bluuebunny/crossref_metadata_2025 huggingface dataset:
Low Dose Interleukin-7 Supplementation Increases Intrinsic Cord Blood T Cell Survival without Enhancing Proliferative Alloresponses
Authors: Hivert Bánádicte , Pascal Laurent Benjamin , Trauet Jacques , Yakoub-Agha Ibrahim , Labalette Myriam
Published: October 2019

Abstract 4353

Background:

Umbilical cord blood (CB) transplantation is associated with delayed and defective immune reconstitution, in part because recent thymic emigrants, the most abundant subset among CB T cells, have limited intrinsic survival capacity. Interleukin-7 has been reported to increase the initial recovery of the graft-derived T cell compartment. The aim of this in vitro study was to define the optimal supplementation with recombinant human IL-7 (rhIL-7) than can promote the survival of CB T cells without enhancing allogeneic reactions, so as to limit the risk of eliciting an uncontrolled acute GVHD in vivo.

Methods:

Twenty-six CB were obtained immediately after normal-term delivery, using the same procedure as for CB banking, and a freeze-thawing step in order to recapitulate the clinical procedure. CB T cells were cultured for one week alone or with HLA-mismatched monocytes from healthy adults (MLR conditions), in medium supplemented or not with rhIL-7. Cell viability was assessed by flow cytometry by scatter analysis and 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and propidium iodide staining. CB T cell proliferation was assessed by CFSE dilution.

Results:

Under basic culture conditions, a unique high dose of rhIL-7 (1 ng/mL) added on day 0 improved CB T cell survival but also triggered their proliferation. A daily addition of a low dose of rhIL-7 (final concentration of 100 pg/mL) reduced CB T cell apoptosis and increased significantly cell survival after 1 week of culture (p <0.001), without inducing any cell proliferation.

After one week of culture in allogeneic condition, small and large CB T cells were individualized on the cytogramm. Small cells corresponded to nonresponder CB T cells (CFSE dilution < 6,3%), while large CB T cells were allogeneic responder cells which underwent cell division. When allogeneic cultures were run with daily addition of rhIL-7 (100 pg/mL), the number of viable quiescent small CB T cells rose markedly (median 59,279 versus 36,726 without IL-7 supplementation, p=0.01), but the number of dividing cells among the large CB T cells did not increase significantly (median 29, 437 versus 24,471, p=0.19).

Conclusion:

These experimental data show that repeated exposition to low-doses of rhIL-7 can preserve a viable CB T cell compartment, potentially useful in CB transplantation both for T cell reconstitution and T cell recruitment after primary infections. In these IL-7 exposition conditions, recent thymic emigrants could survive better without undergoing an uncontrolled expansion that would be deleterious in increasing the risk of acute GVHD.

These results indicate that clinical low dose IL-7 administration in umbilical cord blood transplanted patients, could improve post-transplant immune reconstitution, without potentiating risk of acute GVHD.

Disclosures:

No relevant conflicts of interest to declare.
Kiler Immunoglobulin Receptor(KIR) Ligands Are Associated with At Least Partial (HLA A*03, B*44) or Poorer (HLA B*51, C*04) Response to Thalidomide Among Patients with Multiple Myeloma Treated in Two Turkish Centers
Authors: Suyani Elif , Bakanay Sule Mine , Dalva Klara , Cakmak Ezgi , Haznedar Rauf , Sucak Gulsan , Beksac Meral
Published: October 2019

Abstract 4904

Aim:

We have previously presented HLA DRB1*13 and 11 tobe associated with response (IMMW, Paris, 2011). In this analysis we have expanded the sample size and have included Killer Immunoglobulin Receptor (KIR) ligands HLA A, Bw4 and C epitopes to further understand the mechanisms underlying this association.

Patients and Methods:

115 patients diagnosed and treated in Ankara and Gazi University Hospitals and who have received a minimum of two months of Thalidomide (T), either alone or within a combination for the treatment of newly diagnosed or relapse myeloma were included. Treatment was given as either part of induction or consolidation: monotherapy (n= 54) or combination (n= 61), at a dose of median 100 mg (50–600 mg) for a median of 7 months (2–114 mo). Following consent HLA typing was done using Sequence-Specific Oligonucleotide primers. IMWG criteria was used for response assessment. Statistics were performed using the SPPS 15. 0 program. The most frequently observed three alleles at each loci and KIR ligands (C epitopes 1, 2 and Bw4) were evaluated to determine effects on response to T. Logistic regression analysis was used for univariateand multivariate analysis to evaluate the factors affecting response to thalidomide.

Results:

Fifty-two (45%) of the patients achieved at least PR. ≥PR and <PR responders were similar in respect to age, time from the diagnosis to initiation of thalidomide, gender, serum β2-microglobulin, albumin, LDH, and creatinin levels, paraprotein characteristics, bone marrow plasma cell ratio, presence of ASCT or allo-HCT, response to induction treatments except for the median number of chemotherapy lines prior to T use which was higher in the less responding group compared to ≥PR responders (p=0. 016). Comparison of these KIR ligand frequencies revealed HLA A*03, B*51, C*04 and DRB1*13 to be associated with response significantly (Tables). The frequency of A*03∼B*44∼DRB1*13, A*03∼ DRB1*13 or A*03∼ B*44, B*44∼ DRB1*13 (contains ≥PR response associated specificities) patients responded frequently:1/1, 7/9, 4/5 or 3/3. Similarly A*11∼B*51∼C*04 or A*11∼B*51∼DRB1*11, A*11∼B*51 or B*51∼DRB1*11 (combining <PR response associated specificities) were not responding as well: 1/4, 0/2, 1/5 or 2/13. The OR for HLA A*03∼DRB1*13, B*51∼DRB1*11, B*51∼C*04, but not C*04 ∼DRB1*11 showed a synergistic effect of the combination (Tables).

Conclusion:

Host related factors such as HLA DRB1 and KIR ligands are found to exert either inhibitory or activating effects on response to T. In addition to the number of previous lines of therapy, HLA, deserves to be included into the list of factors effecting response.

Disclosures:

No relevant conflicts of interest to declare.
Valuation of MIP-1α and MR-Proadm in Serum of Patients with Systemic AL Amyloidosis
Authors: Antolino Giusy , Moscetti Alessandro , Resci Federica , De Benedittis Daniela , Naso Virginia , Bianchi Maria Paola , Festuccia Francescaromana , Salerno Gerardo , Corsetti Maria Teresa , Cardelli Patrizia , Ferranti Giovanna , La Verde Giacinto
Published: October 2019

Abstract 4995

Background.

AL systemic amyloidosis is the most common and lethal form of amyloidosis. Macrophage inflammatory protein-1 alpha (MIP-1α) is a member of the CC chemokine family which is primarily associated with cell adhesion and migration. Adrenomedullin, and more the mid-regional fragment of proadrenomedullin (MR-proADM), comprising amino acids 45–92, have immune modulating, metabolic and vascular actions.

Aims and Methods.

Aim of the study was to evaluate MIP-1α and MR-proADM serum levels in patients with systemic AL λ amyloidosis at presentation to find out potential differences useful to define a characteristic inflammatory pattern. Blood samples were collected from 7 patients with systemic AL amyloidosis (median age 68 yrs) and from 10 age-matched healthy control individuals referred to our Unit and analyzed for serum MIP-1 α and MR-proADM levels. For every patient 1 sample of peripheral blood have been obtained. The blood was separated into plasma at the time of blood draw and frozen to −80°C. Two-group comparisons were performed using the Mann-Whitney U test and paired t test. Correlation analyses were performed using Spearman rank correlation. All statistical tests were two tailed and p < 0. 05 was considered statistically significant.

Results.

Serum MIP-1α levels were significantly higher in AL amyloidosis patients (median 25. 04 pg/mL; IQR 12. 77) compared to the control group (median 2. 54 pg/mL; IQR 0. 34; p=0. 0007). Also serum MR-proADM levels were significantly increased in AL amyloidosis patients (median 1. 15 nmol/L, IQR 0. 6 vs median 0. 42 nmol/L, IQR 0. 18; p=0. 0008). In addition, a positive correlation between MIP-1α and MR-proADM has been observed in the group of patients with systemic amyloidosis (r2=0. 82, p=0. 034).

Conclusions.

The increase of MIP-1α and MR-proADM serum levels in patients with systemic AL amyloidosis at presentation is indicative of active basal inflammation which can contribute to organ damage, in particular heart and kidneys, due to microvascular impairment. On the basis of our results, MIP-1α and MR-proADM could be used as new serum markers of inflammation in AL amyloidosis patients, with possible role in monitoring of organ damage.

Disclosures:

No relevant conflicts of interest to declare.
Five-Years' Follow-up Study for Frequency of Monoclonal Gammopathy of Undetermined Significance (MGUS) in a Korean Elderly Urban Cohort
Authors: Bang Soo-Mee , Lee Jeong-Ok , Song Jung Han , Kim Tae-Hee , Kim Ki Woong , Lee Jong -Seok
Published: October 2019

Abstract 5007

We previously reported the prevalence of MGUS in a Korean Elderly Urban Cohort recruited from 2005 to 2006 (First Wave, Park HK Am J Hematol. 2011;86:752–5). Their plasma samples were screened using immunofixation and free light chain (FLC) assays. Age and gender-adjusted prevalence rates of MGUS were estimated as 3. 3% (95% CI=2. 0–4. 6%), and the age-adjusted prevalence of MGUS was 4. 3% in men (95% CI=1. 9–6. 6%) and 2. 6% in women (95% CI=1. 0–4. 2%). And then we followed them and collected their serum between 2010 and 2011(Second Wave). Among 1, 000 participants of First Wave, 497 participated to the Second Wave and 419 agreed with the donation of serum for protein electrophoresis, immunofixation and FLC assays. Causes of nonattendance were death in 200 (40%), refusal in 197 (40%), move to other area in 69 (14%), and impossible contact in 37 (7%). The frequency of MGUS in Second Wave was 3. 10% (95% CI=1. 44–4. 76%) in all, 4. 27% (95% CI=1. 54–6. 99%) in men, and 1. 92% (95% CI=0. 06–3. 79%) in women. Among 35 MGUS patients in First Wave, 11 were followed. Eight of 11 had persistent MGUS and other 2 showed the disappearance of M protein in Second Wave. The last one showed mild anemia with persistent M protein of 1. 4g/dL suggestive of progression to MM, but was not confirmed because of early death just after Second Wave. Additional 4 MGUS newly developed in Second Wave among 408 persons without MGUS at First Wave. The mean amount of M protein in 13 patients with MGUS was 0. 55g/dL (range: 0. 2∼1. 4). Subtypes of M protein were predominantly A and G in 8 and 5 patients. Light chain was lambda, kappa and none in 8, 4, and 1 patient. Abnormal ratio of FLC was correlated with the presence of MGUS (p=0. 000). In conclusions, the frequency of MGUS is persistently lower in elderly Koreans (3. 1%) than other races.

Disclosures:

No relevant conflicts of interest to declare.
Induction of CRBN(Cereblon) mRNA Expression by Baicalein
Authors: Ma Zi , Zhang Rui bo , He Li , Xu Chao ping , Zhou Ding xin , Ji Jing na , Huang Jianying , Zhang Yicheng , Liu Shang qin
Published: October 2019

Abstract 5025

The CRBN gene that encodes the cereblon protein is found on the short arm at position p26. 3 of human chromosome 3. Cereblon is a primary target of thalidomide teratogenicity and required for the anti-myeloma activity of lenalidomide and pomalidomide. CRBN depletion myeloma cells become highly resistant to both lenalidomide and pomalidomide. Baicalein, a component of Scutellaria radix from HLJDT, not only suppressed proliferation and induced apoptosis of myeloma cells by down-regulating interleukin −6(IL-6) and XIAP gene expression, but also inhibited the signaling cascades mediated by IL-6 and facilitated myeloma cell inhibition induced by dexamethasone. In clinic, we found that treatment of thlidomide- or lenalidomide-resistant myeloma patients by applying Huang-Lian-Jie-Du-Tang (HLJDT) can induce hematological remission. The precise molecular mechanism of HLJDT exerts its anti-tumor effects remains unclear. Here, by RT-PCR, we demonstrated that treatment of U266 cells and primary myeloma cells with 20μM baicalein can induce CRBN mRNA expression in time-dependent manner. As lenalidomide and thlidomide are effective drugs for maintenance therapy with the advantage of oral administration. It was particularly active in patients with higher cereblon expression. Thus, the combination of HLJDT with thlidomide or lenalidomide may be a novel strategy of maintenance therapy for myeloma patients.

Disclosures:

No relevant conflicts of interest to declare.
A Novel DNM2 Mutation Displaying Embryonic Lethality and Impaired Transferrin Uptake Identified in a Mouse ENU Mutagenesis Screen for Genes Perturbing Erythropoiesis
Authors: Brown Fiona C , Collett Michael , Robinson Phillip J , Whisstock James C , Hilton Douglas J. , Kile Benjamin T. , Jane Stephen M , Curtis David J.
Published: October 2019

Abstract 608

Forward-genetic screens have become a powerful method to study the pathogenesis of human disease and gene function. Chemical mutagenesis in mice using the mutagen, N-ethyl N-nitrosourea (ENU), has shown to be highly successful in elucidating novel genes or alleles in a variety of biological pathways, describing new functions of existing genes, and establishing mouse models that accurately recapitulate human disease. Advances in mapping strategies and deep sequencing technologies has dramatically simplified mutation detection, making ENU mutagenesis screens a feasible tool to study specific organ systems. To identify novel alleles regulating erythropoiesis, our laboratory has undertaken a dominant ENU mutagenesis screen. In this screen, the G1 progeny were screened at seven weeks of age for abnormalities in red cell indices (MCV, MCH, and HCT) using an automated hematological analyser. Here, we describe the identification of mice with a missense mutation of the large GTPase Dynamin 2 (DNM2) leading to an amino acid substitution V235G, predicted to lie within the nucleotide binding pocket for GTP. Western blot analysis for DNM2 protein revealed 50% protein levels in heterozygotes, suggesting that the point mutation leads to loss of protein rather than a dominant negative effect. Inherited DNM2 mutations are associated with autosomal dominant Charcot Tooth Myopathy (CTM) and Centronuclear Myopathy (CNM), but no recognised blood disorders. Heterozygous DNM2V235G displayed hypochromic, microcytic anemia – HGB (15 g/dl compared to 16.5 g/dl in wild type mice), MCV (41.3 fl compared to 45.6 fl in wild type mice) and MCH (12.7 pg compared to 14.5 pg in wild type mice), but no obvious neuropathy or myopathy. Homozygosity was lethal before embryonic (E) day 8.5. DNM2 is an essential component in clathrin-mediated endocytosis, which is required for uptake of transferrin into red cells for incorporation of heme. Accordingly, endocytosis assays for transferrin uptake by FACS and confocal microscopy revealed reduced uptake in heterozygotes, explaining the microcytic hypochromic anemia. Western blot analysis for ferritin demonstrated reduced cellular ferritin, indicating cellular iron deficiency. Thus, this mouse model provides the first in vivo evidence that haplo-insufficiency of DNM2 can lead to iron deficiency anemia.

Disclosures:

No relevant conflicts of interest to declare.
Prevalence of Common Thrombophilia and Antiphospholipid Antibodies in Unexplained Infertility Women Undergoing in Vitro Fertilization (IVF)
Authors: Steinvil Arie , Raz Raanan , Berliner Shlomo A. , Steinberg David M , Zeltser David , Levran David , Shimron Orit , Sella Tal , Chodick Gabriel , Shalev Varda , Salomon Ophira
Published: October 2019

Abstract 628

Assisted reproductive technology (ART) is extensively used as a tool for pregnancy achievement in subfertile couples. Congenital and acquired thrombophilia have been suggested by some investigators to play a role in abnormal embryos implantation and placentation. The objective of this study was to assess the role of common thrombophilia in women with unexplained infertility undergoing in vitro fertilization (IVF). We enrolled five hundred ninety-four women from a large healthcare maintenance organization going through IVF and who had a thrombophilia workup, and compared them for prevalence of thrombophilia to two reference groups consisting of 637 fertile women from previous work and 17,337 women members of the same healthcare organization with no history of venous thromboembolisms. The mean age of the women at the first cycle of IVF was 30.9 years (SD: ±4.1).The mean number of IVF cycles was 7.3 (SD: 5.0), and the mean fertility success rate per woman was 14.6% (SD: 19.0).

None of the common thrombophilia tested was found to be significantly associated with the number of IVF cycles or with lower fertility success rate. Rather, women who had APCR and/or factor V Leiden and lupus anticoagulant had significantly higher live birth rates (12.25% and 12.64%, respectively) in comparison to women who were tested negative (8.98% and 9.7%, respectively). Thus, hypercoagulability is not associated with failure to achieve pregnancy. These data suggest that neither screening for thrombophilia nor anticoagulant treatment is indicated in cases with unexplained reproductive failure.

Disclosures:

No relevant conflicts of interest to declare.
RUNX1 Mutations Are the Most Frequent Leukemia Associated Mutations in Congenital Neutropenia Patients
Authors: Skokowa Julia , Steinemann Doris , Makaryan Vahagn , Klimiankou Maksim , Schnittger Susanne , Kohlmann Alexander , Schlegelberger Brigitte , Zeidler Cornelia , Dale David C. , Welte Karl
Published: October 2019

Abstract 7

Congenital neutropenia is a rare inborn heritable disease with a 20% risk to develop acute myeloid leukemia (AML). One of the most frequently mutated genes in de novo as well as in secondary AML is RUNX1. The aim of our study was to investigate whether patients with congenital neutropenia who developed secondary AML (CN/AML) acquire mutations within the RUNX1 gene prior to the development of AML and whether RUNX1 mutations might be involved in leukemogenesis. Mutation analysis was performed using a sensitive next-generation amplicon deep-sequencing assay (454 Life Sciences, Branford, CT) with minimum coverage of at least 200-fold and confirmed by ABI Sanger sequencing. Using these methods we tested 22 patients with CN/AML for the presence or absence of RUNX1 mutations. Fourteen of the 22 patients (63.6%) demonstrated mutations within the RUNX1 gene. The mutation load was between 8 % and 78 % as assessed by the deep-sequencing read counts. Most of the mutations were detected within the Runt homology/DNA binding domain. Four patients have even two RUNX1 mutations. The RUNX1 mutations consisted of 11 missense mutations, 2 nonsense mutations, 4 frameshift mutations, and one ins/del mutation. Of the 14 patients with RUNX1 mutations 12 were tested positive for inherited congenital neutropenia causing mutations: 10 ELANE mutations, one HAX1 mutation, and one G6PT (SLC37A4) mutation. Cooperating mutations were detected in 8 patients, 7 with CSF3R mutations, one with FLT3-ITD mutation. Cytogenetic analysis of the 14 patients with RUNX1 mutations revealed that 4 patients had monosomy 7 and 3 patients trisomy 21. Sequential analysis of the time prior to overt AML revealed one patient harboring RUNX1 mutation already at birth and a second patient had a RUNX1 mutation 6 months prior to AML. The time course of acquisition of RUNX1 mutations in the remaining 12 patients is presently under investigation. The incidence of RUNX1 and CSF3R mutations in at least 7 patients clearly suggests that these two mutations cooperate in causing leukemia. In conclusion, these data show that patients suffering from congenital neutropenia frequently acquire mutations within the RUNX1 gene prior to development of leukemia.

We thank the Investigators of the Severe Chronic Neutropenia International Registry for supplying us with patient material and clinical data.

Disclosures:

Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Dale:AMGEN: Consultancy.
PKCδ Negatively Regulates Primary Megakaryocyte Proliferation and Differentiation
Authors: Kunapuli Satya P. , Kostyak John C
Published: October 2019

Abstract 853

Megakaryocytes are large, polyploid cells that give rise to platelets in the bone marrow and spleen. Megakaryocytes achieve their size and DNA content through a process known as endomitosis via signaling from the cytokine thrombopoietin (Tpo). We have previously determined that the novel Protein Kinase C isoforms theta (PKCθ) and delta (PKCδ) regulate a number of platelet functions including aggregation and secretion. However, the function of these two PKC isoforms in primary megakaryopoiesis has not yet been elucidated. Therefore we chose to utilize primary mouse megakaryocytes from WT, PKCδ−/− and PKCθ−/− mice to characterize the roles of PKCδ and PKCq in megakaryopoiesis. We were first able to determine via western blotting that megakaryocytes express more PKCd than either mononuclear bone marrow cells (p < 0.05) or progenitor cells isolated from bone marrow (p < 0.05). Deletion of PKCδ in mice caused an increase in white blood cell and platelet counts compared to WT mice (p < 005). However, deletion of PKCθ had no effect on murine blood cell counts. Observed increases in platelet counts in PKCδ−/− mice are due to increased platelet production as PKCδ−/− mice contain more thiazole orange positive platelets than WT mice (p < 0.05). Furthermore, we determined via flow cytometry that PKCδ−/− mice had more bone marrow megakaryocytes than WT mice (p < 0.05), although megakaryocyte DNA content was unaltered. Conversely, there was no alteration in megakaryocyte number or DNA content with PKCθ deletion. Interestingly, the increase in bone marrow-derived megakaryocyte count observed in PKCδ−/− mice was heightened following culture of bone marrow cells in 50ng/mL exogenous Tpo (Figure 1). Furthermore, in similar experiments, megakaryocyte DNA content was also enhanced in PKCδ−/− mice compared to WT mice (p < 0.05). PKCδ is an important pro-apoptotic protein, and heightened megakaryocyte number following culture could be due to reduced apoptosis in PKCδ−/− megakaryocytes. However, neither apoptosis nor necrosis was altered with PKCδ deletion as the number of AnnexinV+/7AAD- cells, and the number of AnnexinV+/7AAD+ cells, were not different from WT. Therefore, the observed increases in megakaryocyte number and DNA content could be due to elevated Tpo-induced signaling as ERK1/2 phosphorylation was heightened in PKCδ−/− megakaryocytes compared to WT, in response to exogenous Tpo (Figure 2). These data suggest that PKCδ is an important megakaryopoietic protein, which negatively regulates signaling induced by Tpo in megakaryocytes, while PKCθ is dispensable for primary mouse megakaryopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Exclusion Criteria In The Dasision and Enestnd Trials: Which Could Their Impact Be On The Front-Line Treatment Of a “Real-life” Patient Population With Chronic Myelogenous Leukemia?
Authors: Latagliata Roberto , Carmosino Ida , Vozella Federico , Volpicelli Paola , Loglisci Giuseppina , Santopietro Michelina , Salaroli Adriano , Romano Angela , Montagna Chiara , Roberto Amanda , Serrao Alessandra , Diverio Daniela , Nanni Mauro , Mancini Marco , Tafuri Agostino , Breccia Massimo , Alimena Giuliana
Published: October 2019

The DASISION and ENESTnd controlled clinical trials have changed the front-line treatment of Chronic Myelogenous Leukemia (CML) leading to the advent of dasatinib and nilotinib in this setting: however, both these company-sponsored trials had many exclusion criteria, with a possible selection bias compared to the real-life CML population. To address the impact of these exclusion criteria on the 1st line treatment in the current clinical practice, we revised 207 unselected newly diagnosed chronic phase CML patients [M/F 108/99, median age 58.8 years, interquartile range (IR) 42.3 – 70.2] treated with front-line imatinib from June 2002 to June 2013 at our Institution and evaluated how many of them would have been excluded from enrolment in the 2 trials. Among these 207 patients, 28 patients should have been excluded by both trials due to polycomorbidities (12 cases), severe cardiopathy (5 cases), age > 80 with frailty (3 cases), drug abuse (2 cases), severe liver impairment, Rendu-Osler disease, active prostatic cancer, chronic obstructive broncopulmonar disease (COPD) + peripheral arterial obstructive disease (PAOD), COPD + arrhythmia, refusal to any marrow examination (1 case each). In addition, 8 patients should have been considered not eligible only for the DASISION due to isolated COPD and 19 patients should have been considered not eligible only for the ENESTnd due to isolated diabetes (10 cases), arrhythmia (4 cases), acute myocardial infarction > 6 months before CML diagnosis (2 cases), chronic pancreatic disease (2 cases), PAOD (1 case). On the whole, 36/207 patients (17.4%) would have been considered not eligible for the DASISION trial and 47/207 (22.7%) for the ENESTnd trial. As expected, these patients potentially not eligible for DASISION and ENESTnd were significantly older and with the imatinib treatment had a worse follow-up in terms of Complete Cytogenetic Response (CCyR), Major Molecular Response (MMolR) and Overall Survival (OS) compared to patients potentially eligible, as shown in the Table.

In conclusion, our data highlight that an important fraction of newly diagnosed patients in a real-life setting would have been excluded by the 2 controlled trials whose results are the current mainstay of the 1st line treatment in CML: thus, an automatic transposition of those results into the current clinical practice should be regarded with caution.

Disclosures:

No relevant conflicts of interest to declare.
Efficacy Analysis With Imatinib Monotherapy Was Superior To Allogeneic Hematopoietic Stem Transplantation In Pediatric Patients With Chronic Phase Of Chronic Myelogenous Leukemia
Authors: Jiang Hao , Liu Daihong , Xu Lanping , Liu Kaiyan , Chen Shanshan , Jiang Bin , Jiang Qian , Chen Huan , Chen Yuhong , Han Wei , Zhang Xiaohui , Qin Yazhen , Lai Yueyun , Liu Yanrong , Huang Xiaojun
Published: October 2019

Introduction

Whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) or imatinib should be first-line therapy for childhood chronic myelogenous leukemia in the chronic phrase (CML-CP) is controversial. This study compared imatinib monotherapy and allo-HSCT for the management of CML-CP in pediatric patients.

Methods

This was a retrospective study of children (aged <18 years) with CML-CP, treated between January 2006 and September 2012 at a hospital in China. Patients were treated with imatinib (260 mg/m2 body surface area) or allo-HSCT. Overall survival (OS), event-free survival (EFS) and adverse events (AEs) were compared.

Results

62 patients (40 males, 22 females) were enrolled: 41 received imatinib, and 21 received allo-HSCT. Median follow-up in the imatinib and allo-HSCT groups was 29 and 56 months, respectively. Imatinib was well tolerated. In the imatinib group, the cumulative complete cytogenetic response (CCyR) and major molecular response (MMR) at 24 months were 96.6% (95%CI, 93.3–99.9%) and 85.6% (95%CI, 78.5–92.7%), respectively; patients achieved CCyR and MMR at a median of 3 and 6 months, respectively. In the allo-HSCT group, allografts were from an HLA-matched sibling (n=3), an HLA mismatched/haploidentical familial donor (n=15) or an unrelated donor (n=3). Twelve patients (57.1%) developed acute graft-versus-host disease (grades 2–6 in 7 patients), and 5 deaths were reported. 5-year OS in the imatinib and allo-HSCT groups was 97.1±2.9% and 73.7±10.3% (P=0.032), respectively, while 5-year EFS was 92.5±4.2% and 63.8±11.1% (P=0.041), respectively.

Conclusion

Treatment with imatinib yielded satisfactory cytogenetic and molecular responses, and superior 5-year OS and EFS to allo-HSCT.

Disclosures:

No relevant conflicts of interest to declare.
Somatic and Germ-Line Molecular Characteristics Of Prefibrotic Myelofibrosis
Authors: Barosi Giovanni , Bonetti Elisa , Catarsi Paolo , Massa Margherita , Campanelli Rita , Poletto Valentina , Viarengo Gianluca , Villani Laura , Fois Gabriela , Biamonte Flavia , Rotunno Giada , Guglielmelli Paola , Magrini Umberto , Vannucchi Alessandro M , Rosti Vittorio
Published: October 2019

Prefibrotic myelofibrosis (pre-MF) is a prodromic variant of primary myelofibrosis (PMF) characterized by prevalence of female sex, young age, high frequency of splanchnic vein thrombosis and indolent disease course (Barosi et al, PLoS One. 2012;7(4):e35631). Here we report the molecular characteristics at diagnosis of 177 consecutive pre-MF patients included in the institutional data-base of PMF patients at the Center for the Study of Myelofibrosis in Pavia from January 2001 through July 2013. As compared with patients with fibrotic type-MF (bone marrow (BM) fibrosis= 1 to 3), the frequency of JAK2V617F mutation in pre-MF patients (BM fibrosis=0) was higher (69.2% vs. 60.9%; P=0.02) but the JAK2V617F allele burden was lower (39% vs. 50.6%; P=<0.001). Patients with pre-MF displayed MPL, EZH2 or ASXL1 mutation at a frequency of 2.6%, 0% and 0% respectively, lower than in fibrotic type-MF (frequency: 7.6%, 4%, 17.4%, respectively). Both 45/1 haplotype of the JAK2 gene (rs12343867), and A3669G polymorphism of the glucocorticoid receptor (GR)- rs6198, known susceptibility alleles for PMF, were over-represented in pre-MF with respect to the normal control population (minor allele frequency, 43.3% vs. 24%, and 22.5% vs. 18.2%, respectively; P<0.001 and P= 0.04, respectively). However, the frequency of the variants were not significantly different in pre-MF with respect to fibrotic type-MF (minor allele frequency, 43.4% vs. 46.9%, and 22.5% vs. 27.1%). In pre-MF JAK2V617F mutated patients, a significant correlation between the burden of the V67F allele and the frequency of 46/1 haplotype (rs 12343867) was documented. By dividing JAK2V617F mutated patients in quartiles of allele burden, the frequency of the minor C allele increased progressively from 0.29% to 0.71% (P<0.001, X2 test for trend). No association was ascertained between the burden of the V617F allele and G allele of CR A3669G polymorphism. In conclusion, pre-MF patients segregated preferentially with JAK2V617F mutated genotype with low allele burden. Pre-MF patients were characterized by an almost complete absence of the acquired somatic mutations (EZH2 and ASXL1) that complement the JAK2V617F mutation in the fibrotic type-MF, and that are known to be associated to poor survival and propensity to blast transformation. Pre-MF patients were associated with susceptibility alleles 46/1 and GR A3669G, and the strength of association was not different from that of the fibrotic type-MF patients.

Disclosures:

No relevant conflicts of interest to declare.
Graft Versus Leukemia With Little Graft Versus Host Disease? Long Term Follow-Up Of a Study Using Transduced Donor T-Cells For Donor Leukocyte Transfusion
Authors: Weissinger Eva M , Borchers Sylvia , Ogonek Justyna , Verma Kriti , Ehrlich Steve , Koenecke Christian , Schweier Patrick , Bonini Chiara , Ciceri Fabio , Dammann Elke , Luther Susanne , Krauter Jürgen , Stadler Michael , Ganser Arnold
Published: October 2019

Fourteen patients with myeloid leukemia (12 with acute and 2 with chronic myelogenous leukemia) were transplanted from their HLA-identical (n = 9) or haploidentical (n = 5) family donors with CD34-enriched stem cells (HSCT) without further immunosuppression. In order to induce a graft versus leukemia (GvL) effect and to investigate the possibility of controlling graft-versus-host disease (GvHD), the standard transplantation protocol was adapted to include transfusion of gene-modified donor T-cells after HSCT in 11 patients, 3 did not receive any T-cells.

Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3 which expresses the herpes simplex thymidine kinase (HSV-Tk) as a suicide gene and the truncated low affinity nerve growth factor receptor (ΔLNGFR) for selection purposes. After transfusion, SFCMM-3 transduced T-cells were detectable in all 11 patients by PCR and FACS analyses immediately after transfusion and during the follow up period (range: 1.1-11 years). Two of 9 patients developed acute GvHD: one of the skin, grade 1, 56 days after transfusion of the transduced cells, the other grade II which was successfully treated with ganciclovir. Loss of bcr-abl gene expression was achieved in one patient after expansion of transduced cells. Donor chimerism was stabilized after transfusion of transduced cells in all patients treated. In one patient a cytomegalovirus-reactivation was treated by the transfusion of gene-modified donor T-cells also indicating effectiveness of treatment. To date, 5 patients have relapsed, and died, one after a second HSCT. Two of the patients without transduced T-cells died due to relapse and 7 patients are alive and well and in complete clinical remission.

Thus we have shown that transfusion of transduced T-cells is effective and safe in a long follow-up of 11 years post transfusion.

Disclosures:

No relevant conflicts of interest to declare.
Characterization Of Full-Length, Recombinant Amsh, USP9x, and USP1: Clinically-Relevant Deubiquitinases Involved In Hematological Malignancies
Authors: Russell Nate , Taylor Trevor , Parmar Kalindi , D'Andrea Alan D. , Brasher Bradley B , Melandri Francesco D
Published: October 2019

Attachment of polyubiquitin to substrate proteins generates important biological signaling cues that are inherent to the linkage type of the polyubiquitin chain. For example, K48-linked polyubiquitin chains result in proteasome-mediated degradation of proteins to which they are attached, whereas K63-linked polyubiquitin chains play roles in various intracellular signaling cascades. An important feature of protein ubiquitination is that it is reversible. Substrate-anchored chains may be edited or removed from proteins by highly specialized proteases called deubiquitinating enzymes (DUBs). There are approximately 90 DUBs identified in humans and many have been identified as potential druggable targets because of their involvement in hematological malignancies such as Fanconi Anemia, human follicular lymphomas and diffuse large B-cell lymphomas. DUB activity is regulated by a variety of cues including specificity for a protein substrate(s) to which polyubiquitin chains are conjugated; the presence of protein cofactor(s) that activate or inhibit DUB function; or preference for a specific polyubiquitin linkage type(s). Thus, understanding the mechanisms, regulation, and substrate preferences for deubiquitinases is of great interest, from both academic and clinical viewpoints. To help address these needs, we produced highly purified recombinant deubiquitinase enzymes to facilitate in vitro biochemical studies and drug-discovery efforts. Herein we report the initial characterization of the following, clinically-relevant enzymes:

In conclusion, we have investigated the in vitro substrate preferences and kinetic profiles of three deubiquitinases of clinical relevance. This data will be valuable in the design and analysis of assays used to identify small-molecule inhibitors of these highly specialized proteases.

Disclosures:

Russell: Boston Biochem Inc: Employment. Taylor:Boston Biochem Inc: Employment. Brasher:Boston Biochem Inc: Employment. Melandri:Boston Biochem Inc: Employment.
Genetic Variations In Immune Regulatory Genes and The Risk For Diffuse Large B-Cell Lymphoma
Authors: Kang Jeong-Eun , Kim In-Suk , Kim Nam-Hee , Kim Min Kyoung
Published: October 2019

Introduction

There is growing evidence linking genetic variations of immune regulation genes to non-Hodgkin lymphoma (NHL) etiology. In a large consortial study, it has been recently reported that an increased risk for NHL, especially the major lymphoma subtype diffuse large B-cell lymphoma (DLBCL), with genetic variations in immune regulatory genes that mediate inflammation and autoimmunity. To complement ongoing agnostic approaches for identifying susceptibility genes, we evaluated 57 genetic variations of 7 candidate genes, and their relation to risk for DLBCL in Koreans.

Methods

The case-control series consisted of 192 de novo DLBCL treated at five hospitals throughout Korea from August 2001 through August 2009 and 192 individuals from the population with age and gender matched healthy volunteers. The DNA samples were genotyped using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (Sequenom, Inc., San Diego, CA). We genotyped 11 haplotype-tagging SNPs (htSNPs) of spleen tyrosine kinase (STK), 9 htSNPs of Fc fragment of IgG, low affinity IIa, receptor (FCGR2A), 3 htSNPs of Fc fragment of IgG, low affinity IIIa, receptor (FCGR3A), 15 htSNPs of interferon regulatory factor 4 (IRF4), 9 htSNPs of interleukin 7 receptor (IL7R), 7 htSNPs of interleukin 10 (IL10), and 3 htSNPs of tumor necrosis factor (TNF) genes. We used logistic regression to evaluate the association between genotypes and haplotypes with DLBCL. For DLBCL subtypes, we conducted polytomous multivariate unconditional logistic regression (adjusted for sex, race, age). We calculated P-trends under the co-dominant model for each SNP. The haplotypes were reconstructed according to the genotyping data and the linkage disequilibrium status of these SNPs.

Results

We did not find significant associations between the htSNPs of FCGR2A, FCGR3A, IRF4, IL7R, and TNF genes and the risk of DLBCL. However, the minor allele heterozygotes of the rs3021094 htSNP of IL10 gene (P-trend = 0.028) and the rs2991216 htSNP of the SYK gene (P-trend = 0.035) showed an increased risk of DLBCL. On 10-million permutation testing, the haplotype including rs3021094 variant allele of IL10 gene was significantly associated with an increased risk of DLBCL (P = 0.001), however, the haplotype including rs2991216 variant allele of STK gene was statistically insignificant (P = 0.055).

Conclusions

This study presents several novel aspects of the genetic susceptibility to develop DLBCL. Although this study did not show statistically significant association between STK htSNPs and risk for DLBCL on 10-millinon permutation testing, we demonstrated that genetic variants and haplotypes of IL10 gene showed the significantly increased risk for DLBCL. Larger studies that focus on the role of the STK and IL10 genes will support to confirm causal variants to develop diffuse large B-cell lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Impact Of Beta-2 Microglobulin In Patients With Non-Gastric Marginal Zone Lymphoma
Authors: Yoo Changhoon , Yoon Dok Hyun , Kim Shin , Huh Jooryung , Park Chan-Jeoung , Lee Sang-wook , Suh Cheolwon
Published: October 2019

Background

Although serum Beta-2 microglobulin (B2M) has been suggested as a prognostic factor for several hematologic malignancies, this was rarely investigated in marginal zone lymphoma (MZL).

Materials

Between January, 2000, and May, 2013, a total of 270 patients with non-gastric (NG)-MZL were identified from database of Asan Medical Center, Seoul, Korea. Among them, pretreatment baseline serum B2M was available in 204 patients. Progression-free survival (PFS) and overall survival (OS) were compared according to the level of B2M with cut-off value of 2.5 mg/L.

Results

Median age of study population was 51 year-old (range, 16-81) and 85 (42%) patients were male. Thirty (15%) patients had nodal MZL and 174 (85%) had extranodal MALToma. B2M ≥2.5 mg/L was related with more adverse clinical features, such as poor performance status, ≥2 extranodal sites, advanced stage (III-IV), anemia, elevated LDH, bone marrow invasion, and higher IPI risk group. In univariate analysis, serum B2M (<2.5 mg/L vs ≥2.5 mg/L) was significantly associated with PFS (p<0.001) and OS (p<0.001). In multivariate analysis including B2M and International Prognostic Index (IPI), serum B2M ≥2.5 mg/L was an independent adverse prognostic factor in terms of PFS (hazard ratio [HR]=3.7, 95% CI, 1.5-9.1; p=0.005) and OS (HR=7.5, 95% CI, 1.7-32.7; p=0.008). In analysis including Korean MZL Prognostic Index (MZLPI: nodal MZL, ECOG performance status ≥2 and advanced stage), B2MG ≥2.5 mg/L was also significant for PFS (HR=3.5, 95% CI, 1.5-7.7; p=0.003) and OS (HR=7.3, 95% CI 1.9-28.2; p=0.004).

Conclusion

In patients with NG-MZL, baseline serum B2M is a powerful prognostic factor for PFS and OS, independent of validated prognostic indexes, such as IPI and MZLPI.

Disclosures:

No relevant conflicts of interest to declare.
FDC Like Cell Line HK With IL-2, IL-4 and OX40 Ligand Supports The Growth Of ATLL Cells
Authors: Kagami Yoshitoyo , Chihara Dai , Kato Harumi , Yoshida Noriaki , Kinoshita Tomohiro , Seto Masao
Published: October 2019

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive neoplasm derived from CD4+ T-cells with HTLV-I infection, and its mechanisms of tumorigenesis still remain to be elucidated. The fact that tumor cells rarely proliferate in vitro is one of the most important problems to be solved. The establishment of cell line from ATLL patient samples has been difficult even in the presence of interleukins. Previously we established one cell line (HU-ATTAK) from acute or lymphoma types of 10 ATLL cases which did not proliferate in the presence of IL-2 and/or IL-4. HU-ATTAK is critically dependent on IL-2 and human umbilical cord vein endothelial cells (HUVEC) as feeder cells. In HU-ATTAK, adding anti-OX40 ligand antibody into the culture system completely inhibited its proliferation. So, OX40 ligand as well as L-2 and/or IL-4 is suggested be necessary for the proliferation of ATLL cells, and feeder cells may also confer the favorable environment. As the substitute of HUVEC, follicular dendritic cell like cell line HK which expresses OX40 ligand was used by introducing human OX40 ligand cDNA (HK-OX40L). When 9 ATLL patient samples were co-cultured with HK-OX40L in the presence of L-2 and/or IL-4, two ATLL cells proliferate vigorously only in the presence of both IL-2 and IL-4 simultaneously. These cell lines were confirmed to be derived from original tumor cells by array CGH analysis, and continued the growth for more than a year. Depletion of IL-2 and IL-4 made these cell lines stop growing within 6 days even on HK-OX40L. In the presence of IL-2 and IL-4, the conditions such as HK alone without OX40 ligand or OX40 ligand alone without HK made the cell lines growing for three months at most. In the presence of IL-2 and IL-4 without HK-OX40L, these cell lines vigorously proliferated for more than three months but finally stopped growing. These data suggested that for the growth of these two cell lines, the cell division is dependent on IL-2 and IL-4, and the maintenance of immortalization is dependent on OX40 ligand and HK cells. This culture analysis would provide important factors for cell growth of ATLL which will explore new targets for ATLL treatment.

*HK cells are kindly provided by Dr. Young S Choi at Ochsner Cancer Center, New Orleans.

Disclosures:

No relevant conflicts of interest to declare.
Unmasking Lymphoma Immune Reconstitution Inflammatory Syndrome Among HIV-Infected Individuals In The Center For AIDS Research Network Of Integrated Clinical Systems
Authors: Gopal Satish , Patel Monita , Yanik Elizabeth , Burkholder Greer , Reid Erin , Rodriguez Benigno , Deeks Steven , Mayer Kenneth , Moore Richard , Kitahata Mari , Eron Joseph , Richards Kristy L.
Published: October 2019

Introduction

Cohort studies have demonstrated increased incidence of Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) among HIV-infected individuals during the first 6 months after antiretroviral therapy (ART) initiation, perhaps due to unmasking immune reconstitution inflammatory syndrome (IRIS). Unmasking IRIS is characterized by the diagnosis of a new HIV-associated condition soon after ART that was not apparent prior to ART, coupled with evidence of ART effectiveness. It has been well described for opportunistic infections and Kaposi sarcoma. However, clinical characteristics and survival for unmasking lymphoma IRIS have not been described.

Methods

We studied lymphoma patients in the Centers for AIDS Research Network of Integrated Clinical Systems (CNICS) from 1996 until 2011. Unmasking lymphoma IRIS was defined as HL or NHL occurring within 6 months after ART initiation accompanied by a ≥0.5 log10copies/mL reduction in HIV RNA between values taken prior to ART and at lymphoma diagnosis. Differences in presentation and survival were examined between lymphoma IRIS and non-IRIS cases.

Results

Of 482 lymphoma patients, 48 (10%) met criteria for unmasking lymphoma IRIS. Of these, 10 (21%) had HL, 19 (40%) diffuse large B-cell lymphoma (DLBCL), 4 (8%) Burkitt lymphoma (BL), 9 (19%) primary central nervous system lymphoma (PCNSL), and 6 (12%) other NHL (Table). Median CD4 cell count at lymphoma diagnosis among IRIS cases was 163 cells/mL (interquartile range 67-302), and 54% had suppressed HIV RNA (< 400 copies/ml). No significant differences were identified between lymphoma IRIS and non-IRIS cases, with the exception of possible earlier stage (47% stage I/II versus 24%, p=0.03), more frequent hepatitis B/C co-infection (31% versus 19%, p=0.05), and more frequent prior AIDS illness (92% versus 79%, p=0.05), as well as expected lower HIV RNA at lymphoma diagnosis likely resulting from the IRIS case definition. Additionally, no differences in cumulative mortality 5 years after lymphoma diagnosis were identified between IRIS and non-IRIS cases, although there was a suggestion of increased early mortality among IRIS cases (Figure).

Conclusions

In a large HIV-associated lymphoma cohort in the United States, 10% of patients met a standardized unmasking lymphoma IRIS case definition. No major differences between lymphoma IRIS and non-IRIS cases were identified. Possible effects of subclinical lymphoma leading to care seeking behavior and subsequent ART initiation prior to lymphoma diagnosis could not be excluded.

Disclosures:

No relevant conflicts of interest to declare.