bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidemiology and Management of Immune Thrombocytopenia: A Nationwide Population-Based Study in Korea
Authors: Lee Ji Yun , Lee Ju Hyun , Lee Jung-Ok , Bang Soo-Mee
Published: October 2019

The epidemiology of immune thrombocytopenia (ITP) is not well characterized in the general Korean population. ITP patients from 2010 to 2014 were identified using the Korean Health Insurance Review and Assessment Service (HIRA) database. Patients with diagnosis codes for secondary thrombocytopenia or secondary ITP were excluded if those codes coexisted with ITP code of D693.0 or D693.8. The prevalent cases before 1st July 2010 and incident cases from 1st July 2014 were also excluded to calculate the exact incidence of ITP. Overall age-standardized incidence rates of ITP were 5.42/100,000 person-years (95% CI: 5.33-5.51; women, 6.17 [95% CI, 6.03-6.30]; men, 4.69 [95% CI, 4.57-4.81]).The overall incidence rates of ITP appeared to have a bimodal distribution, with peaks among children (< 10 years) and in those > 70 years of age. Among adults, the age-specific incidence rates increased with age for man, but those were observed to be constant for women (figure 1). Overall, 75.2% of patients needed treatment with the median time from the diagnosis to treatment of 1.7 months (range, 0-53.3 months). First-line therapy consisted of steroids and/or immunoglobulin in 83.6%, immunoglobulin only in 14.5%, and other immunosuppressive agents such as cyclosporine A, azathioprine, mycophenolate mofetil, and danazol in 1.9%. Only 1.0% of patients underwent splenectomy and laparoscopic procedure accounted for 90.5% of these 108 patients. The median age was 46 years (range, 9-77 years) at the time of splenectomy, and 77.8% of patients were female. 58.8% of patients received treatment beyond 6months after initial therapy. Among these patients, the most frequently used therapy was steroid and/or immunoglobulin in 92.6% followed by other immunosuppressive agents in 6.9% and immunoglobulin in 0.5%. Platelet transfusions were given 13.7% of patients and the median numbers of platelets transfused were 12 units (range, 1-2,484 units) during study periods. The usage of thrombopoietin receptor agonists or rituximab couldn't be analyzed because these drugs have not been reimbursed for the treatment of ITP before 2016 in Korea. This population-based study firstly describes the incidence rates of ITP and treatment reality for patients with ITP in Korea.

Figure Age/gender-specific incidence of ITP in Korea during the period from mid-2010 to mid-2014. Figure. Age/gender-specific incidence of ITP in Korea during the period from mid-2010 to mid-2014.

Disclosures

No relevant conflicts of interest to declare.
Dosing of Rviii-Singlechain Based on Clinical Bleeding Phenotypes Results in Low Bleeding Rates and Low Consumption in Patients Treated with Prophylaxis Two or Three Times Weekly
Authors: St Ledger Katie , Blackman Nicole , Veldman Alex , Pabinger Ingrid
Published: October 2019

Introduction: Safety and efficacy of rVIII-SingleChain (AFSTYLA) was investigated in two pivotal and one extension study, comprising the Affinity program. The study design and instructions provided to the investigators allowed the physician to freely choose the dose assignment and regimen, based on the FVIII treatment regimen used prior to enrollment, and the subject's clinical bleeding phenotype. Adjustments of dose and schedule were allowed at any time during the study.

Methods: This analysis investigates initial and final dose assignment and the corresponding total and spontaneous bleeding rates in subjects in the adult and adolescent study assigned to prophylaxis two or three times weekly (>80% of the subjects on prophylaxis therapy) in dosing brackets of 20 to <30 IU/kg, 30 to <40 IU/kg and 40 to ≤50 IU/kg.

Results: In the final dose assignment, 43 subjects were assigned a dose of 20 to <30 IU/kg, 62 subjects were assigned a dose of 30 to <40 IU/kg, and 38 subjects were assigned a dose of 40 to ≤50 IU/kg. No differences were observed in the analysis by dose brackets in the adult and adolescent (12-65) population (complete or stratified by age); neither in the initial nor the final dose assignment analysis. The median annualized bleeding rate for the dosing brackets of 20 to <30 IU/kg, 30 to <40 IU/kg and 40 to ≤50 IU/kg was 1.93, 1.05 and 2.21, respectively. The median annualized spontaneous bleeding rate was 0, 0 and 0.50, respectively (final dose assignment).

Conclusions: This data shows that dosing of rVIII-SingleChain based on clinical bleeding phenotypes results in low bleeding rates and low consumption in patients treated with prophylaxis two or three times weekly, with more than 70% of subjects being maintained at doses of <40 IU/kg.

Disclosures

St Ledger: CSL Behring: Employment. Blackman:CSL Behring: Employment. Veldman:CSL Behring: Employment. Pabinger:CSL Behring: Consultancy.
Superimposed Hypercoagulable and Hypocoagulable States: Elevated Von Willebrand Factor (vWF) Levels Secondary to Polycystic Ovary Syndrome Masking Von Willebrand Disease
Authors: Scheitler Kristen M , Coberly Emily A , Hossain A. Mosharraf , Hammer Richard D
Published: October 2019

Von Willebrand Disease (vWD) is the most common inherited bleeding diathesis and can present with easy bruising, cutaneous bleeding, prolonged bleeding from mucosal surfaces after surgical procedures, and menorrhagia. However, laboratory confirmation of vWD can be challenging, as vWF and Factor VIII are acute-phase reactants, and levels will be elevated in inflammation, stress, physical activity, estrogen therapy, and hyperthyroidism.

Polycystic Ovary Syndrome (PCOS) induces a state of both chronic inflammation and hyperestrogenism. PCOS is further characterized by a prothrombotic state, with elevated circulating levels of vWF, fibrinogen, and PAI-1. The precise origin of PCOS-induced hypercoagulability is yet to be elucidated but is most likely multifactorial, with insulin resistance, hyperestrogenism, hyperandrogenism, LH excess, age, and BMI imparting varying degrees of influence and synergism.

We present a case of a morbidly obese 23 year-old female with PCOS presenting with menorrhagia. She has a history of prolonged bleeding after surgical procedures and was self-referred due to concern for bleeding risk during an upcoming dental extraction. Laboratory evaluation revealed she was blood group A positive with a normal PT and aPTT. Evaluation for vWD was within normal limits, with vWF antigen of 84% (55-200%), vWF activity of 68% (55-200%), Factor VIII activity assay of 75% (55-200%), and normal vWF multimers. Factor IX assay was significantly elevated at 218% (60-150%). Thromboelastography (TEG) showed elevated MA and G, consistent with platelet hypercoagulability and increased clot strength.

Subsequent review of remote external records determined that the patient had a previous evaluation for a bleeding disorder at age three following prolonged bleeding after tonsillectomy. At that time, she had a prolonged aPTT of 36.1 sec (21.0-31.0 sec) that corrected after mixing study to 29.9 sec. vWF antigen and ristocetin cofactor levels were both low. Factors VIII, IX, XI, and XII were within normal limits. The patient was diagnosed with type I von Willebrand disease at that time.

PCOS is a hypercoagulable state associated with hyperestrogenism and elevated vWF levels, which may lead to a false negative workup for vWD. In this case, a patient with PCOS and a remote diagnosis of type I vWD had elevation of vWF levels into the normal range on current laboratory studies. The patient's current laboratory studies are consistent with a hypercoagulable state with an elevated Factor IX level and do not reflect the underlying bleeding diathesis. Management of such patients with coexisting hypercoagulable and hypocoagulable states, particularly perioperatively, may be complex.

Disclosures

No relevant conflicts of interest to declare.
Ginkgo Biloba Extract Blunts the Prothrombotic Effect of Thalidomide on Human Umbilical Endothelial Cells
Authors: Lu Rui-Nan , Wang Ling , Zhou Li , Li Jianyong , Lu Hua
Published: October 2019

Patients with multiple myeloma (MM) are at increased risk of developing venous thromboembolism (VTE) compared to general individuals. With the introduction of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide, substantially elevates the incidence of VTE. It was reported that patients with MM have significantly increased plasminogen activator inhibitor (PAI-1) level resulting in a decrease of fibrinolytic activity. To prevent VTE in MM patients receiving IMiDs, different prophylactic strategies such as low molecular weight heparin, aspirin and warfarin have been recommended. But most of those recommendations are based on limited evidence.

Ginkgo biloba extract (GBE) is one of the most widely used herbal supplements, which has been used in thrombosis prevention. It prompts us to investigate the impact of thalidomide on fibrinolysis and whether GBE has affection on fibrinolysis. In this study, the human umbilical vein endothelial cells (HUVECs) were cultured alone or co-cultured with human multiple myeloma cell line (MMCs) in the medium with or without thalidomide and in the presence or absence of GBE. After 48 hours, culture supernatants were collected and protein concentration of t-PA and PAI-1 were analyzed by enzyme-linked immunosorbent assay.

Results showed that MMCs and thalidomide can both altered the profibrinolytic potential of HUVECs by decreasing t-PA and increasing PAI-1 levels. When co-cultured with MMCs, thalidomide can further elevate PAI-1 levels but not further reduce t-PA. Whether HUVECs alone or co-cultured with MMCs, GBE was able to counteract these effects caused by thalidomide through increasing t-PA and decreasing PAI-1 levels.

Results in this study indicate that GBE blunts the prothrombotic effect of thalidomide on HUVECs. It supports GBE clinic use for the prevention of VTE induced by IMiDs.

Disclosures

No relevant conflicts of interest to declare.
Lef1 Is a Critical Mediator of Wnt/β-Catenin Signaling in T-Cell Acute Lymphoblastic Leukemia (T-ALL)
Authors: Giambra Vincenzo , Gusscott Sam , Gracias Deanne , Song Raymond , Weng Andrew P.
Published: October 2019

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy characterized by an uncontrolled proliferation of immature T-cells. While current therapies cure ~80% of pediatric patients, adults fare more poorly with ~40% overall survival. Refractory cases and relapses are presumably due to the ineffective targeting of leukemia stem cells (LSC), previously described in human and in mouse models of T-ALL, and thought to be resistant to standard treatments.

Recently, we have reported that active signaling through the Wnt/β-catenin pathway is a defining feature of LSC in T-ALL, and that interruption of this signaling pathway abrogates disease propagation in vivo. Using an integrated, real-time reporter of Wnt/β-catenin signaling (7TGC; composed of 7 Tcf/Lef-binding sites upstream of a minimal promoter and GFP marker), we identified leukemia-initiating cell (LIC) activity to reside asymmetrically within the minor proportion of Wnt-active, GFP+ cells in primary mouse NOTCH1-induced T-cell leukemias. Moreover, inducible deletion of β-catenin in this context eliminated LIC activity.

Here, we report that LIC activity in this Wnt-active subpopulation is dependent on Lef1. Using Lef1loxP/loxP animals, we show that inducible Cre-mediated deletion of Lef1 in established leukemias extinguishes both Wnt/GFP reporter expression and LIC activity. To explore mechanisms underlying asymmetry of LIC activity within the tumor population, we have also investigated the differential expression of various Lef1 protein isoforms in Wnt-active (GFP+) vs. Wnt-inactive (GFP-) leukemic subsets and assessed their function in supporting LIC activity. These results suggest that β-catenin acts via Lef1 to support asymmetric LIC activity within the Wnt-active subset of leukemia cells.

Disclosures

No relevant conflicts of interest to declare.
High-Expressing Cftr Interacts with HDAC2 to Promote the Development of Ph+ Leukemia through HDAC2-Mediated PENT Pathway
Authors: Yan Tianyou , Gong Yuping
Published: October 2019

The cystic fibrosis transembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily and mediates the transportion of Cl- and HCO3-. Recent studies have reported that CFTR is not only an anion channel protein, but also is used as a regulator to regulate the function of other proteins through interaction with other proteins. Our previous study found that CFTR protein expression in Ph+ acute leukemia cells K562, SUP-B15 and Ph+ primary leukemia blasts was significantly higher than that in normal control cells, and CFTRinh-172 (a CFTR specific inhibitor) had the significant anti-proliferative, apoptosis- inducing and cell-cycle-arrest effect on the CFTR-high expression Ph+ leukemia cells, suggesting that high-CFTR expression might be involved in the incidence and development of Ph+ acute leukemia. At the present study, we found with co-immunoprecipitation (co-IP) and LC-MC assay that HDAC2 protein existed in the fragment pulled down by CFTR antibody in K562 cells; Western Blot assay further confirmed that pretreatment with CFTRinh-172 reduced the expression of HDAC2 protein, but not HDAC2 mRNA in the K562 and SUP-B15: Immunfluorescence analysis testified the co-localization of CFTR and HDAC2 in situ in K562 and SUP-B15 cells; What's more, high expression of HDAC2 protein not mRNA were exhibited in the HEK 273 cell with CFTR cDNA transfect, meanwhile, CFTRinh-172 reduced the expression of HDAC2 protein not mRNA in the CFTR-transfect HEK 293 cells. Moreover, MG132, a proteasome inhibitor, recovered partly the expression of HDAC2 reduced by CFTRinh-172. These results demonstrated that CFTR could interact with HDAC2, and the combination of two proteins protected HDAC2 from the degradation via ubiquitin-proteasome pathway and keep HDAC2 high-expression; Next, Chip/qPCR assay were used to check the relation of HDAC2 and PENT transcript in two Ph+ leukemia cells, the results showed that HDAC2 protein bound in the promote region of PENT gene, and the mRNA and protein of PENT decreased when pre-treated by HDAC2 inhibitor as well as CFTRinh-172, in the meantime, PDK1 and mTOR (down-dream signaling of PENT) activity also decreased. At last, MTT test revealed that CFTRinh-172 raised the anti-proliferation of HDAC2 inhibitor. In conclusion The research has provided the strong evidence that high-expression CFTR plays an important role in the development of Ph+ leukemia through HDAC2-mediated PTEN pathway. The work was supported by the Foundation of the Science & Technology Department of Sichuan Province (NO. 2015SZ0234-5)

Disclosures

No relevant conflicts of interest to declare.
Felipe Samaniego
Authors: Jain Neeraj , Sehgal Lalit , Davis R. Eric , Shuttleworth Stephen Joseph , Samaniego Felipe
Published: October 2019

Background: Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and approximately 30% of the patients develop relapsed/refractory disease that becomes a major cause of mortality and morbidity. Several reports indicated that the BTK inhibitor ibrutinib successfully blocks B-cell receptor signaling and shows clinical benefit in leukemia and lymphomas, including mantle cell lymphoma [23422267] and DLBCL [26193343], for which ibrutinib is FDA-approved. In phase I/II clinical trials, ibrutinib elicited an overall response rate of 68% in patients with relapsed/refractory MCL. However, in spite of these encouraging results, responses are variable and generally incomplete, acquired resistance is common, and recurrence is anticipated [26430726]. We undertook a study of factors underlying acquired ibrutinib resistance (IR) in initially ibrutinib-sensitive DLBCL cell lines.

Methods: IR DLBCL cell lines were generated by continuous culture of parental (PT) cell lines in increasing concentrations of ibrutinib, up to a maximum concentration of 10µM. Once established, IR cell lines were removed from ibrutinib, expanded, and cultured under the same conditions as the PT cell lines for further experiments. Gene expression profiling (GEP) of IR and PT populations was performed on Agilent 4 x 44K gene microarrays.

Results: Of five ABC DLBCL cell lines tested, two (OCI-LY3, U2932) were initially resistant to ibrutinib (IC50>10µM).Three (TMD8, OCI-LY10, HBL1) were initially sensitive (IC50 < 10 nM), but chronic exposure to ibrutinib generated syngeneic versions with IC50 > 5µM. In comparison to PT versions of these cell lines, IR cells did not form clumps in suspension cultures, displayed irregular cell morphology, elevated colony formation ability in methylcellulose matrix, and had higher proliferation rate. Western blots and GEP data showed increased expression by IR cell lines of IAP family members survivin, cIAP2, and oncogenic BCL2 and BCL6. Reduced B-cell receptor signaling, and enhanced PI3K-Akt activity was identified in IR cell lines. Analysis of PI3K isoforms revealed up-regulation of PI3Kα and PI3Kβ with decreased expression of PI3Kδ and PTEN (PI3K negative regulator). Given the enhanced PI3K isoform expression with IR, we treated cell lines with KA2237, a PI3Kβ/δ isoform targeting drug, and observed reduced metabolic activity (survival) of IR cells compared to PT cell lines.

Conclusion: This study highlights that changes in a regulator (PTEN) and mediator (p110β) of PI3K/AKT signaling have important roles in the development of ibrutinib resistance in DLBCL. Treatment with KA2237 may provide a better outcome for ibrutinib-resistant DLBCL.

Disclosures

Samaniego: Karus Therapuetics: Research Funding.
Erythropoietin Signaling Regulates Heme Biosynthesis
Authors: Chung Jacky , Wittig Johannes G. , Ghamari Alireza , Maeda Manami , Lodish Harvey , Bauer Daniel E. , Orkin Stuart H. , Cantor Alan B. , Maeda Takahiro , Dailey Harry A. , Paw Barry H.
Published: October 2019

Heme plays a fundamental role in a diverse array of cellular processes and is required for the survival of all cells. During erythropoiesis, heme production is drastically upregulated to support the production of oxygen-carrying hemoglobin. This increase in heme production is mediated by transcriptional induction of heme metabolism genes including ferrochelatase (FECH), which is the enzyme that catalyzes the rate-limiting insertion of ferrous iron into protoporphyrin IX in the mitochondria of erythroid cells. However, how heme production is coordinately regulated by extracellular cues is currently unknown.

Here, using complementary biochemical and genetics approaches, we show that erythropoietin (EPO) signaling regulates heme biosynthesis via a protein kinase A (PKA)-dependent mechanism. In its inactive state, PKA is a tetrameric complex consisting of two catalytic subunits (C) that are bound to and inhibited by two regulatory subunits (R). The C subunits become activated to phosphorylate downstream target proteins when they dissociate from the R subunits. We demonstrate that EPO-induced JAK2 (janus kinase 2) activity leads to release of the C subunits from the R subunits. We also find that phosphorylated STAT5 (signal transducer and activator of transcription 5) forms a molecular complex with PKA-C. This suggests that phospho-STAT5 can outcompete PKA-R to release PKA-C to directly phosphorylate FECH at a highly conserved threonine residue located in the catalytic site. We examined the importance of FECH phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. Erythroid cells harboring the homozygous Thr115Ala Fech mutation exhibited a block in hemoglobin production and concomitant intracellular accumulation of upstream protoporphyrin intermediates. Strikingly, this phenotype bears resemblance to erythropoietic protoporphyria (EPP), a human hematologic disorder typically associated with FECHmutations.

Together, our results support a model where EPO signaling during erythroid maturation activates PKA by a previously unrecognized JAK2/STAT5-dependent mechanism. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Furthermore, our data implicates aberrant EPO/JAK2/PKA signaling in the pathogenesis of human EPP.

Figure Figure.

Disclosures

No relevant conflicts of interest to declare.
Aspirin-Platelet Sensitivity in Patients with Polycythemia Vera and Human Platelet Antigen Genotype: Which Are Assays Valid?
Authors: Cacciola Emma , Gentilini Cacciola Elio , Cacciola Rossella Rosari
Published: October 2019

Polycythemia vera (PV) is a myeloid neoplasm characterized by thrombotic risk related to high hematocrit (HCT) and to platelet hyperactivation. According to PVSG, the antithrombotic therapy include low dose aspirin (ASA). However, there are insensitive ASA PV patients. It is debated if enherited thrombophilia is responsible to affect platelet activation and, hence, to cause ASA insensitivity. Therefore, we evaluated gene polymorphism human platelet antigen-1 (HPA-1) as thrombophilic indicator associated with abnormal platelet activity, HCT, platelet count, b-TG and PF4, as markers of platelet activation, the platelet functional activity (PFA) and the maximum clot firmness (MCF), as direct and indirect indicators of ASA sensitivity. We studied 40 patients (28 men, 12 women; mean age 64 years, range 35-85 years) with PV according to WHO criteria. Fifty subjects served as controls. The mean duration of disease was 9 years. All patients were on phlebotomy and ASA (100 mg once daily). Platelets and HCT were measured by automated analyzer. b-TG and PF4 were determined by ELISA. PFA and MCF were measured by Platelet Function Analyzer (PFA-100) and by ROTEM delta. Of 40 patients, 24 were homozygous HPA-1a/a and 16 heterozigous HPA-1a/b. The mean HCT value was 47±3% and platelets were 428±180x109/L. All patients had high b-TG and PF4 (113±47 IU/ml vs 20±11 IU/ml and 45±21 IU/ml vs 6±2 IU/ml, respectively), prolonged C/EPI closure time (CT, unit: s, n.v. 84-160 s) (233±65 s) and normal MCF (MCF, unit: mm, n.v. 50-72 mm) (65±5 mm). These findings suggest that PFA-100 and Thromboelastometric assays may be an useful tool to detect ASA platelet sensitivity in PV patients.

Disclosures

No relevant conflicts of interest to declare.
Antibody Response to Pneumococcal Conjugate Vaccine (PCV13) in Chronic Lymphocytic Leukemia Patients Receiving Ibrutinib
Authors: Andrick Benjamin , Alwhaibi Abulrahman , DeRemer David , Quershi Sameera , Khan Rahil , Shenoy Somanath , Pantin Jeremy M
Published: October 2019

Introduction

Due to immune dysregulation and subsequent increased risk of infection associated with chronic lymphocytic leukemia (CLL), both the CDC and ACIP recommend patients with CLL receive the 13- valent pneumococcal conjugate vaccination (PCV13). The effect of ibrutinib, a selective inhibitor ofBruton'styrosine kinase (BTK) which effects B-antigen receptor (BCR) signaling, on IgG antibody responses to pneumococcal vaccination in patients with CLL has not been evaluated. The aim of this study was to evaluate the immune response to PCV13 in patients receiving in ibrutinib and CLL control patients (no active treatment).

Methods

Study patients were enrolled on an IRB approved protocol at Georgia Cancer Center. Specific inclusion criteria a) histological confirmed CLL as defined by WHO classification b) no current therapy (CLL control) c) ibrutinib 420 mg/day (active) d) ECOG ≤ 2. Exclusion criteria a) Previous vaccination with PCV13 within 2 years b) anti-CD20 therapy within last 6 months c) IVIG therapy within 6 weeks. Serum pneumococcal antibody assessment was performed by microsphere photometry on subjects at Day 0 and Day 30 days following vaccination. Antibody specific serotypes (1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23) were determined by ELISA. Pre- and post- vaccination expression of BTK and HACS1 (hematopoietic adapter containing SH3 and SAM protein) of CD19+ lymphocytes was determined by Western Blot analysis. The primary objective was to determine whether concurrent administration of PCV13 in patients receiving ibrutinib generates a ≥ 2 fold increase in ≥ 3 of pneumococcal serotypes as compared to the control group. Secondary objectives were to evaluate expression BTK and HACS in both study groups.

Results

At interim analysis, 9 patients have been enrolled to date, with 8 patients (n=4 ibrutinib, n=4 CLL control) having completed pre and post-vaccination assessments. The median patient age was 69 yo(range: 53-77 yo) with 75% ≥ 65 yo. All CLL control patients (4/4) generated a ≥ 2 fold increase in ≥ 3 of pneumococcal serotypes, whereas (0/4) of ibrutinib patients generated an adequate immune response to PCV13 (p=0.029; Fisher exact). See Table 1 for mean serotype changes in both patient cohorts.

Conclusions

Preliminary data suggests that patients who are receiving ibrutinib group do not generate an effective immune response to PCV13 vaccination compared to untreated CLL controls. This may have an impact on susceptibility and response to infection with pneumococcal infection as well.

Disclosures

No relevant conflicts of interest to declare.
MK2 Is a Therapeutic Target for High-Risk Multiple Myeloma
Authors: Gu Chunyan , Yang Hongbao , Peng Hailin , Xu Zhiye , Chen Ruini , Yang Ye
Published: October 2019

p38 mitogen-activated protein kinase (MAPK) is constitutively activated in human multiple myeloma (MM). However, a major substrate of p38, MAPKAPK2 (MK2) has received little attention in MM. In this study, we disclosed that elevation of MK2 on DNA, mRNA and protein levels in MM cells compared to normal plasma control cells by array-based comparative genomic hybridization (aCGH), Gene expression profiling (GEP) and immunohistochemistry (IHC) respectively. High MK2 expression prognosticated inferior outcome of newly diagnosed myelomas, particularly in patients with high-risk disease based on GEP. Follow-up study demonstrated that enforced overexpressing MK2 promoted MM cell proliferation, drug-resistance and xenograft tumorigenesis in vivo, while knockdown MK2 expression by shRNA abrogated these characteristics. Further mechanism study showed MK2 directly bound and activated AKT signaling in MM, while specific MK2 inhibitor inhibited MM cell growth and clonogenicity through suppressing AKT phosphorylation. Our findings not only indicate that MK2 is a potential therapeutic target in high-risk MM but also suggest the clinical relevance of MK2 inhibition in MM.

Disclosures

No relevant conflicts of interest to declare.
Bendamustine-Bortezomib-Desametasone (BVD) in the Management of Relapsed and Refractory Multiple Myeloma : A REAL-Life Experience
Authors: Cerchione Claudio , Catalano Lucio , Pareto Anna Emanuele , Basile Santina , Marano Luana , Peluso Ilaria , Simeone Luigia , Vitagliano Orsola , Nappi Davide , Palmieri Salvatore , Rocco Stefano , Ferrara Felicetto , Pane Fabrizio
Published: October 2019

Introduction :

Bendamustine is a bifunctional alkylating agent, with low toxicity, proved to be effective in relapsed, refractory and in new diagnosed Multiple Myeloma (MM).

It has been evaluated efficacy and tolerance of Bendamustine, in combination with bortezomib-dexamethasone (BVD) in patients with relapsed and refractory MM (rrMM), whose prognosis is particularly severe. A regional retrospective real-life analysis of patients with rrMM who had been treated with BVD as salvage therapy has been performed.

Methods :

56 patients (31 M/25 F), with rrMM, median age at diagnosis 57.3 years (r. 36-82), median age at start of treatment 61.8 years (r.37-83) treated with several lines of treatments (median 6, r. 2-11), every refractory to all the drugs previously received (also Bortezomib), received BVD (Bendamustine 90 mg/sqm days 1,2; Bortezomib 1.3 mg/sqm days 1,4,8,11, Dexamethasone 20 mg days 1,2,4,5,8,9,11,12, Pegfilgrastim day +4) every 28 days, until progression.

ISS was equally distributed, and cytogenetic was evaluable in 12 patients, and in particular one del13q and one t(11;14). All the patients had previously been treated with schedule containing bortezomib and IMIDs, and 30% had also received radiotherapy. 67% of them had undergone at least to a single auSCT. All patients were relapsed and refractory to last therapies received before BVD.

Results :

Bendamustine was well tolerated, with grade 3 transfusion-dependent anemia in 41% of patients, and 37% grade 3 neutropenia (no ospedalization was required, no septic shocks were observed). No severe extrahematologic toxicity was observed, only grade 1 gastrointestinal side effect (nausea), treated by common antiemetic drugs.

According to IMWG, after a median follow-up of 14 months (r.2-36), ORR was 64% (36/56: 4 CR, 7 VGPR, 16 PR, 9 MR) with 8 PD and 12 patients in SD, which can be considered as an impressive result in this subset of rrMM patients. In particular, for 11 patients, BVD was, after having achieved at least a PR, a bridge to second auSCT, and for two patients a bridge to alloSCT.

Median time to response was 1.2 months (range, 1-3), median OS from diagnosis was 62.7 months (range, 6-151), median OS from start of Bendamustine was 9.8 months (range 2-36).

Conclusion :

BVD has shown significant efficacy in a particularly severe setting of patients, relapsed and refractory to all available therapeutic resources, and, in particular cases, it could be considered as a bridge to a second autologous or allogenic SCT.

Disclosures

No relevant conflicts of interest to declare.
Allogeneic Hematopoietic Cell Transplantation for Lymphoma: Baseline and Post-Transplant Prognostic Factors
Authors: Ko Sun-Hye , Lee Jung-Hee , Lee Je-Hwan , Park Han-Seung , Choi Eun-Ji , Lee Young-Shin , Kang Young-Ah , Lee Kyoo-Hyung
Published: October 2019

Background: Allogeneic hematopoietic cell transplantation (HCT) has been increasingly used for the treatment of lymphoma. The outcomes of HCT in lymphoma depend on baseline patient characteristics including chemosensitivity and International Prognostic Index (IPI). Several studies have shown that post-transplant occurrence of acute or chronic graft-versus-host disease (GVHD) and immune recovery might be associated with the outcomes. In this retrospective study, we investigated baseline and post-transplant prognostic factors in lymphoma patients receiving allogeneic HCT.

Patients and methods: Between May 1998 and December 2015, a total of 61 patients underwent allogeneic HCT for lymphoma and the median age was 39 years (range, 16-62 years). Thirty four patients (55.7%) had chemo-sensitive disease and 24 patients had received autologous HCT. Fifty-six of 61 patients received reduced-intensity conditioning regimens. We evaluated tumor response, overall survival (OS), progression-free survival (PFS), non-relapse mortality (NRM), and event-free survival (EFS) after allogeneic HCT along with potential prognostic factors including GVHD and immune reconstitution.

Results: Objective tumor response after HCT was observed in 41 (67.2%; complete 30 and partial 11). The 5-year probabilities of OS, NRM, PFS, and EFS were 30.7%, 23.5%, 41.3%, and 24.0%, respectively. Among the baseline characteristics, chemosensitivity had a significant impact on OS, NRM, and EFS. Persistent disease status at allogeneic HCT had an adverse effect on OS and low IPI risk category at HCT was associated with longer OS and EFS. HCT co-morbidity index ≥1 was an independent prognostic factor for higher NRM. Grade III-IV acute GVHD was associated with lower OS and higher NRM. Severe chronic GVHD had higher OS (60.0% vs. 19.8%, P=0.002), PFS (68.8% vs.35.9%, P <0.001), and EFS (46.7% vs. 19.1%, P<0.001) than those without severe chronic GVHD. Patients who achieved higher CD3+, CD4+ or CD8+ cell counts on post-transplant 1 month showed significantly higher OS and EFS than those who did not.

Conclusion: Chemosensitivity was the most important prognostic factor in allogeneic HCT for lymphoma. Acute GVHD had unfavorable impact, whereas, chronic GVHD had favorable impact on post allogeneic HCT outcomes. Early immune recovery could predict allogeneic HCT outcomes.

Disclosures

No relevant conflicts of interest to declare.
Opposing Effects of PD-1/PD-L1/L2 Engagement and IFN-γ/TNF-α in the Treatment of AML w/ Anti-CD33 Chimeric Antigen Receptor-Modified T Cells
Authors: Basham Jacob Halum , Geiger Terrence L.
Published: October 2019

Chimeric antigen receptor-modified T lymphocytes (CART cells) have shown benefit as an adjuvant immunotherapy in the treatment of B cell malignancies. This success of re-targeted T cells has not been extended to other hematologic malignancies. We have developed an immunotherapeutic approach to treat acute myeloid leukemia (AML) using CAR T cells re-directed against the myeloid-specific antigen CD33 (CART-33). CART-33 cells are potent and specific in eliminating AML cells in vitro and in vivo. Despite this, CART-33 cells have shown poor in vivo expansion and persistence in NOD-SCID IL2rγ (-/-) (NSG) AML xenograft models. To address the reason for this, we assessed the impact of AML-expressed programmed death ligands 1 & 2 (PD-L1/2) on CART-33 cell activity. PD-L1 inhibits T cell functions upon binding PD-1, which is upregulated with T cell activation. Less is known about PD-L2's effect. Interferon-gamma (IFN-γ), a primary effector cytokine secreted by CD4+ and CD8+ effector T cells, is a known potent inducer of PD-L1 on AML blasts. Using AML cell lines U937, Oci-AML3, CMK, and MV4-11 we show that IFN-γ, TNF-α, and activated CART-33 supernatant can induce up-regulation of PD-L1 and PD-L2 on AML. IFN-γ and TNF-α synergize strongly in up-regulating PD-1 ligands on AML. The kinetics and induction of PD-L2 are distinct from that of PD-L1. Although PD-L1 is well documented to suppress T cell function via ligation of T cell expressed PD-1, induction of PD-L1/L2 had no effect on the cytolytic activity of CART-33 cells against AML in short term (<48 h) cultures. Paradoxically, 24 hr pre-treatment of AML with either IFN-γ or CART-33 supernatant increased AML susceptibility to killing by CART-33 cells despite elevated expression of PD-L1/L2 by AML. Our results highlight the regulatory complexity of AML cytolysis by re-targeted T lymphocytes, and argue that tumor-expressed PD-L1 and PD-L2 impacts the sustainability, but not short-term killing activity, of adoptively transferred CAR T cells in the treatment of AML.

Disclosures

No relevant conflicts of interest to declare.
The Development of a qPCR Assay for the Evaluation of the Dendritic Cell Chimeric Antigen Receptor Transcriptome
Authors: Leyfman Yan , Daniyan Anthony , Brentjens Renier J.
Published: October 2019

Chronic lymphocytic leukemia (CLL) is a hematological malignancy of B cells that accounts for a third of all new leukemia cases, but is incurable with standard first-line chemotherapies. Hematopoietic stem cell transplants offer the only curative treatment but are restricted to those with a matching donor. The recent advent of adoptive transfer therapy utilizing Chimeric Antigen Receptor (CAR) T cells, which are synthetic receptors that combine the target-specificity of antibodies to the effector capabilities of T cells, have emerged as a promising anticancer therapy due to its targeted tumoricidal activity. Clinical trials utilizing CD19-targeted CAR T cells have demonstrated strong efficacy against acute B-cell leukemia, but those performed in CLL have not been as successful for unclear reasons. This may be due to the dysfunctional tumor microenvironment which renders key immune effectors ineffective. Dendritic cells (DCs), a potent antigen presenting cell that functions as a bridge between the innate and adaptive immune systems, are rendered functionally and quantitatively deranged with an impaired ability to present antigen and thereby stimulate immunogenic responses. These limitations can potentially be rectified through an effective recruitment of an endogenous antitumor immune response and thus we hypothesize that genetically modified DCs should be capable of repolarizing the tolerogenic microenvironment induced by CLL toward one of immunogenicity to promote tumor eradication. In this study, we developed a qPCR assay to assess the DC CAR transcriptome. Because our CD19-targeted DC CARs with a CD40 intracellular domain signal via NF-κB to activate downstream elements, we needed to first assess the fidelity of our assay. Using the immortalized mouse DC cell line JAWS II, which lacks CD40, we used a potent TLR4 agonist, MPL, to stimulate our DCs through MyD88, an alternative pathway that also activates NF-κB. We saw elevated expression of downstream NF-κB elements, including IL-6, IL-10, IRF3, and IRF7. Establishing the sensitivity of our assay, we next evaluated the transcriptome of DC CARs by coculturing our CD19-targeted DC CARs containing a CD40 intracellular domain with HEK 293 cells, a human embryonic kidney cell line, retrovirally transduced with a human CD19 antigen to activate our CAR DCs and assess downstream gene expression. We observed expected elevation of downstream NF-κB elements including IL-6, IL-10, NOS2, IRF3, IFN-γ, TNFα, IL12p40, and TGF-β, indicating the viability of this assay. These positive results confirm the multidisciplinary potential of our assay as a promising technology that will allow us to better augment future CARs.

Disclosures

Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Regulation of T-Cell Activation and Differentiation
Authors: Vahedi Golnaz
Published: October 2019

Enhancers regulate spatiotemporal gene expression and impart cell-specific transcriptional outputs that drive cell identity. Super-enhancers (SEs), also known as stretch-enhancers, are a subset of enhancers especially important for genes associated with cell identity and genetic risk of disease. CD4+ T cells are critical for host defence and autoimmunity. Here we analysed maps of mouse T-cell SEs as a non-biased means of identifying key regulatory nodes involved in cell specification. We found that cytokines and cytokine receptors were the dominant class of genes exhibiting SE architecture in T cells. Nonetheless, the locus encoding Bach2, a key negative regulator of effector differentiation, emerged as the most prominent T-cell SE, revealing a network in which SE-associated genes critical for T-cell biology are repressed by BACH2. Disease-associated single-nucleotide polymorphisms for immune-mediated disorders, including rheumatoid arthritis, were highly enriched for T-cell SEs versus typical enhancers or SEs in other cell lineages. Intriguingly, treatment of T cells with the Janus kinase (JAK) inhibitor tofacitinib disproportionately altered the expression of rheumatoid arthritis risk genes with SE structures. Together, these results indicate that genes with SE architecture in T cells encompass a variety of cytokines and cytokine receptors but are controlled by a 'guardian' transcription factor, itself endowed with an SE. Thus, enumeration of SEs allows the unbiased determination of key regulatory nodes in T cells, which are preferentially modulated by pharmacological intervention.

Disclosures

No relevant conflicts of interest to declare.
Surviving Change: The Metabolic Journey of Hematopoietic Stem Cells
Authors: Passegue Emmanuelle
Published: October 2019

Hematopoietic stem cell (HSC) activation is accompanied by mitochondria activation and a shift in metabolic activity from glycolysis to OXPHOS, which provides energy and increases the production of ROS and other mitochondrial metabolites that can act both as signaling molecules and substrates/co-activators for epigenetic enzymes. Metabolically activated HSCs are poised to undergo lineage priming and produce different lineage-biased multipotent progenitors (MPP). However, activated HSCs must also return to quiescence to maintain the stem cell pool. In this context, autophagy plays an essential role by clearing activated mitochondria to allow OXPHOS-driven HSCs to efficiently revert to a mostly glycolysis-based quiescence metabolism. Without autophagy, HSCs display an overactive OXPHOS-driven metabolism that promotes myeloid-biased differentiation and loss of stemness, likely as a direct consequence of epigenetic reprogramming. At steady state, blood production reflects the differential generation by HSCs of a small number of myeloid-biased MPP2/3 and a large number of lymphoid-biased MPP4, which both contribute to myeloid output. In contrast, during blood regeneration, activated HSCs are induced to overproduce MPP2/3, and MPP4 are reprogrammed towards almost exclusive myeloid output in large part due to cytokine stimulations and the triggering of specific regulatory pathways. Altogether, the metabolic activation of HSCs and the remodeling of the MPP compartment represent emergency myelopoiesis pathways that are transiently activated during regeneration, and are continuously triggered in myeloid disease conditions.

Disclosures

No relevant conflicts of interest to declare.
Specialized Ribosomes: A New Frontier in Gene Regulation, Organismal Biology, & Evolution
Authors: Barna Maria
Published: October 2019

The central dogma of molecular biology has for decades served as an explanation for the flow of genetic information within a biological system. In so far as the normal flow of biological information from mRNA to protein, the ribosome has been perceived to decode the genome with essentially machine-like precision; serving as an integral but largely passive participant in the synthesis of all effector proteins across all kingdoms of life. Importantly, a large class of human diseases collectively known as 'ribosomopathies' are characterized by mutations in ribosome components that lead to devastating human conditions including bone marrow failure for which the underlying molecular basis remains poorly understood. In this respect, our research has changed the view that ribosomes carry out largely rote-like functions by demonstrating that not all of the millions of ribosomes within each cell are the same and that ribosome heterogeneity provides a novel means for diversity of the proteins that can be produced in specific cells, tissues, and organisms. I will present our work centered on providing a roadmap for the characterization of ribosome composition at a single cell level and during cellular differentiation. We employed a highly quantitative mass spectrometry-based approach to precisely quantify the abundance of each ribosomal protein (RP) as well as a large cohort of auxiliary ribosome associating factors belonging to actively translating ribosomes within embryonic stem cells. This led to the identification of a subset of ribosomes that are heterogeneous for RP composition. To further address the functional role of ribosome heterogeneity in translational control of the mammalian genome, we employed CRISPR/Cas9 to endogenously tag and purify heterogeneous ribosome populations. We then developed an adapted ribosome profiling method to precisely quantify and characterize the nature of mRNAs translated by distinct heterogenous ribosomes genome-wide. This led to the identification of subpools of transcripts, critical for key cellular processes including cell signaling, metabolism, growth, proliferation and survival, which are selectively translated by specific types of ribosomes. Most remarkably, there are specific metabolic pathways where almost every single component is selectively translated by specialized ribosomes demarcated by a single RP. I will further present recent findings on the mechanisms by which ribosome-mediated control of gene expression is encoded by structured RNA regulons within 5'UTRs. Together, these studies reveal a critical link between ribosome heterogeneity and specialized translational control of the mammalian genome, which adds an important layer of control to the post-transcriptional circuitry of gene regulation and may be critically perturbed in human diseases.

Disclosures

No relevant conflicts of interest to declare.
Nucleic Acids and Protein Metabolism in Acute Leukemia Cells
Authors: GAVOSTO FELICE , MARAINI GIOVANNI , PILERI ALESSANDRO
Published: October 2019

DNA, RNA and protein metabolism was investigated by means of a high resolution autoradiographic technic in normal and acute leukemia blast cells by studying the incorporation of tritiated thymidine, uridine, leucine and phenylalanine. A strikingly lower percentage of cells labelled with thymidine was demonstrated in acute leukemia and was interpreted as evidence of a decreased proliferative capacity. A very significantly lower uptake of uridine, leucine and phenylalanine was detected in acute leukemia cells.

In normal and leukemic cells, amino acid incorporation occurred both in the nucleus and in the cytoplasm; uridine was incorporated exclusively in the nucleus during the first hour of incubation and the cytoplasm became labelled only in a later period.

The constant ratio between uridine and amino acid incorporation detected in normal myeloblasts was always altered in acute leukemia cells.

The lower RNA and protein metabolism and its dissociation in acute leukemia cells was discussed as related to the well-known maturation defect of these cells.
Studies on Agranulocytosis. III. The Reduced Glutathione (GSH) Content of Leukocytes of Normals and Patients Recovered from Agranulocytosis
Authors: PISCIOTTA ANTHONY V. , DALY MARY
Published: October 2019

1. A method for the direct determination of GSH in leukocytes is described. Treatment with alkali (0.5 M. NaOH) effects complete solution of the white cells and after deproteinization (5 per cent HPO3), the GSH is determined by the sensitive "alloxan 305" procedure. Control studies showed that under the conditions of the test, GSH is not affected by the alkali and no splitting of soluble minus SH from protein occurs.

2. Using this method, the amount of reduced glutathione content of normal leukocytes was found to be 5.2 ± 1 mg, per 1010 WBC.

3. No differences from normal levels were detected for the leukocytic GSH of patients with mental disease and those susceptible to agranulocytosis. Incubation of whole blood with the drug which caused agranulocytosis had no effect upon the GSH content of leukocytes.
Serum Protein Binding of Myoglobin, Hemoglobin and Hematin
Authors: WHEBY MUNSEY S. , BARRETT O'NEILL , CROSBY WILLIAM H.
Published: October 2019

Using paper and starch-gel electrophoresis we have demonstrated binding of hemoglobin, myoglobin and hematin by human serum protein which is not a haptoglobin. This protein was present in all sera tested except that from two patients with hemolytic anemia and was also present in serum genetically deficient in haptoglobin.

The binding capacity of this protein is low and contributes only slightly to the total hemoglobin binding capacity of normal serum. However, it may represent most or all of the binding in sera from patients with hemolytic disease.
Au Unusual Lymphomatous Disease Associated with Intracytoplasmic Crystals in Lymphoplasmocytoid Cells
Authors: GOLDBERG ARTHUR F.
Published: October 2019

A case of an unusual lymphomatous disease associated with lymphoplasmocytoid cells containing intracytoplasmic crystals is presented. These crystals are considered to be the secretory product of these cells. They are discussed in the light of recent studies on the secretory activity of plasmocytes and related cells.

A specific diagnosis of the patient's disease is not made although the main cell type present closely resembles that occurring in Waldenström’s macroglobulinemia.