bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin E2 Stimulates Human Brain Endothelial Cell Migration Via a Signaling Pathway Involving Activation of Rho-Kinase II
Authors: Khan Gausal , Rizzo Maria Theresa
Published: April 2020

Prostaglandin E2 (PGE2) plays a crucial role in angiogenesis as well as in ischemic and inflammatory disorders of the brain associated with breakdown of the blood-brain barrier. However, the effects of PGE2 on brain endothelial cell migration, a key process in the angiogenic response and blood-brain barrier stability, are not well defined. Exposure of human brain endothelial cells (HBECs) to PGE2 elicited a chemotactic response in a time- and dose-dependent manner. The maximum migratory response was detected following 8 hours exposure of HBECs to PGE2 (100 nM). Migration of HBECs in response to PGE2 was accompanied by profound changes in the reorganization of actin filaments. Fluorescence microscopy examination of NBD-phallacidin-labeled endothelial cells showed increased formation of stress fibers, lamellipodia and podosomes after treatment with PGE2 (100 nM) compared to control. Based on these results, we hypothesized that Rho-kinase (ROCK), an enzyme involved in regulation of actin dynamics and cell migration, mediated the effects of PGE2 on HBEC migration. Western blot analyses revealed that ROCK II (type alpha), but not ROCK I (type beta), was expressed in HBECs. To examine ROCK II activation, we performed immunocomplex kinase assays using myosin light chain (MLC) as a substrate. PGE2 (100 nM) induced a 2-fold increase of 32P-incorporation into MLC indicating activation of ROCK II. Pretreatment of HBECs with the selective ROCK inhibitor, Y27632 (150 nM), blunted HBEC migration in response to PGE2 but had no effect on migration induced by fetal bovine serum (10%). Knockdown of ROCK II by siRNA also abrogated the migratory response of HBECs to PGE2. In contrast, similar treatment had no effect of HBEC migration stimulated by hepatocyte growth factor. Taken together, these results are consistent with the hypothesis that stimulation of HBECs with PGE2 leads to activation of ROCK II, reorganization of the actin cytoskeleton and ultimately migration. A better characterization of the molecular events that regulate migration of brain endothelial cells is critical for the development of novel strategies to treat cerebrovascular diseases associated with deregulated angiogenesis.
Genes Involved in Acute Leukemias Are Favored Targets of HIV Vector Integration
Authors: Appelt Jens U. , Giordano Frank A. , Zimmermann Marcel , Weinhard Stephan , Grund Nadja , Hotz-Wagenblatt Agnes , Zeller W. Jens , Allgayer Heike , Fruehauf Stefan , Laufs Stephanie
Published: April 2020

Insertional mutagenesis and development of leukemia following retroviral gene therapy has created intense interest in assessing the safety of viral vectors for further gene therapy trials. Using the gtsg.org database we analyzed more than 14,900 different viral integration sites of ASLV, FIV, FV, HIV, MLV and SIV based vectors in terms of insertions into fragile sites, cancer genes, transcription factor binding sites, CpG islands, and repetitive elements (SINE, LINE, LTR elements). When we compared these data with our newly generated random set, containing 1,000,000 random integrations, we discovered that the gene density on fragile sites strongly correlates to the HIV vector insertion frequency. Furthermore, we report a up to a five fold increased frequency of HIV, MLV and SIV insertions in cancer genes. The majority of cancer genes preferentially hit by HIV viruses were found associated to acute leukemias, while MLV and SIV vector insertion sites are seen more evenly spread over the cancer gene repertoire. When analyzing different cell entities, it turned out that CD34+ hematopoetic stem cells had highest rates of intragenic insertions and hosted significantly more HIV and FV insertions in cancer genes than other cell types, such as HeLa, T cells, 293T cells, macrophages, fibroblasts, or SupT1 cells.
A Prospective Randomised Trial of Intravenous Iron Therapy Versus Oral Iron for Iron Deficiency Anaemia in Pregnant Australian Women
Authors: Khalafallah Alhossain A. , Dennis Amanda , Bates Joan , Bates Gerald , Smith Lauern , Shaw Susie , Seaton David , Berteram John , Robertson Iain , Ball Madeleine , Beamish Michael , Rooney Kim , Brain Terry
Published: April 2020

To date, limited data is available regarding prevalence of iron deficiency during pregnancy in Australia. There is little if any data currently available regarding comparative efficacy of IV iron versus oral iron therapy in pregnant women. In Australia the prevalence of iron deficiency anaemia (IDA) is approximately 5% in the general population with higher rates in pregnant women. At a single site, the Launceston General Hospital (LGH), we prospectively investigated 200 pregnant women between January and July 2007 with FBC and iron studies at the first or second antenatal visit. Among those, 40 women (20%) had iron deficiency anaemia, and were recruited to a prospective randomised trial to determine whether intravenous iron therapy (iron polymaltose) is superior to oral iron (ferrous sulphate) for the management of IDA associated with pregnancy. The patients’ median age was 28 years old (range; 19–40) with a median gestational age at recruitment of 27 weeks (range; 13–29) and a median body weight of 74 kg (range; 47–130). At recruitment median Hb was 106 g/L (range; 90–114, normal range; 120–160 g/L), while median serum ferritin was 11.5 μg/L and mean ferritin was 19 μg/L normal range; 30–460). After four weeks of treatment the Hb level increased by a mean of 6g/L on oral iron and by 10.5 g/L after IV iron. Mean/Median serum ferritin did not increase significantly in women on oral iron, but increased to a median of 96.5 μg/L and a mean of 224 μg/L in those received IV iron. Multi-variate analysis using general linear modelling for continuous variables showed a significant increase in serum ferritin after treatment with IV iron versus oral iron (p=0.017). There was no statistically significant difference in terms of Hb increment, patients’ parity, weight, and date of last pregnancy between groups. Analysis of quality of life questionnaires to assess patients’ well-being, ability to perform activities, and symptoms of anaemia showed improvement in both groups of patients with a trend to greater and faster improvement after IV iron. Both treatments were well tolerated without major side effects. The preliminary data indicate IDA is a common finding during pregnancy in the LGH population, and intravenous iron therapy appears a safe and effective treatment in this cohort of patients. This research received a grant from the Clifford Craig Medical Research Trust, Tasmania, Australia.
HMGB1 Expression in Sickle Cell Disease: A Pro-Inflammatory Cytokine and Potential Therapeutic Target.
Authors: Barginear Myra F. , Chandok Anu G. , Sison Cristina , Yang Lihong , Budman Daniel R. , D’Olimpio James , Tracey Kevin J. , Bradley Thomas
Published: April 2020

Background: HMGB1 is traditionally known as a DNA binding protein involved in gene transcription. When released into the extracellular milieu, HMGB1 activates vascular endothelial cells and macrophages/monocytes to express proinflammatory cytokines, chemokines, and adhesion molecules. Therefore, HMGB1 may be a good marker for disease activity in Sickle Cell Disease (SCD), a chronic inflammatory state.

Methods: After informed consent as per Institutional guidelines, plasma was obtained, frozen, and batch run for HMGB1 levels by ELISA technique in patients with SCD during non-crisis and crisis states. The detection limit of HMGB1 was achieved at concentrations above 1 ng/ml. Each experiment was repeated at least five times to ensure reproducibility.

Results: 12 patients enrolled, 6 patients have gone into crisis. HMGB1 levels were measured on consecutive days for 5 five days during a crisis. We established mean HMGB1 expression levels of 32.83 ± 27.13ng/ml during a non-crisis state and 52.57± 15.22 ng/ml during a crisis, respectively (p< .028). In noninflammatory states, HMGB1 is < 5 ng/ml.

Conclusion: We have demonstrated, for the first time, a persistent expression of HMGB1 in SCD patients. Although recruitment is on going, this data is consistent with pre-clinical data indicating that HMGB1 may be an important diffusible signal of necrosis-mediated inflammation. Additionally, our laboratory has previously demonstrated therapeutic benefit by neutralization of HMGB1 in sepsis. Thus if confirmed in a larger cohort, anti-HMGB1 therapy to reduce the effects of inflammation may be an attractive therapeutic target in sickle cell disease.
An Enriched Multigen Low-Density Array To Predict Outcome in Advanced Hodgkin Lymphoma Paraffin-Embedded Samples.
Authors: Sanchez-Espiridion Beatriz , Sanchez-Aguilera Abel , Montalban Carlos , Garcia-Cosio Monica , Bellas Carmen , Romagosa Vicente , Menarguez Javier , Fresno Manuel F. , Tomas Jose F. , Morente Manuel M. , Piris Miguel A. , Garcia Juan F.
Published: April 2020

Despite the major advances in the treatment of classical Hodgkin Lymphoma (cHL) patients, around 30% to 40% of cases in advanced stages may relapse or die as result of the disease, and current markers to predict prognosis are rather unreliable. The identification of molecular events and biological processes associated with treatment failure are essential to develop new predictive tools. We used gene expression data from 29 samples of advanced cHL patients and HL-derived cell lines in order to identify transcriptional patterns from both tumoral cells and cell microenvironment. Student t-test was used to detect genes differentially overexpressed in cell lines and in tumor samples, thus creating two databases that report for genes expressed by the tumor HRS cells and genes expressed by the microenvironment. Using Gene Set Enrichment analysis (GSEA) we identified specific gene sets enriched in both databases in patients with favorable and unfavorable outcome, respectively. To validate these pathways we designed a novel Taqman low-density array (LDA) to examine the expression of the most relevant genes in 60 formalin-fixed, paraffin embedded (FFPE) tissue samples, and correlated the results with treatment outcome. Functional pathways related to unfavorable outcome significantly enriched in the HRS cells included the regulation of the G2/M checkpoint of the cell cycle, S phase and G1/S transition, chaperons, histone modification and other signaling pathways with an important representation of the MAPK pathway. On the other hand, genes reporting for specific T-cell populations (T-cytotoxic and T-regulatory cells) and macrophage activation were found to be overexpressed in the microenvironment. The final model presents a balanced representation of these genes, including also genes encoding factors implicated in drug resistance (RRM2, TYMS and TOP2A). RNA extracted from FFPE sections yielded analyzable data for 80% of samples. LDA analysis of the genes included in the model confirmed the feasibility of this approach, and the capacity for identifying cases with increased risk of failure.LDA provides an effective technique for analyzing gene expression in FFPE tissues, and it can be used for clinical prediction in diagnostic samples, using a selection of genes identified after GSEA analysis of the initial molecular signatures. This novel Taqman LDA will be used to develop a new molecular predictor of the outcome of patients with advanced cHL.
Bortezomib Alone or in Combinination with Doxil Is Active in Adult Refractory Multi-System Langerhans Cell Histiocytosis (LCH).
Authors: Haddad Philip A.
Published: April 2020

Langerhans cell histiocytosis (LCH) is a clonal disorder of activated Langerhans cells. LCH is characterized by increased proliferation index, Ki-67, as a result of dysregulation of anti-apoptotic pathways (Bcl-2) and cell cycle regulators (p53-p21 and p16-Rb pathways). Although current available therapies are very effective at inducing remission, treatment of LCH remains problematic due to multiple recurrences and eventual fatalities in a significant proportion of young patients. More effective therapies based on the pathogenesis of LCH are needed. We investigated the use of Bortezomib, a proteosome inhibitor with a reported antiproliferative effect on plasma cells and lymphocytes, in a heavily pre-treated patient with recurrent multi-system LCH involving lungs, bones, skin and pituitary gland and who was previously treated with and progressed on Vinblastine, Steroids, Peg-Interferon, Methotrexate/ 6MP. The patient received Bortezomib initially at 1.3 mg/m2 days 1, 4, 8, 11 Q 21 days (cycles 1&2) which was reduced to 1.0 mg/m2 days 1, 4, 8, 11 Q21 days (cycles 3&4) and then to 1.0 mg/m2 weekly for cycle#5 due to grade 3 exacerbation of his baseline Vinblastine induced peripheral neuropathy. Patient’s skin disease achieved very early response after cycle 1 with PR as maximum response after cycle 2. Such response was maintained until the patient was switched to weekly dose. Stable disease was observed at other sites especially the pituitary mass. Upon flaring of his skin disease, patient was started back on Bortezomib 1.0 mg/m2 days 1, 4, 8, 11 along with Doxil 30mg/m2 on day 4 Q21 days, to explore their reported synergy. After cycle 1, patient achieved a very good PR as far as his skin disease and CR after the 3rd cycle. His pituitary LCH mass was also noted to decrease by 20% with visual improvement. Unfortunately, due to grade 3 fatigue and anorexia, therapy was stopped. However, responses are still maintained for 3 months so far. Hematologic toxicities were limited to Grade 1 neutropenia, anemia, and thrombocytopenia. Bortezomib has activity on its own and synergies with Doxil in recurrent and heavily pre-treated multisystem LCH. Its incorporation into front-line treatment of patients with multi-system LCH needs further study.
Incidence and Risk Factors for Agranulocytosis in Latin American Countries - The LATIN Study.
Authors: Hamerschlak Nelson , Maluf Eliane , Eluf Jose , Cavalcante Alexandre B. , Pasquini Ricardo , Lorand-Metze Irene , Falcao Roberto P.
Published: April 2020

The association of agranulocytosis with the use of many different drugs has been documented. LATIN is a case-control study designed to identify risk factors for agranulocytosis and aplastic anemia, including drugs, and to estimate the incidence rates of both diseases in some Latin American countries. This report will cover just agranulocytosis. In 4 years, 52 cases of agranulocytosis were diagnosed. The overall incidence rate was 0.38 cases per 1 million inhabitant-years (0.35 for Brazil, 2.09 for Argentina, and no case verified in Monterrey, Mexico). Agranulocytosis patients more often took medications already associated with agranulocytosis than controls (76.7% of cases and 52.5% of controls; OR 3.7, 95% CI 1.3–12.5; p = 0.01), the most important being methimazole (OR 44.2, 95% CI 6.8 to infinite). The population attributable risk percent (etiologic fraction) was 56%. The use of nutrients supplements was more frequent among agranulocytosis patients than controls (p = 0.03). The agranulocytosis incidence in Latin America was lower than that of European countries and Israel. The rarity of the disease indicates that it is not a public health problem, and there is no reason for major protection measures other than improving diagnostic tools and making earlier agranulocytosis diagnosis.
The Prognostic Role of Clinical and Microbiological Factors on Mortality Related to Candida Bloodstream Infections in Hospitalized Patients with Hematologic Malignancies.
Authors: Bigni Ricardo S. , Velasco Eduardo D. , Dobbin Jane A.
Published: April 2020

Between January 2001 and June 2005 a prospective cohort study of hospitalized patients with hematological malignancies including 47 adults and 30 children with candidemia was conducted at a tertiary oncology care center in Brazil in order to compare the epidemiological characteristics, concurrent illnesses and the clinical microbiological data of both groups that may influence the outcome. The crude mortality was higher in the adult population than in children (46,8% vs. 20,0%) (figure 1). A univariate analysis indicated that in the adult population were lymphoma, neutropenia, presence of comorbidities, a non-removed central venous catheter (CVC), a poor performance status, lack of CVC, use of steroid, hepatic dysfunction, previous surgery, hypotension and severe respiratory dysfunction were risk factors significantly associated with death. Among children the predictors of mortality were acute leukemia, neutropenia, presence of comorbidities, lack of CVC, poor performance status, hypotension, concomitant infected sites, pulmonary infiltrates and severe respiratory dysfunction. Although no major differences was detected in survival rates following fungemia with C. albicans and all Candida non-albicans species, episodes with Candida glabrata, krusei and tropicalis subgroup species had the highest crude death rate compared with C. albicans and other isolates (59,4% vs. 35,3% vs. 10,7%; P<0.01) (figure 2). Candidemia due to C. parapsilosis was associated with the lowest mortality rate. Two variables remained statistically associated with mortality among adults in the multivariate analysis: CVC retention (OR 6.41; 95% CI 1.04–39.55) and presence of comorbidities (OR 2.17; 95% CI 1.33–3.53). Among children only the presence of comorbidities (OR 2.61; 95% CI 1.46–4.66) affected independently the outcome. Our data demonstrate children had a significant lower mortality rate than adults, despite the higher incidence of candidemia in this lower age subjects. There were significant differences of epidemiological, clinical characteristics and other risk factors between both groups. Concurrent comorbidities were the most important independent prognostic factor in both groups of patients.
An Individual Platelet Count Set-Point in ITP: A Concept Learned from Patients with Mild Thrombocytopenia and a Good Response to IVIg or Corticosteroids.
Authors: Arnold Donald M. , Moore Jane C. , Smith James W. , Kelton John G.
Published: April 2020

Background: Immune thrombocytopenic purpura (ITP) is a heterogeneous disease caused by both increased platelet destruction and decreased platelet production. Thrombocytopenia is typically severe, and in the absence of a sensitive and specific test, a platelet count response to intravenous immune globulin (IVIg) or corticosteroids is diagnostic. Patients with mild thrombocytopenia (platelets 50 – 150 × 109/L) may have ITP, non-immune thrombocytopenia or a low-normal platelet count. The diagnostic value of platelet autoantibodies for such patients is not known.

Methods: We studied the platelet count responses of 6 patients with mild thrombocytopenia who were treated with either IVIg or corticosteroids. Baseline platelet count was defined as the mean of the 2 lowest, consecutive platelet counts that were within 15% of each other. The peak platelet count was the highest recorded platelet count measured within 1 month of treatment. For most patients, the dose of IVIg was 1g/kg ×1, and the dose of prednisone was 1mg/kg for 2 – 4 weeks. Complete response was defined as a peak platelet count that was at least 2x baseline; a partial response was defined as a peak platelet count 1.5 – 2x baseline; below that was not considered a response. Platelet glycoprotein IIb/IIIa and Ib/IX autoantibodies were determined by the antigen capture assay using platelet lysates prepared from samples collected prior to treatment. An OD >0.4 was considered positive.

Results: Five patients with mild thrombocytopenia received 5 treatments with IVIg and 2 treatments with corticosteroid (2 patients received IVIg on 2 separate occasions). The indications for treatment were: planned invasive procedure (n=5), pregnancy/delivery (n=1), and treatment of multiple sclerosis (n=1). Patients were followed for a median of 1.6 years (range 0.6 – 3 years). Median baseline platelet count prior to treatment was 70 ×109/L (range 57 – 79 ×109/L). A platelet count response was observed following all 7 treatments; including 5 complete responses and 2 partial responses. Median peak platelet count was 180 ×109/L (range 115 – 297 ×109/L). Post-treatment platelet counts returned to within 15% of pre-treatment values following 6 of 7 treatments after a median of 3 months (range 1 week – 5 months). Of the 3 patient tested, none had anti-GP IIb/IIIa or anti-Ib/IX autoantibodies.

Interpretation: A good response to IVIg (or steroid) treatment confirmed the immune nature of mild thrombocytopenia in this cohort and should be used as the gold standard to evaluate the test characteristics of platelet autoantibodies. We observed that some patients with mild ITP have an individual platelet count “set-point” which remained relatively stable over time. Although further testing is required, this concept implies that in some patients, there is a regulated balance between platelet destruction and underproduction.
Distribution of Hypercoagulation Laboratory Diagnoses among Patients with History of Thromboembolic Disease.
Authors: Bern Murray M. , McCarthy Nancy , Bonner Jamie
Published: April 2020

This abstract demonstrates the distribution of hypercoagulation diagnosis among patients with histories of thromboembolic disease (TED) among a group of patients detected at surgery prescreening clinic or through other referral sources. The consulting hematologists determined which laboratory tests were ordered; thus not all patients had all tests. This abstract describes the results of those clinical consultations. For this study the hospital’s computer logs were probed for patients having had measurements of protein C and factor V Leiden from 11/7/01until 8/1/07. The laboratory records of identified patients were searched for additional hypercoagulation laboratory parameters. A total of 383 patients have been identified, among whom abnormal diagnostic results were found for 231. Genomic assays were performed often for the commonly found defects (i.e., factor V Leiden and prothrombin 20210) and selectively for other situations, such 4G/5G for patients with elevated plasminogen activator inhibitor 1 (PAI-1) and unresolved venous thrombus, or methylene tetrahydrofolate (MTH) reductace for unexplained elevation of homocysteine. The table demonstrate the distribution of these laboratory diagnoses. The risk of having TED associated with these results will be stratified to emphasize the increased risk associated with the more severe abnormalities of protein C, protein S, ATIII, PAI-1, and homocysteine. These results demonstrate that laboratory explanations for TED may be found in a large proportion of patients with TED, which thereafter can be used to design prophylactic programs for at risk patients upon entry to hospital, especially elective surgery.

Hypercoagulation Parameters Patients Protein C* (<60%) Protein S* (<60%) AT III (<80%) Homocysteine (>12 um/L) Lupus anticoagulant Anti-phospholipid syndrome** * excludes patients taking warfarin; includes functional and antigen assays. ** combines anticardiolipin; anti-beta2, glycoprotein1; and anti-phosphotidyl -serine, -ethanolamine and -choline antibodies. total n 350 358 244 252 166 234 abnormal n (%) 7(2) 64(18) 29 (12) 89(35) 18 (11) 41 (18) PAI-1 (>42) ng/ml) APC Resistance (<2.1) VIII:c & VIII:vW (>180%) Factor V Leiden Prothrombin 20210 MTH Folate Reductace 4G/5G 36 75 61 225 219 10 9 22 (61) 10 (13) 11 (18) 37 (16) 16 (7) 8 (80) 8 (90)
Characterization of Heparin Binding Site Residues on the Catalytic Domain of Factor XIa
Authors: Yang Likui , Sun Mao-fu , Manithody Chandrashekhara , Gailani David , Rezaie Alireza R.
Published: April 2020

The basic residues of the 170-helix (chymotrypsinogen numbering) in the catalytic domain of the procoagulant serine proteases are known to interact with heparin and facilitate their rapid inhibition by specific plasma serpins. The homologous loop of factor XIa possesses three basic residues (Lys-170, Arg-171 and Arg-173). To investigate the role of these residues in the heparin-mediated inhibition of fXIa by C1-inhibitor (C1-INH) and antithrombin (AT), we expressed the monomeric light chain fragment of fXIa containing only the catalytic domain of the protease in wild-type and mutant forms in which the three basic residues of the 170-helix have been substituted individually, or in combinations, with Ala. The catalytic properties of fXIa derivatives were compared to wild-type fXIa (fXIa-WT) with respect to their ability to hydrolyze the chromogenic substrate S2366 and to undergo inhibition by C1-INH and AT in the absence and presence of heparin. All mutants exhibited normal amidolytic activity, hydrolyzing S2366 with catalytic efficiencies similar to fXIa-WT. All mutants were also inhibited by both C1-INH and AT with normal rate constants in the absence of heparin. However, the heparincatalyzed inhibition of mutants by both serpins differed to varying degrees relative to fXIa-WT, with the Arg-171 to Ala substitution mutant exhibiting ~3-fold lower rate of inhibition by C1-INH and requiring ~3-fold higher concentration of heparin for observing optimal inhibitory effect. In reactions with AT, all three basic residues appeared to make similar contributions to the heparin-catalyzed inhibition of fXIa by the serpin as evidenced by all three mutants exhibiting dramatically lower inhibition rate constants in the presence of heparin. A bell-shaped heparin concentration dependence for the fXIa inhibition by both serpins suggested that the template effect of heparin primarily accounts for the acceleration mechanism. However, a comparison of the heparin bell-shaped dependence inhibition of the dimeric plasma-derived wild-type fXIa with that of the isolated single chain catalytic domain suggested that the optimal concentration of heparin for the acceleration of the protease has been increased from ~50 nM for the dimeric protease to ~250 nM for the isolated catalytic domain, possibly suggesting further contribution to affinity of the fXIaheparin interaction from the Apple-3 domain of the non-catalytic domain on which a binding-site for heparin has been reported.
Predicting Response to Immunosuppressive Therapy in Childhood Aplastic Anemia.
Authors: Yoshida Nao , Yagasaki Hiroshi , Kosaka Yoshiyuki , Kobayashi Ryoji , Yabe Hiromasa , Kaneko Takashi , Tsuchida Masahiro , Ohara Akira , Nakahata Tatsutoshi , Kojima Seiji
Published: April 2020

Aplastic anemia (AA) is defined as a pancytopenia caused by bone marrow failure; its pathogenesis is thought to involve autoimmune processes. Immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CyA) provides response rates of 50–70% for children with AA, but predictive markers of response to therapy have not been well defined. We previously investigated the clinical relevance of HLA, a minor population of paroxysmal nocturnal hemoglobinuria-type cells, and a specific auto-antibody associated with AA in pediatric patients and reported that there was no correlation between these markers and response to therapy (Yoshida N, et al. Br J Haematol, 2008). In the current study, we prospectively evaluated whether clinical and laboratory findings before treatment, including age, sex, interval between diagnosis and treatment, etiology, severity of disease, white blood cell (WBC) count, neutrophil count, hemoglobin level, reticulocyte count, and platelet count, could predict the IST response at 6 months. Subjects included a large population of pediatric AA patients enrolled in a multicenter AA-97 study conducted by the Japan Childhood Aplastic Anemia Study Group. Between October 1997 and September 2006, 312 children (186 boys and 126 girls) younger than 18 years who were newly diagnosed with AA were enrolled in the study and treated with a combination of ATG and CyA. The median age at diagnosis was 8 years (range, 1–17 years). Of the 312 patients, 261 had idiopathic AA, 44 had hepatitis-associated AA, and 7 had AA from other causes. In terms of severity, 156 patients had very severe disease, 107 had severe disease, and 49 had moderate disease. The median interval between diagnosis and treatment was 15 days (range, 1–180 days). The overall response rate was 56%. In multivariate analyses, lower WBC count, shorter interval between diagnosis and therapy, and male sex were predictive markers of better response. Patients with WBC <2500/μl had a significantly higher response rate than those with WBC ≥2500/μl (61 vs. 48%; P=0.008); this was the strongest predictor of response. Notably, response rate was inversely related to the interval between diagnosis and treatment; response rates of patients with an interval between diagnosis and treatment of <30 and ≥30 days were 60% and 43%, respectively (P=0.02). Boys had a better response compared with girls (62 vs. 48%; P=0.02). In conclusion, pretreatment clinical and laboratory findings influence the response rate to IST. Response is well correlated with WBC count rather than neutrophil count or severity of disease. IST should be started as soon as possible after diagnosis of AA, given that the response rate worsens as the interval between diagnosis and treatment increases.
De Novo Deletion 17p13.1 Chronic Lymphocytic Leukemia Shows Significant Clinical Heterogeneity: The MD Anderson/Mayo Clinic Experience.
Authors: Tam Constantine S. , Shanafelt Tait D. , Wierda William G. , Abruzzo Lynne V , Van Dyke Daniel L. , Lerner Susan , Lynn Alice , Kay Neil E. , Keating Michael J
Published: April 2020

Figure Figure

In order to characterize the clinical course of patients (pts) with deletion 17p13.1 (17p-) CLL, we identified 100 consecutive treatment-naïve pts with 17p- from the MD Anderson Cancer Center (MDACC, n=61) and Mayo Clinic (n=39) clinical databases. The diagnosis of 17p- was established by FISH with a median time from initial diagnosis to FISH documenting 17p- of 9 months. Median age was 64 yrs (60 for MDACC pts & 70 for Mayo pts) and Rai stage was 0, 1–2 and 3–4 in 34%, 49% and 17% pts respectively. 17p- was the sole FISH abnormality in 29%. Unmutated IgVH and ZAP-70 positivity were present in 30/62 and 54/84 patients respectively. <<>> 69 asymptomatic pts were followed until they developed progressive disease requiring therapy as defined by the NCIWG 96 criteria. The 2 year actuarial risk of progression to initiation of therapy was 43% (46% for MDACC pts & 36% for Mayo pts). Only 2 of 10 pts with stable disease >2 years progressed on prolonged follow-up of up to 54 months. Two pts with baseline 17p- in 10% and 13% of nuclei lost their 17p- clone after 23 and 41 months. Independent predictors of shorter time to treatment were Rai stage > 0 (hazard ratio 7.2) and unmutated IgVH (HR 5.3). Pts with 0, 1 or 2 of these factors had a 2 year progression rate of 0%, 48% and >75% respectively (p<0.01). Other prognostic parameters such as ZAP-70 and CD38 did not have independent prognostic utility. <<>> 41 pts from MDACC were assessable for response to first therapy. Overall (OR) and complete response (CR) rates were: rituximab without chemotherapy (n=6), OR 67% CR 17%; purine analogue + rituximab combinations (n=20), OR 75% CR 30%; purine analogue, rituximab + alemtuzumab combinations (n=13), OR 85% CR 54%. The pt who achieved CR after rituximab was negative for minimal residual disease assessed by flow cytometry and consensus primer IgH PCR, and has remained in remission at 29 months. At 2 years, 75% of complete responders remained relapse free. <<>> Two year survival was 78%. Independent predictors of inferior survival were age 60+ (HR 3.4), B-symptoms (HR 3.7) and 17p- in nuclei 70+% (HR 6.0). Considering age 60+ and 17p- in nuclei 70+% in a prognostic model, 2 year survival was 100%, 82% and 55% for pts with 0, 1 and 2 risk factors respectively. Other prognostic parameters such as ZAP-70, IgVH mutation status and CD38 did not have independent prognostic utility. <<>> Pts with de novo 17p- CLL exhibit marked clinical heterogeneity, with some pts experiencing an indolent course with prolonged survival. Survival and time to treatment among patients with 17p- can be predicted using clinical and biological characteristics.
Enhanced Formation and Survival of Regulatory T Cells in CLL.
Authors: Jak Margot , Mous Rogier , Remmerswaal Ester BM , Spijker René , Jaspers Annelieke , Yagüe Adriana , Eldering Eric , van Lier René A.W. , van Oers Marinus H. J.
Published: April 2020

Background. In healthy individuals it has been shown that regulatory T cells (Treg) differentiate from rapidly proliferating memory T cells. Rapid proliferation in combination with high susceptibility to apoptosis, make the Treg population highly dynamic. Tregs are thought to play an important role in immune evasion by malignancies. High Treg numbers in patients with malignancies are often associated with poor prognosis. An important question to be answered is whether tumor cells can promote the expansion and activation of Tregs. Recently it has been described that chronic lymphocytic leukemia (CLL) patients have increased numbers of Treg, with highest Treg frequencies in progressive patients1. The mechanism of this expansion however remains unknown. In the present study, we analysed the mechanism behind Treg expansion in CLL.

Results. Neither analysis of the T cell receptor (TCR) repertoire nor CD45 isoform expression of Treg from CLL patients provided evidence for chronic (tumor) antigenic stimulation as a possible cause for Treg expansion in CLL. Furthermore, we found no correlation with CMV serology, which has been demonstrated to influence the CD8+ T cell repertoire in CLL. However, in line with recent studies demonstrating that CD70+ NHL cells are strong inducers of Treg, we found evidence for increased formation of Treg in CLL via CD70 costimulation. We observed that CD40 ligand activated CLL cells (which might be considered a model for lymph node CLL cells2) strongly induced CD70-dependent formation of Treg. RT-MLPA expression analysis of 34 apoptosis-regulating genes showed that in comparison to other CD4+ T cells, Treg of both healthy individuals and CLL patients had a high expression of pro-apoptotic Noxa and a low expression of anti-apoptotic Bcl-2. Strikingly, Bcl-2 levels of Treg in CLL patients were significantly higher than in healthy individuals. Finally, the different apoptotic profile resulted in differences at the functional level, since Treg from CLL patients were more resistant to drug-induced apoptosis than Treg from healthy individuals.

Conclusion. Treg in CLL may accumulate both by increased formation, facilitated by CD27-CD70 interaction in the lymph node proliferation centres, and decreased sensitivity to apoptosis due to a shifted Noxa-Bcl-2 balance. Since high Treg numbers might negatively affect the course of disease, targeting these mechanisms may provide additional therapeutic approaches in CLL.
Mtss1 Suppresses BCR-ABL Induced Cell Migration and Is Downregulated in CML Stem Cells.
Authors: Schemionek Mirle , Agrawal Shuchi , Stehling Martin , Tenen Daniel G. , Hamilton Ashley , Holyoake Tessa , Steidl Ulrich , Berdel Wolfgang E. , Schwab Albrecht , Müller-Tidow Carsten , Koschmieder Steffen
Published: April 2020

Migration and adhesion properties of hematopoietic stem cells (HSC) are disrupted in chronic myeloid leukemia (CML). Egression of these cells from the bone marrow is associated with cytoskeletal changes including actin remodeling. In a microarray screen of differentially regulated genes in HSC from BCR-ABL positive transgenic mice, we found downregulation of multiple genes involved in actin-associated changes of cell structure, adhesion, and migration (i.e. intersectin-1, cortactin, Mtss1, synaptopodin, and Gem GTPase). Mtss1 was further studied since it has been described to be a binding partner of Rac, which is essential for BCR-ABL mediated transformation, and a potential tumor suppressor. Using quanitative RT-PCR, Mtss1 downregulation (6-fold) was confirmed in HSC and unfractionated bone marrow and spleen cells from SCLtTA/BCR-ABL transgenic mice after 3 weeks of BCR-ABL induction as well as in human BCR-ABL positive cell lines. Treatment of BCR-ABL positive (32D/BCR-ABL, K562, KYO-1) but not BCR-ABL negative (32D, U937) cell lines with 5μ M Imatinib led to upregulation of Mtss1 mRNA (5- to 10-fold) and protein, suggesting that downregulation of Mtss1 is dependent on BCR-ABL kinase activity. Retroviral transduction of Mtss1 into 32D/BCR-ABL cells almost completely inhibited BCR-ABL induced cell motility of individual cells seeded on murine bone marrow stromal cells in time-lapse video experiments over the course of two hours. Interestingly, we found that retroviral transduction of Mtss1 into 32D/BCR-ABL cells completely suppressed migration of these cells to extra-hematopoietic sites in vivo upon intravenous transplantation into syngeneic C3H mice. Moreover, when Mtss1-transduced cells were injected subcutaneously, the size of the tumors was significantly decreased as compared to empty vector-transduced 32D/BCR-ABL cells (p<0.05), confirming that Mtss1 may be a tumor suppressor.

These results demonstrate that Mtss1 antagonizes BCR-ABL induced cell migration and is downregulated by BCR-ABL in CML stem cells, suggesting that downregulation of Mtss1 and other cytoskeletal adaptor proteins may be required for egression of CML stem cells from the bone marrow niche. Since the same Mtss1 protein domain is responsible for both Rac and actin binding, Mtss1 may interfere with Rac function and thereby inhibit the effects of Rac on migration and cytoskeletal dynamics of HSC.
HLA-Identical Allogeneic Stem Cell Transplantation (alloSCT) for Patients with Primary Cutaneous T-Cell Lymphoma (CTCL): A Retrospective Analysis of the Lymphoma Working Party of the European Group for Blood and Marrow Transplantation (EBMT-LWP).
Authors: Duarte Rafael , Canals Carme , Onida Francesco , Olavarria Eduardo , Arcese William , Ferrant Augustin , Narni Franco , Bacigalupo Andrea , Bunjes Donald , Rowe Jacob , Lambertenghi Giorgio , Schmitz Norbert , Sureda Anna
Published: April 2020

Patients with aggressive forms of Mycosis Fungoides (MF), Sézary syndrome (SS) and other less common subtypes of primary CTCL have a dismal prognosis with conventional therapy. A potential role for alloSCT in these patients has been suggested by a number of case-reports and small series. We conducted a retrospective registry analysis of 64 recipients who received an alloSCT from HLA-identical donors (52 related, 12 unrelated) for advanced CTCL reported to EBMT between 1997 and 2006: 23 MF, 21 SS, 16 primary cutaneous anaplastic large cell lymphoma and 4 subcutaneous panniculitis-like T-cell lymphoma; 38 patients were male, 26 were female; median age was 46 years (5–65); median follow up of survivors was 28 months. AlloSCT was performed at a median of 22 (3–313) months from initial diagnosis, following a median of 3 (1–8) prior lines of therapy, the stem cell source was PB in 54 cases and BM in 10 cases, reduced-intensity conditioning (RIC) was used in 41 (64%) cases. Twenty-three percent of patients were in complete response (CR) and 26% in partial response (PR) at the time of alloSCT. All other patients were primary refractory (RE, 25%) or progressed after a previous response (PG, 26%). Non-relapse mortality was 24% at 3 years, and appeared higher in patients receiving myeloablative conditioning (MAC) than in those receiving RIC alloSCT (35% vs 16%; p=0.06). Acute graft versus host disease (GVHD) occurred in 37% of patients at risk, and chronic GVHD in 69% of patients alive at day +100. The cumulative incidence of disease relapse (36% at 3 years) was significantly higher in patients with advanced disease status at alloSCT (RE/PG 46% vs CR/PR 21%; p=0.01), but similar in patients with related and unrelated donors (p=0.39) and receiving MAC or RIC (p=0.6). Progression free survival (PFS) at 3 years was 41%, with a significant advantage for patients in CR/PR at alloSCT (58% vs 26%; p<0.01), but not for type of conditioning (MAC 45% vs RIC 44%; p=0.4) or donor type (related 46% vs unrelated 33%; p=0.2). Out of 22 patients who relapsed, 10 remain alive and 4 of these were in CR at last follow up, possibly indicating that a proportion of patients achieved a better control of CTCL following relapse after alloSCT. Overall survival for the whole series was 55% at 3 years, with a trend towards an improved OS for patients with disease in CR/PR at transplant (65% vs 46%; p=0.1). Overall, outcome after allogeneic transplantation appears superior to that expected for such patients under conventional therapy. Our analysis reinforces the important role of alloSCT in advanced stage CTCL in a relatively large registry-based series.
Graft Versus Host Disease (GVHD) Prophylaxis with Everolimus and Tacrolimus Is Associated with a High Incidence of Sinusoidal Obstruction Syndrome and Microangiopathy – Results of the EVTAC Trial
Authors: Platzbecker Uwe , Bonin Malte , Goekkurt Eray , Radke Joergen , Kiani Alexander , Stoehlmacher Jan , Schetelig Johannes , Ehninger Gerhard , Bornhauser Martin
Published: April 2020

Beyond disease biology, the success of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematological malignancies is mainly determined by the occurrence and extent of graft versus host disease (GVHD). Therefore, prevention of GVHD is the major goal and challenge in clinical HSCT. A calcineurin-inhibitor combined with methotrexate is the standard graft versus host disease (GVHD) prophylaxis after allogeneic hematopoietic stem cell transplantation (HSCT). Everolimus is a derivative of sirolimus, which also seems to mediate anti-leukemia effects. Given the potential synergism and favourable toxicity profile of everolimus and tacrolimus (EVTAC) after allogeneic HSCT we sought to investigate the efficacy of this combination in patients with either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). We report a combination of everolimus (days 0–56) and Tacrolimus (from day −1 on) in 24 patients (pts, median age 62 years) with either MDS (n=17) or AML (n=7) undergoing intensive busulfan-based conditioning followed by HSCT from related (n=4) or unrelated matched (n=12) or 1-allele mismatched (n=8) donors. All pts engrafted and only one experienced grade IV mucositis. However, although everolimus was scheduled to be administered up to day 56, patients received the drug a median of 44 days (range 10–56) only. The reason for premature discontinuation (50%) were either occurrence of early-onset (day 6) GVHD associated hyperbilirubinemia CTC grade 4 (n=1), transplantation-associated microangiopathy (TMA, n=3), sinusoidal obstructive syndrome (SOS) of the liver (n=6) or a drop of platelets after engraftment by at least 50% (n=2). Nine pts (37%) developed grade II–IV acute GVHD, however, chronic extensive GVHD was observed in 11 of 17 (64%) evaluable pts. TMA occurred in 7 pts (29%) with two cases of acute renal failure. In five out of seven patients with TMA either tacrolimus (n=4) or everolimus (n=1) blood through levels were slightly above the upper target level at the time of TMA appearance. The study was terminated prematurely because additional 6 pts (25%) developed SOS, which was fatal in two cases. With a median follow-up of 26 months, the 2-year overall survival rate is 47%. In conclusion, although this new combination appears to be effective as prophylactic regimen for acute GVHD, the incidence of TMA and SOS seems to be higher compared to other regimens. As a result this combination cannot be recommended as prophylactic regimen after busulfan-based intensive conditioning. However, studies in the context of TBI-based or reduced-intensity conditioning regimens might come to a different conclusion.
The Zebrafish Ortholog of Hemojuvelin Participates in Notochord and Somite Development, but Fails to Regulate Embryonic Hepcidin Expression
Authors: Gibert Yann , Lattanzi Victoria J. , Holzheimer Jason , Burnett Sarah F. , Fraenkel Paula G.
Published: April 2020

Hemojuvelin (hjv), a member of the repulsive-guidance molecule (RGM) family upregulates the iron regulatory hormone hepcidin in a BMP-dependent manner in mammalian cells. A mutation interfering with hjv’s ability to bind neogenin has been identified in patients with juvenile hemochromatosis, while furin cleavage of hjv has also been implicated in its function. Previously, we demonstrated that hepcidin expression in zebrafish embryos increases in response to iron loading or activation of the BMP pathway. We hypothesized that hjv would regulate hepcidin expression in zebrafish embryos and used whole mount in situ hybridization and morpholino knockdowns to study the expression and function of hjv. We found that hjv is strongly and sequentially expressed in the notochord and somites and that knockdown of hjv resulted in severe defects in these structures. Hjv was not expressed in the liver and knockdown of hjv failed to affect the timing, intensity, or location of hepcidin expression. Furthermore, knockdown of hjv failed to prevent the upregulation of hepcidin expression caused by overexpression of BMP2b. Zebrafish hjv exhibits conservation at the site required for binding neogenin, however zebrafish hjv and all nonmammalian RGM’s lack the furin cleavage motif. We found that morpholino knockdown of the zebrafish orthologs of neogenin or furin failed to affect hepcidin expression. Taken together, these data indicate that

regulation of hepcidin expression in the zebrafish embryo is BMP-responsive, but independent of hjv, furin, or neogenin zebrafish hjv participates in notochord and somite development, which we propose as the ancestral function of hjv.
Distinct Expression Patterns in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) Characterized by Uniparental Disomy.
Authors: Bullinger Lars , Kronke Jan , Botzenhardt Ursula , Heinrich Sabrina , Urlbauer Katja , Miller Simone , Schon Christian , Schlenk Richard F. , Holzmann Karlheinz , Dohner Konstanze , Dohner Hartmut
Published: April 2020

Genome-wide single nucleotide polymorphism (SNP) analyses have revealed uniparental disomy (UPD) to be a common event in cytogenetically normal acute myeloid leukemia (CN-AML) occurring in approximately 20% of cases. Acquired UPD results in copy number neutral loss of heterozygosity (LOH). Comparing matched tumor and germline DNA samples recurrent acquired UPDs affecting chromosomes 11p and 13q were identified. As DNA microarray-based gene expression profiling (GEP) has recently been shown to powerfully capture the molecular heterogeneity of leukemia, we sought to identify gene expression patterns associated with recurrent UPD in CN-AML.

We profiled a set of clinically annotated CN-AML specimens (n=66) entered on a multicenter trial for patients <60 years (AMLSG 07-04) which had been characterized by either 50k or 500k Affymetrix SNP microarrays. All cases were analyzed using Affymetrix microarrays (Human Genome U133 Plus 2.0 Arrays). In this data set we investigated 12 UPDs (affecting chromosomes 1p, 2p, 6p, 11p, 13q and 19q) and applied supervised analyses to define gene-expression patterns associated with UPDs on chromosome 11p and 13q.

For the case with an acquired UPD on 19q a gene dosage effect could be demonstrated. Genes located in the 36 Mb large UPD region showed a significantly lower average expression (p<0.001; t-test). Similarly, we observed a gene dosage effect in one of the UPDs observed on chromosome 1 (p=0.0097; t-test), whereas for the other UPDs no significant association between LOH and gene expression levels could be identified. Despite small sample numbers supervised analyses revealed a biologically meaningful gene expression signatures associated with acquired UPD 11p and 13q. In accordance with the association of UPD 13q with FLT3-ITD, the UPD13q gene expression signature was enriched for genes associated with FLT3-ITD. The UPD11p expression pattern was characterized by genes found to be down-regulated in CEBPAmut CN-AML cases, such as down-regulation of homeobox genes HOXA9, HOXA10, HOXB2, and MEIS1. Notably, the UPD11p signature was also characterized by the expression of e.g. UGT2B28, P2RX5, PGDS, CAPN1, NDFIP1, and TRIB2, an expression profile that has been shown to be associated with CEBPAmut CN-AML as well as AML cases with epigenetic CEBPA silencing.

Thus, our findings represent a starting point to further dissect CN-AML characterized by recurrent UPD, and ongoing analyses will provide additional insights into leukemia biology.