bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-4 Expression and Constitutive Activation of Erk Signaling In Vivo in Follicular Lymphoma.
Authors: Calvo Katherine R. , Dabir Bhavana , Bandle Russell , Bond Amelia , Jaffe Elaine S.
Published: October 2019

Follicular lymphoma (FL) is characterized by constitutive expression of the Bcl-2 oncoprotein as a consequence of the t(14;18) BCL2/JH translocation in germinal center B-cells. While Bcl-2 confers survival advantage through anti-apoptosis mechanisms, the t(14;18) alone is not sufficient for lymphomagenesis. Evidence suggests that additional factors in the lymph node microenvironment, related to tumor infiltrating T-cells and macrophages, may cooperate with Bcl-2 in orchestrating the malignant phenotype in vivo. In this study we generated proteomic cytokine profiles of patient lymph node tissue lysates from FL (N=53) grades 1–3, and the non-malignant normal counterpart follicular hyperplasia (FH; N=24). Using an ultrasensitive multiplex elisa system we assayed a panel of 11 cytokines known to be secreted by T-cells and/or macrophages including IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-12p70, TNF-alpha, and INF gamma. With the exception of IL-4, all cytokines measured showed higher concentrations in FH than FL. In particular, IL-1 beta demonstrated levels 20 times higher in FH; IL-8 levels were 30 times higher; and IL-13 levels were 9 times higher in FH than in FL (p values < 0.005). In contrast, IL-4 levels were 5 times higher in FL than in FH (p < 0.0054). Interestingly, IL-4 levels were highest in FL grade 1 (N=20) in comparison to grade 2 (N=18) and grade 3 (N=12). Immunohistochemical stains for CD3 showed no significant difference in the numbers of CD3+ T-cells in FL samples over FH lymph nodes; or in FL grade 1 vs. grade 2 or grade 3. Hence the differences observed are not due to numerical differences in the total number of T-cells present in FL and FH. We evaluated the activation state of the mitogen activated protein (MAP) kinase signaling pathway, which is activated in response to stimulation by most cytokines in our panel via PI3Kinase. Western blot analysis showed that Erk was constitutively phosphorylated in 14 out of 15 (93%) of FL cases in contrast to only 3 out of 9 (33%) of FH cases. These findings demonstrate that tumor growth in FL is characterized overall by low levels of cytokines within the lymph node microenvironment in comparison to growth associated with the benign counterpart FH. The T-cell secreted cytokine, IL-4, which drives B-cell proliferation, was the only cytokine that showed statistically significant higher expression in the lymph node microenvironment of FL in comparison to FH. Moreover, the intracellular MAP kinase signaling pathway was constitutively activated in vivo in patient FL samples as compared to FH, despite higher overall cytokine levels in FH. These findings suggest that the IL-4 cytokine, and intracellular Erk activation in FL cells, may 1) play a role, in cooperation with Bcl-2, in driving the malignant phenotype in FL and 2) may serve as effective molecular targets for future therapies in FL.
Transferrin Acts as an Iron Sensor Upstream of BMP2b in the Zebrafish.
Authors: Fraenkel Paula G. , Burnett Sarah , Holzheimer Jason , Dooley Kim A. , Wingert Rebecca , Hammerschmidt Matthias , Zon Leonard I.
Published: October 2019

The BMP pathway is important in modulating expression of the iron regulatory hormone hepcidin in response to iron, however how iron levels are sensed is unknown. The zebrafish mutant gavi was identified in a large-scale chemical mutagenesis screen for embryonic anemia. Two alleles were identified: gaviHE067 and gaviIT029, both of which have a homozygous recessive pattern of inheritance. Sequencing of transferrin cDNA from the mutants revealed that each allele of gavi produces an aberrant message and virtually absent transferrin expression. Our previous work demonstrated that hepcidin expression is upregulated following iron dextran injection of the zebrafish embryo. This effect was not impaired by a defect in ferroportin1 (Fraenkel et al., (2005) J Clin Invest115:1532–1541), an iron exporter and ligand for hepcidin. To evaluate the effects of iron loading on transferrin-deficient mutants, gaviIT029 homozygotes at 48 hours post-fertilization were either anesthetized and injected with iron dextran, or anesthetized but not injected. GaviIT029 homozygote embryos exhibited low levels of hepcidin expression that failed to increase following iron injection. Morpholino knockdown of transferrin or transferrin receptor 2 abrogated hepcidin expression, even following iron injection, while knockdown of transferrin receptor 1a (erythroid-specific transferrin receptor 1) or transferrin receptor 1b (a ubiquitously expressed form of transferrin receptor 1) failed to produce this effect. Overexpression of BMP2b, however, rescued hepcidin expression following transferrin knockdown. These findings provide the first evidence that transferrin acts as an in vivo iron sensor upstream of the BMP pathway.
A Randomized Comparison of Total-Marrow Irradiation, Busulfan and Cyclophosphamide with Tandem High-Dose Melphalan in Patients with Multiple Myeloma.
Authors: Knop Stefan , Bauer Katja , Hebart Holger , Wandt Hannes , Trumper Lorenz , Liebisch Peter , Maschmeyer Georg , Peest Dietrich , Wolf Hans H. , Kroger Nikolaus , Straka Christian , Pfreundschuh Michael , Coser Paolo , Meisner Christoph , Kanz Lothar , Einsele Hermann
Published: October 2019

Background Myeloablative chemotherapy with support of autologous peripheral blood stem cells (APBSC) has widely been accepted as a standard of care in patients (pts) with newly diagnosed multiple myeloma (MM). High-dose (HD) melphalan (Mel) 200 mg/m2 was considered superior to total-body irradiaton (TBI) plus Mel 140 mg/m2 for toxicity reasons. Since MM plasma cells are inherently responsive to irradiation, our group evaluated TBI aimed at reduced organ toxicity by shielding lungs and liver (total-marrow irradiation [TMI], 9 Gy) combined with busulfan (Bu, 12 mg/kg) and cyclophosphamide (Cy, 120 mg/kg) in a previous phase I/II trial (Einsele et al, Bone Marrow Transplant, 2003).

Patients and methods In the current study (DSMM I), subjects with previously untreated MM in Durie-Salmon stages II/III were randomly assigned to either receive one course of TMI/Bu/Cy versus two cycles of HD Mel 200 mg/m2 each with APBSC transplantation if having had an adequate number of stem cells collected and at least stable disease. Primary end point was event-free survival (EFS), secondary end points overall (OS) and disease-free survival (DFS).

Results A total of 294 pts (median age, 54 years), 246 of whom completed stem cell harvest were enrolled between 8/1998 and 1/2002 by 46 centres. Eventually, 198 (n=100 TMI/Bu/Cy and n=98 HD Mel) pts were randomized and included into the ITT population. The safety population (n=80 TMI and n=118 HD Mel, due to 18 pts switching to Mel) was analyzed for toxicity and response. CR rate before HD therapy was 7.0% (7/100) in the TMI and 6.1% (6/98) in the Mel arm respectively. Significantly more pts receiving TMI/Bu/Cy experienced WHO grades 3 and 4 pulmonary and gastrointestinal toxicity and pain. Following HDT, CR rate increased to 17.5% (14/80, TMI) and 32.2% (38/118, HD Mel; p=.022) respectively. After a median follow-up of 1447 days, median EFS in the TMI group was 1161 days versus 1090 days for HD Mel (p=.812). Probability of 4-year OS was 72.7% (95%-CI: 62.1–80.7) with TMI and also 72.7% (95%-CI: 61.7–81.1) after HD Mel (p=.754). For pts in CR following HD therapy, probability of 4 year DFS was 62.4% (95%-CI: 33.6–81.6) for TMI vs. 50.4% (95%-CI: 30.6–67.3) for HD-Mel (p=.138).

Conclusion In this randomized trial on pts with newly diagnosed MM, the irradiation-based regimen was associated with more pulmonary and GI toxicity when compared to HD Mel. Incidences of other toxicities including hepatotoxicity, however, were not different between the two treatment arms. CR rate was superior for HD-Mel, while there was no difference in OS and EFS between the two treatment arms. Subjects achieving CR may be more likely to enjoy prolonged DFS after TMI/Bu/Cy.
Clinical Relevance of a Pharmacogenetic Approach Using Multiple Candidate Gene Polymorphisms To Predict Response and Resistance to Imatinib Mesylate Therapy in Chronic Myeloid Leukemia.
Authors: Kim Dong Hwan (Dennis) , Sriharsha Lakshmi , Xu Wei , Kamel-Reid Suzanne , Liu Xiangdong , Siminovitch Katherine , Messner Hans , Lipton Jeffrey
Published: October 2019

Background: Imatinib resistance (IR) is a well known cause of treatment failure in chronic myeloid leukemia (CML) patients being treated with imatinib mesylate (IM). Several cellular and genetic mechanisms of IR have been proposed including amplification and overexpression of the BCR-ABL gene, the presence of specific point mutations and MDR1 gene overexpression.

Methods: We investigated the impact of 16 single nucleotide polymorphisms (SNPs) in 5 genes potentially associated with pharmacogenetics of IM (ABCB1, multidrug resistance 1; ABCG2, breast-cancer resistance protein; CYP3A5, cytochrome P450 3A5; HOCT1, human organic cation transporter 1; AGP1, alpha-1-acid glycoprotein-1, plasma protein binding to IM). The major endpoints included:

response to IM: cytogenetic (CyR) or molecular response (MoR); resistance to IM: loss of response (LOR), treatment-failure (including primary resistance or LOR); progression to accelerated phase (AP) or blast crisis (BC), or death; and need for IM dose escalation to overcome resistance or LOR. The DNAs from peripheral blood samples were genotyped using the Sequenom MassARRAY system based on MALDI-TOF technique.

The study population included 229 patients whose clinical outcomes following IM therapy were evaluated from January 2000 to January 2007 (male:female 96:133; median age at start of IM, 53 years-old; white:non-white 170:59; chronic phase:AP:BC, 199:23:3).

Results: The frequencies of genotypes in 16 SNPs are summarized in Table 1. The GG allele in ABCG2 (rs2231137), AA allele in CYP3A5 (rs776746) and advanced stage were significantly associated with poor response to IM, while GG allele at HOCT1 (rs683369) and advanced stage correlated with high rate of LOR or treatment failure. The CC allele in ABCG2 (rs2231142) was also identified as an independent predictor of more frequent need for IM dose escalation. The results of multivariate analyses are summarized in Table 2.

Conclusion: Using a novel, multiple candidate gene approach based on the pharmacogenetics of Imatinib mesylate, we identified several SNP candidates in patients with CML that are potential predictors of clinical response to IM (rs2231137, ABCG2 or rs776746, CYP3A5), resistance to IM (rs683369, HOCT1) and need for IM dose escalation (rs2231142, ABCG2). Further studies are warranted to validate the role of these SNPs in the early identification of individuals with CML who may not respond optimally to standard IM therapy.

Table 1. The genotype frequency and clinical significance in 16 SNPs Table 2. Multivariate regression model based on Cox's proportional hazard model for each-point
Th-17 Lymphocytes, Not Regulatory T Cells, Mediate Immune Regulation of Bone Marrow Infiltrating Lymphocytes and Induce Osteoclast Activation in Multiple Myeloma.
Authors: Noonan Kimberly A. , Anderson Judy , Mgebroff Stephanie , Serafini Paolo , Roodman David , Borrello Ivan
Published: October 2019

The bone marrow of myeloma patients is a reservoir of marrow infiltrating lymphocytes (MILs) that upon activation with anti-CD3 and anti-CD28 antibodies show significant tumor specificity against both mature myeloma plasma cells and their clonotypic precursors, whereas activated peripheral blood lymphocytes (PBLs) fail to demonstrate any tumor specificity. Here we examine the major differences between MILs and PBLs of both myeloma patients and normal donors. In myeloma, CD4+/CD25+ MILs lack a regulatory T-cell (Treg) phenotype as shown by the absence of FoxP3 expression and the inability to suppress tumor-specific proliferation of effector T cells. In contrast, the CD4+/CD25+ PBLs express FoxP3 and suppress tumor specific proliferation. Analysis of MILs and PBLs from normal individuals reveals the exact opposite (more Tregs in the marrow than blood). Considering the abundance of plasma cell-derived IL-6 in myeloma marrow, we hypothesize that IL-6 skewing of MILs towards the effector Th17 phenotype could explain the reciprocal paucity of Tregs in myeloma and relative abundance in normal bone marrows. Intracellular staining shows increased expression of IL-17 in myeloma CD4 MILs (2.7%) as compared to either myeloma PBLs (0.02%) or normal MILs (0.7%). The Th17 cytokine secretion pattern also appears more pronounced in myeloma-derived MILs as compared to either myeloma PBLs or normal MILs respectively (see table). Th17 T cells have recently been implicated in osteoclastogenesis of rheumatoid arthritis. In fact, bone marrow serum IL-17 levels correlate with the degree of lytic bone disease. In experiments utilizing M-CSF and RANK-L as the osteoclast differentiation medium, we show that the addition of IL-17 to the culture medium increases the number of mature osteoclasts by 2.3 fold whereas γIFN reduces mature osteoclast numbers by 80%. These findings explain the profound differences seen in MILs of normal and myeloma patients and implicate Th17 MILs in the generation of lytic bone disease. Activation of myeloma MILs with anti-CD3/CD28 skews the population from Th17 to a γIFN-producing Th1 phenotype. As such, targeting Th17 T cells or utilizing adoptive T cell therapy with activated, γIFN-producing MILs may represent a novel therapeutic mechanism to target osteoclastogenesis and reduce bone disease in myeloma.

Th-17 Cytokine Profile N=56 MM MILs MM PBLs NL MILs IL-6 (pg/ml) 30 3.7 6.5 IL-23 (pg/ml) 246 19.6 35.9 IL-17 (pg/ml) 12.2 0.4 5.1
Isoprostane (8-Epiprostaglandin F2α) Plasma Concentration and Aspirin Resistance in Patients after Ischaemic Stroke.
Authors: Zawilska Krystyna M. , Zytkiewicz Marcin M. , Hanszke Ewelina E. , Turowiecka Zofia Z. , Gielwanowska Liwia L. , Psuja Piotr P.
Published: October 2019

Background The limited efficacy of secondary prevention for ischaemic stroke may be partially related to aspirin resistance leading to continuous generation of intraplatelet thromboxane A2. Besides other underlying metabolic mechanisms, an oxidant stress along with nonenzymatic biosynthesis of isoprostanes supporting the platelet activation has been suggested. Aims We analyzed the incidence of aspirin resistance in survivors of ischaemic stroke and compared the usefulness of some platelet tests designed for its laboratory exploration. Also we searched for relationship between isoprostane concentration and results of platelet function tests.

Material and Methods Forty four patients, at least a month after acute onset of ischaemic stroke were included into the study. All of them have been receiving 75–150 mg aspirin daily at least for a month. The control group for 11-dehydro Thromboxane B2 measurements consisted of 12 healthy volunteers. The platelet function was investigated by platelet aggregation tests induced by either ADP (3.5 and 5.0 μM), collagen (2 μg/ml) or arachidonic acid (AA) (0.6 mM) and by measurement of closure time in PFA-100 analyzer. Thromboxane A2 metabolite - 11-dehydro Thromboxane B2 (11-dTxB2) and isoprostane - 8-epi Prostaglandin F2α (8-epiPgF2α) plasma concentration by immunoenzymatic method (EIA Kits from Cayman Chemicals) were also determined. The aspirin ingestion was controlled by diminished intraplatelet concentration of malonyldialdehyde. Aspirin resistance has been determined by the following criteria: the intensity of platelet aggregation induced by ADP >60%, collagen >70%, AA >20%, PFA-100 closure time <165 s and 11-dTxB2 concentration >mean of the control group minus SD.

Results Table 1 Statistically significant inverse correlations have been found between the plasma concentration of 11-dTxB2 and PFA-100 (Col/Epi) closure time (r= −0.31; p= 0.039) as well as between plasma concentration of 8-epiPgF2α and PFA-100 (Col/Epi) closure time (r= −0.36; p= 0.019).

Summary/conclusions

Laboratory platelet function tests reveal aspirin resistance in almost half of patients after ischaemic stroke. The most significant correlation has been found between plasma concentration of 11-dehydro Thromboxane B2 and PFA-100 closure time. An important interrelationship observed between PFA-100 closure time and plasma concentration of 8-epi Prostaglandin F2α may support the hypothesis of nonenzymatic production of isoprostanoids with platelet proaggregatory activity, playing a role in aspirin resistance.

The frequency of aspirin resistance in patients after ischaemic stroke ADP 3.5μM ADP 5.0μM Collagen Arachidonic acid PFA-100 11-dTxB2 45% 52% 20% 7% 52% 43%
Sequential VAD (Vincristine, Adriamycin, Dexamethasone) and VTD (VELCADE®, Thalidomide, Dexamethasone) Induction Followed by High-Dose Therapy with Autologous Stem Cell Transplantation and Maintenance Treatment with VELCADE for Newly Diagnosed Multiple Myeloma: Interim Results of Phase II Trial.
Authors: Yoon Sung-Soo , Kim Hye Jin , Lee Dong Soon , Eom Hyeon Seok , Jang Jun Ho , Chung Ju Seup , Kang Hye Jin , Kim Cheol Soo , Kim Kihyun , Lee Jae Hoon , Lee Sang Jae , Min Yu Hong , Seong Chu Myong , Sohn Sang Kyun , Suh Cheolwon , Won Jong Ho
Published: October 2019

Introduction Effective reduction of myeloma before autologous stem cell transplantation (ASCT) prolongs survival in multiple myeloma patients. Recently, incorporation of novel agents resulted in improved response rate and reduced side effect in newly diagnosed multiple myeloma.

Method: Patients are planned to receive 2 cycles of VAD (vincristine 0.4mg D1-4, adriamycin 9mg/m2 D1-4, dexamethasone 40mg D1-4, 9–12 every 3 weeks), and VTD (bortezomib 1.3mg/m2 D1, 4, 8, 11, thalidomide 100mg daily, dexamethasone 40mg D1-4, 9–12 every 3 weeks). High dose melphalan (200mg/m2) is used as a conditioning regimen for ASCT. Bortezomib (1.3mg/m2) as a maintenance treatment is administered weekly x 4 times every 6 weeks for 4 cycles after ASCT. Response was assessed by EBMT criteria, with additional category of nCR. Adverse events were graded by the NCI-CTCAE, Version 3.0.

Result: At this interim analysis, 60 patients have been entered into the ongoing trial, and efficacy could be assessed in 53 patients. After 2 cycles of VAD, response rate was 70%. After VTD, two patients showed further improvement with additional CR, and an overall response was 97% with 14% CR. Especially, patients with poor prognostic cytogenetics (n=6) all responded after VTD. So far, autologous stem cells were successfully collected in all 28 patients with a median CD34+ count of 7.8 x 106/kg (range, 2.17–44.7 x 106/kg). In 24 patients who underwent autologous stem cell transplantation, five patients gained additional CR. There was no progression in patients completed bortezomib maintenance (n=9, CR 77%). The median follow-up duration was 6 months, median time to response was 1.4 months, and median overall survival was not reached. Grade 3,4 hematologic toxicity was more frequently observed after VAD than VTD (anemia 15.8%, 4.6%, neutropenia 7.9%, 3.5%), and incidence of grade 2,3 peripheral neuropathy was low (VAD 3.5%, VTD 7%).

Conclusion: Sequential VAD and VTD induction therapy in newly diagnosed multiple myeloma was highly effective, even in patients with poor prognostic cytogenetics, and did not prejudice stem cell collection. VTD could have contributed to increased RR and minimized side effects. An updated results will be presented at the ASH meeting.

*Protocol Number: KMM51-NCT00378755.
Anemia and Iron Deficiency in Strenuously Trained Adolescents.
Authors: Merkel Drorit , Huerta Michael , Grotto Itamar , Rachmilewitz Eliezer A. , Fibach Eitan , Epstein Yoram , Shpilberg Ofer
Published: October 2019

Background: Healthy soldiers serving in combat units have a higher prevalence of anemia than age- and sex-matched civilians. This may be a “pseudo-anemia” caused by the hemodilution typical among training athletes, or a “true anemia” due to reduced total body iron stores.

Objective: To investigate the incidence of iron-deficiency anemia in recruits to the Israel Defence Forces during their first 6 months of intense combat training. This is a follow-up study to previous publication of measured values on induction.

Methods: Blood was collected from new recruits to an elite infantry unit before training. After 6 months, 153 paired samples were collected from the initial group. Total blood count and serum iron, transferrin and ferritin were measured at both time points. Soluble transferrin receptor (sTfR) was measured in 119 of the paired samples, and sTfR/log ferritin ratio was calculated.

Results: At recruitment, mean hemoglobin concentration was 14.7±0.9 g/dL (range 11.5–16.8). Iron-transferrin saturation was 34.1±13.6%, and mean ferritin concentration was 53.6±33.2 ng/mL. Twenty-seven participants (17.6%) were anemic (Hb<14g/dL), and 14.9% were iron-deficient (ferritin level <22 mg/dL). At the end of the follow-up period, 50.3% of the soldiers examined were anemic, and 27.3% had signs of iron-store depletion. Analysis of the paired samples showed an average reduction of 0.83 g/dL in hemoglobin level, and of 9.8mg/dL in ferritin levels (p<0.001 for both). sTfR increased slightly from 1.9 to 2.1mg/dL (p<0.003) among the recruits who became anemic during the follow-up period.

Conclusion: Nearly half the new recruits studied endure mild anemia after the first 6 months of training. Iron store depletion was observed among 24.5% of the cohort after 6 months, as opposed to 15% at recruitment. Overall, these changes were not accompanied by a significant increase in sTfR, but among the subset of anemic soldiers, there was a slight increase in this index. From the iron status analyses it can be concluded that in half the cases, the observed new-onset anemia was attributable to iron deficiency, and in the remainder, to hemodilution. The high incidence of iron deficiency in young healthy recruits is an important issue. The therapeutic implications of these findings require further evaluation.
Effectiveness of Management of Suspected Deep Vein Thrombosis in General Practice Based on a Clinical Decision Rule Including a Point of Care D-dimer Test.
Authors: Cate-Hoek Arina ten , Toll Diane , van der Velde Eit Frits , Hoes Arno , Buller Harry , Prins Martin
Published: October 2019

Prior studies have evaluated the safety and effectiveness of a diagnostic work up based on combined use of a clinical decision rule and a D-dimer laboratory test in patients with suspected deep vein thrombosis in secondary care facilities. The overall outcome of these studies show that approximately 30% of additional objective evaluation can be avoided without compromising the safety, resulting in failure rates of less than 1%. The objective of our study was to assess the effectiveness of a modified algorithm using a dichotomized clinical decision rule including a point of care D-dimer test in patients suspected of deep vein thrombosis in primary care. The AMUSE study is a prospective cohort study of consecutive patients with clinically suspected acute deep vein thrombosis conducted in 400 general practices coordinated by 3 university affiliated centers in the Netherlands from March 2004 through January 2007. The study population of 1029 patients included 63% women. Patients were categorized as “deep vein thrombosis unlikely” or “deep vein thrombosis likely” using a dichotomized version of a for general practice adapted clinical decision rule based on the Wells rule. Patients classified as unlikely were excluded from further testing. All other patients underwent ultrasonography. Anticoagulants were withheld from patients classified as unlikely. All patients were followed up for 3 months. The main outcome measure was symptomatic or fatal venous thromboembolism (VTE) during 3 months follow-up. Deep vein thrombosis was classified as unlikely in 500 patients (48.6%), all of whom were not treated with anticoagulants; subsequent non fatal VTE occurred in 8 patients (1.6% [95 CI 0.7–3.1%]). In 500 patients categorized as likely, deep vein thrombosis was confirmed in 124 patients (24.8%), 3 patients did not receive objective testing (0.6%) and 373 patients were tested negative (74.6%). Two out of 4 patients not receiving objective testing had confirmed deep vein thrombosis in the 3 month follow-up period. In patients that tested negative 4 non fatal events occurred during the 3 month follow-up (1.1%). The algorithm was completed and allowed a management decision in 97.2% of patients. We conclude that this management strategy is both safe and highly effective in patients with suspected deep vein thrombosis in general practice. Its use is associated with low risk for subsequent fatal and nonfatal VTE and a further increase in the yield of objective diagnostic tests.
Hoxa9 Suppresses Cellular Senescence and Sustains the Development of Leukemic Stem Cells Induced by Fusion Proteins In the Absence of Bmi1.
Authors: Smith Lan-Lan , Yeung Jenny , Zeisig Bernd B , Huijbers Ivo , Popov Nik , Barnes Josephine , Wilson Amanda J , Lugthart Sanne , Delwel Ruud , Gil Jesus , van Lohuizen Maarten , So Chi Wai Eric
Published: October 2019

Abstract 1578

While self renewal is an essential feature for the maintenance of both normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs), very little is known about the underlying molecular pathways. Here we report a critical functional interplay between Bmi1 and Hox in establishment of HSCs and LSCs. Using Bmi1-/- bone marrow cells, we observe that leukemia-associated fusion proteins have distinctive Bmi1 requirements. AML1-ETO (AE) and PLZF-RARα (PR) fail to transform Bmi1-/- primary hematopoietic cells, and induce expression of p16/Arf leading to oncogene-induced senescence (OIS). In contrast, MLL-AF9 driving expression of multiple Hox genes can bypass oncogene-induced senescence and exhibits modest Bmi1-dependence for establishment of LSCs, which can induce leukemia upon serial transplants. Since members of Hox genes with proclaimed self-renewal property are specifically up-regulated by MLL fusions in patient samples and our murine models, we asked the question if these Hox genes may partly compensate the functions associated with the loss of Bmi1. To this end, we generated compound Bmi1-/-Hoxa9-/- mice, which have even more compromised hematopoietic stem cell/progenitor compartments than those of Bmi1-/- or Hoxa9-/- mice. Bmi1-/-Hoxa9-/- mice have a greater than eight-fold reduction in the absolute number of Lin-Sca+kit+ (LSK) in the bone marrow as compared to Bmi1-/- mice and a very significant forty-fold reduction for long term hematopoietic stem cells (LT-HSC). More importantly, while MAF9 is able to transform wild type, Bmi1-/- and Hoxa9-/-, it fails to transform Bmi1-/-Hoxa9-/- cells for establishment of LSCs, which can however be resurrected by re-expression of either Bmi1 or Hoxa9, indicating a critical functional interplay between these protein in development of MLL LSCs. Consistent with the known function of Bmi1 in suppressing cellular senescence and the expression of p16/Arf loci, we showed that Hoxa9 alone can also inhibit replicative senescence and Ras-induced senescence in primary human fibroblast. Forced expression of Hoxa9 can suppress p16/Arf expression, as well as cellular senescence induced by AE and PR in Bmi1-/- cells. Together, these results reveal a previously unrecognized functional interplay between Hox and Bmi1 in regulating cell senescence and development of LSCs induced by fusion proteins, which also suggests that synergistic targeting of both molecules may be required for certain LSCs.

Disclosures:

No relevant conflicts of interest to declare.
MixL1 Expression Identifies Putative Hematopoietic Stem Cells In the Murine Allantois.
Authors: Wolfe Adam D , Downs Karen
Published: October 2019

Abstract 1591

MixL1, a paired-type homeodomain transcription factor, is implicated in pre-hematopoietic commitment of stem cell populations. In poorly-differentiated human lymphoma and leukemia lines, MixL1 is inappropriately over-expressed (Drakos et al., Human Pathol 2007:38:500). When constitutively expressed in mice, MixL1 is sufficient to induce Acute Myeloid Leukemia (Glaser et al., PNAS 2006:103:16460). On the basis of these observations, we hypothesize that MixL1 plays an important role in gating the cellular decision to remain in a poorly differentiated and proliferative phase rather than proceeding to definitive hematopoietic stem cell (HSC) identity.

Several years ago, the placenta was shown to be a major site of hematopoiesis (Gekas et al., Dev Cell 2005:8:365; Ottersbach and Dzierzak, Dev Cell 2005:8:377). The placenta is composed of the chorionic disc and the allantois, the latter of which matures into the umbilical component of the placenta. The allantois exhibits definitive hematopoietic potential (Ziegler et al., Development 2006:133:4183; Corbel et al., Dev Biol 2007:301:478), and has recently been demonstrated to contain a core of stem cells referred to as the Allantoic Core Domain, or ACD, where potential placental hematopoietic activity may originate (Downs et al., Dev Dyn 2009:238:532).

Our immediate goal is to evaluate whether MixL1 is expressed in the allantois, and to establish its precise spatiotemporal whereabouts with respect to early markers of hematopoietic cells, such as Runx1 (Chen et al., Nature 2009:457:887). Using immunohistochemistry in conjunction with the LacZ/Runx1 reporter mouse (North et al., Development 1999:126:2563), we have demonstrated that MixL1 is strongly expressed in the blood islands of the yolk sac, as well as in a broad, contiguous posterior domain of the embryo that extends to include the ACD stem cell core of the allantois. This domain does not include Runx1, and MixL1 expression temporally precedes that of Runx1 in the allantois.

As development proceeds, the MixL1 signal becomes most prominent in putative nascent blood cells budding off from the poorly-described blood vessel common to the allantois, yolk sac and dorsal aortae, which we have called the “Vessel of Confluence” (VOC). Here, fetal blood is shuttled into the umbilical cord to the chorion for exchange with the mother. Shortly thereafter, Runx1 begins to appear within VOC, and is co-expressed with MixL1.

These findings provide preliminary evidence that MixL1 is expressed within the allantois, and within nascent blood cells derived from a specific arterial site common to the allantois, yolk sac, and fetus. Moreover, MixL1 expression appears to precede that of Runx1. Thus, MixL1 may identify one of the earliest hematopoietic precursor cell populations thus far known in mammals. Further, these data provide additional evidence that the allantois is a promising model system for the study of definitive hematopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
the Utility of CD200 Expression In the Differentiation of Mantle Cell Lymphoma and B-Cell Chronic Lymphocytic Leukaemia: A Single Centre Experience.
Authors: Bhatnagar Neha , Wright Faith , McAleese Dimpna , McNamara Christopher J
Published: October 2019

Abstract 1673

Introduction:

Mature B-cell lymphoproliferative disorders that present with a leukemic phase may have overlapping morphological and immunophenotypic features. Accurate diagnosis is essential to determine prognosis and therapy. B-cell chronic lymphocytic leukaemia (B-CLL) and mantle cell lymphoma (MCL) both express CD5 but usually have divergent clinical courses. Conventional markers are able to discriminate in many cases but a significant proportion of cases require more detailed analysis. CD200 is a membrane glycoprotein that has been reported to differentiate between B-CLL and MCL. It is expressed on resting and activated T and B cells, but not on NK cells, monocytes, granulocytes or platelets. We prospectively assessed the utility of this antibody in a single centre, servicing haematology units in North London.

Methods:

Over a 12 month period we prospectively assessed the expression of CD200 (Clone MRC OX-104, BD Pharmingen) on neoplastic cells of patients with presenting with mature B-cell lymphoproliferative disorders. EDTA anti-coagulated peripheral blood and bone marrow underwent a lyse/wash technique before being acquired on a BD FACS Canto II flow cytometer equipped with DIVA 6.1.2 software. A lymphoid gating strategy was used to analyse the data and expression of more than 20% of nucleated cells referenced to a negative internal control was considered positive. A diagnosis was made using all available data according to the WHO classification of haematopoietic tumours, including the 5 point CLL score. The predictive value of CD200 expression was subsequently assessed.

Results:

There were 100 patients (58M: 42F) with a median age of 70 years (range 29–92). CD200 was present on the neoplastic cells of all 78 patients with B-CLL; in all 7 MCL patients CD200 was negative; 15 patients had CD5 negative B-cell lymphoproliferative disorder with variable expression of CD200. The specificity of CD200 negativity in the setting of CD5 positive B-cell clone was 100% for MCL. The positive predictive value of CD200 for CLL was 97.5% with a sensitivity of 100% and a Pearson's correlation coefficient of 1.

Conclusion:

Our results confirm CD200 expression in the neoplastic cells of B-CLL patients but not in MCL. This antibody may contribute to the accurate differentiation between B-CLL and MCL in patients presenting with the diagnostic problem of a CD5- positive lymphoproliferative disorder. We propose to add CD200 to our diagnostic B-cell lymphoma panel. Further studies are needed to determine whether CD200 could replace other immunophenotypic strategies currently used in this situation, such as CD23, FMC7 and intensity of CD22/CD79b expression.

Disclosures:

No relevant conflicts of interest to declare.
Vitamin D Triggers Killing of Burkitt Lymphoma Cells by Human Macrophages
Authors: Bruns Heiko , Mougiakakos Dimitrios , Fabri Mario , Pasemann Shirin , Jitschin Regina , Amann Kerstin , Büttner Maike , Mackensen Andreas , Gerbitz Armin
Published: October 2019

Abstract 1035

Introduction:

Distinct macrophage subsets have been linked with either protective or pathogenic roles in cancer. M2 macrophages have been shown to suppress adaptive tumour-specific immune responses and promote tumour growth, while M1 macrophages eliminate neoplastic cells and activate tumour-killing mechanisms. Macrophages isolated from solid tumours (tumor-associated macrophages, TAM) reveal a suppressive and tumor promoting M2-like phenotype. However, it remains unclear which tumoricidal effector mechanisms are compromised in theses immune cells. While infilatration of macrophages is a characteristic morphological hallmark in Burkitts' lymphoma (BL), the phenotype and functional properties of TAM as part of the stroma are poorly understood. In this study we investigated the phenotype of lymphoma associated macrophages (LAM) and the mechanisms by which macrophages are able to modulate the growth of tumor cells.

Methods:

We included 19 patients diagnosed with BL and 20 patients with benign reactive lymphadenopathy as a control group. Phenotypic characterizations of LAM were evaluated by immunohistochemistry and by qPCR in paraffin embedded tissues. To investigate anti lymphoma cytotoxicity of distinct macrophage subsets we generated macrophages from PBMC either by GM-CSF to create the M1 phenotype, or by M-CSF to obtain the M2 phenotype. M1 and M2 macrophages were coincubated them with several BL cell lines and cytotoxicity was analyzed by flow cytometry, qPCR and immunofluorescence.

Results:

First, we could demonstrate that BL infiltrating macrophages displayed an anti-inflammatory M2-phenotype characterized by expression of surface markers such as CD68 and CD163. Second, we identified impaired vitamin D metabolism in LAM and M2 macrophages as demonstrated by low expression of vitamin D receptor and Cyp27B1, and increased expression of Cyp24A1. Third, macrophages revealed a reduced cytotoxic potential towards lymphoma cells which was dependent on the expression of the vitamin D dependent peptide cathelicidin. Finally, we could demonstrate that excess supplementation of calcitriol led to increased cytotoxicity of M1- and M2 macrophages towards BL cells.

Conclusions:

These results suggest a mechanism in which vitamin D is required for innate immunity to overcome the ability of lymphoma cells to evade macrophage-mediated antitumoral responses. The present findings underscore the importance of vitamin D for sustaining innate immunity and imply that the therapeutic activation of the vitamin D pathway may even result in triggering tumoricidal effector mechanisms of LAM.

Disclosures:

No relevant conflicts of interest to declare.
HIV Triggers Interleukin 21-Mediated Induction of Granzyme B-Secreting B Cells with Regulatory and Antiviral Potential
Authors: Kaltenmeier Christof , Gawanbacht Ali , Lindner Stefanie , Beyer Thamara , Haerter Georg , Gruener Beate , Kern Peter , Kirchhoff Frank , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: October 2019

Abstract 1042

Certain regulatory lymphocyte subpopulations including plasmacytoid dendritic cells and regulatory T cells secrete granzyme B (GrB), thereby suppressing T cell expansion. Recently, we found that B cells can also produce GrB and acquire regulatory potential in response to interleukin (IL-)21. Since HIV has been shown to be associated with elevated serum IL-21 levels, we hypothesized that GrB-expressing B cells may be induced during HIV infection. Here, we show that infection of CD4+ T cells with HIV 1 (NL4-3), but not mock infection, induces strong expression of IL-21 without upregulation of CD40 ligand. Importantly, we further demonstrate that such IL-21+CD40Llow T cells induce GrB in cocultured B cells in an IL-21-dependent fashion, rather than supporting their differentiation into plasma cells. In support of these findings, serum levels of both IL-21 and GrB are significantly higher in HIV-infected patients before HAART as compared to healthy controls. Up to 60% of freshly isolated B cells (36.2% ± 12.9%) from patients infected with HIV, but not B cells isolated from healthy individuals, express GrB. Of note, coculture of HIV-infected CD4+ T cells with GrB+ B cells result in GrB transfer, and strongly suppresses both, proliferation of T cells and viral replication as indicated by significantly reduced p24 levels in coculture supernatants. The observed effects are enhanced by IL-21, and reduced by GrB inhibition. In summary, we demonstrate that HIV infection induces IL-21 in CD4+ T cells, thereby indirectly triggering the development of GrB-secreting B cells with antiretroviral properties. GrB-secreting B cells may play a so far unappreciated role in decelerating HIV expansion, particularly in the early phase of infection. On the other hand, induction of GrB in HIV-specific B cells may interfere with their terminal differentiation into plasma cells, which may explain the lack of an efficient anti-HIV humoral immune response in HIV-infected patients.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of PI3K/Akt and Erk Signaling Pathways by BCR-ABL Tyrosine Kinase Inhibitors up-Regulates the Immunosuppressive Receptor Osteoactivin in Monocyte-Derived Dendritic Cells
Authors: Gutknecht Michael , Joas Simone , Kanz Lothar , Salih Helmut R , Rittig Susanne M , Gruenebach Frank
Published: October 2019

Abstract 1050

Osteoactivin (GPNMB, DC-HIL) is a type I transmembrane glycoprotein that is expressed in dendritic cells (DC). Osteoactivin/syndecan-4 (SD-4) interaction was previously shown to inhibit T cell activation by antigen-presenting cells. We recently demonstrated that exposure of human peripheral blood monocytes to IL-10 or pharmacological levels of the BCR-ABL tyrosine kinase inhibitors (TKI) imatinib or nilotinib during differentiation into monocyte-derived DC (moDC) causes up-regulation of osteoactivin at the transcript and protein level in vitro (Schwarzbich et al., 2012). Here we aimed to elucidate the molecular mechanisms responsible for osteoactivin up-regulation in moDC upon exposure to IL-10 or TKI (imatinib, nilotinib). moDC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Every second day, starting from the first day of culture, IL-10 or TKI were added to the culture medium. Alternatively, specific inhibitors of PI3K, Akt, Erk and/or C-Raf signal transduction pathways were added to the cell cultures. Cells were harvested on day 7 of culture for immunophenotyping and osteoactivin expression analysis using FACS, western-blotting and real-time qRT-PCR. The expression and activity of signal transduction molecules was monitored by western-blotting and pathway protein arrays. Analysis of moDC function was performed using mixed lymphocyte reactions (MLR). Our in vitro analysis revealed that IL-10 and BCR-ABL TKI inhibit the PI3K/Akt and, to a lesser extent, the Erk pathway in moDC. Specific inhibition of these signal transduction cascades resulted in profound up-regulation of osteoactivin expression in moDC confirming the involvement of PI3K/Akt and Erk in the regulation of osteoactivin expression. In line, TKI treatment and inhibition of PI3K/Akt and Erk comparably affected the phenotype of moDC. Moreover, the inhibition of these signalling cascades resulted in reduced stimulatory capacity of moDC in MLR with allogenic T cells, and this could be restored by addition of blocking osteoactivin antibody. Our data reveal that TKI exert immunosuppressive effects in moDC by interfering with pathways involved in IL-10 receptor signaling. Inhibition of osteoactivin expression or function may thus constitute a promising strategy in combinatory approaches using TKI and DC-based immunotherapy and may enhance the efficacy of immunotherapeutic interventions in cancer patients.

Disclosures:

No relevant conflicts of interest to declare.
Human-Cl Rhfviii Effectively and Safely Prevents Bleeding Episodes in Previously Treated Adult Patients with Severe Haemophilia A
Authors: Knaub Sigurd , Lissitchkov Toshko , Hampton Kingsley , Von Depka Mario , Rangarajan Savita , Hay Charles R.M. , Tuddenham Edward GD , Collins Peter , Holstein Katharina , Huth-Kühne Angela , Pabinger Ingrid , Oldenburg Johannes , Bichler Johann
Published: October 2019

Abstract 1132

The main purpose of this prospective, multi-center, open-label phase 3 study was to assess the efficacy of prophylactic treatment with Human-cl rhFVIII, the first human cell-line derived recombinant FVIII, in previously treated patients (PTPs) with severe haemophilia A.

Patients were to receive 30–40 international units (IU) FVIII of Human-cl rhFVIII per kg every other day for 6 months. Efficacy of preventing and treating bleeds were judged using objective criteria taking the monthly bleeding rate and the number of infusions needed to manage a break-through bleed into account. In-vivo recovery (IVR) was determined at the beginning of the study and after 3 and 6 months. FVIII:C was measured by validated chromogenic (CHR) and one-stage (OS) assays in a central laboratory, which also assigned drug potencies. Inhibitor activity was determined using the Nijmegen modification of the Bethesda assay before the first administration and at defined intervals thereafter. Thirty-two patients between 18 and 75 years of age were enrolled from 11 centres in Europe and treated prophylactically for 6.0±0.9 months (mean ± SD) with a mean prophylactic dose of 32.8 IU/kg. Sixteen patients never bled, 11 patients bled once and 5 more than once. The mean total and spontaneous monthly bleeding rate was 0.188±0.307 and 0.095±0.211, respectively. Efficacy of the prophylactic treatment was “excellent” in all patients for spontaneous BEs and “excellent” or “good” in all patients but one for all types of bleeds. All treatments of bleeds were rated as “excellent” (71.4%) or “good” (28.6%). The IVR at baseline was 2.6±0.5 % per IU/kg for the CHR and 2.2±0.5 % per IU/kg for the OS assay and remained stable during the study. A total of 2921 infusions were given in the study. Human-cl rhFVIII was well tolerated and no patient experienced a related serious adverse event. No FVIII inhibitors were detected.

Conclusion:

The data indicate that Human-cl rh FVIII is safe and efficacious in preventing and treating bleeds in PTPs with severe haemophlia A.

Disclosures:

Knaub: Octapharma AG: Employment. Lissitchkov:Octapharma AG: PI Other. Tuddenham:College London: Consultancy, Employment, Gene therapy for hemophilia A, Gene therapy for hemophilia A Patents & Royalties, Research Funding. Collins:Octapharma AG: Consultancy. Oldenburg:d and e: Baxter, Bayer, Biotest, CSL-Behring, Grifols, Inspiration, NovoNordisk, Octapharma, Pfizer e: Biogen IDec, Swedish Orphan Biovitrum: Honoraria, Research Funding. Bichler:Octapharma AG: Employment.
Temporal Incidence Trends of Pediatric Venous Thromboembolism: A Population-Based Cohort Study
Authors: Sabapathy Christine A. , Kahn Susan R. , Platt Robert W , Tagalakis Vicky
Published: October 2019

Abstract 1138

Background:

Pediatric venous thromboembolism (VTE), although rare, is associated with significant morbidity and mortality. Published incidence rates in this age group vary from 0.07 to 0.49 VTE per 10 000 children/year and there is currently a paucity of studies evaluating temporal incidence trends.

Objectives:

To describe the age-adjusted incidence rates of pediatric VTE and its trend over time in a large pediatric cohort.

Methods:

A retrospective cohort of all children (ages 1–17 inclusive) with a first time diagnosis of VTE in the province of Quebec, Canada over an eleven-year period, from January 1st, 1994 to December 31st, 2004, was obtained from a comprehensive administrative hospital database (Med-Echo). Quebec census estimates were used to calculate age-standardized incidence rates (IR) of pediatric VTE. The incidence rate trend was then analyzed over the eleven-year study period using Poisson linear regression. Sex differences in incidence rates at the population level stratified by age group as a confounder as well as baseline characteristics of the cases were also evaluated.

Results:

In total, 487 incident cases of VTE in children 1–17 years of age were documented during the study period. Based on the estimated provincial census person-years during the study period, the age-standardized IR was 0.29 VTE per 10 000 person-years (95% confidence interval (CI) 0.26–0.31). Females overall had a statistically significant higher VTE incidence rate with an incidence rate ratio of 1.75 (95% CI 1.46–2.11) when controlled for age groups, as compared to males. When analyzed by age group, the age-standardized IRs were as follows: 1–5 year olds 0.04 VTE per 10 000 person-years (95% CI 0.03–0.05); 6–10 year olds 0.03 VTE per 10 000 person-years (95% CI 0.02–0.04); 11–14 year olds 0.06 VTE per 10 000 person-years (95% CI 0.05–0.07); 15–17 year olds 0.16 VTE per 10 000 person-years (95% CI 0.14–0.18).

Trend analysis of the age-standardized IRs over the 11-year period showed no significant change in incidence rates whether using time as a continuous (yearly) or categorical variable (time-periods).

Conclusions:

Pediatric VTE is more frequent than previously described, however the rate is stable. As shown by others, children in their late-teen years have a higher risk of VTE than primary school-aged children. Unlike prior studies, females were more prone to VTE than males. Future studies that address sex differences in the incidence of pediatric VTE are needed to help determine effective primary thromboprophylaxis strategies in children at high risk for VTE.

Disclosures:

No relevant conflicts of interest to declare.