bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Atorvastatin on Hematological and Inflammatory Parameters.
Authors: Kadikoylu Gurhan , Uysal Hilal Bektas , Yenisey Cigdem , Yavasoglu Irfan , Bolaman Zahit
Published: April 2020

Background: Chronic inflammation may play role in the development of atherosclerosis and its complications. In hypercholesterolemia, the evidences of chronic inflammation such as the stimulation of chemokines and cytokines, increase in endothelial adhesion molecules, and the immune reactions against oxidants on lipoproteins are detected. It was shown that statins had some beneficial effects on lipids, thrombosis, endothelial dysfunction, and smooth muscle proliferation.

Aim: To investigate the effects of atorvastatin on hematological and inflammatory parameters in prospective study.

Material and methods: Forty patients (14 male and 26 female) with primary hypercholesterolemia were treated with 20 mg/day atorvastatin for 12 weeks, according to Adult Treatment Panel for Third Report of National Cholesterol Education Program. All patients received a detailed description of this study and signed consent prior to enrollment. National and local ethical committees approved this study. The exclusion criteria were secondary hypercholesterolemia, acute coronary syndromes, liver and renal dysfunctions, diabetes mellitus, acute/chronic infection and inflammatory diseases, pregnancy, lactation, malignancy, and tendency to bleeding. At baseline, and 12th weeks, lipid levels such as low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), very-low-density lipoprotein cholesterol (VLDL-C), triglycerides (TGs), and hematological parameters such as whole blood cell counts, hemoglobin and fibrinogen levels, CD3, CD4, CD5, CD8, CD14, CD16, CD19, CD40, CD45 using flow-cytometry, inflammatory parameters such as interleukin-1 (IL-1), IL-6, IL-18, tumor necrosis factor-alpha (TNF-a), interferon-gamma, soluble CD-40, intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1, high-sensitive CRP, sedimentation rate, and enzymes including CK, AST, ALT were evaluated. The results were compared with two-paired student’s-t test.

Results: At the end of study, atorvastatin decreased TC (p<0.001), LDL-C (p<0.001), TGs (p=0.006), VLDL-C (p=0.012), and HDL-C (p<0.001). While absolute lymphocyte (p=0.003) and platelet counts (p=0.001) were decreased with atorvastatin treatment, absolute monocyte count increased (p=0.002). On flow-cytometric examination, the expressions of CD14 (p=0.015) and CD19 (p=0.039) on lymphocytes were decreased with atorvastatin. Moreover, atorvastatin decreased the levels of TNF-a (p<0.001), sCD40 (p<0.001), ICAM-1 (p<0.001), and IL-18 (p=0.024). ALT increased at the end of treatment (p=0.041). But important adverse events were not seen in the patients.

Conclusion: The anti-platelet and anti-inflammatory effects of atorvastatin, independent from lipid-lowering effects, may play an important role on the prevention of atherosclerosis, in addition to its beneficial effects on lipid parameters.
Identification of a Diagnostic Gene Signature for SGN-40, Anti-CD40 Monoclonal Antibody, in Pre-Clinical NHL Models and the Role of FAS in SGN-40 Mediated Apoptosis.
Authors: Shi Xiaoyan , Burington Bart , Januario Tom , Lau Jeffrey T , Yu Shang-Fan , Ebens Allen , Dornan David
Published: April 2020

SGN-40, an anti-CD40 monoclonal antibody, is a humanized IgG1 antibody that binds to CD40, mediates effector cell functions (ADCC/ADCP) and activates downstream signaling pathways. SGN-40 has shown activity in a phase I single agent multi-dose trial in non Hodgkin’s lymphoma, with greatest activity in diffuse large B-cell lymphoma (Advani et al. 2008, ICML). Previous in vitro studies implicated down-regulation of the germinal center expressed protein Bcl-6, upregulation of p53 family member TAp63a, and FAS death receptor induction as potential mechanisms leading to lymphoma cell death (Lewis et al. 2007, AACR, ASH). In order to further define the apoptosis signaling mechanism, we assessed the ability of SGN-40 to inhibit proliferation and promote apoptosis across a large panel of non-Hodgkin’s lymphoma cell lines. SGN-40 reduced cell viability in 58% (18/31) of cell lines tested. To identify the genes that may be promoting apoptosis and/or inhibiting proliferation, we compared gene expression levels before and after SGN-40 exposure in both sensitive and resistant cell lines, as well as in normal B-cells. SGN-40 strikingly and specifically upregulated FAS on the cell surface of sensitive cell lines. Furthermore, the addition of soluble FAS-Fc dampened SGN-40-induced apoptosis in a subset of sensitive cell lines, suggesting a dependence on a FAS-FASL interaction. These data imply that FAS-dependent apoptosis may directly contribute to the anti-tumor effect of SGN-40. Our data also demonstrate that SGN-40 sensitivity is dependent on the point in B-cell development at which the NHL cell lines were transformed. Sensitive cell lines had a gene signature characteristic of minimal activation of CD40 signaling prior to SGN-40 exposure, whereas resistant cell lines had a signature consistent with prior constitutive signaling downstream of CD40. Thus, SGN-40 appears to elicit its apoptotic properties through activation of CD40 signaling in NHL cell lines not previously exposed to CD40L signaling in the germinal center environment at the time of lymphocyte transformation (GCB lymphomas). In order to develop a clinically feasible assay from FFPE tissue, we developed a 14-gene signature by Stepwise Linear Modeling, utilizing genes from the CD40 pathway activation and GCB gene sets; the classifier gave >96% accuracy (30/31) on the ‘training’ set of cell lines and 75% (3/4) accuracy on a ‘test’ set of xenografts. Overall, our data provides unique insights into SGN-40 mechanisms of action, provides a testable hypothesis of the clinical mechanism of action, and a potential diagnostic test to identify patients more likely to benefit from SGN-40. Efforts are currently underway to test the clinical relevance of these findings.
The Heterogeneous Prognosis of Patients with Myelodysplastic Syndrome (MDS) and Chromosome 5 Abnormalities: How Does It Relate to the Original Lenalidomide Experience in MDS?.
Authors: Kantarjian Hagop M , O’Brien Susan , Ravandi Farhad , Borthakur Gautam , Faderl Stefan , Bueso-Ramos Carlos , Abruzzo Lynne , Pierce Sherry , Shan Jianqin , Issa Jean-Pierre , Garcia-Manero Guillermo
Published: April 2020

Purpose. The positive lenalidomide experience in lower risk myelodysplastic syndrome (MDS) with deletion 5q and red blood cells transfusion dependence resulted in its broad use in MDS. The study aim was to define the prognosis in MDS and deletion 5q with or without other cytogenetic abnormalities.

Patients and Methods. Patients with MDS (<20% blasts) and evaluable cytogenetic studies (1966-present) were reviewed. Outcome of patients with deletion 5q or with loss of chromosome 5 (monosomy 5) was evaluated by the presence or absence of additional chromosomal abnormalities, overall, and in “lower risk” MDS.

Results. Of 2743 patients analyzed, 287 (10%) had deletion 5q, and 216 (8%) had monosomy 5 abnormalities. The median survival was 9 months with deletion 5q, 6 months with monosomy 5, and 17 months without a chromosome 5 abnormality. Considering patients with deletion 5q, the median survival was 33 months with deletion 5q alone, 17 months with deletion 5q and one additional abnormality, but only 6–12 months with deletion 5q and ≥ 2 abnormalities or chromosome 7 abnormality. Only 93 patients (3.4% of total) had lower risk MDS (International Prognostic Scoring System low or intermediate 1) and deletion 5q; their median survival 29 months (2-year survival 60%), and ranged from 13 to 41 months depending on the presence or absence of additional chromosomal abnormalities. Among 58 patients with lower risk MDS deletion 5q with identical entry criteria to the original lenalidomide study, the median survival was 44 months and the 2-year survival rate 80%. Among 198 patients with MDS with deletion 5q and < 10% blasts (a subset now often offered lenalidomide) the median survival was 12 months. Monosomy 5 was significantly more frequently associated with advanced MDS and with other chromosomal abnormalities. Considering only patients with IPSS intermediate 2 or high risk, the survival was better with deletion 5q versus monosomy 5 (medians 7 versus 6 months; p = 0.005).

Conclusions. The prognosis of MDS with chromosome 5 abnormalities is heterogeneous. This study provides baseline expectations of outcome in different MDS subsets and deletion 5q, which could be used for comparative purposes with the lenalidomide experience.
Bortezomib Targets Stroma-Mediated APO2L/TRAIL Apoptosis Resistance in Multiple Myeloma.
Authors: Perez Lia , Parquet Nancy , Anasetti Claudio , Dalton William
Published: April 2020

TRAIL mediates graft versus tumor responses while sparing normal tissues. Previous studies in our laboratory showed that myeloma:stroma microenvironment interactions confer resistance to TRAIL-induced apoptosis, a process referred to as environmental mediated-immune resistance (EM-IR) by increasing synthesis of the anti-apoptotic protein c-FLIP (J Immunol, 180: 3; 2008). Since bortezomib influences TRAIL signaling proteins and targets stroma induced survival signals, we studied whether it reverses TRAIL EM-IR. Myeloma cells (RPMI 8226 and U266) were treated either adhered to HS5 stroma cells or exposed to HS5-released soluble factors in a transwell system with myeloma cells in the upper well and HS-5 stroma cells in the lower well. Pretreatment with bortezomib (10nM for 20 hours) not only induced synergistic cytotoxicity in myeloma but did overcome TRAIL (10mg/mL for 4 hours) apoptosis resistance induced by HS5-released soluble factors. Mechanistic studies showed that bortezomib is not cytotoxic to HS5 stroma cells and does not alter HS5 release of IL-6, a cytokine known to mediate APO2L/TRAIL EM-IR (J Immunol, 180: 3; 2008). Cytokine array testing showed that stroma-myeloma co-culture lead to a significant increase in IL-10 levels but bortezomib had no effect on IL-10. Bortezomib only decreased MCP-2 and MCP-3 levels without affecting other inflammatory cytokines tested. Expression of DR5 receptor (agonistic) and DCR1-DCR2 receptors (antagonist) were increased at 20 hours bortezomib treatment. Pretreatment with DR4/DR5 blocking antibodies suggested that TRAIL signals trough DR4, not DR5, in these cells and increased of decoy receptors suggest that receptor expression could not predict TRAIL sensitivity. Among the proteins involved in TRAIL signaling that are bortezomib targets, HS5-released factors increased only c-FLIP expression, without affecting Heat Shock proteins, BcL-2 and/or IAP protein families. In the presence of HS5 stroma cells, bortezomib reduced c-FLIP thereby contributing to overcome apoptosis resistance. Since c-FLIP is a NF-κB target gene, we studied whether stroma cells lead to NF-κB activation. Gel shift assay showed increased NF-κB activation in myeloma cells treated with HS5 stroma. Bortezomib decreased NF-κB activation in myeloma cells in suspension, but was not effective in the presence of stroma. Since bortezomib abrogates cytokine-induce kinase activation and understanding that c-FLIP may be under control of these kinases, we next studied the effect of stroma and/or bortezomib on proteins involved in IL-6 signaling. HS5-released factor(s) induced STAT-3 and ERK phosphorylation but not AKT activation, although bortezomib effects on c-FLIP shown to be independent of these pathways. Our findings provide the rationale to combine bortezomib to APO2L/TRAIL treatment to abrogate the positive influence of the stroma microenvironment on myeloma cell survival.
Genomic Complexity and Recurrent Aberration in Multiple Myeloma: Analysis of a Compendium of aCGH and Gene Expression Profiles.
Authors: Chng Wee-Joo , Lee Ian , Jimenez-zepeda Victor , Braggio Esteban , Keats Jonathan , Bergsagel Peter Leif , Fonseca Rafael
Published: April 2020

Multiple Myeloma (MM) is the second most prevalent haematological disorder in the US with great recent progress propelled by various technological and therapeutic advances. Nevertheless, the complexity of MM genomes remains inadequately characterized. We have combined high-resolution (44K Agilent) array comparative genomic hybridization (aCGH) and gene expression data with clinical outcomes in an initial attempt at indexing genomic complexity in MM and its clinical implication in 100 MM patients. We enumerated the Number of Aberrations per Tumor (NAPT) as defined by the ADM-1 algorithm available within CGH Analytics. There appear to be no difference in degree of genetic complexity between hyperdiploid and non-hyperdiploid myeloma. Generally, t(11;14) have low NAPT whereas D2 tumors have high NAPT. Amongst the high-risk genetic subtypes, tumors with maf translocations have relatively low NAPT whereas t(4;14) have high NAPT. Whilst a Multivariate Cox Proportional Hazards regression of survival outcome showed little statistical support for popular indicators such as ploidy and TC classification, results for NAPT were considerably more encouraging (p~ 0.07, median survival of 40 months in low complexity vs 18 months in high). Log-rank tests further supported prognosis based on NAPT independent of clinical status and ploidy. To characterize complexity, we clustered aCGH profiles by clinical status, ploidy and TC classification. Clear patterns of aggregation and recurrent aberration were observed suggesting that they were possibly instrument to aberrant gene expression. Indeed, expression of a large proportion of genes approximately 2Mb of the recurrent breakpoints varied significantly between patients carrying aberrations vis-a-viz others that did not. Comparison against known gene sets showed an enrichment for cell-cycle and apoptosis genes. We are presently characterizing several such breakpoint-associated genes for their functional significance.
A Pre-Existing Plasma Cell Disorder Occurs in Most Patients with Multiple Myeloma.
Authors: Weiss Brendan M , Verma Pramvir , Abadie Jude , Howard Robin , Kuehl Michael
Published: April 2020

Background. A pre-existing plasma cell disorder (PPCD), such as monoclonal gammopathy of undetermined significance (MGUS), is thought to be present in at least one-third of patients presenting with symptomatic multiple myeloma (MM). However, no study has comprehensively evaluated the proportion of patients with MM that had a PPCD by laboratory testing on pre-diagnostic sera.

Methods. The Walter Reed Army Medical Center autologous stem cell transplant database was cross-referenced with the Department of Defense Serum Repository (DoDSR) database, which catalogs serum samples collected every 2 years on over 4 million active-duty service members. All samples 32 years prior to the diagnosis of MM were retrieved. Serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE) and serum free light chain analysis (sFLC) (The Binding Site, San Diego, CA) were performed on all samples. A PPCD was defined as a positive SPEP, IFE or abnormal sFLC ratio.

Results. Serum samples prior to the diagnosis of MM were available for 30/90 patients, and the median number of samples per patient was 3.5 (range, 1–14). The median age at diagnosis of MM was 48.1 yrs (29–67), with 96% male, 53% Caucasian, and 47% African-American. The Ig isotype of MM was IgG 76%, IgD 10%, light-chain 7%, and non-secretory 7%. A PPCD was detected in 27/30 patients (90%, 95% CI 74–97%). The initial PPCD was detected by sFLC alone in 6/27 (22.2%), IFE alone 2/27 (7.4%), SPEP+IFE 5/27 (18.5%), SPEP+IFE+sFLC 13/27 (48.1%) and IFE+sFLC 1/27 (3.7%). There were 4 patients whose only positive sera was 2.5–3.5 years prior to diagnosis, with all preceding sera negative.

Conclusions. First, a pre-existing plasma cell disorder is present in most MM patients at least 2.5 years prior to diagnosis. Second, consistent with published evidence for a small fraction of patients with high risk MGUS, 4/30 patients were documented to progress rapidly through an MGUS phase to MM. Third, 4/4 patients with light chain only or non-secretory MM had a PPCD that was detected only by sFLC, thereby indicating that all these tumors are preceded by a light chain only or non-secretory PPCD.
Decreased Thymosin Beta 4 Expression Results in Poor Prognosis and Decreased Survival in Multiple Myeloma.
Authors: Caers Jo , Hose Dirk , Kuipers Ine , Bos Tomas Jan , Van Valckenborgh Els , Menu Eline , De Bruyne Elke , Goldschmidt Hartmut , Van Camp Ben , Klein Bernard , Vanderkerken Karin
Published: April 2020

Thymosin beta 4 (Tβ4) is a small cytoplasmic and nuclear protein involved in different cellular processes including differentiation, migration and proliferation. It is overexpressed in several malignant cells, resulting in an increased angiogenic respons and metastatic potential of tumor cells. In the current study we wanted to evaluate the expression of Tβ4 in primary human multiple myeloma cells (MM) and murine 5TMM cells. Microarray analysis and qRT-PCR of 171 MM patients treated with high dose therapy and autologous stem cell transplantation, showed that MM cells display a decreased Tβ4 expression compared to plasma cells from healthy individuals. We investigated whether Tβ4 expression in MM cells influences the event free (EFS) and overall survival (OAS) of these patients. As Tβ4 was expressed in all MM cells, we compared survival of patients with Tβ4 expression in MM cells in the upper thirtile (Tβ4high MMC) against the lowest thirtile (Tβ4low MMC). Patients with Tβ4high MMC had an increased EFS (P< 0.05) and also their OAS tended to be longer. A similar observation was made in the 5TMM model where qRT-PCR and ELISA revealed a decreased expression of Tβ4 in BM samples from 5T33MM and 5T2MM diseased mice compared to control mice. To study the functionality of Tβ4, we overexpressed Tβ4 in 5T33MMvitro (5T33MMvt) cells by lentiviral transduction. These 5T33MMvt-Tβ4+ cells demonstrated a significantly decreased proliferative capacitity and an increased sensitivity to different anti-myeloma agents (bortezomib, melphalan, dexamethasone) compared to control 5T33MMvt cells. Furthermore, injection of 5T33MMvt-Tβ4+ cells in C57BlKaLwRij mice resulted in a significant decreased tumor formation (paraprotein concentration was 1.92 g/dl versus 3.74 g/dl for untransduced cells, p<0.05) and a prolonged survival compared to mice injected with untransduced 5T33MMvt cells (respectively 65.9 days versus 88.9 days, p< 0.05). In conclusion, we found a decreased Tβ4 expression in human and murine MM cells compared to normal plasma cells. In our cohort of MM patients treated with high-dose chemotherapy, low expression of TB4 identifies a group of patients with adverse prognosis while in the murine 5TMM33vt model, overexpression of Tβ4 resulted in an inhibitory effect on tumor formation suggesting a role of Tβ4 as a tumor suppressor gene in MM.
A Multicenter Phase I/II Dose Escalation Study of Lenalidomide in Combination with Melphalan and Dexamethasone in Subjects with Newly-Diagnosed Light-Chain (AL)-Amyloidosis.
Authors: Moreau Philippe , Jaccard Arnaud , Benboubker Lotfi , Royer Bruno , Coiteux Valerie , Bridoux Franck , Harousseau Jean-Luc , Traulle Catherine , Grosbois Bernard , Leblond Veronique , Alakl May , Hermine Olivier , Fermand Jean-Paul
Published: April 2020

A recent prospective randomized trial comparing standard-dose to high-dose melphalan in patients presenting with newly diagnosed AL-amyloidosis showed that oral melphalan-dexamethasone (M-dex) given monthly could be considered the current standard of care, with a median survival of 56 months (Jaccard, N Engl J Med 2007). The use of a combination of lenalidomide and dexamethasone has also been tested in patients with symptomatic AL-amyloidosis. The initial dose of lenalidomide at 25 mg/day was poorly tolerated. However, a 15 mg/day dose regimen was well tolerated and effective, with both hematologic and organ responses (Dispenzieri, Blood 2007 & Sanchorawala, Blood 2007). Combining M-dex with lenalidomide (M-dex-rev) could increase the response rate but the toxicity of this regimen is still unknown. Thus we have initiated a multicenter single-arm open-label phase I/II dose escalation study of lenalidomide administered in combination with M-dex. The primary endpoint was the incidence of dose limiting toxicities (DLT) during the first cycle of lenalidomide at a given dose level in order to determine the maximum tolerated dose (MTD). In addition to melphalan 0.18mg/kg/day from day 1–4 of each 28 day cycle and dexamethasone 40mg/day from day 1– 4 of each 28 day cycle, 3 cohorts of 3 subjects were successively exposed to escalating doses of lenalidomide (5, 10 and 15mg once daily on days 1–21 of a 28 day cycle). DLT was defined using National Cancer Institute (NCI) common toxicity criteria during the first 4 weeks of treatment (one cycle) as the following: at least grade 2 cardiac arrhythmia, at least grade 3 non hematologic toxicity, grade 4 neutropenia lasting >7 days or any other Grade 4 hematologic toxicity, or treatment delay due to toxicity that occurred during the first cycle. No DLT was observed among the first 3 patients treated at 5, 10 and 15 mg lenalidomide/day. 9 additional subjects will be enrolled at 15 mg/day to better define the safety profile and estimate the hematologic and organ response rate. Final results regarding both toxicity and efficacy will be presented during the meeting.
Similar Rate of Thrombosis in Essential Thrombocythemia and Polycythemia Vera Patients after Stratification for JAK2 V617F Allele Burden.
Authors: Carobbio Alessandra , Finazzi Guido , Antonioli Elisabetta , Guglielmelli Paola , Vannucchi Alessandro M. , Delaini Federica , Rambaldi Alessandro , Barbui Tiziano
Published: April 2020

Patients with Essential Thrombocythemia (ET) can be categorized as either JAK2 V617F mutated (V617F+) or wild type (V617F−). Mutated patients display multiple features resembling Polycythemia Vera (PV), with significantly higher hemoglobin level and neutrophil counts, lower platelet count, more pronounced bone marrow erythropoiesis and granulopoiesis and higher tendency to transform in PV. Presence of the mutation and/or allele burden has been variably associated with the rate of vascular complications in ET and PV, but a direct comparison between the two disorders under this respect has not been performed. To tackle this issue, we compared the rate of major thrombosis in 867 ET patients (57% were JAK2 V617F+) with that in 415 PV patients (all V617F+). The median follow-up was 4.9 (0 – 39) and 3.8 (0 – 26) years in ET and PV, respectively. High risk ET patients (age ≥ 60 years and/or previous thrombosis) received Hydroxyurea whereas the vast majority of low-risk remained untreated. PV patients were treated according to the current risk-stratified recommendations. Thrombotic episodes were recorded over time and calculated as rates % per patient/year (pt/yr). After adjusting for age, the thrombosis-free survival curves of JAK2 V617F+ and V617F− ET patients were superimposable until 10 years after the diagnosis, then they diverged so that the actuarial probability of major thrombosis in mutated ET patients reached that of PV (48% vs 55%, test for trend p=0.05). We found that JAK2 V617F+ allele burden measured by real-time quantitative PCR influenced these rates in a comparable way in both ET and PV. Actually, in JAK2 wild type ET (n=376, 43%) the rate was 1.4% pt/yr. In ET patients with JAK2 V617F+ allele burden ranging from 1 to 25% (N=190; 49%) the rate was 1.9 % pt/yr compared to 1.2 in PV patients (N=64, 19%); in the group with 26–50% the rate was 2.0 % pt/yr in ET (N=177; 45%) and 3.0 in PV patients (N=118, 36%); in cases of V617F+ allele burden greater than 50% the rate was 3.8 % pt/yr in ET (N=23; 6%) and 2.9 in PV patients (N=147, 45%). In conclusion, from this retrospective analysis, we conclude that in patients with ET harboring JAK2 V617F mutation the rate of stroke, myocardial infarction and venous thromboembolic complications is similar to that of PV patients and increases in dependence of V617F allele burden, supporting the hypothesis that ET and PV may be viewed as a continuum also in terms of vascular complications
Molecular Mechanisms of JAK2V617F Oncogenic Activation: The JAK2- V617F Mutant Utilizes the SH2 Domain for Constitutive Kinase Activity in the Presence of Unstimulated Heterodimeric Cytokine Receptor.
Authors: Gorantla Sivahari P , Dechow Tobias , Grundler Rebekka , Peschel Christian , Duyster Justus
Published: April 2020

The JAK2-V617F mutation has been reported in the majority of MPDs including PV, ET, and IMF. This mutation leads to the constitutive activation of the JAK2 tyrosine kinase activity and overexpression of JAK2V617F renders hematopoietic cell lines growth factor-independent. However, the molecular mechanism leading to constitutive activation of JAK2V617F is largely unclear and the requirement of homodimeric or heterodimeric cytokine receptors needs to be determined. Here we show that oncogenic JAK2-V617F requires an intact SH2 domain for constitutive kinase activity. To this end we mutated the conserved arginine 426 within the SH2 domain to a lysine. Ba/F3 cells expressing JAK2V617F grew IL-3-independent and showed constitutive activation of JAK2, STAT5, and ERK1/2. In contrast, introduction of the SH2 mutation in JAK2V617F abrogated both transformation as well as constitutive activation of downstream signaling pathways. Accordingly, reconstitution of JAK2 mutants in a JAK2-negative cell line with IL-3R co-expression revealed reduced activation of JAK2 when the SH2 domain was mutated. It has been reported that JAK2 binding to homodimeric type I cytokine receptor may facilitate JAK2V617F-mediated transformation. Interestingly, co-expression of the homodomeric EpoR with SH2 mutated JAK2V617F rescues the phenotype indicating that the SH2 domain is required for JAK2 signaling in the presence of heterodimeric but not homodimeric cytokine receptors. Membrane localization studies showed equal membrane distribution of SH2-mutated and unmutated JAK2-V617F indicating that the SH2 domain mutation does not affect subcellular distribution of JAK2. However, co-IP experiments revealed a possible role for the SH2 domain in the dimerization and transphosphorylation of JAK2. Consequently, reduced transphosphorylation was seen in IL-3R- but not in EpoR-expressing cells. In a BM transplantation model we found that an intact SH2 domain in JAK2V617F was required for the induction of a MPD-like disease. Thus, our results points to an important role of the SH2 domain for the constitutive activation of JAK2V617F in cells expressing heterodimeric cytokine receptors.
Deletions of Chromosomes 6q and 13q, and Trisomy 4 Are the Most Common Cytogenetic Abnormalities in Waldenstrom Macroglobulinemia. Preliminary Results of a Multicentric Study.
Authors: Nguyen-Khac Florence , Chapiro Elise , Mould Sarah , Barin Carole , Daudignon Agnes , Gachard Nathalie , Struski Stéphanie , Henry Catherine , Penther Dominique , Mossafa Hossein , Andrieux Joris , Perissel Bernard , Eclache Virginie , Bilhou-Nabera Chrystele , Luquet Isabelle , Terre Christine , Baranger Laurence , Mugneret Francine , Gregoire Marie-Jose , Chiesa Jean , Ramond Sylvie , Mozziconacci Marie-Joelle , Callet-Bauchu Evelyne , Merle-Beral Helene , Leblond Veronique
Published: April 2020

The genetic bases of Waldenstrom Macroglobulinemia (WM) are poorly understood. We have studied a cohort of 139 untreated WM patients, enrolled in a prospective randomized trial from the French Cooperative Group on Chronic Lymphocytic Leukemia and Waldenstrom Macroglobulinemia (FCG-CLL/WM), by conventional cytogenetic (CC) and Fluorescence in situ hybridization (FISH). The sex ratio was 2.2M/1F, the average age at inclusion was 66 years [40–85]. The mean percentage of lymphoplasmacytic cells was 53% [8–97]. CC was systematically performed on bone marrow or peripheral blood, and FISH analysis carried out using 7 probes CEP4, CEP12, 13q14, 11q22 ( ATM), 17p13 (TP53), IGH Abbott, 6q21 Q-Biogene, on metaphases and interphase nuclei. Out of 110/137 successful karyotypes, 37% showed clonal abnormalities (9 additional cases had numerical changes of sexual chromosomes). Among abnormal karyotypes, there were 14 (34%) 6q deletions, 5 (12%) trisomy 4/partial trisomy 4, 4 (10%) trisomy 18, 3 (7%) trisomy 12. Using FISH, deletions of 6q21 were observed in 23/86 cases (27%), 13q14 in 12/89 cases (13%), TP53 9/85 cases (11%), ATM 6/81 cases (7%). Trisomy 4 was present in 9/82 cases (11%), and trisomy 12 in 4/86 cases (5%). No rearrangement of IGH was observed in the first 27 analyzed cases. The 6q deletion is the most frequent reported cytogenetic abnormality in WM. We found 27% with deletions of 6q21 (FISH), a low percentage compared to the literature [39–54%]. This could be explained either by the difference in the probe used or by the absence of selection of lymphoplasmacytic cells before cytogenetic analyses. Interestingly, we confirmed our recent observation that trisomy 4 was frequent in WM (12% if partial trisomy 4 was included). Furthermore we observed in this large series a frequent 13q14 deletion (13%). In conclusion, cytogenetic abnormalities in WM differ from those commonly reported in other B-cell neoplasms and confirm the originality of this disease. 6q deletion is frequent compared to CLL or marginal zone lymphoma (MZL) and 13q14 deletion is rare compared to CLL. In our series trisomy 12 is rare compared to atypical-CLL and MZL. We didn’t observe cytogenetic involvement of the IGH locus, which is frequent in multiple myeloma or lymphoplasmocytic lymphoma. Finally trisomy 4 is present in WM but not reported in other B-cell malignancies. Searches for correlations with clinical and other biological parameters are ongoing.
CS-1 Is Expressed in Nasal Type NK/T Cell Lymphomas and Angioimmunoblastic T-Cell Lymphomas: Implications for Targeted Therapy with Elotuzumab (HuLuc63).
Authors: Hsi Eric D , Steinle Roxanne , Balasa Balaji , Rice Audie , Ko Young-Hyeh , Afar Daniel E.H.
Published: April 2020

Background: CS-1 (CRACC, SLAMF7, CD319) is a member of the signaling lymphocyte activating molecule-related receptor family. It is highly and uniformly expressed on the cell surface of benign and malignant plasma cells. We have recently reported the generation of elotuzumab (formerly known as HuLuc63), a humanized antibody targeting CS-1, which is currently in phase 1 trials in relapsed multiple myeloma. Lower levels of CS-1 have also been reported on NK cells and NK-like T-cells (NK/T). CS-1 expression in NK and T-cell lymphomas - aggressive lymphomas for which no effective therapy exists - is unknown. Here, we examined the expression of CS-1 in normal NK/T cells and in a series of NK and peripheral T-cell lymphomas (PTCL).

Methods: CS-1 expression in normal NK and T-cells were assessed by gene expression profiling. Flow cytometry (FACSCalibur, Becton Dickinson) was performed on blood from normal samples using a directly conjugated Alexa-488 elotuzumab. Archival formalin-fixed, paraffin-embedded tissues from PTCLs, including angioimmunoblastic T-cell lymphomas (AITL) and nasal type NK/T cell lymphomas were tested for CS-1 expression using the a paraffin-reactive 1G9 monoclonal antibody and automated immunohistochemistry (IHC, Ventana Medical Systems).

Results: Gene expression profiling showed CS-1 expression in purified NK and NK/T cells. We confirmed cell surface expression of CS-1 protein on normal blood NK and NK/T cells (n=18 samples) by flow cytometry with Alexa-488-HuLuc63. The majority of normal NK and NK/T cells expressed CS-1 (mean% positive and standard deviation of 96% +/− 4% and 71 % +/− 24%, respectively). We then evaluated tumor samples from patients with nasal type NK/T cell lymphoma as well as other peripheral T-cell lymphomas by IHC. Biopsies from 13 patients (5 from the United States, 8 from Korea) with nasal type NK/T cell lymphomas were evaluated by IHC. 12 of 13 (92%) patient samples expressed CS-1 with most cases showing a majority of cells positive. 46 PTCLs were also evaluated (including 9 AITL). Overall, 8/46 (17%) of the PTCL cases expressed CS-1. However, of the AITLs, 4 of 9 (44%) expressed CS-1.

Conclusions: CS-1 is expressed on nearly all nasal type NK/T cell lymphomas and in a substantial proportion of AITLs. These results provide the rationale for exploring elotuzumab in the targeted treatment of NK/T-cell malignancies.
The Role of Protein Tyrosine Phosphatase, Shp2, in FLT3-ITD-Induced Leukemogenesis.
Authors: Nabinger Sarah C , Fukuda Seiji , Kapur Reuben , Chan Rebecca
Published: April 2020

Internal tandem duplications of the FMS-like receptor tyrosine kinase (FLT3-ITDs), an in-fame insertion of several amino acids within the juxtamembrane domain, are present in 25% of acute myeloid leukemia (AML) patients and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperactivation of Erk and promiscuous activation of STAT5; however, the molecular mechanisms underlying aberrant activation of these signaling molecules is largely unknown. Tyrosine 599 (Y599) of WT FLT3 recruits the protein tyrosine phosphatase, Shp2, upon stimulation with FL, resulting Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73- FLT3, via Y599, results in enhanced Shp2 activation and contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation, Erk hyperactivation, and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT FLT3, N51-FLT3, or N73- FLT3, co-immunoprecipitation assays demonstrated that Shp2 is phosphorylated and associates with WT FLT3 in a FL-dependent manner. However, in contrast, Shp2 is constitutively hyperphosphorylated and associated with FLT3-N51 and FLT3-N73 independent of FL stimulation. To investigate the functional role of Shp2 in Flt3-ITD-induced leukemogenesis, Baf3 cells expressing WT FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III– directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Western blot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3- ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias.
Levels of Inflammatory Markers and the Development of the Post Thrombotic Syndrome.
Authors: Shbaklo Hadia , Holcroft Christina A , Kahn Susan R
Published: April 2020

Background: The post-thrombotic syndrome (PTS) occurs frequently after deep vein thrombosis (DVT) despite appropriate anticoagulant therapy. A close relationship between inflammation and thrombosis exists. While the inflammatory process at the time of DVT appears to improve thrombus resolution, it may promote destruction of venous valves, valvular reflux and subsequent development of PTS.

Objective: We prospectively evaluated the association between levels of four cytokines (IL-6, IL-8, IL-10 and MCP-1), two adhesion molecules (ICAM-1 and VCAM-1) and the development of PTS in a well-defined cohort of patients with DVT.

Methods: The study population consisted of 387 patients with objectively diagnosed symptomatic DVT who participated in the Venous Thrombosis Outcomes (VETO) Study and were followed for two years to determine the incidence of PTS. For this substudy, plasma samples frozen at the 4 month visit in 307 study patients who consented to provide blood samples were thawed and analyzed for the above inflammatory markers using the Luminex beads technology. Associations between marker levels and development of the PTS during follow-up were evaluated.

Results: Mean levels of IL-6 were significantly higher in patients with PTS compared to patients without PTS (7.35 pg/ml ± 14.26 vs. 4.60 pg/ml ± 4.90;p=0.03). Logistic regression analyses showed significant associations between PTS and levels above vs. below the median of IL-6 (OR 1.66; 95% CI 1.05, 2.62 (p=0.03)) and ICAM-1 (OR 1.63; 95% CI 1.03, 2.58 (p=0.04)). None of the other markers showed any association with PTS.

Conclusion: Our study suggests the presence of significant associations between markers of inflammation such as IL-6 and ICAM-1 and the development of PTS. Further work is needed to evaluate this relationship and to analyze other candidate markers that could be implicated etiologically in the association between DVT and PTS. If confirmed, this could lead to identification of new therapeutic targets for preventing PTS after DVT.
Prothrombin 20209C>T: Thirteen New Cases and Association with the 19911A>G Polymorphism.
Authors: Warshawsky Ilka , Makkar Vinit , Kottke-Marchant Kandice
Published: April 2020

During routine screening for the prothrombin 20210G>A mutation as a risk factor for venous thrombosis, we and others have reported cases of prothrombin 20209C>T heterozygotes that are enriched in individuals of non-Caucasian descent who have had thrombotic events and/or obstetrical complications. The prothrombin 20209C>T variant, as well as six other variants close to position 20210, have been found by melting curve analysis using the LightCycler Instrument (Roche Molecular Biochemicals) and hybridization probes. Unlike assays which use restriction enzymes or allele-specific PCR, melting curve analysis can distinguish multiple base changes that lie within the region of a hybridization probe as long as the base change causes a significant change in melting temperature from expected results. We describe thirteen new prothrombin 20209C>T heterozygous cases from unrelated individuals and one family study which were found during routine screening of clinical samples for the prothrombin 20210G>A mutation. Ethnic and clinical information were available in 11/13 cases: Ten individuals were of African descent while one was Hispanic. Eight individuals had thrombotic events (venous thrombosis, pulmonary embolism, and/or stroke), one had a pregnancy loss, one had bilateral below the knee amputations due to severe peripheral vascular disease, and one had a questionable pulmonary embolism. We next examined the prevalence of the prothrombin 19911A>G polymorphism in 20209C>T heterozygotes since alleles 19911A and 20210A are in full linkage and since the 19911G allele has been associated with increases in both plasma prothrombin activity and venous thrombosis risk. Of twenty 20209C>T heterozygotes, eighteen were 19911GG homozygous and two were 19911AG heterozygous. These results are consistent with linkage of alleles 20209T and 19911G and contrasts with the linkage of alleles 20210A and 19911A. Since the 19911G allele was found in all 20209C>T patients, it is possible that the 19911G-20209T haplotype increases the risk for thrombosis and/or obstetric complications, especially among individuals of African descent.
TLT-1 (TREM-Like transcript-1) Protects against Hemorrhage Associated with Inflammation by Facilitating Platelet Aggregation.
Authors: Washington A. Valance , Gibot Sébastien , Acevedo Ismael , De La Mota Alina , Gattis James , Rivera Linette , Quigley Laura , Gomez-Rodriguez Julio , Cheng Jun , Dutra Amalia , Lubkowski Jacek , Pak Evgenia , Hunter Robert , Schwartzberg Pamela L , McVicar Daniel W
Published: April 2020

TREM-like transcript-1 (TLT-1) is a novel, platelet-specific, membrane protein that is stored in a-granules and brought to the platelet surface upon activation. Little is known about its function. Here we build upon our recent findings that a soluble fragment of TLT-1 (sTLT) is released upon platelet activation and can be detected in the serum, but not the plasma, of healthy individuals. Evaluation of patients admitted to the University Hospital, Ramon Ruiz Arnau, in Bayamon Puerto Rico with the diagnosis of sepsis for the presence of plasma sTLT demonstrated significant levels of sTLT in septic individuals as compared to controls, indicating that TLT-1 plays a role in the early stages of sepsis. To directly assess this possibility we generated TLT-1 knockout mice and characterized their hemostatic function. Compared to wild type mice, TLT-1−/− platelets displayed decreased aggregation when treated with thrombin, collagen, ADP, or U46619 in vitro. The reduced platelet aggregation of TLT-1−/− mice translates to a 30% increase in bleeding time in tail snip assays compared to controls, supporting TLT-1’s role in vascular homeostasis. To evaluate TLT-1’s role in sepsis we challenged mice with lipopolysaccharide (LPS). LPS challenged TLT-1 null mice die faster and have a significantly lower rate of survival than wild type mice. We used the localized Shwartzman reaction to model the pathology associated with sepsis in a controlled lesion. Although neutrophil infiltration, thrombus formation, and the formation of microclots was not significantly higher in TLT-1−/− mice, the hemorrhage associated with Shwartzman lesions of these mice was nearly double and lesion size was greater than three times that of wild type mice. Moreover, we demonstrate a recombinant sTLT augments platelet aggregation in vitro suggesting a mechanistic explanation for the increased hemorrhage in TLT-1−/− mice. Thus these studies reveal an important role for TLT-1 in the maintenance of vascular hemostasis by facilitating platelet aggregation through incorporation of sTLT into the forming clot. This work suggests that therapeutic intervention using tools such as anti-TLT-1 single chain antibodies that inhibit platelet aggregation may provide a novel approach to the control of the thrombotic response.
Genetic Modifiers of Homozygous Beta Zero Thalassemia.
Authors: Galanello Renzo , Sanna Serena , Perseu Lucia , Sollaino Maria Carla , Satta Stefania , Uda Manuela , Usala Gianluca , Abecasis Goncalo , Schlessinger David , Cao Antonio
Published: April 2020

Introduction: Extensive studies over the last 50 years have derived two major modifiers of the clinical expression of beta-thalassemia: innate ability to produce fetal hemoglobin (Hb F) and co-inheritance of alpha-thalassemia. Recently genetic variants at the BCL11A locus and HBS1L-MYB intergenic region, that can modulate Hb F levels have been identified ( Thein et al 2007, Menzel et al 2007). Moreover, BCL11A variant has been shown to moderate the phenotype of homozygous beta-thalassemia (Uda et al 2008). In this study we evaluated the contribution of these Hb F modulating genetic markers and of alpha-thalassemia in ameliorating the severity of homozygous beta-thalassemia.

Methods: We studied 50 patients with the mild non-tranfusion dependent thalassemia intermedia phenotype and 75 with the severe tranfusion dependent thalassemia major. All patients were homozygotes for the beta 39 non-sense C → T mutation and were negative for the Xmn I -158 Gγ polymorphism. Alpha thalassemia was detected by GAP-PCR technique (deletion defects) and restriction enzyme digestion (nondeletion defects).Genotyping of HbF modulating genetic variants was performed using TaqMan® SNP genotyping assay (Applied Biosystems, Warrington, UK) according to the manufacturer’s protocol. Evaluation of the association, OR calculation and test for interaction were performed fitting a logistic regression.

Results: We found that the frequency of the BCL11A and HBS1L-MYB Hb F modulating markers and of co-inherited alpha- thalassemia is significantly increased (p= 2.05 x 10−5, p= 8.1 x 10−4, and p= 9.9 x 10−5 respectively) in the group of patients with thalassemia intermedia. Interestingly, the contribution of BCL 11A gene appears to be greater than HBS 1L-MYB locus ( OR= 5.15 and 4.61 respectively) and both are striking larger than the effect attributable to the co-inheritance of alpha-thalassemia (OR= 3.32). Furthermore, to visualize the combined effects of all loci, we considered a score variable defined as the number of positive alleles (i.e. those associated with the amelioration of the phenotype) carried from each patient. When considered all together the three loci explain 79% of the variation, evaluated as the correlation between fitted and observed values in the logistic regression. All three analyzed loci act in an additive fashion, where each copy of the minor allele contributes to the amelioration of the phenotype expression.

Conclusions: Molecular genetic analysis and particularly genome-wide association scans are producing rapid advances in defining the genetic variants for complex diseases and quantitative traits. The information reported are relevant in understanding the correlation between genotype and phenotype in thalassemia syndromes.
Hydroxyurea in Children with Beta Thalassemia Intermedia.
Authors: Marwaha Ram Kumar , Naranje K. , Panigrahi Inusha , Das R.
Published: April 2020

The beneficial effects of hydroxyurea (HU) have been well documented in sickle cell disease. Its usefulness in thalassemia syndromes has not been clearly elucidated. The aim of the study was to analyse the efficacy and side effects of HU in children with beta thalassemia intermedia (TI). The data of 73 children with TI were retrospectively analysed. Response to HU was assessed by change in hemoglobin (Hb) level and by a fall in transfusion requirements. An increase in Hb level was observed in 46 (63.0%) of 73 patients; the response was categorised as good in 43 and partial in 13 cases. There was a poor or no response in the remaining 27 (37.0%) children. Significant rise in Hb levels were documented after HU therapy; the responses were better in patients diagnosed beyond the age of 3 years (p=0.001). A fall in transfusion requirements by >50% was observed in 28 (66.7%) of the 42 patients who received transfusion prior to referral. There were no serious side-effects inspite of prolonged use. Transient neutropenia (<1.5x109/L) was observed in 9 patients. A solitary case had severe myelotoxicity requiring discontinuation of HU. Over 60% of our patients showed good or partial response in terms of hemoglobin level and/or transfusion requirements. The study has drawbacks and limitations of retrospective analysis. The preliminary observations suggest the need for evaluating larger number of patients in a prospective manner of determine the optimum dose and duration of therapy.