22 is a haploinsufficient tumor suppressor in T acute lymphoblastic leukemia/lymphoma (T-ALL). Loss one Rpl22 allele accelerates T-cell lymphomagenesis in the murine Akt transgenic mouse model, by inducing the stem cell factor Lin28B in an NFkB-dependent manner. Here we employed two distinct murine T lymphoma models to uncover a new role for Rpl22 in lymphoma progression through effects on migration and angiogenesis. Compared with Rpl22+/+ mice, mice deficient for Rpl22 (Rpl22-/-) exhibited significantly increased thymic tumor size, which was associated with markedly enhanced angiogenesis through the activation of VEGF signaling. Unlike Rpl22 sufficient tumors, Rpl22-deficient lymphomas were retained in the thymus as a large mediastinal mass that did not disseminate to the periphery due to a defect in migration. The migration defect in Rpl22-/- tumors results from attenuation of S1P1 receptor expression through downregulation of the KLF2 transcriptional factor, which is responsible for its expression. Indeed, re-expression of S1P1 receptor in Rpl22 deficient tumor cells rescues the migration defect. Re-expression of Rpl22 into Rpl22 deficient cells rescues expression of both KLF2 and its target, S1P1 receptor, indicating that Rpl22 is regulating the KLF2/S1P1 receptor axis, rather than the changes in expression being an indirect consequence of transformation. Consistent with this idea, human T-ALL lines with in which Rpl22 is mutated, exhibit reduced expression of both KLF2 and S1P1 receptor, relative to that in T-ALL lines with intact RPL22 alleles. Collectively, we have defined a new role of Rpl22 in tumorigenesis through regulation of migration.

Disclosures

No relevant conflicts of interest to declare.
PET Adapted Salvage Therapy for Advanced Hodgkin Lymphomas: Towards the Suppression of Positive Interim PET Unfavorable Prognosis? a Report of the Goelams Multicentric Phase 2 Trial LH2007
Authors: Carras Sylvain , Dubois Benjamin , Senecal Delphine , Quittet Philippe , Vuillez jean-Philippe , peoc'H Michel , Jais jean-Philippe , Lamy Thierry , Molina Lysiane
Published: November 2019

Introduction: Continuous improvement in the management of Hodgkin Lymphoma (HL) has brought to favorable outcome for the majority of patients. However, patients with advanced stages still present unsatisfactory results with ABVD. The GOELAMS group conducted a prospective multicentric phase 2 trial for advanced stage HL with front-line therapy by VABEM and early salvage therapy followed by ASCT in case of absence of complete metabolic response to test a global PET adapted strategy with early salvage (LH 2007 trial, NCT00920153).

Patients & Methods: Patients with advanced stage according to prognostic system score (PSS>3) received VABEM in front-line and interim 18F-FDG PET restaging using visual analysis after 2 courses. In case of negative PET-2 patients continued their therapy with a third course. In case of positive interim PET they underwent early salvage therapy by platin, gemcitabine and dexamethasone combination followed by intensification in case of metabolic response.

Results: 51 patients were included in the advanced arm. Median age was 40.5 years (range 18.2-64.8). Ann Arbor stage was IV in 35 (78%). B symptoms were observed in 44 (88%) patients; bulky disease in 22 (43%) of cases. The complete remission rate at the end of the strategy was 88% (n=45/51), with 34/37 patients in CR after 3 VABEM courses and 11/12 patients in CR after salvage therapy and ASCT. The metabolic complete response rate after 2 VABEM courses was 76% (37 above 49 assessable patients). The median follow up is 4.2 years (range: 0.02-5.8). Estimated 2-yrs EFS and OS (n=51) were respectively 82.3% (IC95% 72.5% to 93.5%) and 92.1% (IC95% 85% to 99.8%). 9 events occurred during the first 2 years and led to 4 deaths. The comparison by the one sample log-rank test of the observed EFS to a reference curve with a 2years EFS of 70% was statistcally significant (p=0.0004). No difference was observed in 2yrs-EFS and OS depending on interim PET-2 status. 2yrs-EFS and 2yrs-OS were respectively 83.7% (IC 95 72.6%-96.5%) and 97.2% (IC95 92.0%-100.0%) for PET-2 negative population vs 91.7% (IC 95 77.3%-100%) and 91.7% (IC95 77.3%-100.0%) for PET-2 positive population.

Discussion & conclusion: VABEM is an efficient first line treatment choice for Advanced HL. We demonstrate here that a PET-guided strategy with early salvage therapy and intensification for interim PET-2 positive patients is safe and feasible and that this global strategy permits to obtain interesting results in a population of patients associated with poor prognosis. Finally, we suggest that salvage by non-cross-resistant drugs and intensification may erase the unfavourable prognostic of positive interim PET-2 status in HL.

Disclosures

No relevant conflicts of interest to declare.
Modulation of the β2-Integrin Axis in Inflammatory Cell Trafficking By Fucosylated Ligands
Authors: Buffone Alex , Nasirikenari Mehrab , Neelamegham Sriram , Lau Joseph T.
Published: November 2019

Leukocyte trafficking to a site of inflammation, in which selectin-mediated recruitment is the well-established initial step in mouse and humans, requires α1,3-fucosylated carbohydrates as canonical selectin ligands. Disrupting these ligands mitigates leukocyte recruitment, and this pathway presents a choice target in managing inflammatory conditions. Here we report that fucose can act paradoxically in cell recruitment to select sites of inflammation. α1,3-Fucose deficient neutrophils lacking the fucosyltransferases Fut4 and Fut7 have severely impaired recruitment in the well-established selectin dependent acute peritonitis. By contrast they are preferentially recruited to inflamed airways, because α1,3-fucose deficiency augments CXCR2 mediated Rap1-GTP signaling and enhances the β2 integrin - ICAM-1 axis to promote neutrophil recruitment. Recognition of this newly identified role for α1,3-fucoslated carbohydrates in modulating the integrin axis of inflammatory cell recruitment illuminates the unique pathophysiology of the airway and new insights into therapeutic anti-inflammatory approaches.

Disclosures

No relevant conflicts of interest to declare.
Genetic Defects in 10 Index Cases of Inherited Vitamin K Dependent Clotting Factors Deficiency
Authors: Naz Arshi , Watzaska Mattiha , Oldenburg Johannes , Kristin Inga , Waryah Ali mohammad , Raza Ali , Nadeem Mohammad , Ahmed Nisar , Shamsi Tahir
Published: November 2019

Introduction: Vitamin K dependent factors i.e II, VII, IX and X plays an important role in the clotting mechanisms of the human body. Deficiency or defect in any of these factors, Vitamin K or Gamma glutamyl Carboxylase (GC) and Vitamin K Epoxide Reductase (VKOR) involved in the metabolism of these factors will compromise the clotting mechanisms and can lead to the bleeding disorder. 28 rare single mutations in VKORC1 coding sequence have been reported so far.

Objective: To investigate the biochemical, haemostatic and molecular basis of the Vitamin K dependent factor deficiency in Pakistani subjects.

Setting: NIBD &BMT, University of Bonn and Children hospital.

Method: For this purpose 10 subjects, 9 males and 1 female with age ranging from 7 months to 13years were included from selected region of Pakistan who had a history of bleeding disorder and low levels of all the Vitamin K dependent clotting factors. BT, PT, APTT, Factor II, VII, IX, X,Platelet count and fasted serum bile acids were analysed for exclusion of thromobocytopenia which cause of bleeding and bile acid for cholestatic liver disease respectively. DNA sequencing was done for the GCCX and VKOR encoding gene i.e. GCCX-1 and VKORC-1 respectively to understand the etiology at molecular level.

Results: Platelet counts of all the subjects were normal only in one case it was mild low 100x103/ul. Mean± S.E.M value of platelet was 440x103 ± 67. Factor II, VII, IX and X mean levels were 12.7%, 6.9%, 11.2%, 7.1% respectively. Out of ten subjects mean bile acid levels were increased in 3 patients (20 µmole/l) while 3 had low levels (1.2 µmole/l). 3 novel mutations identified in GCCX-1 gene in those patients who had normal levels of bile acids.

Conclusion: Out of ten only in three patients novel mutations were identified in GCCX-1 while other 6 patients had abnormal levels of bile acid may be a cause of vitamin K clotting factors deficiency.Better strategies for the diagnosis of vitamin K deficiency are needed.

Disclosures

Oldenburg: SOBI: Consultancy.
Haemophilia Care in Martinique: From 1982 to 2015
Authors: Pierre-Louis Olivier , Baudin Jacqueline Véronique , Joachim Clarisse Contaret , Marie-Nadiège Yerro , Sabine Molcard , Schved Jean-Francois , Pierre-Louis Serge
Published: November 2019

Introduction: Hemophilia A (HA) and B (HB) are the most common congenital bleeding disorders, characterised by missing clotting factor VIII (FVIII) for HA or factor IX (FIX) for HB, associated with specific mutations for the corresponding genes. This is the first article to focus on a localised specific hemophiliac population in the French West Indies.

Methods: Clinical, biological, genetic and socio-demographic data were collected at the Martinique Regional Centre for the Treatment of Hemophilia.

Results: Nowadays patients with hemophilia and von Willebrand diseases can benefit from clinical and paramedical care comparable to what prevails in mainland France. 130 hemophilia patients were diagnosed in Martinique. This study provides the first global report on epidemiological characteristics of hemophilia in Martinique. Hemophilia treatment center was established in 2000, implementation of care services, number and diagnoses of hemophilia patients, frequency of complications such as inhibitors, HIV and hepatitis C infection, and underlying genetic mutations are described.

Conclusion : These original data support that the French West Indies island of Martinique has the highest rate of HA and HB in the world compared to other countries. The establishment of a treatment center now provides standard of care to this population and may be a model for similar approaches in other French overseas regions.

Disclosures

No relevant conflicts of interest to declare.
The First Registry: Follow-up in Rivaroxaban Patients in the Setting of Venous Thromboembolism
Authors: Lang Kathryn Jane , Solis Vicente , Patel Jignesh , Czuprynska Julia , Roberts Lara N , Patel Raj , Arya Roopen
Published: November 2019

The direct oral anticoagulants (DOACs) are revolutionising the management of acute venous thromboembolism (VTE). Although favourable short term outcomes have been demonstrated with these agents, there is a growing need to improve our understanding of biological and clinical predictors of longer-term outcomes with all DOACs. In addition to reducing the need for dual anticoagulant coverage, the use of oral rivaroxaban, has accelerated the implementation of outpatient and early discharge treatment protocols for VTE, in particular for pulmonary embolism (PE).

Although clinical trials provide evidence of efficacy and safety, there is a need for ongoing assessment of outcomes in unselected populations treated in routine 'real-world' clinical practice. As for most drugs, conditions requiring special attention include advanced age, impaired renal or liver function, extremes of body weight, presence of multiple co-morbidities, and the need for concomitant therapies. Such conditions commonly co-exist, particularly in elderly patients. In clinical practice patients aged 80 years or older and requiring anticoagulation are becoming increasingly common. In addition, trials do not report long-term adverse outcomes after VTE; aside from bleeding, currently there are no long-term data that assess the impact of the DOACs as part of a single-drug treatment algorithm on long-term recurrence.

The FIRST registry will assess long-term outcomes of patients with acute VTE treated with rivaroxaban from diagnosis - our hypothesis is that the long-term outcomes for such patients will be favourable due to consistent anticoagulation intensity, particularly during the acute phase of thrombosis. There are currently no data describing post-thrombotic syndrome (PTS) or chronic thromboembolic pulmonary hypertension (CTEPH) incidence rates in patients treated with DOACs. Patients enrolled onto the FIRST registry will be followed up for five years with primary end-points defined as incidence of long-term sequelae (PTS and CTEPH, defined clinically) and secondary endpoints including recurrence, bleeding and anticoagulation related satisfaction and adherence scores. Analysis of these data may also contribute to improved risk stratification strategies to identify patients who have the greatest risk of adverse bleeding events, recurrence and long-term VTE-related morbidity. Recruitment is currently underway at 20 UK hospitals, with a target of 1500 patients, to be achieved by summer 2017.

Disclosures

No relevant conflicts of interest to declare.
The Role of SMAD7 Downstream of TEL-AML1 Signalling in t(12;21) Acute Lymphoblastic Leukemia
Authors: Sundaresh Aishwarya , Mangolini Maurizio , de Boer Jasper , Hubank Mike , Goulden Nicholas , Williams Owen
Published: November 2019

The single most frequent chromosomal translocation associated with childhood ALL is the t(12;21) rearrangement that creates a fusion gene between TEL (ETV6) and AML1 (RUNX1). Although TEL-AML1+ patients have very good prognoses, relapses occur in up to 20% of patients and many patients face long-term side effects of chemotherapy.

Recent data has shown that TEL-AML1 has a direct role in inducing signal transducer and activator of transcription 3 (STAT3) activation in human t(12;21) leukemia. This activation has been shown to transcriptionally induce MYC and is critical for survival of TEL-AML+ leukemia cells. Here, we demonstrate that STAT3 also regulates SMAD7 gene expression. SMAD7 is an antagonist of TGF-β signaling, functioning through a negative feedback mechanism, but is also known to function in other biological pathways. Interestingly, SMAD7 has also been shown to play a role in promoting self-renewal of hematopoietic stem cells. We show that both pharmacological and mechanistic inhibition of STAT3 results in down regulation of SMAD7 gene expression in TEL-AML1+ cell lines. This result was specific to TEL-AML1+ cells and not found in cells of other ALL subtypes. To understand the role played by SMAD7 in TEL-AML1+ cells, we used lentiviral vectors expressing shRNA targeting SMAD7. Interestingly, SMAD7 silencing was found to inhibit proliferation of TEL-AML1+ cell lines, eventually leading to growth arrest and apoptosis. Furthermore, we have established that this effect is not mediated through TGF-β signalling. This poster highlights the results of RNA-seq performed on TEL-AML1+ cells with SMAD7 knockdown and in vivo xenograft model of SMAD7 shRNA in TEL-AML+ ALL.

Disclosures

No relevant conflicts of interest to declare.
Effect of Expression of Multidrug Resistance Genes on Efficacy the Treatment of Multiple Myeloma By Proteasome Inhibitor
Authors: Chernykh Yulia , Golenkov Anatoly
Published: November 2019

Background: Increased expression of efflux protein, such as P-glycoprotein, multidrug resistance protein, lung resistance protein, breast cancer resistance protein are a major reason for resistance to existing or previously effective therapy multiple myeloma (MM). Effect of the expression of genes of multidrug resistance (MDR) - MDR1, MRP1, LRP, and BCRP on the efficacy of therapy with proteasome inhibitor is currently not well understood.

Aim: Determination of mRNA expression of genes responsible for the development of drug resistance, such as MDR1, MRP1, LRP, BCRP in bone marrow aspirates in patients with newly diagnosed and refractory / relapsed MM before start bortezomib-containing chemotherapy programs.

Materials and methods: We studied bone marrow aspirates of 29 patients (12 men and 17 women) aged 48 to 77 years (median 60 years) with stage 3 MM by classification Durie-Salmon. 14 of studied patients made a group of newly diagnosed MM. 15 patients made a clinically refractory / relapsed group MM. The bone marrow in this group of patients was studied after treatment with alkylating agents at the time of registration of resistance to the given therapy. In the future, all patients were treated by bortezomib - containing chemtheapy regimens. mRNA expression studied genes was determined by semi-quantitative polymerase chain reaction reverse transcription.

Results: In both groups of patients had comparable expression of all studied MDR`s genes. In the group of newly diagnose patients the MDR 1 mRNA expression was detected in 93% (13/14) of the samples, the mRNA of the gene MRP 1 was detected in 79% (11/14). The BCRP mRNA expression was detected in 100% of the investigated samples. The expression of mRNA of the gene LRP was identified in 71% of the studied cases (10/14). In the group of patients after treatment with alkylating agents the expression of mRNA of MDR1 was detected in 87% (13/15) of samples, MRP 1 was detected in 93% (14/15), BCRP was in 100% of cases, and the LRP mRNA was detected in 13/15 (87%) of the samples.Treatment efficacy was assessed by Iinternational Unified Response Criteria for multiple myeloma after the 6 cycles of treatment. Evaluation of treatment efficacy in patients previously non-treated with chemotherapy as well as in the group of clinical resistance patients showed comparable results by the number of objective response rate (57% and 60% respectively).

Conclusions: The expression of MDR genes does not determine the resistance of tumor cells to bortezomib. The proteasome inhibitor bortezomib can overcome the MDR gene activity, attaching it to alkylating drugs increases the sensitivity to them of resistant tumor cells.

Disclosures

Golenkov: Novartis: Consultancy.