bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of ID4 as a Cooperating Second Hit for T Cell Lymphoma Development in PU.1 UREΔ/Δ Mice.
Authors: Owens Bronwyn M. , Yu Li , Steidl Ulrich , Kutok Jeffrey L. , Clayton Linda K. , Wagner Katharina , Iwasaki Hiromi , Liu Chunhui , Hackanson Björn , Akashi Koichi , Plass Christoph , Tenen Daniel G. , Rosenbauer Frank
Published: April 2020

The transcription factor PU.1 is a key regulator of hematopoiesis and is essential for normal hematopoietic stem cell (HSC) function and myeloid and lymphoid differentiation. Knockout mice are embryonic or neonatal lethal and exhibit an early differentiation block at the transition from HSC to CMP and CLP. Mice with a deletion of a critical upstream regulatory element (URE) at −14kb has been deleted exhibited decreased PU.1 expression in HSC, progenitors and B cells to 20% of normal levels. In contrast, an increase of PU.1 expression was detected in early thymic progenitors. UREΔ/Δ animals exhibited a block in normal thymocyte differentiation and frequently progressed to fatal T cell lymphomas between 3 and 8 months of age. Tumors were clonal and transplantable into NOD-SCID recipients. Since aberrant PU.1 expression constituted the initiating mutation, we sought to identify cooperating mutations contributing to tumor development by surveying the genome for hypermethylated promoter regions using restriction landmark global scanning. Methylation patterns revealed a characteristic epigenetic footprint leading to selective tumor suppressor gene silencing in transformation of lymphoid but not myeloid UREΔ/Δ progenitors, since DNA methylation was significantly lower in the AML samples compared with lymphomas. One of the corresponding methylated genes that was silenced in all lymphomas analyzed but none of the AMLs was identified as Idb4 (encoding ID4, inhibitor of DNA binding 4). This observation is consistent with data describing ID4 as a strong tumor suppressor gene which is frequently silenced in neoplasms. The expression of Idb4 in 6 additional lymphomas was assessed by quantitative RT-PCR and showed a strong correlation between promoter hypermethylation and Idb4 downregulation. To test whether Idb4 silencing through promoter hypermethylation can be pharmacologically reversed, a UREΔ/Δ lymphoma cell line was established and treated with 5-aza-2′deoxycytidine. We found that Idb4 expression was restored to normal levels following increasing concentrations of this demethylating agent. Finally, we analyzed the functional relevance of the Idb4 downregulation by restoring Idb4 expression in the UREΔ/Δ tumor line. UREΔ/Δ lymphoma cells reexpressing Idb4 had a marked reduction in both the in vitro growth rate and the ability to cause tumors after transplantation into NOD/SCID recipient mice, indicating that Idb4 is a potent suppressor of UREΔ/Δ thymic tumorigenicity. Taken together, we provide evidence that epigenetic DNA modifications contribute significantly and selectively to lymphoid transformation in UREΔ/Δ mice, and suggest a role for Idb4 repression as a second event in PU.1-initiated transformation of lymphoid precursors.
The BCL6 Oncoprotein Forms Distinct Transcriptional Repression Complexes on Cohorts of Target Genes Involved in Specific Cellular Functions.
Authors: Polo Jose M. , Ranuncolo Stella Maris , Parek Samir , Arcaya Jose , Melnick Ari M.
Published: April 2020

The BCL6 (B-Cell-Lymphoma-6) oncoprotein is required for formation of germinal centers by normal B-cells and is frequently constitutively activated in B-cell lymphomas. BCL6 is a transcriptional repressor of the BTB/POZ - zinc finger family of proteins. Transcriptional repressors are generally believed to function by binding to specific DNA sequences in target promoters and then recruiting a cohort of corepressor proteins that physically alter the chromatin structure of the locus leading to silencing. BCL6 can recruit several different corepressors. Accordingly, N-CoR, SMRT and BCoR bind to the BTB domain of BCL6, the NuRD complex binds to the second repression domain and ETO binds to the C-terminal zinc finger domain. These proteins recruit additional corepressors such as HDACS, thus forming multi-protein complexes. BCL6 is involved in both licensing germinal center B-cells for survival as well as blocking differentiation to memory or plasma cells. However, we found that blockade of the BCL6 BTB domain with a specific inhibitor causes only apoptosis but not differentiation of B-cells. In order to identify BCL6 target genes and the mechanism through which they are silenced, we performed extensive ChIP on chip analysis of BCL6, its corepressors, and their chromatin signatures using tiled oligonucleotide arrays containing 1.5 KB of 24,000 promoters. These were performed in the presence or absence of BCL6 shRNA and other BCL6 inhibitor molecules, in tandem with expression arrays as a functional readout. We also performed in depth ChIP on chip experiments using custom arrays, where sets of entire BCL6 target loci were tiled through with overlapping oligos. From these studies we i) identified a large cohort of direct BCL6 target genes involved in apoptosis, cell damage, protein degradation and differentiation that provide critical insight into the mechanism of action of BCL6 in normal and malignant B-cells; ii) discovered that cohorts of BCL6 direct target genes involved in different pathways are regulated by specific corepressors, which are mutually exclusive in their binding to BCL6 target loci, iii) that BCL6 can repress genes through a variety of different mechanisms with unique chromatin signatures. Thus, previously unrecognized mechanisms exist in transcriptional repression, that determine formation of specific corepressor complexes. Using this information, we have been able to re-activate discrete cohorts of BCL6 target genes controlled by specific corepressors, resulting in specific biological effects such as apoptosis or differentiation of lymphoma cells. These results provide fundamental insights into the transcriptional and biological mechanism of action of BCL6 in B-cells.
cJun Is Phosphorylated at Ser73 and Contributes to Cell Cycle Progression in Anaplastic Large Cell Lymphoma.
Authors: Leventaki Vassiliki , Drakos Elias , Claret Francois-Xavier , Medeiros L. Jeffrey , Rassidakis George Z.
Published: April 2020

Anaplastic Large Cell Lymphoma (ALCL) frequently carries the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). cJun is a member of the activator protein-1 (AP-1) family, which is a group of transcription factors that control cell proliferation, differentiation, growth and apoptosis. The activity of cJun can be regulated by phosphorylation at serine 73 (Ser73) and serine 63 (Ser63) residues of the N-terminal domain. It is believed that cJun promotes cell cycle progression, in part, through downregulation of the cyclin-dependent kinase inhibitor p21. Previous studies have shown high AP-1 activity and cJun overexpression in Hodgkin lymphoma and ALCL (Mathas et al, EMBO J2002; 21:4104). In this study, we assessed for expression of cJun and its Ser73- and Ser63-phosphorylated forms in two ALK+ (Karpas 299 and SU-DHL-1) and one ALK- (Mac2A) ALCL cell lines by western blot analysis, and in 31 ALCL tumors (15 ALK+, 16 ALK-) by immunohistochemistry using tissue microarrays and specific antibodies. To examine the role of cJun in cell survival and proliferation in our in vitro system, ALCL cells were transiently transfected with small interfering RNA (siRNA) specific for cJun. Cell viability, proliferation of viable cells and cell cycle progression from G1 to S-phase were assessed by trypan blue exclusion, MTS and BrdU assays, respectively. All three ALCL cell lines expressed total cJun and Ser73-phosphorylated cJun (Ser73p-cJun) at a high level, whereas Ser63-phosphorylated cJun was expressed at a low level. In addition, all 31 ALCL tumors expressed total cJun in most neoplastic cells. Ser73p-cJun was also detected in all ALCL tumors at a variable level with the percentage of Ser73p-cJun-positive tumor cells ranging from 5% to 95%. By contrast, Ser63p-cJun was detected rarely in tumor cells. Transient transfection of ALCL cells with specific siRNA resulted in almost complete silencing of total cJun expression and absence of Ser73p-cJun expression, which was associated with decreased cell viability and a substantial (40%) decrease of cell growth. cJun silencing also resulted in cell cycle arrest as shown by decreased S-phase fraction. These cell cycle changes were associated with a marked increase of p21 levels and downregulation of cyclin D2 and D3. In conclusion, cJun is highly phosphorylated at serine 73 in ALCL cell lines and tumors and may contribute to cell cycle progression. Targeting cJun expression or phosphorylation using gene therapy approaches may represent a novel therapeutic strategy for patients with ALCL.
Increased Risk of Cerebrovascular Events Is Associated with Elevated Factor XI Activity.
Authors: Yang David T. , Flanders Michele M. , Rodgers George M.
Published: April 2020

High levels of factor XI as measured by antigenic methods have been implicated as a risk factor for deep venous thrombosis, and there is limited evidence that increased factor XI activity as measured by a functional assay is associated with cardiovascular disease. In the current study, we evaluated factor XI antigen, factor XI functional activity and high-sensitivity C-reactive protein (hs-CRP) from 123 patients under the age of 55 with normal prothrombin and partial thromboplastin times undergoing evaluation for a hypercoagulable state. Of the 123 patients, 80 had a history suggestive of arterial thrombosis (65 with stroke symptoms, 13 with transient ischemic attack symptoms, and 2 with other arterial thrombi), 17 had a history of venous thrombosis, and 26 had indeterminate histories for arterial or venous thrombosis. 40 age- matched healthy subjects were used to determine the upper limit of normal for factor XI activity as defined by the 95th percentile value (141%). 17/80 (21%) of patients with arterial thrombosis and 3/17 (18%) of the patients with venous thrombosis had above normal factor XI activity levels. Based on this, those with elevated factor XI activity have a relative risk of 5.3 for a cerebrovascular event. Regression analysis demonstrates that factor XI activity appears to correlate with factor XI antigen level (slope=0.79 and R=0.67), but there is no correlation between factor XI activity or factor XI antigen levels and hs-CRP (R= −.003 and R=.096 respectively). Our findings suggest that elevated factor XI activity is associated with an increased risk for cerebrovascular events and confirms that elevated factor XI levels are also associated with increased risk for venous thrombosis. In addition, assessment of factor XI levels by two methods, both functional activity and antigenic level, appear to correlate with one another to a fair degree suggesting that increased activity is likely related to increased levels of the protein itself. Lastly, lack of correlation with hs-CRP suggests that factor XI is not an acute-phase reactant.

Population Characteristics n Mean Age Median Age Range Male:Female Normals 40 39±9 41 23–55 15:25 Arterial Thrombosis 80 42±8 43 20–55 36:44 Venous Thrombosis 17 38±11 37 20–54 7:10 Factor XI ActivityLevels Normal Arterial Thrombosis Venous Thrombosis Factor XI Activity (%) Mean±SD 101±23 136±111 111±36 Median 100 117 113 Range 57–155 55–675 71–196 95th percentile 141 # Cases above 95th percentile 2/40 (5%) 17/80 (21.3%) 3/17 (17.6%)
Evidence for a Novel Splice Variant of the Gene for Platelet Activating Factor Acetylhydrolase 1B2 (PAFAH1B2), an Important Signal Terminator in the Platelet Activating Factor (PAF) Pathway.
Authors: Olson Nels , Scott Bruce , Long George , Bovill Edwin
Published: April 2020

Introduction: A genome-wide scan of linkage with venous thrombosis in a thrombophilic kindred with type I protein C deficiency identified a candidate region on chromosome 11q23 (P<0.0001). Located at the linkage peak is PAFAH1B2, the β-subunit of the intracellular form of PAFAH. Intracellular PAFAH is a heterodimer of alpha and beta subunits both of which have catalytic activity. PAFAH reduces intracellular PAF levels and a reduction in active PAFAH likely leads to an enhanced inflammatory response at the vascular wall. Resequencing experiments revealed a novel splice variant for the intracellular form of PAFAH. The current study characterizes this novel splice variant that results in the deletion of exon 6 and its catalytic domain and the introduction of an alternative 7th exon.

Methods: Identification of novel mRNA splice variants was achieved by sequencing PCR products obtained from amplifying known regions of PAFAH 1B2 mRNA. Confirmation of the exon 5 to 7 splice and exon 7 polyadenylation were obtained from sequencing 3′RACE products. Protein extracts were resolved by SDS- PAGE and Western blotted.

Results: An alternative splice form of PAFAH 1B2 was isolated and sequenced. This isoform was observed in cDNA from human liver, moncytes, and T and B lymphocytes using PCR. This variant contains the first five exons of the native form protein and replaces exon 6 with an alternative 7th exon located downstream of exon 6. Exon 6 contains two residues involved in the protein’s active site triad, Asp and His, and are absent in exon 7. Computer modeling suggests replacement of exon 6 by exon 7 would abolish the active site and possibly alter the proteins’ ability to dimerize. Preliminary Western blots from selected tissues demonstrated the presence in liver of a new band consistent with the predicted size of the variant.

Conclusions: We hypothesize that this alternative splice likely decreases the proteins ability to function as a regulator of inflammation. We are presently carrying out gene expression analysis to determine the relative levels of the splice variant in different tissues and changes in expression associated with activation of leukocytes.
Endothelial Microparticles, Platelet and Leukocyte Activation and Cellular Interactions in Patients with Non-Valvular Atrial Fibrillation.
Authors: Chirinos Julio A. , Castrellon Aurelio , Jy Wenche , Horstmann Lawrence L. , Jimenez Joaquin J. , Willens Howard J. , Castellanos Agustin , Myerburg Robert J. , Ahn Yeon S.
Published: April 2020

Background: Atrial fibrillation (AF) is associated with a systemic pro-coagulant state. Coagulation involves complex interactions between endothelial and circulating cells, cell-derived microparticles and plasma proteins.

Methods: We investigated the levels of endothelial and platelet microparticles, leukocyte and platelet activation, and cell interactions in 48 patients with non-valvular AF and compared to normal controls. We performed flow cytometry in periheral venous blood to measure (1) Platelet expression of P-selectin, (2) Platelet microparticles; (3) Platelet-leukocyte conjugates; (4) Leukocyte expression of CD11b; (5) Endothelial microparticles (EMP) positive for CD31 and negative for CD42 (EMP31+), EMP positive for E-selectin (EMP62E+), CD54 (EMP54+), or CD51 (EMP51+); (6) EMP54+- and EMP62E+-leukocyte conjugates. Atrial fibrillation was confirmed electrocardiographically and left atrial volume was estimated by echocardiography. The mean age of patients with AF was 61.3±12 years. Congestive heart failure was present in 54.2% of patients, hypertension in 72.9%, and diabetes mellitus in 22.9%. Most patients (81%) were receiving anticoagulant therapy and 62.5% were receiving antiplatelet medications. Calcium channel blockers or digoxin were given to 43.8% and 54.2% of patients, respectively. The mean ventricular rate was 85.5±17 beats per minute, and the mean left atrial volume was 97.9 ml.

Results: Patients with AF had significantly higher levels of platelet P-selectin expression and platelet-leukocyte conjugates. P-selectin expression strongly correlated with platelet-leukocyte conjugate levels (r=0.77; p<0.001; Figure 1). Patients with AF also demonstrated increased levels of free EMP31+, EMP62E+ and EMP51+, as well as EMP62E+-lymphocyte, -monocyte and -neutrophil conjugates. Levels of free EMP54+ were lower, whereas EMP54+- neutrophil and EMP54+-lymphocyte conjugates were higher among patients with AF. EMP62E+-neutrophil conjugates correlated with leukocyte expression of activation marker CD11b (r=0.42; p=0.003; Figure 2) and predicted CD11b expression independently of platelet-leukocyte conjugates (r2 increase=16%; p=0.003).

Conclusions: Patients with AF have increased levels of platelet and leukocyte activation, circulating endothelial microparticles, and binding of endothelial microparticles and platelets to leukocytes. Platelet activation is a determinant of platelet-leukocyte conjugate formation in AF. The formation of EMP62E-neutrophil conjugates is a major determining factor for circulating leukocyte activation in AF. Further studies are needed to assess the correlation of these markers with the risk of thromboembolism in AF.

Figure 1. Figure 1. Figure 2. Figure 2.
ADAMTS-13 Binds Platelets in a Specific, Divalent Cation and Activation Dependent Manner.
Authors: Artoni Andrea , Garagiola Isabella , Lombardi Rossana , Peyvandi Flora , Mannucci Pier Mannuccio
Published: April 2020

ADAMTS-13 cleaves high molecular weight von Willebrand factor (VWF) realeased by the endothelium in order to prevent massive intravascular platelets adhesion and aggregation as pathologically observed in thrombotic thrombocytopenic purpura. ADAMTS-13 is present at low levels in plasma. We therefore surmised that platelets are able to specifically bind the metalloprotease on their surface, hereby concentrating the enzyme where it is most required. With this as background, 96 wells polystyrene NUNC plates were coated either with albumin or fibrinogen or VWF or recombinant ADAMTS-13 (each at 10 μg/ml) and then blocked with 5% albumin overnight. Washed platelets (100,000/μl), incubated with divalent cations, were then let adhere to the wells for 1 hr at 37°C. After extensive washing adherent platelets were lysed, p-nitrophenilphosphate was added and the reaction was stopped with NaOH 2M. Detection was done by assessing optical density at 405 nm. Binding of washed platelets preincubated with 2mM CaCl2 and/or 2mM MgCl2 to wells covered with immobilized recombinant ADAMTS-13 was significantly higher than binding to wells coated with albumin (p<0.001), was at the same levels of the binding to wells covered with recombinant VWF and approximately half of binding to the wells coated with fibrinogen. When washed platelets were preincubated with EDTA 2mM binding to the wells coated with fibrinogen or recombinant ADAMTS-13 decreased at the same degree of the wells covered with albumin. Preincubation of platelets with antibodies against αIIbβ3 (7E3, 10 μg/ml) or GpIb (10 μg/ml) did not effect the levels of binding to recombinant ADAMTS-13 while the binding to fibrinogen and VWF was totally abolished. Activation of platelets, obtained by preincubating platelets with ADP (5 μM) or collagen (10 μg/ml), significantly increased their binding to recombinant ADAMTS-13 (p<0.001) as compared to the binding of non-activated platelets. Immunofluorescent studies were then performed to see whether or not ADAMTS-13 bound to the platelet plasma membrane using as primary antibody a murine anti-human ADAMTS-13 monoclonal antibody (13E2). A positive membrane fluorescent signal was detected using as a source of platelets either a normal donor or a patient with type III Von Willebrand disease, demonstrating that ADAMTS-13 is located on the platelet surface independently from VWF. In conclusion ADAMTS-13 binds to the surface of platelets, the binding is specific, activation and divalent cations dependent, inhibited by EDTA, and not mediated by VWF on the membrane of platelets; the binding site does not appear to be αIIbβ3 or GpIb. Hence it can be hypothesized that ADAMTS-13 on platelet membrane might cleave high molecular weight VWF.
Mechanisms of Static and Shear-Induced Platelet Adhesion: Differences in the Adhesion Supported by MMRN1 Comparisons with Other β3 Integrin Ligands.
Authors: Adam Frederic , Zheng Shilun , Santos Aurelio V. , Kelton John G. , Hayward Catherine P.M.
Published: April 2020

Platelet adhesion and aggregation at the site of vascular injury are key events in hemostasis and thrombosis. These processes are supported by interactions between platelet glycoprotein (GP) receptors (including integrin αIIbβ3, GP Ib-IX-V and GPVI) and ligands that include von Willebrand factor (VWF), collagen, and fibrinogen (Fg). Recently, the polymeric protein multimerin 1 (MMRN1) was identified to bind β3 integrins. Normally, MMRN1 is sequestered within secretion granules of platelets, megakaryocytes, and endothelium until its release. In static adhesion assays, MMRN1 supports platelet adhesion to integrins αIIbβ3 and αvβ3 by an RGD-dependent mechanism.

Study goals and methods: To further determine the mechanisms of MMRN1 binding to platelets, we investigated (i) the importance of platelet activation in MMRN1 binding to platelets, (ii) the ability of MMRN1 to support platelet adhesion compared to other adhesive ligands (Fg, VWF), and (iii) the role of β3 integrins in MMRN1 binding to platelets at low and high shear.

Results: The binding of secreted platelet MMRN1 to thrombin activated platelets was significantly reduced by antibody inhibitors of ligand binding to αIIbβ3 and αvβ3. Thrombasthenic platelets (GT), deficient in αIIbβ3 and αvβ3 integrins, stored normal quantities of MMRN1 but after activation, they retained 34% less MMRN1 than normal platelets. Platelet adhesion experiments confirmed that αIIbβ3/αvβ3 and other binding sites supported MMRN1 binding to platelets. MMRN1 supported platelet adhesion at both low (150 s-1) and high (1500 s-1) shear rates. Like platelet adhesion to VWF, platelet adhesion to MMRN1 was greater at high shear rates. Platelet stimulation by agonists was essential to induce platelet binding to MMRN1 in static and shear adhesion assays, but not to induce platelet adhesion to Fg and vWF. While platelets activated by ADP and TRAP showed similar adhesion to Fg and VWF, TRAP induced more platelet adhesion to MMRN1 at high shear rates than ADP. Inhibitors of ligand binding to αIIbβ3 and αvβ3 had greater effects on high compared to low shear platelet adhesion to MMRN1.

Conclusions: These data indicate interesting differences in the mechanisms that support platelet adhesion to MMRN1 and other β3 ligands, which could be important for molecular events in hemostasis and thrombosis. The activation-dependent binding of MMRN1 to platelets, augmented by high shear flow, may reflect the unique recognition properties of platelet β3 integrins and/or exposure of other binding sites (e.g. phosphatidylserine) on activated platelets that promote adhesive interactions with MMRN1.
A Rapid Test for the Diagnosis of Thrombotic Thrombocytopenic Purpura Using SELDI-TOF-Mass Spectrometry.
Authors: Wu Haifeng , Cataland Spero , Bissell Michael , Jin Ming
Published: April 2020

Thrombotic Thrombocytopenic Purpura (TTP), a life-threatening microangiopathic thrombotic disorder, requires immediate diagnosis and plasma exchange therapy. Development of TTP is related to functional deficiency of a metalloprotease, ADAMTS13, that leads to accumulation of ultra large von Willebrand factor (vWF) and subsequent platelet thrombosis. Currently no clinical test is available for a rapid detection of functional ADAMTS13 activity. In this study, a recombinant vWF peptide containing the ADAMTS13 cleavage site and a 6 X Histidine tag was used as a substrate for ADAMTS13. ADAMTS13 cleaved the substrate in a dose-dependent manner, generating a ~7739 Dalton peptide containing a 6 X Histidine tag. This cleaved peptide, bound to an IMAC/Nickel ProteinChip, was quantified using Surface Enhanced Laser Desorption/Ionization Time-of-flight Mass Spectrometry (SELDI-TOF-MS). The assay is capable of quantifying ADAMTS13 activity as low as 2.5 % in plasma within 3–4 hours. When the cleaved peptide was quantified as a ratio of an internal control peptide, the test displayed excellent reproducibility, with an average inter-assay CV of < 33 %. Further validation of the test in healthy donors (n=39) revealed normal ADAMTS13 activity with a mean of 92.5% + 16.6. Sixteen patients with idiopathic TTP displayed mean ADAMTS13 activity of 1.73% + 3.62 at presentation, prior to any therapy. Further utility of this novel method includes functional determination of ADAMTS13 antibody titers in cases of acquired TTP. In summary, we have devised a novel SELDI-TOF-MS assay that offers a rapid, cost-effective, and functionally relevant test for timely diagnosis and management of TTP patients.
Combined Modality Treatment of Two or Four Cycles of ABVD Followed by Involved Field Radiotherapy in the Treatment of Patients with Early Stage Hodgkin’s Lymphoma: Update Interim Analysis of the Randomised HD10 Study of the German Hodgkin Study Group (GHSG).
Authors: Engert Andreas , Pluetschow Annette , Eich Hans T. , Herrmann Richard , Doerken Bernd , Kanz Lothar , Greil Richard , Markova Jana , Pfistner Beate , Josting Andreas , Mueller-Hermelink Konrad , Mueller Ralf P. , Diehl Volker
Published: April 2020

Combined modality treatment is regarded as standard by most study groups for patients with early-stage Hodgkin’s lymphoma (HL). However, the optimal chemotherapy, the number of cycles needed and the optimal radiotherapy dose is still unclear. The GHSG thus conducted a randomised study for patients with early stage favourable Hodgkin’s lymphoma (HD10) in which these questions were addressed.

A total of 1370 patients were randomised from 5/1998 to 1/2003 between two or four cycles of ABVD and independently to 20Gy or 30Gy involved field (IF) radiotherapy.

For the second interim analysis at a median follow up of 28 months, 847 patients were available. Patients were equally balanced for age, gender, stage, histology, performance status and risk factors. Compared with two cycles, there was more toxicity in patients receiving four cycles of ABVD for leucopenia, hair loss and infection. Concerning radiotherapy dose, there was more toxicity associated with 30Gy for dysphagia, mucositis and leucopenia. The rate of complete remissions ranged between 98% and 99% with no significant differences among treatment arms. Freedom from treatment failure (FFTF) and overall survival showed no differences between the four treatment arms. The curves for overall survival and FFTF were nearly superimposable for all four arms.

This analysis suggests that 2 chemotherapy cycles with involved field radiotherapy may be sufficient for patients with early favourable HL, but a reliable assessment must await the final analysis including all randomised patients and with adequate follow-up. The results of the third interim analysis (10/2005) including 1110 patients with a median follow up of more than 3 years will be presented.
Leucopenia during Treatment Is a Good Prognostic Risk Factor for Patients Undergoing Chemotherapy for Hodgkin’s Lymphoma: An Analysis of the German Hodgkin Study Group (GHSG).
Authors: Klimm Beate , Reineke Thorsten , Haverkamp Heinz , Eich Hans T. , Behringer Karolin , Josting Andreas , Pfistner Beate , Diehl Volker , Engert Andreas
Published: April 2020

Purpose: A systematic analysis of gender-specific factors in Hodgkin’s lymphoma (HL) studies previously revealed a better outcome for female HL patients. Factors contributing to better prognosis among female patients not only included more favorable risk factors at diagnosis, but also more treatment-induced severe leuopenia (WHO grade III/IV). The purpose of the current analysis was to characterize and investigate the protective role of severe leucopenia for both, male and female HL patients.

Patients and Methods: 4626 HL patients including 2050 females (f) and 2576 males (m) of all prognostic risk groups who were enrolled into the multicenter studies HD4-HD9 of the GHSG were evaluated. The median follow-up after treatment was 5.5 years.

Results: Female patients had a significantly better freedom from treatment failure (FFTF) with 81% at 66 months (95% confidence interval 79% – 82%), when compared with male patients (74%; 72% – 76%; p<.0001). This was in part related to the occurrence of more acute chemotherapy-related leucopenia (WHO grade III/IV): 69.9% in female patients and 55.2% in male patients (p<.0001). Furthermore, severe leucopenia during chemotherapy was strongly associated with better FFTF for both, male and female patients. Separate multivariate analyses showed that severe leucopenia remained significant when only male patients were included (0.011).

In addition, patients who developed severe leucopenia within the first two cycles of chemotherapy also had a significantly better FFTF (p=.0002) compared with all other patients (Klimm et al, JCO in press). When considering patients with advanced HL undergoing BEACOPP in baseline or escalated dose, the presence of severe leucopenia during chemotherapy (p=.0074) was associated with a better outcome. This finding was also confirmed when only the first two cycles of treatment were analyzed (p =.0205).

Conclusion: The protective role of severe leucopenia suggests evaluating of a more individualized therapy in future trials, that may be tailored in a response-adapted manner depending on the individual toxicity profile within the first cycles.
Cisplatin-Based Protocols as Salvage Therapy for Relapsed or Refractory Hodgkin’s Lymphoma Patients.
Authors: Alvarez Iván , Sureda Anna , Arranz Reyes , Córdoba Raúl , Xicoy Blanca , Sánchez Blanca , Bernal María Teresa , Roncero María Isabel G. , Martí Edelmira , Pliego Alberto , Fernández-Abellán Pascual , Ribera Josep María , Fernández-Rañada Jose María , Sierra Jorge
Published: April 2020

We have analyzed the results in terms of response rate and peripheral blood progenitor cell (PBPC) mobilization ability of two cisplatin-based chemotherapy salvage regimens [DHAP (cisplatin 100 mg/m2 iv day 1, ara-c 4 g/m2 iv day 2 and dexamethasone 40 mg po days 1-4) and ESHAP (etoposide 40 mg/m2 iv days 1–4, cisplatin 25 mg/m2 iv days 1–4, ara-c 2 g/m2 iv day 5 and methilprednisolone 500 mg iv days 1–5)] in a group of 110 patients with Hodgkin’s lymphoma (HL) in first or subsequent relapses. There were 70 males and 40 females with a median age at diagnosis of 29 years (range, 10 – 79). Sixty seven patients (64%) presented with advanced stage, 54% of them had B symptoms, 32% bulky mediastinal mass and 38%, extranodal disease. Disease status at the time of administration of the salvage protocol was primary refractory disease (PRD) (n = 16, 17%), first relapse (n = 65, 69%), second or subsequent relapse (n = 10, 11%) and relapse after a prior autologous stem cell transplantation (ASCT) (n = 3, 3%). Twenty-seven patients (26%) received the DHAP protocol and the remaining 78 (74%), the ESHAP protocol. Median time from diagnosis to the salvage therapy was 15 (range, 1 – 249) mo. In patients treated after a first relapse, the median duration of the first complete remission (CR) was 7 (range, 1 – 77) mo, with 18% of the patients relapsing after a long first CR (> 12 mo). Median number of cycles administered per patient was 2 (range, 1 – 3) with a median time interval between cycles of 28 (range, 21 – 76) days. PBPCs were collected after the second and the third cycle of chemotherapy in 35% and 49% of the patients respectively and was successful (> 2.0 x 106 CD34+ cells/kg) in 97% of them. Grade 3–4 hematological toxicity was seen in 20% of the cycles with severe neutropenia (< 0.5 x 109/l granulocytes) and thrombocytopenia (< 20 x 109/l platelets) developing in 26% and 7% of the cycles, respectively. The most frequent grade 3–4 extra-hematological toxicity was nausea/vomiting (30%). Procedure related mortality was 2%. Response rate (RR) was 78% [CR: 39%, partial remission (PR): 39%] with significant differences between patients treated for PRD [RR of 41% (CR: 12%, PR: 29%)] and those treated for disease relapse [RR of 86% (CR: 44%, PR: 42%)] (p = 0.005). Cisplatin-based regimens are a useful therapeutical strategy in the treatment of refractory or relapsed HL due to a high overall RR and an adequate PBPC mobilization ability. Results in terms of disease response are significantly worse in patients treated for PRD.
STAT5-Induced Self-Renewal and Impaired Myelopoiesis of Human Hematopoietic Stem/Progenitor Cells Involves Downmodulation of C/EBPα.
Authors: Schuringa Jan Jacob , Wierenga Bart-Jan , Schepers Hein , Moore Malcolm A.S. , Vellenga Edo
Published: April 2020

Previously, we demonstrated that enforced activation of STAT5 in human cord blood (CB)-derived stem/progenitor cells results in enhanced long-term stem cell self-renewal and impaired myelopoiesis (J.J.Schuringa et al, J.Exp.Med. 2004;200:623). Now, C/EBPα was identified as a critical transcription factor that is downregulated by STAT5. Affymetrix microarray analysis on STAT5A(1*6)-transduced CD34+ cells identified C/EBPα as the most prominently downregulated gene (−3.3 fold), and these data were confirmed by RT-PCR and Western blotting. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPα-ER protein was co-expressed with the STAT5A(1*6) mutant in CB CD34+ cells by using a retroviral approach. Re-expression of C/EBPα in STAT5A(1*6) cells resulted in a marked restoration of myelopoiesis as determined by morphological analyses, FACS analyses for myeloid markers such as CD11b, CD14 and CD15, and RT-PCR for myeloid-restricted genes such as g-csfr. While enforced activation of STAT5A resulted in accelerated erythropoiesis, this was blocked when C/EBPα was re-introduced into STAT5A(1*6) cells. Similarly, the proliferative advantage imposed on CD34+ cells by STAT5A(1*6) depended on the downmodulation of C/EBP as reintroduction of C/EBPα in these cells induced a quick cell cycle arrest and the onset of myeloid differentiation. At the stem/progenitor cell level, LTC-IC frequencies were elevated from 0.5% to 11% by STAT5A(1*6) as compared to controls, but these elevated LTC-IC frequencies were strongly reduced when C/EBPα was reintroduced in STAT5A(1*6) cells. Enumeration of progenitors in methylcellulose assays revealed similar results, the number of CFCs was reduced over 10-fold when C/EBPα was expressed in STAT5A(1*6) cells. Also, secondary CAFCs and long-term cultures could only be generated from STAT5A(1*6) expressing cells, but not from cells that co-expressed STAT5A(1*6) and C/EBPα. Taken together, these data indicate that STAT5-induced self-renewal and impaired myelopoiesis involves downmodulation of C/EBPα.
Hepcidin Expression Is Correlated with Erythropoietic Indexes and Non-Transferrin-Bound Iron Levels in Patients with Thalassemia Major.
Authors: Kattamis Antonios , Papassotiriou Ioannis , Palaiologou Danai , Drakaki Kalliopi , Apostolopoulou Filia , Papanikolaou George
Published: April 2020

Hepcidin plays a central role in iron homeostasis. Hepcidin seems to be the common final mediator of both erythroid and stores regulators, and coordinates intestinal iron absorption and iron release from reticuloendothelial macrophages. The erythroid regulator probably dominates over the stores regulator. Iron overload in thalassemia major is attributed mainly to blood transfusions and partly to increased iron absorption. Urine hepcidin levels in regularly-transfused thalassemia patients are inappropriately low in regards to their iron stores. Liver hepcidin expression is suppressed in the murine model of human thalassemia (Hbbth3/+). We evaluated the correlation between indexes of iron stores and of erythropoiesis and liver hepcidin expression in patients with thalassemia major. Nineteen transfusion-dependent thalassemic patients (14 females) of 20±7.2 years of age underwent liver biopsy. Fourteen patients were seronegative for hepatitis C. Liver iron concentration (LIC) was estimated by atomic absorption spectrometry. Hepcidin mRNA expression levels were estimated by quantitative Real-Time PCR (Lightcycler, Roche) from isolated RNA from liver tissue. Hematologic and blood chemistry parameters were determined by standard methods. NTBI was measured in 13 patients by atomic absorption spectrometry. Statistical analysis was performed using non-parametric tests. Hepcidin expression ranged from 0.08 to 38.4 (median 1.13) arbitrary units. The most significant correlations between hepcidin and indexes of erythropoesis and of iron load are shown on the table.

Variable median (range) hepcidin LIC NTBI r = Spearman’s rho, n.s. = non statistical Ferritin (μg/L) 2174 (990–5963) n.s. n.s. n.s. Hb (g/dL) 12 (11.2 – 13.4) r:0.55, P:.017 r:-0.43, P:.071 n.s. sTfR (mg/L) 2.64 (0.75 – 5.75) r:-0.59, P:.01 r:0.51, P:.03 r:0.71, P:.006 EPO (IU/L) 21.6 (2.9 – 106) r:-0.61, P:.007 r:0.56, P:.015 r:0.63, P:.02 NTBI (μmol/L) 3.1 (0.9 – 4.5) r:0.56, P:.047 r:0.67, P:.012 LIC (μg Fe/d.w.tissue) 8.3 (3.1 – 18.9) n.s.

The correlations between hepcidin and Hb, sTfR, EPO were stronger when patients with infectious hepatitis were excluded from analysis. Hepcidin did not correlate with any indexes of iron load, including LIC, ferritin, serum iron, transferrin saturation and annual transfusional iron load. Our results provide additional evidence that increased erythropoietic activity down-regulates hepcidin expression. The lack of correlation between iron stores and hepcidin expression is in consistency with the hypothesis that increased erythropoietic activity dominates over iron stores in the regulation of hepcidin expression in patients with thalassemia major. Furthermore, the negative correlation between NTBI and hepcidin RNA levels underlies the role of hepcidin in iron body trafficking even in hemosiderotic patients.
Altered Expression of Transcription and Chromatin Remodeling Factors in Deletional HPFH.
Authors: Costa Fernando F. , de Andrade Tiago G. , Cunha Anderson F. , Fattori André , Saad Sara T.O.
Published: April 2020

Hereditary Persistence of Fetal Hemoglobin is a rare, heterogeneous and benign group of hereditary disorders with an abnormal switch from fetal to adult hemoglobin, resulting in high levels of Hb F in the adult stage. A total of six deletions related to HPFH have been described, associated with increased levels of both gamma chains. Three main hypotheses have been proposed to explain the relationship between these deletions and the non-suppression of gamma genes: the removal of competitive regions that interact with the LCR; the juxtaposition of enhancer elements; and the removal of silencers. Despite evidence to support these hypotheses, however, they are not conclusive. Recently, Xiang and cols (Abstract #1215, 2004 ASH Meeting; Blood, Volume 104, issue 11, November 16, 2004) developed a YAC construct with the whole beta-globin locus containing a deletion of approximately 83.5 Kb responsible for the HPFH-2. Unexpectedly, the gamma gene was completely silenced in the adult transgenic mice. These data suggest that other mechanisms could be involved in the increased levels of HbF in these conditions. The authors speculate that other regions upstream from the cluster may harbor this activity. We, herein, investigate the possible involvement of transcription factors, using the subtractive hybridization method to identify differentially expressed transcripts in reticulocytes from a normal subject and a HPFH-2 subject. We have identified 56 and 106 unique genes in the normal and HPFH-2 cDNA libraries, respectively. Some of these are transcription (zinc fingers and homeobox proteins) and chromatin remodeling (NAP and SWI like proteins) factors that could participate in globin gene regulation. These genes are located in cis or in trans to the deletion and their altered gene expression has been confirmed by Quantitative Real-time PCR in other two HPFH subjects. The data may present new clues about globin gene regulation, the increased expression of gamma gene in deletional HPFH and the dynamic organization of genes and chromosomes in cells.
High Prevalence of Fractures and Bone Pain in Thalassemia: The Thalassemia Clinical Research Network Experience.
Authors: Vogiatzi Maria , Macklin Eric , Schneider Robert , Lane Joseph , Olivieri Nancy , Kirby Melanie , Cheung Angela , Vichinsky Elliott , Fung Ellen , Cohen Alan , Kwiatkowski Janet , Stallings Virginia , Holm Ingrid , Neufeld Ellis , Giardina Patricia J.
Published: April 2020

Historically fractures and bone pain have been frequent problems in Thalassemia. Recently a cross-sectional observational study conducted by the North American Thalassemia Clinical Research Network (TCRN) found a high prevalence of low bone mass among the various Thalassemia syndromes. In addition, the prevalence of fractures and bone pain with contributing factors were determined and reported below. The study population consisted of 369 patients: 65% had Thalassemia Major (TM), 12% Thalassemia Intermedia (TI), 12% Beta E (βE), 5% hemoglobin H (HbH), 5% hemoglobin H/Constant Spring (HbH/CS) and 1% Homozygous alpha (Hα). A 35% prevalence of fractures which increased with age was identified (6–10 yrs: 1.1/100 pt-yrs; 11–19 yrs: 1.2/100 pt-yrs; 20+ yrs: 2.1/100 pt-yrs). Fractures occurred more frequently in males vs. females (40% vs. 31%), and among patients with Beta Thal (TM: 40%, TI: 49%, βE: 19%, HbH: 11%, HbH/CS: 5%, Hα: 0%) even after controlling for differences in age distribution. Prevalence of fractures increased with decreasing bone mass (AP spine Z or T scores measured by DXA) and increasing weight Z scores but was not associated with height or BMI Z scores. Prevalence of fractures was higher among patients treated for hypogonadism (59% vs. 26%) or hypothyroidism (65% vs. 32%) after controlling for age and gender. Traumatic fractures were most commonly reported (91%). Increased physical activity was associated with fractures among children ≤11y but not among other age groups. Rates of traumatic fractures among Beta Thal were higher than among other diagnosis (2.3/100 pt-yrs vs. 0.8/100 pt-yrs) after controlling for age. Rates of non-traumatic fractures among Beta Thal were elevated but not significantly higher than among other diagnosis (0.23/100 pt-yrs vs. 0.07/100 pt-yrs). 34% of patients reported bone pain during the 30 days prior to enrollment. Of those, 17% used prescription pain medications and an additional 36% used over-the-counter analgesics. The prevalence of bone pain increased with age, was higher in females (males: 28%, females: 40%), was higher in TM and Hα (TM: 40%, TI: 16%, βE: 19%, HbH: 37%, HbH/CS: 35%, Hα: 67%) and did not correlate with bone mass, height, weight and BMI Z scores or any reported endocrinopathies. In summary, fractures continue to occur frequently despite current treatment practices, especially among the older patients with Beta Thal. Bone pain remains a frequent complaint among the Thalassemia syndrome subjects.
RTP801 Is a Novel Retinoic Acid Responsive Gene Possibly Involved in Myeloid Differentiation.
Authors: Gery Sigal , Park Dorothy J. , Vuong Peter T. , Koeffler H. Phillip
Published: April 2020

Retinoic acid (RA) promotes terminal differentiation of both normal hematopoietic cells and acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid genes. To identify additional RA target genes, we used representational difference analysis (RDA) with RNA derived from a PML/RARα inducible U937 myeloid cell line. From this screen we identified a novel early responsive RA target gene, RTP801 (REDD1). Recent studies showed that RTP801 is a critical transducer of several cellular stress signals, including hypoxia and energy depletion, through the TSC-mTOR pathway. We show that All-trans retinoic acid (ATRA) induces RTP801 mRNA in AML cell lines in a dose- and time-dependent manner. ATRA regulation of RTP801 is direct and does not require protein synthesis. Inhibition of endogenous RTP801 in U937 cells by siRNA abrogates ATRA-induced dephosphorylatioin of 4E-BP1, a key mTOR substrate. Overexpression of RTP801 in these cells results in growth arrest and apoptosis. RTP801 is differently expressed during maturation of normal CD34+ cells, suggesting it is involved in this process. We performed a yeast two-hybrid screen using a leucocyte cDNA library and identified the myeloid-specific protease, neutrophil elastase, as a binding partner of RTP801. Taken together, RTP801 is a novel ATRA target gene possibly involved in ATRA-induced differentiation of myeloid cells.
Two Single Nucleotide Polymorphisms of the ETS2 Transcriptional Factor Gene Predispose Individuals to High-Risk Acute Myelogenous Leukemia (AML).
Authors: Lee Il-Kwon , Choi Jeong-Hwa , Kim Yeo-Kyeoung , Kim Hee Nam , Park Kyeong-Soo , Lee Je-Jung , Shin Myung Geun , Choi Chan , Kook Hoon , Hwang Tae Ju , Park Moo Rim , Jo Deog Yeon , Won Jong Ho , Kim Hyeoung-Joon
Published: April 2020

ETS2 (v-ets avian erythroblastosis virus E2 oncogene homolog 2) is a transcription factor located in the human chromosomal region 1q22.3 encoding a 56-kDa protein. In this study we carefully selected a set of haplotype-tagging SNPs (htSNP) and genotyped their frequencies in order to identify polymorphic variants that contributes to the inter-individual differences in susceptibility to disease phenotypes. Here we report polymorphisms of ETS2, a hematopoietic transcription factor gene is associated with increased risk to AML. Seven SNPs derived from genomic region of the ETS2 gene, rs1209953, rs3746882, rs2298560, rs457705, rs2070531, rs711 and rs5307 were genotyped to estimate allele frequency, haplotype block and linkage disequilibrium (LD) map. Among those rs711 and rs530 were shown to be associated with increased risk to AML with statistical significance. The odds ratio (OR) for rs711 and rs530 relative to wild type genotypes are 1.76 (95% C.I. 1.2–2.5, p=0.0019) and 1.67 (95% C.I. 1.3–2.2, p=0.0003) respectively. Cumulative frequencies of four common haplotypes are 72%, among which T-T-G-T-C-G-T was the most ancestral haplotype comprising 36% of total haplotypes. We also examined the possibility of haplotype association, but no association was found. When we compared LD map with LDs constructed from the International HapMap project, Korean LD map was similar to Japanese LD, but least similarity was shown with LDs from African(Yoruba in Ibadan, Nigeria). Since these two SNPs are located in the 3′ UTR region we simulated the change in secondary structure in silico of the 3′ UTR region with two variants sequence. Severe change in the secondary structure was observed in the rs530 containing domain suggesting this change might affects stability of mRNA. Real time Q-PCR and western blot revealed that expression of ETS2 decreased in a dosage-dependant manner, showing most abundant expression when homozygously T/T at rs530, least expression with the homozygous A/A and intermediate level of expression when the heterozygous genotype. Our results suggest that polymorphic variants in the 3′ UTR region predispose individual to high-risk AML by altering the secondary structure of mRNA.