bluuebunny/crossref_metadata_2025 huggingface dataset:
Bcl-xS Is an Important Pro-Apoptotic Bcl-2 Family Member in Chronic Lymphocytic Leukaemia and Its Expression Is Regulated by SAM68, hnRNP-F and hnRNP-H.
Authors: Willimott Shaun , Wagner Simon D.
Published: October 2019

We have previously shown that the pro-apoptotic Bcl-x isoform Bcl-xS is induced in chronic lymphocytic leukaemia cells during spontaneous and drug induced apoptosis to a greater extent than other pro-apoptotic Bcl-2 family members including Bim, Puma and Noxa. Puma, Noxa and also Bid were induced strongly by stimulation with CD40 ligand, in culture conditions causing CLL cell proliferation and survival, and associated with strong induction of the pro-survival Bcl-x isoform, Bcl-xL. Immunohistochemistry demonstrates Bcl-xL expression in lymph node proliferation centres. We, therefore, conclude that splicing of Bcl-x mRNA is important for regulation of CLL survival such that Bcl-xL is required during proliferation in the lymph node or bone marrow microenvironment, but Bcl-xS is induced during apoptosis. Splicing of Bcl-x mRNA in HeLa cells is known to be controlled by RNA binding proteins including SAM68, heterogeneous ribonucleoprotein-F (hnRNP-F) and hnRNP-H, but expression and regulation of these molecules has not been investigated in CLL. We have demonstrated that expression of hnRNP-F and hnRNP-H decline in association with an increase in Bcl-xS expression. Conversely expression of the ribonucleoproteins is maintained (relative to expression in freshly isolated peripheral blood CLL cells) or increases on CD40 stimulation. SAM68 showed a similar pattern of expression with increased expression on CD40 stimulation and decreased under conditions of apoptosis. Phosphorylation of SAM68 can control the RNA binding activity of this molecule. We immunoprecipitated SAM68 from freshly isolated CLL cells and cells that had been cultured with CD40 ligand (to promote proliferation) or on tissue culture plastic (to promote apoptosis) for 2 days. Westerns of the immunoprecipitated protein were probed with an anti-phosphotyrosine antibody. We found variable patterns: phosphorylation was uniformly present in cells cultured on plastic (4/4 cases) but was only detectable in freshly isolated cells in 1 case and on CD40 stimulation in 2 cases. We conclude that the RNA binding proteins hnRNP-F, hnRNP-H and SAM68 have important roles in controlling Bcl-x splicing in CLL cells, and that their effects are largely due to variation in expression level rather than phosphorylation. Strategies to bias production of pro-apoptotic Bcl-xS using siRNA either directed against Bcl-xL mRNA or against RNA binding proteins may be therapeutically useful.
High Level EBV Persistence in the IgM+ IgD+ CD27+ B Cell Subset in Patients with X-Linked Lymphoproliferative Disease Lacking Isotype-Switched Memory B Cells.
Authors: Chaganti Sridhar , Ma Cindy , Bell Andrew , Croom-Carter Debbie , Hislop Andrew , Tangye Stuart , Rickinson Alan
Published: October 2019

Epstein-Barr virus (EBV) infects >90% of population world wide and, in healthy virus carriers, establishes life long persistence in the immunoglobulin (Ig)Dneg, CD27+ (“class-switched”) memory B cell compartment normally produced by antigen stimulation and transit through germinal centres. Patients with the X-linked lymphoproliferative disease (XLP) cannot make such class-switched memory B cells due to an inherited mutation in the slam-associated protein (SAP) gene involved in the maturation of antibody responses. Interestingly, XLP patients are highly susceptible to severe primary EBV infection and develop a fulminant infectious mononucleosis (IM) which is often fatal and where the symptoms progress to resemble those of a different (non-familial) disease, EBV-associated haemophagocytosis syndrome (EBV-AHS), caused by virus entry into the NK or T cell system. Some XLP patients survive their primary infection, but the nature of EBV carriage in these individuals (lacking conventional memory B cells) remains unresolved. To investigate this further we obtained blood samples from 8 such XLP patients. EBV load in total peripheral blood mononuclear cells (PBMC), determined by quantitative PCR, occupied a broad range but on average was 2 to 3-fold fold higher than that of healthy controls. The virus was concentrated within the B cell (CD19+) compartment, as in healthy carriers, and not within T or NK cells, as typically seen in EBV-AHS. We then confirmed that these XLP patients indeed lacked conventional class-switched memory B cells but did carry a small population of IgM+, IgD+, CD27+ (“non-switched”) memory cells; however their circulating B cell pool was dominated by naïve (IgM+, IgD+, CD27neg) cells and by expanded numbers of immature “transitional” (CD10+ CD27neg) cells. In each of 4 cases studied by cell sorting, EBV was concentrated in this small subset of “non-switched” memory B cells. To see if the high virus load detected in these patients indicated true virus persistence as opposed to recent or recurrent infection, serial samples obtained over a 3 year period from two XLP patients were assayed. Virus load was stable and, in one case with the highest load, screening with markers of virus polymorphism detected the same resident strain over time. Our results in XLP patients make it clear that EBV can persist in the absence of a conventional class-switched memory B cell compartment. Instead, the virus is sequestered in a small population of “non-class-switched memory” cells with Ig gene mutations. The origin of such cells, which are also detectable in the blood of normal donors, is uncertain; however their existence in XLP patients suggests that such cells arise independently of germinal centre activity and hence that EBV may be able to colonise its host without exploiting germinal centre transit.
Population Pharmacokinetics (PK) of Oral Posaconazole (POS) for Antifungal Prophylaxis in Neutropenic Patients with Hematologic Malignancies.
Authors: AbuTarif Malaz A. , Krishna Gopal , Statkevich Paul
Published: October 2019

In a recent large, randomized, multicenter study in neutropenic patients undergoing remission-induction chemotherapy for acute myelogenous leukemia or myelodysplastic syndrome, prophylaxis with POS resulted in significantly fewer invasive fungal infections (IFIs) (2% vs 8%, P<0.001) and longer survival (P=0.04) vs fluconazole or itraconazole (Cornely et al New Engl J Med2007;356:348–59). Using data from this study, the relationship between demographic/baseline covariates and apparent volume of distribution (V/F) of POS was evaluated in a population modeling approach. In the full study, 304 patients were administered POS (200 mg tid) and 298 patients received fluconazole (400 mg qd) or itraconazole (200 mg bid) for up to 12 weeks. For the population PK analysis, data from 215 POS patients were analyzed using nonlinear mixed-effect modeling. The effect on POS V/F of IFI occurrence, demographic characteristics, and baseline variables (occurring at or before day 7) including mucositis, neutropenia, vomiting, diarrhea, proton pump inhibitor (PPI) or H2-receptor antagonist usage, bilirubin ≥2 × upper limit of normal (ULN) and gamma-glutamyl transferase (GGT) ≥2 × ULN was examined. A 2-stage “stepwise forward addition” followed by a “stepwise backward elimination” approach was used to build the covariate model. The model was validated by bootstrapping data with 85% and 50% partial sampling. POS V/F, and thus exposure, was statistically associated with diarrhea, PPI intake, bilirubin ≥2 × ULN, GGT ≥2 × ULN, and race (white vs nonwhite). None of these covariates predominated in patients with proven or probable IFI (n=7). Validation demonstrated the robustness of this model. A significant relationship was found between POS V/F (or exposure) and the following covariates: diarrhea, PPI intake, bilirubin ≥2 × ULN, GGT ≥2 × ULN, and race. However, the influence of these covariates on POS exposure is unlikely to be clinically relevant as none of the covariates predominated in the patients who developed IFI, and plasma POS levels were attained that were adequate for successful prophylaxis against IFIs. Thus, no dosage adjustments are recommended based on these covariates.
Inhibition of the TGF-β Receptor I Can Stimulate Hematopoiesis in Primary Myelodysplastic Syndrome Progenitors as Well as in TGF-β Driven Transgenic Mouse Model of Bone Marrow Failure.
Authors: Zhou L. , Nguyen A. , Pahanish P. , Hayman J. , Gundabolu K. , Chubak A. , Parmar S. , Garry D. , Wickrema A. , Navas T. , Higgins L. , Friedman E. , List A. , Bitzer M. , Verma A.
Published: October 2019

Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. TGF-β is a myelosuppressive cytokine that has been indirectly linked to the pathogenesis of some subsets of MDS and acute leukemias. We demonstrate by immunohistochemistry that smad2, a component of the TGF-β signaling pathway, is constitutively activated in MDS bone marrows. This activation was seen in both low and high grade cases of MDS when compared to anemic controls and provides the first direct evidence of activation of TGF-β signaling in MDS. To demonstrate the functional role of TGF-β signaling in MDS, a GFP expressing lentiviral siRNA construct was designed to knockdown TGF receptor I (TBRI) expression. Stable expression of this lentivirus in a variety of hematopoietic cells resulted in >70% inhibition of TBRI mRNA by qPCR. Lentiviral siRNA Knockdown of TBRI in primary CD34+ cells resulted in an increase in GFP+ erythroid colony formation, indicating an inhibitory role of TGF-β in human hematopoiesis. To further test the efficacy of TBRI inhibition in stimulating hematopoiesis, we used a specific and potent inhibitor of TBRI (ALK5) kinase, SD-208. SD-208 was effectively able to inhibit TGF-β mediated activation of smad-2 and potently inhibit TGF-β mediated gene expression in bone marrow stromal cells. SD-208 treatment also led to reversal of TGF-β induced inhibition of primary CD34 cell proliferation and both erythroid and myeloid colony formation in short term assays. Most importantly, treatment with SD-208 led to significant dose dependent increases in erythroid and myeloid colony formation from primary MDS bone marrow derived hematopoietic progenitors. Furthermore, we tested the efficacy and specificity of SD-208 in a TGF-overexpressing transgenic mouse. This mouse constitutively expresses TGF-β through an albumin promoter, develops progressive anemia and dysplasia and thus serves as a novel model of human bone marrow failure. Treatment with SD-208 by oral lavage at 30mg/kg/d for two weeks led to significant increases in both myeloid and erythroid colony formation from murine bone marrows and led to a trend of increasing hemoglobin and hematocrit. Taken together, these studies demonstrate a role of TBRI inhibition in stimulating hematopoiesis in human bone marrow failure and should lead to future studies with SD-208 and other inhibitors of TGF signaling pathways in MDS.
Lentiviral Insertion Site Analysis of Corrected Mouse Model of β Thalassemia Following Lentiviral Transduction of Embryonic Stem Cells.
Authors: Yang Rui , McConnell Sean C. , Huo Yongliang , Ulrey Clayton , Liu Shan-Run , Zhang Ting-Ting , Ryan Thomas M.
Published: October 2019

The delivery of therapeutic transgenes via lentiviral transduction of stem cells holds great promise for future cell based therapies for inherited genetic disorders. The regulation of any transgene integrated into patient derived embryonic stem (ES) cells (generated via nuclear transfer or reprogramming) needs to be studied in great detail prior to their differentiation and transplantation back into the patient. Because genetically manipulated ES cells can be clonally isolated, expanded to great numbers in an undifferentiated state, and differentiated to specific cell types, they can be carefully tested in vitro prior to their reintroduction back into patients. ES cells derived from a mouse model of β thalassemia were transduced with a recombinant, self inactivating (SIN) lentiviral vector containing a 2.3 kb human β-globin gene and a 3.2 kb Locus Control Region composed of regulatory elements; HS2, HS3, and HS4. Clonal populations of transfected β thalassemic ES cells were isolated, archived, and utilized to produce genetically identical offspring by injection into tetraploid blastocysts or eight-cell embryos. Real-Time QPCR expression analyses demonstrated high levels of human β-globin gene expression in the peripheral blood of five of the six lines produced. The average expression level per transgene copy number ranged from 13% to 62% of endogenous mouse α-globin levels. Peripheral blood hemolysates analyzed by HPLC confirmed the high level production of human β-globin chains. Measurement of the red blood indices in these five lines showed that the anemia was corrected. In order to examine the influence of chromosomal position effects on individual transgene expression, the lentiviral insertion sites were mapped by linear amplification (LAM) PCR. Cloned mice from the six lines were bred in order to segregate each of their individual lentiviral transgenes. Offspring containing single insertion sites were analyzed for human β-globin transgene expression and synthesis by Real-Time QPCR of blood RNA and by HPLC of hemolysates, respectively. These analyses demonstrate that there is a preference for provirus integration in ES cells; all transgene insertions are not expressed equally; lentiviral transduction of a human β-globin gene into ES cells can cure a severe hemoglobinopathy; and that the delivery of therapeutic transgenes via lentiviral transduction of ES cells did not harm the developmental potential of the cells to produce an entire mouse.
STAT5 Signaling Is Associated with the Outcome of Follicular and Mantle Cell Lymphoma Patients Treated with Immunochemotherapy.
Authors: Leppä Sirpa , Taskinen Minna , Sommarhem Antti , Elonen Erkki , Nykter Matti , Monni Outi , Timonen Tuomo , Hautaniemi Sampsa , Karjalainen-Lindsberg Marja-Liisa , Meri Seppo
Published: October 2019

Addition of rituximab (R) to chemotherapy (immunochemotherapy) has significantly improved the outcome of B-cell lymphoma patients. However, responses are still unpredictable due to heterogeneous nature of these diseases. The aim of this study was to determine if differences in gene expression in follicular lymphoma (FL) and mantle cell lymphoma (MCL) tissue correlate with outcome in response to immunochemotherapy. 24 FL and 15 MCL patients were classified into complete (CR) or other responders after immunochemotherapy, and genes capable of separating the groups were identified using oligonucleotide-based microarray and supervised learning technique. One of the transcripts associated with increased CR rates in FL was signal transducer and activator of transcription 5a (STAT5a), a transcription factor and a mediator of cellular responses after cytokine stimulation. According to Kaplan Meier estimates, high STAT5a mRNA levels were associated with better progression free survival (PFS). Immunohistochemical analyses of FL samples demonstrated that predominant STAT5a activity localized to resident T-lymphocytes but only 20% of the samples were STAT5a positive. In a validation set of 79 FL patients, a better PFS was observed among patients with high STAT5a protein expression. In contrast, high STAT5a mRNA and protein levels were associated with poor outcome in MCL patients. In the MCL tissue, STAT5a expression localized to nonmalignant T-lymphocytes but also to tumor cells. In comparison to FL, T-lymphocytes were more uniformly positive for STAT5a. In sum, the data show that nonmalignant tumor cells have a profound prognostic impact both in FL and MCL, but also suggest that depending on the lymphoma subtype, the activity of a certain signaling pathway can have either positive or negative consequences on the survival of immunochemotherapy-treated lymphoma patients.
Variants of Recombinant Factor IX with Enhanced Functional Properties.
Authors: Hartmann Rudolf , Dockal Michael , Kammlander Willibald , Panholzer Erwin , Scheiflinger Friedrich
Published: October 2019

Activated human factor IX (FIXa) exhibits very low catalytic activity towards its physiologic substrate factor X (FX), unless the cofactors, activated factor VIII (FVIIIa), procoagulant phospholipid membranes and Ca2+ are present. We were interested in the elucidation of the structural basis for the FVIIIa dependency of FIXa activity. The surface loop 99 of FIXa is important for regulation of FIXa activity. In FIXa not bound to FVIIIa, this loop is stabilized in an inactive conformation and limits access of substrate to the catalytic cleft. Aiming to improve the catalytic activity in the absence of FVIIIa, amino acid exchanges supposedly influencing the 99-loop were introduced into FIX’s catalytic domain (Hopfner KP et al. EMBO J1997;16:6626–35; Sichler K et al. JBC2003;278:4121–26). One of the 3 most promising mutants generated was FIX-Y94F/K98T (FIX-C), with 2 residues exchanged in the 99-loop. Both residues are known to restrict access of FX to the S2 and S4 substrate binding pockets. In FIX-Y94F/K98T/Y177F/I213V/E219G (FIX-L) two additional sites were targeted. By hydrogen bond formation with Asn97 and Asn100, Y177 locks the 99-loop in an inactive conformation, which is released by binding of FVIIIa to FIXa. I213 and E219 at the S1 site are conserved in FIX whereas V213 and G219 are conserved among most other trypsin-like proteases. In FIX-Y94F/A95aK/K98T/Y177F/I213V/E219G (FIX-M) the mutation A95aK is thought to support the interaction between loop 99 and 60, so that conformations similar to equivalent loops of FXa can be adopted. All full-length mutants and wild type FIX were expressed in HEK293 cells, purified and preactivated FIX was removed by chromatographic steps. The resulting proteins were tested for their amidolytic activity and further assessed in FX activation assays in the absence and the presence of FVIIIa. Activated partial thromboplastin time (aPTT) assays were carried out, using plasmas depleted of FVIII or FIX, and FVIII-inhibitor patient plasma. The rationally designed proteins showed not only improved catalytic properties but also some FVIIIa independent activity (table 1). In an aPTT assay, FIX-M activity was equivalent to 1.6% FVIII in FVIII-depleted plasma and to 162 mU/ml FEIBA in FVIII inhibitor patient plasma whereas the combination of mutations Y94F/K98T as exemplified in FIX-C resulted in a molecule with ∼2.5-fold higher specific activity than plasma derived FIX when assayed in FIX-depleted plasma.

Characterization of rationally designed FIX proteins. Amidolytic Activity FX activation aPTT +FVIIIa −FVIIIa Ratio FIX-DP FVIII-DP FVIII-IP kcat / KM kcat / KM +FVIIIa / −FVIIIa mM−1min−1 ×10−3 mM−1min−1 mM−1min−1 ×10−4 ×103 mU/mg mU FVIII /5μg FIX mU FEIBA / 5μg FIX (Abbreviations: DP, depleted plasma; IP, inhibitor patient plasma) pdFIX 6 10 2 52 194 0 0 FIX-WT 9 9 3 37 216 1 15 FIX-C 22 12 4 29 498 15 114 FIX-L 15 32 33 10 180 12 115 FIX-M 7 9 11 8 208 16 162
Purified MHC-Matched Hematopoietic Stem Cell Transplantation Following Total Lymphoid Irradiation and Anti-Thymocyte Globulin (TLI+ATG) Blocks EAE Pathogenesis.
Authors: Lee Li-Fen , Burge Matthew , Ho Peggy , Sobel Raymond , Shizuru Judith
Published: October 2019

In prior studies, we demonstrated that transplantation of major histocompatibility complex (MHC) matched and mismatched purified hematopoietic cells (HSCs) can block diabetes pathogenesis in pre-diabetic NOD mice. Here we applied this treatment to experimental allergic encephalomyelitis (EAE), an animal model for the human autoimmune disease multiple sclerosis (MS). C57BL/6 (B6) strains congenic for the CD45 locus were immunized with MOG 35–55 to induce disease. Similar to our studies in NOD mice we observed that disease response after transplantation of MHC-mismatched hematopoietic cells in EAE affected mice was superior to those that received congenic cells. Importantly, mice transplanted with MHC-matched HSC or BM showed no significant improvement in disease symptoms when compared with congenic recipients or disease controls. This result was markedly different from the positive response observed in NOD mice transplanted with MHC-matched HSC. We then sought to optimize translation of this approach by giving total lymphoid irradiation (TLI) plus anti-thymocyte globulin (ATG), a non-myeloablative regimen and permits allogeneic hematopoietic cell engraftment with significant protective effect against GVHD. We found that TLI/ATG treatment in wild type C57BL/6 mice permit engraftment of purified HSC isolated from MHC-matched AKR/B donors and improve disease severity significantly. The level of stable mixed chimerism correlated with the clinical score. While the prior studies demonstrated that a class of regulatory cells that express both nature killer cell and T cell markers (NK-T) are present at high levels in TLI/ATG treated mice, we determined that regulatory CD4+CD25+Foxp3+ cells are also present at a disproportionately high levels as compared to wild type animals. To study the role of regulatory T cell in disease protection, a flexible lentiviral vector platform supporting constitutive expression of a Tet-transactivating component with a selection marker and coordinated conditional expression of miR-shRNA targeting Foxp3 gene products in stem cells is being developed. The detailed mechanism will be discussed. We hope a MHC-matched hematopoietic graft in conjunction with TLI/ATG conditioning shown here to successfully ameliorate EAE can be translated to the treatment of human autoimmune disease.
Effects of a Novel, Selective Jak2 Inhibitor, AZ60, on STAT5 Signaling and Cellular Growth in Jak2 V617F Cell Lines.
Authors: Gozgit Joseph M. , Bebernitz Geraldine A. , Patil Pankaj , Ye Minwei , Wu Jiaquan , Ioannidis Stephanos , Davies Audrey , Wang Tao , Huszar Dennis , Zinda Michael
Published: October 2019

A role for Jak2 in the etiology of the myeloproliferative diseases (MPDs) was discovered with the identification of a single activating point mutation, V617F, in the pseudokinase domain of JAK2. We have developed a Jak2 inhibitor, AZ60, which inhibits in vitro JAK2 enzyme activity with a Ki of 0.45 nM. AZ60 demonstrates inhibition of STAT5 phosphorylation and proliferation in a Tel-Jak2 engineered cell line with IC50 values of 18 and 23 nM, respectively. To understand the selectivity versus other Jak kinase family members we engineered three additional cell lines containing Tel fusions with the kinase domains of Jak1, Jak3 and Tyk2. Under these settings, AZ60 demonstrates a 15 to 30-fold selectivity for Tel-Jak2 driven STAT5 phosphorylation when compared to other Jak kinase family members. AZ60 was also tested for its ability to inhibit STAT5 phosphorylation and cellular proliferation in two human hematological cell lines, Set-2 and Hel. Set-2 expresses both wt and V617F Jak2, while Hel is homozygous for the Jak2 V617F mutation. AZ60 decreased phospho-STAT5 levels in a dose-dependent manner in both Set-2 and Hel cells with IC50 values of 15 and 25 nM, respectively. Complete inhibition of proliferation and a marked induction of apoptosis were observed in both cell lines following treatment with AZ60. Induction of apoptosis by AZ60 was characterized by a time- and dose-dependent increase in caspase 3/7 activities and PARP-cleavage. These data demonstrate AZ60 is a potent and selective inhibitor of Jak2 and may help decipher the mechanisms underlying Jak2-driven myeloproliferative disease.
Clinical, Prognostic and Possible Therapeutic Relevance of Angiogenesis in Non-Hodgkin’s Lymphoma.
Authors: Etto Leina Y. , Lacerda Emilio C.A. , Baiocchi Otavio C.G. , Silva Vanderleia C. , Dalboni Maria Aparecida , Inaoka Riguel J. , Corbi Fernanda C. , Alves Antonio C. , Vettore Andre L. , Colleoni Gisele W.B.
Published: October 2019

Considering that angiogenesis may have importance in the pathogenesis of non-Hodgkin’s lymphomas (NHL), the aims of this study were to correlate hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF) serum levels and microvessel density (MVD) with cell origin, biological behavior, tumor load and prognosis in NHL.

Materials and methods: Eighty-seven consecutive previously untreated NHL patients had serum samples collected and were included in the study. Thirty-seven (42%) of them also had additional serum follow-up samples. Control group was composed by ten healthy blood donors. HGF, VEGF and FGF serum levels were determined by ELISA. MVD was measured by CD34 staining in patients with available paraffin blocks.

Results: HGF mean serum level was significantly higher in both early and advanced NHL Ann Arbor stages when compared to control group. HGF was also significantly higher in both aggressive and indolent NHL when compared to control group. Furthermore, mean serum level of HGF in aggressive NHL was significantly higher than in indolent NHL. Regarding International Prognostic Index (IPI), mean serum levels of HGF at diagnosis was significantly higher for patients with IPI > 2 when compared to IPI <=2. FGF mean serum level was also significantly higher in early and advanced NHL stages when compared to controls. We also observed that FGF mean serum level was significantly higher in indolent NHL when compared to control group. Regarding VEGF, serum levels were significantly higher in aggressive NHL when compared to control group. Sequential analyses of HGF, VEGF and FGF serum levels in NHL showed that serum HGF and VEGF levels decreased significantly after 6 months of treatment completion. When we analyzed MVD, no associations between patient’s groups were found, even when stage, biologic behavior or cell origin were taken into account. We did not find correlation among MVD and serum levels of HGF, VEGF and FGF at diagnosis either.

Conclusions: Our findings suggest that HGF seems to play an important role in NHL pathogenesis because:

its serum level is associated to tumor load and aggressiveness; it can be correlated to treatment response.

In face of these results, we could suggest that HGF is a potential target for therapeutic intervention in NHL.
The Effect of Hemoglobin (Hb) Level at ESA Initiation on Outcomes in Cancer Patients (pts) with Chemotherapy-Induced Anemia (CIA): A Patient-Based Meta-Analysis from 6 Randomized, Placebo-Controlled Trials (RCTs) of Darbepoetin alfa (DA).
Authors: Crawford J. , Henry D. , Vansteenkiste J. , Ludwig H. , Lillie T. , Fleishman A. , Glaspy J.
Published: October 2019

Introduction: ESAs are associated with an increased and quantifiable risk of venous thromboembolism (VTE); however, the treatment alternative, RBC transfusion, has only a transient effect and the associated risks are often more numerous and less predictable. Repeated transfusions also diminish patients’ personal independence. Transfusion risks include mistransfusions, hemolytic reactions, exposure to infectious agents, immune-mediated reactions, and iron and circulatory overload. Previous studies have suggested that in order to maximally reduce transfusion requirements, ESAs should be initiated when anemia is mild. We conducted a retrospective analysis to assess the effect of hemoglobin (Hb) level at ESA initiation on the risk of transfusions and safety outcomes.

Methods: A combined analysis of patient-level data from randomized, blinded, placebo-controlled studies in which the ESA darbepoetin alfa (DA) was administered subcutaneously in patients with nonmyeloid tumors and CIA (hemoglobin ≤11 g/dL at study screening) was performed. Patients had received at least 1 prior cycle of chemotherapy and had further chemotherapy planned with or without radiotherapy. Endpoints were analyzed by baseline Hb level and included transfusions, embolic/thrombotic events, death, and death or disease progression. Hazards ratios (HR, with 95% confidence interval limits) were used to describe the risk of these endpoints in the DA group relative to placebo.

Results: Patients (n=2112) were mostly white, with a mean age of 62 years. Nearly half were ≥65 years. Baseline characteristics were similar, with the most frequent tumor types in both treatment groups being lung and hematologic cancers, and most with stage III or higher disease. For both groups, patients with lower baseline Hb levels had a higher probability of transfusions; yet the reduced risk of transfusions in the DA group relative to placebo was apparent at each baseline Hb level. The known increased risk of VTEs in the DA group did not appear to be associated with baseline Hb level. There was no increased risk of death or progression at any baseline Hb level.

Conclusions: Results from this patient-level meta-analysis in CIA patients suggest that baseline Hb is a predictor of transfusion risk, and that earlier ESA intervention lowers the risk of future transfusions. Initiating DA at Hb ≥ 9 to < 10 g/dL resulted in a rate of transfusion of 32%; this rate was substantially lowered when DA was initiated at Hb ≥ 10 to < 11 g/dL. There did not appear to be an increased risk of death or VTEs as a result of initiating DA therapy at the higher Hb levels. Therefore, delaying the start of therapy may only increase the transfusion requirements, resulting in increased risk to the patient, and additional strain on the national blood supply.
Connexin-43 Mediates Fcγ Receptor-Induced Calcium Signaling by Acting as an NAD/Cyclic ADP-Ribose Transporter.
Authors: Song Eun-Kyung , Lee Young-Rae , Yu Hong-Nu , Rah So-Young , Kim Uh-Hyun , Lee Seun-Jin , Kim Jong-Suk , Han Myung-Kwan
Published: October 2019

Cyclic ADP-ribose (cADPR) is produced from NAD by CD38, a leukocyte receptor and ectoenzyme with its catalytic domain positioned outside of cells, and mobilizes Ca2+ from ryanodine sensitive Ca2+ stores. It has long been questioned how NAD approach CD38 and extracellularly generated cADPR reaches its intracellular target Ca2+ stores. Here we address this question by identifying connexin 43 hemichannels (Cx43) as a transporter of cyclic ADP-ribose as well as NAD in Fcg receptor (FcgR) triggering-induced calcium signaling. FcgR clustering triggers a Ca2+ transient via three sequential steps, Cx43-mediated NAD export, CD38-mediated cADPR production and subsequent Cx43-mediated cADPR import in murine macrophages. FcgR clustering induces Cx43 phosphorylation, thereby opening Cx43 and transporting NAD and cADPR. After cADPR-mediated Ca2+ transient, Cx43 is immediately dephosphorylated and closed, blocking further cADPR transport. This finding is the first evidence showing the involvement of Cx43 in cADPR-mediated Ca2+ release induced by physiological ligand where the apparent topological paradox of extracellular cADPR synthesis and its intracellular action exist. Our results provide new insight into the mechanism of intracellular calcium release triggered by the extracellularly generated calcium mobilizer cADPR, which is involved in (FcgR)-mediated calcium signaling in murine macrophages.
Lymphocyte Depletion in Breast Cancer Patients Receiving Adjuvant Dose-Dense Chemotherapy.
Authors: Angitapalli Revathi , Mashtare Terry L. , Janarthanan Bagirathan
Published: October 2019

Background: Dose-intense chemotherapy regimens are reportedly known to cause lymphocyte depletion. Increased incidence of pneumocystis carinii pneumonia (PCP) was reported in patients with diffuse large B-cell lymphoma treated with dose-dense CHOP plus rituximab. No studies have been conducted to date to assess the lymphocyte depletion in breast cancer patients receiving dose-dense chemotherapy. We retrospectively compared the lymphocyte recovery in breast cancer patients treated with dose-dense (q 2 weekly) doxorubicin/cyclophosphamide (AC) to those treated with AC in a non-dose-dense (q 3 weekly) method.

Methods: Newly diagnosed breast cancer patients treated at our institution between 2002 and 2006, with at least 4 cycles of AC in an adjuvant/neoadjuvant setting were analyzed. Complete blood count (CBC) with differential including white blood cells (WBC), hemoglobin (Hg), platelets, neutrophils, lymphocytes, monocytes, eosinophils and basophils were collected at baseline before the start of chemotherapy, before each ensuing cycle of chemotherapy and after the 4th cycle of chemotherapy.

Results: A total of 103 patients were analyzed of which 50 patients were treated with dose-dense AC and 53 patients with non-dose-dense AC. After the 4th cycle of chemotherapy, mean absolute lymphocyte count (ALC) was 780 ± 440/mm3 in the dose-dense group versus 950 ± 600/mm3 in the non-dose-dense group (p = .4617), mean Hg was 11.4 ± 1.2g/dL in the dose-dense group versus 12.2 ± 0.9g/dL in the non-dose-dense group, mean platelet count was 241 ± 85 x109/L in the dose-dense group versus 391 ± 128 x109/L in the non-dose-dense group. No opportunistic infections were noted in the dose-dense group.

Conclusions: These results demonstrate that dose-dense AC results in slightly increased lymphocyte depletion compared to non-dose-dense AC, not statistically significant. This didn’t result in any increased infectious complications. In both groups, baseline ALC was significantly positively related to ALC after the 4th cycle of chemotherapy (p < 0.0001). Dose-dense AC did result in statistically significant platelet depletion.
Regulation of αIIbβ3-Signaling by Myomesin Family of Proteins.
Authors: Reddy Kumar B.
Published: October 2019

Integrins are the type-I transmembrane proteins. They function as heterodimers of α and β subunits. The platelet integrin αIIbβ3 (GPIIb-IIIa) served as the model system in understanding the signaling across integrin receptors. αIIbβ3 exists in a resting state on circulating platelets and can be activated via several inside-out signaling pathways resulting in binding to its ligands, such as fibrinogen (Fg). The inside-out signaling appears to be regulated by several cytoskeletal and signaling proteins, which directly associate with the cytoplasmic tails of αIIbβ3. We have previously identified skelemin/myomesin-I as the cytoskeletal protein that interacted with the cytoplasmic tail of β3-integrins. Skelemin/myomesin-I is very closely related to two other members: myomesins-II and III. Myomesins I-III, along with other proteins such as titin and myosin light chain kinases, are members of a much larger superfamily of proteins. The hallmark of this family of proteins is the presence of a variable number of fibronectin (Fn)-like and immunoglobulin (Ig)-like motifs. Myomesin-I contains five Fn-motifs flanked by a total of seven Ig-like motifs. The interaction between β3-integrin and myomesin-I occurs via the C-terminal Ig motifs 3–7 of myomsin-I. In order to understand the functional consequence of this interaction between myomesin-I and β3-integrin, we measured the ligand binding properties of αIIbβ3 in a CHO cell model system. We found that expression of Ig motifs 3–7 increased Fg binding to the αIIbβ3, comparable to talin-head domain, used as the positive control. However, the expression of smaller and individual Ig motifs 4 and 5 resulted in still significantly higher Fg-binding to αIIbβ3. Ig motifs from myomesin-II also behaved in a similar fashion. We also found that myomesin-II is ubiquitously expressed in all the cell types tested, including platelets, suggesting myomesin-II is the likely candidate among various myomesin family members for the regulation of signaling across αIIbβ3. Our results suggest that the Ig motifs of the myomesin represent novel domains, which can regulate inside-out signaling across αIIbβ3.
Evaluation of Inherited Prothrombotic Risk Factors in Children with Acute Lymphoblastic Leukemia.
Authors: Te Valerie Li Thiao , Favier Remi , Borg Jeanne-Yvonne , Cadet Estelle , Reynaud Jacqueline , Micheli Julie , Rachieru Petronela , Berger Claire , Auvrignon Anne , Schneider Pascale , Leverger Guy , Muchemble Brigitte Pautard
Published: October 2019

This retrospective study was designed to determine the prevalence of inherited prothrombotic risk factors (Factor V Leiden (FV) G1691A and prothrombin G20210A mutations, TT677 genotype of the methylenetetrahydrofolate reductase (MTHFR), protein C, protein S, antithrombin deficiencies) in a population of children with ALL treated according to the FRALLE 2000 study Protocol (High Risk and Standard Risk groups). The study was performed in 5 French Centers including Amiens, Angers, Paris Trousseau, Rouen and Saint-Etienne. From December 2000 to March 2006, 354 children aged 1 to 18 years old were consecutively admitted for ALL and were enrolled in the FRALLE 2000 Protocol. Among them, 281 patients were investigated for hereditary prothrombotic defects at the time of ALL diagnosis. Informed parental consent was required for gene analysis. Abnormal test results for protein S (functional activity and free protein S antigen concentration), protein C and antithrombin were controlled on a second blood sample after induction. In the population studied, the prevalence of one established prothrombotic risk factor was 19,2%: the FV G1691A mutation was diagnosed in 10 patients (3.6%), all heterozygous, 10 patients (3.6%) showed the heterozygous prothrombin G20210A mutation, the TT677 MTHFR genotype was found in 34 children (12.7%), 1 patient showed protein C deficiency (0.4%). No antithrombin deficiency was detected. The prevalence of inherited protein S deficiency could not be evaluated because of missing data in the family medical history. Combined prothrombotic defects were found in 2 patients (0.71%): heterozygous FV G1691A mutation combined with heterozygous prothrombin G20210A mutation in 1 patient and combined with TT677 MTHFR genotype in the second patient. Except for TT677 MTHFR genotype, the prevalence of hereditary prothrombotic risk factors in children with ALL in France were found within the prevalence reported for children treated for ALL (table 1) and comparable to the prevalence in healthy Europeans (Junker et al. 1999, Margaglione et al 2001, Mueller et al. 2005).

Comparison of the prevalence of inherited prothrombotic risk factors in children with ALL Country Population FV G1691A +/− ++ PT G20210A +/− +/+ MTHFR TT677 AT PC AT: antithrombin deficiency ; PC: protein C deficiency ; NE : non evaluated NowakGöttl et al 1999 (n=301) Germany ALL children 5.3% 0.3% 2% 0% 7.7% 0.7% 2.3% Mauz-Körholz et al. 2000 (n=108) Germany ALL children 5.6% 0% 2.8% 0% 5.6% 0% 2.7% Mitchell et al. 2002 (n=60) Canada ALL children 3.3% 0% 2% 0% NE NE NE Present study (n=281) France ALL children 3.6% 0% (n=277) 3.6% 0%(n=279) 2.7% (n=268) 0% 0.4%
Efficacy of Recombinant Activated Factor VII (rFVIIa) vs. Activated Prothrombin Complex Concentrate (APCC) in Patients with Hemophilia with Inhibitors: A Bayesian Meta-Analysis.
Authors: Treur Maarten Jacob , McCracken Fay , Heeg Bart , Joshi Ashish V. , Thurston Stephanie , Verheggen Bram , Marc Botteman , de Charro Frank , van Hout Ben
Published: October 2019

INTRODUCTION: The recent FENOC comparative trial (Astermark et al. 2007) reported comparable efficacy for rFVIIa and APCC in the treatment of joint bleeds in patients with hemophilia complicated by inhibitors. A systematic review was combined with a Bayesian meta-regression analysis to place the FENOC results within the context of earlier non-comparative studies and to identify key variables influencing treatment efficacy.

METHODS: A systematic literature search was performed using electronic databases, web based trial registers, and grey literature. Clinical studies and/or trials in hemophilia patients with inhibitors receiving on-demand treatment with rFVIIa or APCC were included. Outcome of interest was the time of successfully stopping joint bleeds taking explicit account of repeated dosing. To model the latter, the regression model included a repeating Gompertz hazard function to compare the efficacy of standard rFVIIa treatment (one 90 μg/kg rFVIIa infusion every 3 hrs) with standard APCC treatment (one 75 IU/kg APCC infusion every 12 hrs). The hazard function (the conditional probability of ending the bleeding) was characterized by a repeated pattern of fixed increases after each infusion and by subsequent decreases thereafter. It was assumed that each sequential infusion had the same effect on the hazard resulting in a shark tooth like function. Covariates included were medication type, medication type combined with dosage (measured in μg/kg for rFVIIa and IU/kg for APCC), efficacy rating scale method, and efficacy rater.

RESULTS: Of the 17 studies included in the systematic review, 14 provided adequate data to be included in the Bayesian model, reporting on almost 2,000 joint bleeds combined (of which 96 were from the FENOC itself). The analysis demonstrated that medication type combined with dosage appears to have a statistically significant effect on efficacy of treatment. For rFVIIa the model estimated that 75%, 94%, and 98% of bleeds would be treated effectively after 12, 24, and 36 hours respectively. In contrast, for APCC the model estimated that 39%, 63%, and 78% of bleeds would be treated effectively after 12, 24 and 36 hours respectively.

CONCLUSIONS: The FENOC study demonstrated similar efficacy levels for treatment with rFVIIa and APCC. In contrast, the present meta-analysis including 14 studies and almost 2,000 joint bleeds estimated that standard treatment with rFVIIa is likely to result in higher efficacy levels at the 12, 24 and 36 hour time points than treatment with APCC. This implies that treatment with rFVIIa may be associated with a faster time to joint bleed resolution than APCC.
Successful Treatment of Factor IX Inhibitor Complicated by Nephrotic Syndrome with Rituxan.
Authors: Bernstein Jonathan , Gupta Neepa , Berkowitz Rebecca
Published: October 2019

Nephrotic syndrome is a well known complication of the development of inhibitors in factor IX patients. Nephrotic syndrome has been treated with Rituxan as have factor IX inhibitors. We present a 4 year old male diagnosed with severe factor IX deficiency at 5 months of age after severe bruising.

History: Between 11/05 and 5/06 we treated a 3 yo male with history of factor IX deficiency and inhibitor as well as nephrotic syndrome with Rituxan. The child received 4 weekly doses of Rituxan at 375 mg/M2, followed by monthly doses at 375 mg/M2, for six months. He received monthly doses of IVIG at 1 gram/M2. In the last 2 months of the course, he was reexposed to factor IX (Alphanine).

Results: He has had no signs of an inhibitor and is on 3x per week dosing. He did redevelop nephrotic syndrome 17 months later but has since been placed in remission on a repeat course of Rituxan.

Conclusions: This is the first report of the successful treatment of both a Factor IX inhibitor and Nephrotic syndrome with Rituxan therapy.
Venous Thromboembolism and Non-Small Cell Lung Cancer: A Pooled Analysis of National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) Trials.
Authors: Hicks Lisa , Cheung Matthew , Hasan Baktiar , Ding Keyue , Seymour Lesley , Leighl Natasha B. , Winton Timothy L. , Shepherd Frances A.
Published: October 2019

Purpose: To determine the incidence of venous thromboembolism (VTE) in patients with early and advanced non-small cell lung cancer (NSCLC); to explore predictive factors for VTE occurrence during trials in these populations, and to investigate the effect of VTE on overall survival in patients with NSCLC.

Patients and Methods: Data from three National Cancer Institute of Canada Clinical Trials Group trials were included in the analyses (n=1987). JBR.10 was a randomized study of post-resection adjuvant vinorelbine/cisplatin versus observation in stage IB/II NSCLC. BR.18 was a randomized study of paclitaxel/carboplatin ± the metalloproteinase inhibitor BMS-275291 in advanced NSCLC (1st line). BR.21 was a randomized study of erlotinib versus placebo in previously treated (2nd or 3rd line) NSCLC. The relationship between VTE, cancer treatment, concomitant medications, and baseline patient characteristics was explored with univariate and multivariate analysis. Cox regression analysis was performed to determine whether VTE was independently associated with survival. Each trial was analyzed separately; in addition a pooled analysis was performed with the advanced disease trials (BR.18 and BR.21).

Results: The incidence of VTE was 0% in the observation arm of JBR.10 and ranged from 3% in the adjuvant chemotherapy arm of JBR.10 to 8% in BR.18. In JBR.10, VTE was independently associated with the administration of adjuvant chemotherapy (p=0.015), baseline obesity (p=.001), and low platelet count (p=.007). For patients with advanced NSCLC, VTE was significantly more common in patients receiving chemotherapy (BR.18) compared to patients receiving erlotinib or placebo (BR.21) (8% vs 2%, p< 0.0001). In BR.18, but not BR.21, VTE was associated with a prior history of VTE (p=0.001). When BR.18 and BR.21 were pooled, prior VTE remained significant. In BR.18 and BR.21, VTE was associated with shorter survival in multivariate analysis (HR=1.69, 95%CI 1.32–2.17, p<.0001); further analyses, including JBR.10, are planned to explore this finding.

Conclusion: VTE is a frequent event in patients with advanced NSCLC and is associated with the administration of chemotherapy. Treatment with metalloproteinase or epidermal growth factor inhibitors does not appear to increase the risk of VTE. In early NSCLC, adjuvant chemotherapy, morbid obesity and a prior history of VTE are associated with increased risk. VTE is associated with shortened survival in patients with advanced NSCLC. Funding for this study was provided by the Canadian Cancer Society.
New Pulmonary Embolism Diagnostic Algorythm and the Thromboprophylaxis in a Hungarian Teaching Hospital.
Authors: Egyed Miklos , Zadori Peter , Rajnics Peter , Varga Csaba , Horvath Anita , Horvath Gyula
Published: October 2019

The aims of our study were to evaluate a new pulmonary embolism (PE) diagnostic algorythm and applied VTE prophylaxis. Therefore we reviewed and analysed the medical records of 507 patients with suspected PE. The patients were classified as having preventable or nonpreventable PE according to the risk and adequacy of the administered prophylaxis, or noncompliance if the indicated prophylaxis had been refused. 507 multislice computer tomograph angiography (MuCTA) were done (27 unsuccesful) with a susppected diagnosis of PE between oct.2004 and oct. 2006. From the 140 positive patients 106 were regularly controlled by physicians in different hospitals and 87 had to receive prophylaxis according to the recommendation of the ACCP 2004 guidelines. 7% of them refused it (noncompliance). 70% received inadequate prophylaxis, 23% of the patients (nonpreventable) received prophylaxis in accordance with the ACCP 2004 guidelines. Both Medical and surgical patients were in the preventable group. From the 61 preventable cases, the reason for the inadequacyof prophylaxiswas the omission of prophylaxis in 41, inadequate dose of prophylaxis in 9, inadequate lenght in 5, inadequate anticoagulant in 5 cases. MuCTA is a very useful method to diagnose PE. From the 480 evaluable suspected cases 140 patients found be positive (29%). Adherence to the prophylaxis guidelines is frightful, 70% of patients with PE for whom prophylaxis had been indicated, could have been prevented. The incidence of PE is still high despite adequate prophylaxis, which indicates that the guidelinesmay be needed to reevaluate. Quantitative d-dimer and CRP have very good negative predictive value for excluding PE.