bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptide Multimers for Binding of Factor VIII Inhibitors.
Authors: Kessel Christoph , Kreuz Wolfhart , Klich Katharina , Vorpahl Frank , Becker-Peters Karin , Dietrich Ursula , Klingebiel Thomas , Königs Christoph
Published: April 2020

Hemophilia A is an X-chromosome linked bleeding disorder resulting from the absence or nonfunctional expression of coagulation factor VIII (FVIII). About 30% of severe hemophiliacs develop neutralizing antibodies (inhibitors) to FVIII upon treatment with exogenous factor preparations. Specificity of these antibodies is often restricted to functional determinants in the A2 and C2 domain. Phage displayed random peptide libraries were screened with various plasma samples of high titer inhibitor patients and inhibitor specific peptide ligands were selected. In silco mapping of consensus amino acid motifs among peptides revealed conformational epitopes in A2 and C2. Selected ligands partially restored FVIII activity. Equimolar combination of these ligands enhanced blocking of inhibitors in autologous and heterologous patients’ plasma. Peptide ligands were fused to the C-terminal multimerization domain of the C4bp alpha-chain and expressed as multimers in 293T cells. Peptide multimers revealed improved binding of anti-FVIII IgG and prolonged in vitro half-life in comparison to single synthetic peptides. Selected peptide ligands were combined in heteromultimers by co-transfection of respective vectors, resulting in molecules binding both A2- and C2- specific IgG and blocking up to 100% of antibody binding to FVIII. Those molecules could provide a basis for the generation of novel peptide-based therapeutic approaches.
Identification of New CD177 Isoform in NB1 Deficient Individuals
Authors: Bayat Behnaz , Kissel Karin , Werth Silke , Santoso Sentot
Published: April 2020

Human neutrophil-specific antigen CD177 (NB1; HNA-2a) belongs to the member of the cyctein-rich LY-6 superfamily. Transcriptional analysis of this family revealed intron-retaining mechanism, which produce many spliced forms of gene. In some cases, this mis-spliced isoform was the most abundant form suggesting a control mechanism of gene expression. CD177 glycoprotein carries neutrophil antigen HNA-2a, and antibodies to HNA-2a frequently causes transfusion related acute lung injury (TRALI). Recently, CD177 has been found to be associated with the expression of proteinase-3 (PR3) which plays a role in autoantibody mediated Wegener’s granulomatosis. Neutrophils from some people lack CD177 (termed HNA-2a negative), however, the mechanism responsible for this deficiency is not fully understood. In this study, we investigated whether mis-spliced form of CD177 exist in neutrophils. Neutrophil from blood donors were phenotyped for the presence or absence of CD177 by flow cytometry using two mabs 7D8, MEM166 recognising different epitopes on CD177. HNA-2a positive individuals carrying high surface CD177 density and HNA-2a negative individuals were selected. mRNA was isolated from CD177 phenotyped individuals and were analyzed by PCR using different sets of primer set. Amplification of CD177 transcript (bases 98–1401) showed the expected ~1300 bp band encoding for the entire CD177 (isoform 1), and a smaller band (~417bp) with similar intensity in HNA-2a positive as well as in HNA-2a negative individuals. Sequencing analysis of the short transcript showed the presence of intronic sequence between exon 3 and 4, which produces premature stop codon. Transfection of insect cells with related transcript resulted in production of a ~23 kDa protein which is detectable in immunoblot using rabbit polyclonal antibody against synthetic CD177 peptide. This ~23 kDa protein (isoform 4) did not react with mabs 7D8 and MEM166. Furthermore, immunoblotting analysis of other blood cells showed that isoform 4 was exclusively found on neutrophils. In contrast to the isoform 1, isoform 4 was not upregulated on neutrophils of individuals receiving GCSF. Neutrophils treated with fMLP in vitro showed down-regulation of isoform 4 transcript. This phenomenon however, has not been observed in neutrophils treated with LPS and IL-8. In summary, we characterized a new CD177 isoform in neutrophils. The presence of this isoform in both HNA-2a positive and negative CD177 phenotyped individuals and its regulation with fMLP indicate the role of this protein in inflammatory process.
Impact of Gene Expression Profiling on Diagnosis and Prognostication in Cytogenetically Normal AML.
Authors: Bullinger Lars , Hielscher Thomas , Botzenhardt Ursula , Heinrich Sabrina , Schlenk Richard , Pollack Jonathan R. , Dohner Konstanze , Benner Axel , Dohner Hartmut
Published: April 2020

Cytogenetically normal acute myeloid leukemia (CN-AML) comprises a biologically and clinically heterogeneous group of AML. In the past years, molecular markers like FLT3, CEBPA and NPM1 gene mutations have been identified in CN-AML, and the presence of such mutations carries important prognostic information. Furthermore, DNA microarray-based gene expression profiling (GEP) has been shown to capture the molecular heterogeneity of cancers, and has been applied to build classifiers and clinical outcome predictors in AML.

While prior studies have defined gene expression patterns associated with NPM1, CEBPA, and FLT3, we assessed the clinical relevance of gene signatures. We profiled a large set of clinically well annotated CN-AML specimens (n=296 entered on two multicenter trials for patients <60 years (AMLSG HD98A and AMLSG 07-04). The 142 cases from the AMLSG HD98A trial were analyzed using a 40k cDNA microarray platform and the 154 cases from trial AMLSG 07-04 using Affymetrix microarrays (Human Genome U133 Plus 2.0 Arrays). In this data set we applied supervised analyses (LASSO penalized logistic regression) to define gene expression patterns characterizing FLT3 internal tandem duplication (ITD), CEPBA and NPM1 mutations as well as outcome signatures.

We were able to define distinct signatures associated with NPM1, CEBPA, and FLT3 consisting of 39, 27, and 47 genes, respectively. The NPM1 signature revealed a high prediction accuracy of >95% in leave-one-out cross validated classification. Prediction of FLT3-ITD or CEBPA mutation performed less well with accuracies of 80% and 73%, respectively. However, for both CEBPA and FLT3-ITD the predicted mutation class labels performed slightly better than the marker itself with regard to the prognostic impact on overall survival (CEPBA: p=0.006 vs. p=0.007, FLT3-ITD p=9.57e-06 vs. p=5.11e-05; logrank test). In addition, using LASSO we also could define a signature associated with event free survival (EFS) in the cases from the AMLSG 07-04 trial. Adjusted for age, NPM1, and FLT3-ITD mutational status this signature was significantly associated with EFS (p=0.005; Wald test), and validation in our independent cDNA data set also provided significant prognostic information (p=0.02; Wald test).

Thus, GEP-based classification of CN-AML might help to identify alternative genetic changes that either phenocopy or block the effects of common molecular aberrations. Furthermore, gene expression patterns of yet unknown aberrations are reflected in prognostic signatures. Therefore, signature genes also provide a starting point to dissect “mutations” pathways, and our findings underscore the potential clinical utility of a gene expression based measure in CN-AML.
The Efficacy and Safety of Lenalidomide Oral Monotherapy in Patients with Mantle Cell Lymphoma Previously Treated with Bortezomib: Pooled Data from Two Phase II Studies (NHL-002 and NHL-003).
Authors: Reeder Craig B. , Witzig T.E. , Vose Julie M. , Zinzani Pier Luigi , Buckstein Rena , Haioun Corinne , Bouabdallah Reda , Polikoff Jonathan , Guo Pingshan , Ervin-Haynes Annette , Pietronigro Dennis , Zeldis Jerome B. , Czuczman Myron S.
Published: April 2020

Introduction: Mantle-cell lymphoma (MCL) is an aggressive B-cell lymphoma that if not cured with aggressive chemoimmunotherapy and stem cell transplant is usually fatal. The intravenous proteasome inhibitor bortezomib produces an overall response rate (ORR) of 33% in patients with relapsed MCL (J Clin Oncol2006;24(30):4867–74). Unfortunately, most patients eventually relapse and new agents are needed for this disease. Two recently conducted phase II trials (NHL-002 and NHL-003) tested single-agent lenalidomide for patients with relapsed MCL. Fifty-four patients with MCL were enrolled into the two studies; 26% (14/54) had received prior bortezomib and they are the subject of this report.

Methods: Patients with relapsed or refractory MCL and measurable disease 2 cm after at least one prior treatment regimen including prior bortezomib were eligible. Patients received 25 mg of lenalidomide orally once daily on days 1–21 of every 28-day cycle. Therapy was continued as tolerated or until disease progression. The 1999 IWLRC methodology was used to assess response and progression.

Results: The 14 patients in this study were heavily pretreated with a median of 4 (2–6) prior treatments (including bortezomib) and 50% were bortezomib-refractory. Median age was 66 (45–84) years and 6 (43%) patients were female. Median time from diagnosis to lenalidomide treatment was 3.3 (0.7–7.6) years. The ORR with lenalidomide was 57% (8/14), including 21% (3/14) complete response (CR)/unconfirmed CR and 36% (5/14) partial responses. One (7%) patient had stable disease. The most common grade 3 or 4 adverse events were neutropenia (50%), thrombocytopenia (43%), anemia (21%), fatigue (21%), and leukopenia (21%). Neutropenic fever occurred in 7% of patients.

Conclusion: These results demonstrate that lenalidomide oral monotherapy is very effective with manageable side effects in patients with relapsed or refractory MCL who had received prior treatment with bortezomib.
Plitidepsin Is Active in Peripheral T-Cell Lymphoma (PTCL): A Subset Analysis from An Ongoing Multicenter Phase II Trial.
Authors: Ferme Christophe , Mateos Maria Victoria , Szyldergemajn Sergio , Zucca Emanuele , Espinoza Jose , Briones Javier , Morschhauser Franck , Gisselbrecht Christian , Ribrag Vincent
Published: April 2020

Background: Peripheral T-cell lymphomas (PTCL) represent a small (<10%) yet a particularly aggressive subset of NHL. Up to 75% of those patients (pts) eventually become relapsed/refractory, with no effective options available. Thus, a prospective open-label, multicenter phase II study to evaluate the activity of plitidepsin (Aplidin®) in adult pts with relapsed/refractory aggressive lymphomas was planned and is currently ongoing. We report the preliminary results from a cohort of non-cutaneous PTCL.

Patients and methods: As of July 2008, 14 pts were treated with plitidepsin 3.2 mg/m2 i.v. infusion over a 1-h on days 1, 8 and 15 q4wk. Eleven pts are evaluable, one is too early and two were non-evaluable as per protocol criteria: one had a hypersensitivity reaction and one had bone marrow (BM) infiltration only, but achieved a biopsy-documented BM clearance. Pts had a median of 3 (1–6) previous regimens, including 4 pts (33%) with prior autologous transplantation. Lymphoma histology: 8 PTCL-nos, 2 anaplastic large-cell, 2 angioimmunoblastic and 2 NK/T nasal type. Ten pts were male, median age was 56 y (35–74), with performance status 0 in 6 pts, 1 in 5 pts and 2 in 3 pts.

Results: One confirmed CR and 3 PR were observed for a 36% objective response rate (95% CI: 11%–70%). Median duration of response was 4 months (range: 1+ – 12+). Median overall survival was 11 months (range 1+ – 24+). Plitidepsin was tolerable in this heavily pre-treated population, particularly with low hematologic toxicity. Of 2 cases of grade (G) 4 neutropenia, 1 was already present at baseline and only 1 pt developed G3 thrombocytopenia during treatment. Transient and reversible G3 ALT/AST elevations occurred in 7 patients and 2 G3 bilirrubin increases. Clinical toxicities mainly consisted of mild to moderate muscular weakness, myalgia and cramps, plus G1-2 fatigue and nausea in 1/3 of the patients. One pt discontinued due to hypersensitivity reaction before being evaluated for response and one pt was diagnosed with Guillain-Barré syndrome while progressing after 2 cycles.

Conclusion: Plitidepsin (Aplidin®) shows promising activity and an acceptable safety profile in this difficult-to-treat subset of patients. Remarkably, no significant hematologic toxicity was seen in this heavily pretreated cohort. To confirm these preliminary data an expansion of the cohort is currently ongoing. Updated results will be presented.
Leukocytosis at Diagnosis and the Risk of Subsequent Thrombosis in Low-Risk Essential Thrombocythemia and Polycythemia Vera.
Authors: Gangat Naseema , Wolanskyj Alexandra , Schwager Susan , Hanson Curtis A. , Tefferi Ayalew
Published: April 2020

Background: Leukocytosis has recently been implicated as an adverse prognostic feature for thrombosis in both ET and PV. Such an association would be therapeutically most relevant in the context of “low-risk” disease. In the current study, we sought to clarify the relationship between leukocytosis at diagnosis and the subsequent occurrence of either arterial or venous thrombosis, in “low-risk” patients with ET or PV.

Methods: Data was abstracted from the medical records of a consecutive cohort of patients with WHO-defined ET or PV seen at the Mayo Clinic. Low-risk disease was defined by the absence of both thrombosis history and age 3 60 years. Cox proportional hazards model was utilized to determine the impact of clinical and laboratory variables on thrombosis-free survival (TFS). Arterial- or venous-specific TFS curves were constructed by Kaplan-Meier method.

Results: i) Patient characteristics and outcome A total of 407 “low-risk” patients were studied; 153 had PV (median age 48 years; females 43%) and 254 ET (median age 42 years; females 71%). A total of 46 thrombotic events (22 arterial and 24 venous) were recorded in 41 (27%) patients with PV during a median follow up of 130 months (range 2–562 months). The corresponding figures in ET were 54 total thrombotic events (41 arterial and 14 venous) in 47 (19%) patients at a median follow up of 104 months (range 0.25–424 months). Cytoreductive therapy was avoided in the presence of < 1000 x 109/L platelet count but, at the discretion of the treating physician, some patients with higher platelet counts received prophylactic cytoreductive therapy. ii) Correlation between leukocytosis and thrombosis A leukocyte count of 3 15 x 109/L at diagnosis was documented in 42 (27%) patients with PV and 21 (8%) patients with ET; 102 patients (40%) with ET had > 9.4 x 109/L leukocyte count. Leukocyte count considered as either a continuous or categorical variable (using cutoff levels of 15 x 109/L for PV and either 15 x 109/L or 9.4 x 109/L for ET) was not significantly associated with either arterial or venous thrombosis (Figure 1). iii) Correlation between other risk factors and thrombosis In univariate analysis, presence of the JAK2V617F was significantly associated with arterial thrombosis in ET (p=0.049) but significance was lost during multivariable analysis that included age as a covariate. Only advanced age was found to be significantly associated with arterial thrombosis in PV (p=0.04) and higher hemoglobin level with venous thrombosis in ET (p<0.0001).

Conclusion: The current study does not identify leukocyte count at diagnosis as a marker of increased thrombosis risk in low-risk patients with either ET or PV. Instead, the study found an association between arterial thrombosis and advanced age in PV and venous thrombosis and higher hemoglobin level in ET.

Figure Figure
ADAMTS13 is a Risk Factor for MI, Stroke and Vascular Death in the Oxford Vascular Study.
Authors: Eglinton Jennifer , Harrison Paul , Segal Helen , Silver Louise , Mehta Ziyah , Rothwell Peter
Published: April 2020

Normal haemostasis requires a balance between ultra-high MW VWF synthesis/release and its degradation by ADAMTS13. Although a severe acquired/inherited deficiency of ADAMTS13 occurs in TTP, as VWF is an established risk factor for arterial thrombotic disease it has been recently hypothesized that intermediate to low levels of ADAMTS13 could also be an important risk indicator. A new rapid ADAMTS13 Technozym® ELISA activity assay (Technoclone, Vienna, Austria) was therefore used to measure ADAMTS13 activity within plasma samples from a cohort of 521 patients within the Oxford Vascular Study (OXVASC). OXVASC is a large population based study of all patients with acute vascular events (TIA, stroke, acute coronary syndromes and acute peripheral vascular events) and includes a substudy of various risk factors (including platelet function, aspirin responsiveness and classical coagulation markers) for thrombosis. The ADAMTS13 normal range was 68.96 – 144.36% (n = 20, mean = 106.7%, 95% CI = 95.8–115.5%). Within 521 OXVASC samples, the mean ADAMTS13 level was found to be significantly lower (p < 0.0001, Mann-Whitney test) at 72.34% (95% CI = 70.33–74.35%). The minimum and maximum values obtained were 29.17% and 201.86% respectively. Although plasma VWF levels were significantly elevated, they were not inversely proportional to ADAMTS13 activity (r = 0.093). Furthermore low ADAMTS13 levels were not significantly associated with aspirin non-responsiveness (p = 0.0759) as defined by the Collagen/Epinephrine cartridge on the PFA-100, although VWF levels were significantly higher in non-responsive patients (p = <0.0001). Increased VWF levels were predictive of the combined outcome of stroke, MI or vascular death (HR per decile increase 1.12, 95% CI 1.06–1.19, p = 0.0002), whereas lower ADAMTS13 levels were associated with increased risk (HR per decile decrease 0.91 95% CI, 0.85–0.97, p = 0.004). In conclusion, low ADAMTS13 activity was not associated with high VWF levels and there was no association with aspirin non-responsiveness in a high shear dependent assay (PFA-100). However, low levels of ADAMTS13 were predictive of recurrent major vascular events. ADAMTS13 may therefore have some potential as a risk factor in patients with arterial disease.
PI3 Kinase Inhibition Induces Apoptotic Cell Death in Acute Lymphoblastic Leukemia Via the Mitochondrial Pathway.
Authors: von Schwarzenberg Karin , Henkel Marco , Conzelmann Dennis , Stork Björn , Bringmann Anita , Wesselborg Sebastian , Brossart Peter
Published: April 2020

The phosphatidylinositol 3-kinase (PI3K) pathway regulates many cellular processes that are involved in tumor progression. Aberrant activation of the PI3K pathway due to an alteration of its elements like PTEN or Akt occurs quite frequently in malignant cells. Thus, inhibition of this pathway represents a promising option for the treatment of cancer patients. The best characterized PI3K inhibitors are LY294002 and wortmannin that were shown to disrupt downstream signaling and induce apoptotic cell death in tumor cells. Acute lymphoblastic leukemia (ALL) is a malignancy mainly found in young children and elderly with constitutive activation of PI3K pathway.

In our study we analyzed the effect of PI3K inhibition in cell lines deduced from ALL (Jurkat, BV173, SD1, T-2) and primary leukemic cells by incubating them with increasing concentrations of inhibitors of the PI3K signaling. We found that treatment of ALL cells with LY294002, the mTOR inhibitor rapamycin or Akt inhibitor SH5 induced apoptotic cell death that was accompanied by caspase-3 activation and PARP-cleavage and interfered with intracellular PI3K/Akt signaling as analyzed by phosphorylation and expression of mTOR or P70S6K. In line with these results apoptotic cell death could be inhibited by the pan-caspase inhibitor zVAD. In order to determine the pathway of apoptosis induction we took advantage of Jurkat cells (T-ALL) overexpressing or lacking molecules involved in apoptotic pathways such as FADD, an adaptor molecule recruited to the death receptor upon ligand binding, Caspase-8, Caspase-9 or Bcl-2, an anti-apoptotic protein that prevents the release of cytochrom c from mitochondria. PI3K inhibition by LY294002 induced apoptotic cell death in cells deficient of FADD or caspase-8 with no difference to wild type cells. In contrast, cells overexpressing Bcl2 or lacking caspase-9 were resistant to apoptotic death indicating that PI3-kinase inhibition is independent of the external death receptor signaling and is mediated via the mitochondrial pathway. These results were confirmed by analyzing PARP cleavage and caspase-3 activation in utilized leukemic cell lines. Furthermore, we found that the PI3K inhibitor LY294002 induced apoptosis in ALL cells that could be increased by the etoposide, a topoisomerase inhibitor, or TRAIL. In addition, in contrast to etoposide, treatment of ALL cells with TRAIL could overcome the resistance of ALL cells to PI3K inhibition even in caspase-9 deficient Jurkat cells. Our results provide an interesting approach in designing novel therapeutic strategies to target the PI3K pathway in ALL to overcome the resistance to cytotoxic agents.
Expression of Tissue Factor Is up-Regulated in U937/PR9 Cells by the PML/RARα Fusion Protein through Its Interaction with the Regulatory Region of Tissue Factor Promoter.
Authors: Yan Jingsong , Liu Hongchen , Su Xiaoyu , Wang Ping , Ruan Zhen , Wang Kankan , Chen Zhu , Chen Saijuan , Xi Xiaodong
Published: April 2020

Life-threatening bleeding remains a challenging complication of acute promyelocytic leukemia (APL), which is characterized by the t(15;17) translocation producing a fusion gene encoding the PML/RARα fusion protein, and is mainly ascribable to the increased levels of tissue factor (TF) in APL cells. Previous studies have shown that the TF expression levels are elevated in NB4 (APL) cells or in U937 (monoblastic) cells ectopically expressing PML/RARα indicating that the TF expression in these cells is regulated in response to PML/RARα. The molecular mechanisms by which PML/RARα regulates the TF expression are, however, still poorly understood. To examine whether the PML/RARα fusion protein regulates the TF expression through an interaction with TF promoter, a series of cell lines stably expressing luciferase reporter gene under control of different 5’ flanking regions of TF promoter were established with U937/PR9 cells, a U937 cell line expresses Zn-inducible PML/RARα. In luciferase assays a Zn-induced transcriptional activity was present in cells with truncated TF promoters (-2106~+128), (-1386~+128), (-684~+128), (-384~+128) and (-247~+128) but absent in those with a shorter version of TF promoter (-201~+128). These results suggested that the sequences within -247 and -201 of TF promoter were necessary for the regulation of TF gene expression regulated by PML/RARα in U937/PR9 cells. In ChIP experiments a markedly increased amount of PCR products in response to the expression of PML/RARα were amplified by the primers flanking the regulatory region (-247~-201) from the chromatins immunoprecipitated with an anti-RARα antibody, suggesting that the PML/RARα fusion protein interacts with TF promoter concomitant with the elevated luciferase activity. The EMSA data using probes containing distal or proximal AP-1 consensus sequences located in the regulatory region (-247~-201) showed that PML/RARα did not directly bind to this region but enhanced the binding ability of c-Jun and Jun D to the proximal AP-1 site. Collectively, our data showed that the PML/RARα fusion protein up-regulates the expression of TF through the interaction with the regulatory region of TF promoter. The more accurate localization of the regulatory region in TF promoter and the related molecular mechanisms regarding the regulatory effects of the PML/RARα fusion protein on TF expression are under investigation.
Yeast Models of Diamond Blackfan Anemia and Shwachman Diamond Syndrome Differ in Their Effect on the Synthesis and Function of Ribosomal Subunits.
Authors: Ellis Steven R , Moore Joseph B. , Liu Johnson M. , Arceci Robert J. , Farrar Jason E.
Published: April 2020

Nucleolar stress is a frequently invoked mechanism used to describe the pro-apoptotic phenotype of cells affected in human diseases linked to abnormalities of the ribosome. However, the diversity of clinical phenotypes observed in these diseases suggests that there may be different types of nucleolar and/or translational stress that stimulate cell death pathways by alternative mechanisms. We have studied yeast models of Diamond Blackfan anemia (DBA) and Shwachman Diamond syndrome (SDS), two inherited bone marrow failure syndromes linked to defects in ribosome synthesis and/or function, to determine potential underlying molecular mechanisms that distinguish these disease models. To date, all genes identified in DBA encode ribosomal proteins. In contrast, SBDS, the gene affected in SDS encodes a protein that associates with 60S subunits, but is not considered a structural component of the ribosome. We have analyzed the translational capacity of cells harboring mutations in RPL33A and SDO1, yeast orthologs of genes affected in DBA and SDS, respectively. Polysome profiles from cells depleted of Rpl33A have a decrease in the amount of free 60S subunits and the presence of half-mer polysomes, as expected for an essential structural component of the 60S subunit. Polysome profiles from cells depleted of Sdo1 also had half-mer polysomes, but in this case there were significant amounts of free 60S subunits evident. Analysis of the intracellular distribution of 60S subunits by fluorescence microscopy revealed significant differences between the two disease models. In the DBA model, there was no evidence of accumulation of incompletely assembled subunits in the nucleolus indicating that rapid degradation. In contrast, in the SDS model there was significant accumulation of 60S subunits in the nucleoplasm. Thus, the two disease models interfere with the biogenesis of 60S subunits through distinct mechanisms. To determine if these mechanistic differences influence protein synthesis, we analyzed the patterns of proteins synthesized in these two disease models. We found that the expression of the 20S replicon was induced in both models, a sign of general translational stress. However, the two models also showed distinct differences in the synthesis of certain proteins. Thus, the mechanisms by which reductions of Rpl33A or Sdo1 influence levels of functional 60S subunits have differential effects on the patterns of proteins synthesized within cells Together these data indicate that the ribosome-based diseases may result from a composite of effects that include both nucleolar stress mechanisms and changes in translational output. The distinct clinical phenotypes observed in these disorders may result from differences in the relative contributions of either of these two mechanisms.
Very Rapid Clearance after a Joint Bleed Knee Can Not Prevent Adverse Effects on Cartilage and Synovial Tissue: A Canine in Vivo Study.
Authors: Jansen Nathalie W.D. , Roosendaal Goris , Wenting Marion , Hazewinkel Herman A.W. , Bijlsma Johannes W.J. , Theobald Matthias , Lafeber Floris P.J.G.
Published: April 2020

Purpose Joint bleeds lead to joint destruction. In vitro exposure of human and canine cartilage to blood results in long lasting severe adverse changes in cartilage. An in vivo joint haemorrhage in the canine knee joint demonstrates similar adverse effects although less outspoken and long-lasting. We investigated the clearance rate of blood from canine knee joints as a possible explanation for this discrepancy.

Methods Blood was injected into the knee joint of Beagle dogs, either 48h, 24h or 15m before termination. The amount of red and white blood cells present in the joint cavity was determined. Chondrocyte activity and cartilage matrix integrity as well as cartilage destructive activity of synovial tissue were determined biochemically. Additionally, synovial tissue was analyzed by use of histochemistry.

Results Fifteen minutes after the injection of autologous blood, the red blood cell count was 5,7*1012/L, comparable to the amount present in whole blood, and gradually decreased (1,6*1012/L at 24 hours) to 0,2*1012/L within 48 hours (less than 5%). The amount of white blood cells increased in the first 24 hours, and was still increased after 48 hours, although less than after 24 hours. The proteoglycan synthesis rate and -release were adversely affected already within 24 hours (−22% and +24% respectively), and these effects were more severe 48 hours post-injection (−34% and +53% resp.). Synovial tissue culture supernatants demonstrate cartilage destructive properties as expressed by an increased release, a decreased synthesis rate, and decreased content of cartilage proteoglycans; increasing with time after the experimental haemorrhage (+207%/+247%; −58%/−62%; −8%/−28% respectively, for 24/48 hours). Evaluation of the synovial tissue revealed at 15 minutes post-injection countless numbers of intact RBC that were almost completely disappared after 48 hours, withonly limited recruitment of macrophages and iron deposition.

Conclusions Blood is cleared very rapidly from the canine knee joint, but in that short time span already has adverse effects on both cartilage and synovial tissue. This rapid clearance can play a role in the discrepancy between long-term in vitro and in vivo effects of blood-induced joint damage since more than 10% v/v blood for 48 hours is needed induced to long-term adverse effects in vitro. Irrespectively, blood has devastating effects on articular cartilage very rapidly, and in this respect it is important to prevent (traumatic) joint haemorrhages and if they occur, to treat them properly.
Identification of ADAMTS13 Peptide Sequences Recognized by Autoantibodies in Patients with Acquired Thrombotic Thrombocytopenic Purpura.
Authors: Yamaguchi Yusuke , Moriki Takanori , Wada Hideo , Matsumoto Masanori , Fujimura Yoshihiro , Nakagawa Terumichi , Igari Atsuko , Ikeda Yasuo , Murata Mitsuru
Published: April 2020

Anti-ADAMTS13 autoantibodies are considered to play pivotal roles in the pathophysiology of acquired thrombotic thrombocytopenic purpura (TTP). They inhibit the ADAMTS13 function resulting in the appearance of ultra-large von Willebrand factor (VWF) multimers. Major binding sites of the autoantibodies were reported to be in the cysteine-rich/spacer domains. To clarify the precise peptide sequences recognized by anti-ADAMTS13 IgG autoantibodies, we constructed a random cDNA fragment library expressing various peptides of ADAMTS13 on the surface of lambda phage and screened the library using purified IgG from 13 TTP patients. Diverse peptide sequences were obtained from almost entire ADAMTS13 domains such as metalloprotease, disintegrin, TSP1-1, cysteine-rich, spacer, TSP1- 2, 3, 4, 5, 7, 8 and CUB1. In particular, we detected an identical 26 amino-acid epitope sequence in the C-terminus of spacer domain from Gly662 to Val687 (sp662–687) shared by 5 TTP patients. Moreover, the peptide sequence was exactly included in one of the VWF binding epitope sites that we previously determined (Blood110 (11), 795a, 2007). We then assessed the impact of specific autoantibody to ADAMTS13 activity measured by FRETS-VWF73 or EIA and ADAMTS13 inhibitor titer in each of TTP patient plasma. However, both of the ADAMTS13 activity and inhibitor titer seemed not correlated with the existence of specific sp662–687 IgG autoantibody. These observations suggest that the autoantibody to sp662–687 may be one specific feature of TTP, although other epitopes are also involved in the pathogenesis of the disorder.
The MiR23A MicroRNA Cluster Is a Target of PU.1 and Promotes Development of Myeloid Cells at the Expense of B Cells
Authors: Owens Kristin S , Kong Kimi Y , Rogers Jason H , Dahl Richard E.
Published: April 2020

The transcription factor PU.1 is an important regulator of hematopoiesis, directing cell fate decision in a dosage dependent manner with higher levels driving monocytic differentiation and lower levels directing B-cell differentiation. The mechanism by which PU.1 controls this process is not fully understood. Here we show that PU.1 is involved in regulating the expression of several microRNAs. MicroRNAs regulate gene expression at a post-transcriptional level by binding target mRNA through the 3′ UTR and either repressing translation or causing degradation of the mRNA itself. We used microarray expression profiling to assess a large group of microRNAs in PUER cells, a PU.1 −/− cell line that differentiates into macrophages when PU.1 activity is restored. Several miRNAs showed changes in expression four days after restoration of PU.1 activity. We focused on a cluster of microRNAs that includes miR-23a, miR27a, and miR24-2, which we call the miR23a cluster. All three miRNAs are coded for on a single pri-miRNA transcript. Northern blot analysis verified the findings of the microarray–that expression of this microRNA cluster is enhanced by PU.1. The promoter for the cluster contains several conserved predicted binding sites for PU.1. Chromatin immunoprecipitation and EMSA has confirmed that PU.1 binds to these sites. In addition, reporter assays show that the mir23a promoter can be activated by PU.1. The 23a cluster appears to be a critical target gene for PU1 to promote myeloid development. Using in vitro hematopoietic cultures we have observed that expressing this cluster in hematopoietic progenitors promotes myeloid development over B cell development. In addition, data from bone marrow transplant assays will be presented demonstrating the role of this cluster in directing hematopoiesis in vivo. Targetscan and miRanda computer algorithms predict several B cell transcription factors as targets of the miRNAs of the 23a cluster. PU.1 potentially activates expression of the 23a cluster to downregulate B cell transcription factors in order to commit cells to the myeloid cell fate.
The Tyrosine Kinase Src Regulates Adhesion and Survival in Waldenström Macroglobulinemia
Authors: Ngo Hai T. , Farag Mena , Jia Xiaoying , Burwick Nicolas , Melhem Molly R. , Leleu Xavier , Runnels Judith , Azab Abdel Kareem , Roccaro Aldo M. , Azab Feda , Sacco Antonio , Quang Phong , Tai Yu-Tzu , Anderson Kenneth C. , Ghobrial Irene M.
Published: April 2020

BACKGROUND: Tyrosine phosphorylation is a central event in the regulation of a variety of biological processes such as cell proliferation, migration, adhesion, and survival. Waldenström Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy and characterized by widespread involvement of the bone marrow. We sought to determine whether the protein tyrosine kinase Src regulates adhesion and survival in WM.

METHODS: The WM cell lines BCWM1, WM-WSU, and IgM secreting lymphoma cells lines MEC-1 and RL were used in these studies. Primary CD19+ WM cells were obtained from the bone marrow of patients after informed consent. AstraZeneca Biopharmaceuticals (London, England) provided the Src inhibitor AZD0503 (AZD). Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake, respectively. Apoptosis was measured using flow cytometry with Apo 2.7 staining. Western blotting was performed to determine downstream signaling pathways. Migration was performed using the transwell migration assay.

RESULTS: We demonstrated that pSrc is overexpressed in WM cells compared to control B cells. Similarly, phospho-Src protein expression was upregulated in WM cell lines, specifically BCWM.1 but not in WM-WSU. We then showed that pSrc regulates migration and adhesion in response to the chemokine SDF-1, as well as in vivo homing using in vivo flow cytometry. The use of the specific Src inhibitor AZD0530 led to significant inhibition of adhesion and migration in cell lines with pSrc activation, but not in those deficient of Src activation. Similarly, inhibition of Src activity led to significant inhibition of proliferation and survival through inhibition of STAT3, Akt, and ERK/MAPK pathways. The monoclonal antibody rituximab signals through Src kinase, and the combination of AZD0530 and rituximab was synergistic in vitro.

CONCLUSION: Taken together, these studies delineate the role of Src kinase activity in WM and provide the framework for future clinical trials using Src inhibitors in combination with rituximab to improve the outcome of patients with WM.
Confirmation of the Efficacy and Safety of Lenalidomide Oral Monotherapy in Patients with Relapsed or Refractory Diffuse Large-B-Cell Lymphoma: Results of An International Study (NHL-003)
Authors: Czuczman Myron S. , Vose Julie M. , Zinzani Pier Luigi , Reeder Craig B. , Buckstein Rena , Haioun Corinne , Bouabdallah Reda , Polikoff Jonathan , Guo Pingshan , Ervin-Haynes Annette , Pietronigro Dennis , Zeldis Jerome B. , Witzig Thomas E.
Published: April 2020

Introduction: Diffuse large-B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas (NHL). Patients with DLBCL who are not cured by treatment with R-CHOP chemotherapy or high-dose chemotherapy with autologous stem cell rescue have a poor prognosis. These patients reflect a clear unmet need in the treatment of DLBCL. A previous sub-analysis of a phase II trial (NHL-002) of lenalidomide in patients with relapsed or refractory DLBCL, demonstrated a 19% overall response rate (ORR) with a 7-month median duration of response (DR). A confirmatory international phase II trial (NHL-003) of single-agent lenalidomide was initiated for patients with relapsed/refractory aggressive NHL that had received at least one prior treatment and had measurable disease. Herein, we report the data from the DLBCL patients enrolled in this trial.

Methods: Patients received 25 mg oral lenalidomide once daily on days 1–21 of every 28-day cycle and continued therapy until disease progression or toxicity. The 1999 IWLRC methodology was used to assess response and progression.

Results: This report focuses on the 73 DLBCL patients that were enrolled and evaluable for response assessment. The median age was 67 (21–87) years and 49 (67%) patients were male. Median time from diagnosis to lenalidomide treatment was 2 (0–18.6) years, and patients had received a median of 3 (1–6) prior treatment regimens. The ORR to lenalidomide was 29% (21/73), with 4% complete response (3/73) and 25% partial response (18/73). Eleven patients (15%) had stable disease. The most common grade 3 or 4 adverse events were neutropenia (32%), thrombocytopenia (15%), asthenia (8%) and anemia (7%).

Conclusion: These results of this international study confirm that lenalidomide is active in heavily pre-treated patients with relapsed or refractory DLBCL with manageable side effects.
Foxp3 Expression Is Induced in Peripheral Blood Mononuclear Cells from Patients with CTCL and GVHD after Extracorporeal Photopheresis
Authors: Ni Xiao , Shiue Lisa , Arias-Mendoza Paola , Aakhus Erin , Hosing Chitra , Duvic Madeleine
Published: April 2020

Anti-tumor immunity mediated by cytotoxic CD8+ T lymphocytes (CTL) is thought to underlie the efficacy of extracorporeal photopheresis (ECP) for cutaneous T cell lymphoma (CTCL). In contrast, induction of immune tolerance mediated by regulatory T cells (Treg) is hypothesized to underlie the effect of ECP for graft-versus-host disease (GVHD). In this translational research study, peripheral blood mononuclear cells (PBMC) from patients (Pt) with Sézary Syndrome (SS), a leukemic form of CTCL, and GVHD pre- and post-ECP were studied for expression of the CTL cytokine IFN-gamma and of the nuclear transcription factor forkhead box P3 (Foxp3), a specific marker of Treg. Total RNA from PBMCs at pre-ECP, 2 day, 1 month, 3 months, and 6 months post- ECP were quantitated by real-time quantitative reverse transcript-PCR (RT-Q-PCR) for Foxp3 and IFN-gamma mRNA expression levels, normalized to endogenous control gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Foxp3 levels at baseline varied but increased at 2 day and 1 month post-ECP in GVHD by 9.5-fold and 27.6-fold, respectively (Fig.1). Similarly,foxp3 also increased in 3 of 4 CTCL patients (Pt#1, #2 and #4). Foxp3 levels rose steadily in clinical responders, Pt#1 and Pt#2, during the treatment course, and highest levels were detected at 6 months post-ECP with 13.1-fold and 17.4-fold increase, respectively. Pt#1 and #2 experienced partial clinical response and decreased circulating tumor cells with ECP. The levels of interferon-gamma had only < 2-fold up or down over the treatment course from baseline among CTCL post-ECP samples. While the level of IFN-gamma was decreased by half at 2 day and 1 month post-ECP in GVHD compared to that in pre-ECP (Fig.2). These results suggest that Tregs (detected by Foxp3) but not IFN-gamma are induced during ECP in responding CTCL patients as in GVHD. ECP might thus have common mechanisms in these two T cell mediated diseases. The study will continue to enroll patients to further study the effects of ECP.

Fig. 1 Foxp3 expression in PBMCs from CTCL and GVHD pre- and post ECP Fig. 1. Foxp3 expression in PBMCs from CTCL and GVHD pre- and post ECP Fig. 2 IFN-gamma expression in PBMCs from CTCL and GVHD pre- and post ECP Fig. 2. IFN-gamma expression in PBMCs from CTCL and GVHD pre- and post ECP
R2: Preliminary Results of a Phase II Study of Lenalidomide and Rituximab in Relapsed/Refractory Indolent Non-Hodgkin’s Lymphoma (NHL)
Authors: DeRook Ian , Odonnell Robert T , Noble B. , Quirch C. , Tuscano Joseph
Published: April 2020

Background: Lenalidomide (Revlimid®) is a potent immune-modulating, anti-angiogenic agent, approved in the USA for treatment of relapsed/refractory multiple myeloma and myelodysplastic syndromes associated with the 5q- chromosomal deletion. Lenalidomide also demonstrates activity as monotherapy in aggressive and indolent NHL, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), and cutaneous T-cell lymphoma. The current study was designed to evaluate the safety and efficacy of the lenalidomide and rituximab (R2) combination in patients with relapsed/refractory indolent NHL.

Methods: Patients with relapsed/refractory indolent NHL with measurable disease after at least 1 prior treatment regimen were eligible. Lenalidomide was initially administered at a dose of 25mg orally once daily, on days 1–21 on a 28-day cycle, and continued until disease progression. Rituximab 375mg/m2 was infused beginning on day 15 of cycle 1, and repeated weekly for a total of 4 doses. Following development of tumor lysis syndrome (TLS) in two patients, the protocol was amended to reduce the starting dose of lenalidomide to 20mg, and TLS prophylaxis was provided. Response and progression were evaluated using IWLRC methodology.

Results: At the time of this report, 7 patients were enrolled, and 6 had initiated therapy. The median age was 67 (range 54–91) years and 2 were female. Histologies are follicular lymphoma [FL] (n=5), SLL (n=1), and marginal zone lymphoma (n=1). Median time from diagnosis to treatment on study was 7.3 (3.3–19) years and median number of prior treatment regimens was 4 (1–5). All patients had received prior treatment with rituximab. Four patients were evaluable for response, all of whom had rituximab-resistant disease. All four evaluable patients, including two with highly refractory disease, after a median of 2 cycles, exhibited an objective response (1 complete response unconfirmed, 2 partial responses, and 1 minor response). Progression free survival, currently ongoing, is > 102 days. Toxicity included TLS in two refractory patients with FL within the first week of therapy. The most common grade 3 and 4 events were neutropenia, lymphopenia, and hyponatremia.

Conclusion: This is the first study to demonstrate that combination therapy using lenalidomide and rituximab is safe and active in relapsed/refractory indolent NHL. The side effect profile was acceptable with the addition of TLS prophylaxis. With additional analysis, the R2 combination may warrant further study in refractory indolent lymphoma.
HOX-Gene Expression and Inhibition of the Non-Canonical NF-KappaB Pathway in t(7;12) Leukemias
Authors: Wildenhain Sarah , Hauer Julia , Roettgers Silija , Engelmann Hartmut , Novosel Astrid , Binder Vera , Slany Robert K. , Ludwig Wolf-Dieter , Harbott Jochen , Borkhardt Arndt
Published: April 2020

The chromosomal translocation t(7;12) was described recently in MLL negative acute myeloid leukemias of infancy associated with a very poor clinical outcome. The rearrangement involves the genes HLXB9 (7q36) and ETV6 (12p13) with a fusion transcript of exon1/HLXB9 and exon3/ETV6. An alternative in frame splicing variant of exon1/HLXB9 to exon2/ETV6 is also detectable. Leukemic bone marrow samples of 42 infants, diagnosed in Germany with AML, were screened for the fusion transcript HLXB9/ETV6 with RT-PCR. Inclusion criteria were diagnosis of AML, age<2 years, no MLL/AF9, MLL/AF4 or MLL/ENL rearrangement in RT-PCR, no chromosomal 11q23 rearrangement and no trisomy 21. Positive samples were cloned and sequenced. A fusion transcript was found in approx. ~17% of patients. Common features of HLXB9/ETV6 positive leukemias in this cohort are poorly differentiated FAB subtypes, trisomy 19 karyotype of the leukemic cells, coexpression of the T-cell markers CD4 and CD7 as well as high expression of the un-rearranged HLXB9 gene. Quantitative PCR showed no upregulation of HOXA9 and MEIS1 in HLXB9/ETV6 positive patients as reported for MLL+ leukemias. The mechanism of HLXB9/ETV6 induced leukemogenesis remains unknown. We show that in vitro retroviral transduction of murine hematopoietic precursor cells with either HLXB9/ETV6 or HLXB9 or ETV6/RUNX1 was not transforming. Furthermore altered activity of the transcription factor family NF-kappaB has been demonstrated for various types of cancer including acute myeloid leukemias. Therefore we analysed the impact of the HLXB9/ETV6 fusion on the NF-kappaB signaling pathway in a 293T cell-system. HLXB9/ETV6 and HLXB9 showed inhibition of the NF-kappaB dependent transcription signal in a reporter assay, whereas the typical ALL-fusion ETV6/RUNX1 showed no influence. Analysis of nuclear extracts by western blot confirmed, that the inhibition of the NF-kappaB signal is due to a reduced nuclear translocation of the non-canonical NF-kappaB components p52 and RelB, whereas the canonical pathway remains not influenced.

Conclusion: The HLXB9/ETV6 fusion transcript can be found in 17% of infants with MLL-negative AML in a German cohort. HLXB9/ETV6 positive leukemias show no increased expression on HOXA9 and MEIS1, but coexpression of T-cell markers and inhibition of the non-canonical NF-kappaB pathway on protein level. HLXB9/ETV6 did not induce malignant transformation in murine hematopoietic precursor cells.