bluuebunny/crossref_metadata_2025 huggingface dataset:
The BLyS Survival Pathway in NHL-B Cells: Constitutive NF-kB and NFAT Lead to Activation.
Authors: Fu Lingchen , Archito Tamayo , Lin-Lee Yen-Chiu , Pham Lan , Yoshimura Linda , Ford Richard J.
Published: October 2019

B-cell non-Hodgkin’s Lymphomas (NHL-B), neoplasms of the immune system have shown a significant increase in incidence in the USA over the last three decades. While the pathophysiology of the NHL-B is still unclear, the need to identify the relevant genes and critical signaling pathways, and their involvement in the disease processes in NHL-B have begun to be elucidated. Recently, B Lymphocyte Stimulator (BLyS) has been described as a relatively new member of the TNF ligand family, as a potent cell survival factor that is expressed in many hematopoietic cells, including neoplastic B cells. BLyS can bind to three receptors: TACI, BCMA, BAFF-R, and plays a critical role in B cell maturation, differentiation and proliferation. Relatively high levels of BLyS has been found in the serum of NHL-B patients as well as of the patients with autoimmune disease. The mechanisms of BLyS gene expression and regulation is still unclear, but we have recently found that BLyS is constitutively expressed in several NHL-B cell lines and patient tumor samples by RT-PCR, confocal microscopy, realtime PCR and flow cytometry (FCM). We detected high levels and differential expression of BLyS receptors (TACI, BCMA, BAFF-R) in several NHL-B cell lines by flow cytometry, RT-PCR and realtime PCR in both NHL-B cell lines and patient tumor samples. We have identified a single binding site for NF-kB and two binding sites for NFAT in the BLyS promoter. We also show in aggressive lymphoma B cells that constitutive NF-kB and NFAT binds to the BLyS promoter constitutively. Inhibiting NF-kB/NFAT activity levels, using the NF-kB inhibitors, BAY-11 or Velcade (PS-341), can decrease NF-kB binding activity in the BLyS promotor by EMSA. These inhibitors also decrease BLyS and BAFF-R mRNA and protein levels by realtime PCR and flow cytometry. Similarly, when NHL-B cells were transfected with dominant negative NFAT or NF-kB constructs, there is a 50% decrease in BLyS and BAFF-R expressions, demonstrating that both the ligand (BLyS) and the receptor (BAFF-R) expression are regulated by NFAT and NF-kB. Interestingly, follicular (low grade) lymphoma cells do not express constitutive NF-kB/NFAT activation, and barely detectable mRNA and protein levels of BlyS, but can be activated with exogenous CD154/anti IgM in vitro, activating NF-kB/NFAT and promoting binding to the BLyS promoter by EMSA. This results in a significant increase of BLyS protein level by flow cytometry. Our studies indicate that constitutive NF-kB and NFAT are critical transcriptional regulators of the BLyS survival pathway in malignant B cells that may provide a future therapeutic target in the aggressive NHL-B.
Dihydroflavonol BB-1, an Extract of Natural Plant Blumea Balsamifera, Abrogates TRAIL Resistance in Adult T-Cell Leukemia/Lymphoma.
Authors: Hasegawa Hiroo , Yamada Yasuaki , Hayashi Masahiko , Ishibashi Masami , Komiyama Kanki , Murata Ken , Masuda Masato , Takasu Nobuyuki , Tomonaga Masao , Kamihira Shimeru
Published: October 2019

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many transformed cells but not in normal cells, hence TRAIL has recently emerged as a novel anti-cancer agent. Adult T-cell Leukemia/Lymphoma (ATLL) is a neoplasm of T-lymphocyte origin etiologically associated with HTLV-I, and is known to be resistant to standard anti-cancer therapies. In a previous study, we showed that although most ATLL cells express TRAIL-death receptors DR4 and/or DR5, they are resistant to TRAIL. To overcome this resistance, we tried to find natural materials that increase the sensitivity to TRAIL in the present study. Here, we report for the first time that dihydroflavonol from a plant increases TRAIL sensitivity by enhancing death receptor expression.

Materials and methods: We used a TRAIL-resistant ATLL cell line, KOB, for screening, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. The cell surface expression of DR4, DR5, and decoy-receptors DcR1 and DcR2 was examined using flow cytometry (FCM). Cell viability was assessed by MTS assay after incubation with various combinations of natural extracts and TRAIL for 48 hours. Apoptosis was evaluated by Annexin V binding and propidium iodide (PI) staining. We also analyzed caspase−8, − 9 and −3 activation and pro- and anti-apoptotic proteins by Western blot.

Results and Discussion: We screened more than 500 natural products. Among them, the most striking synergism with TRAIL was observed in a dihydroflavonol that was extracted from the Blumea Balsamifera, a family of chrysanthemum from Thailand. It was a methyldihydroquercetin (molecular weight, 318), and we named it BB-1. The viability of KOB cells treated with either TRAIL or BB-1 was 98% and 97%, respectively, and decreased to 60% with concurrent treatment. Interestingly, sequential treatment, pre-treatment with BB-1 followed by TRAIL, further augmented the synergism, and the viability decreased to 38%. Using Annexin V and PI, we confirmed that these are the results of apoptosis. Although it has been reported that anti-apoptotic proteins FLIP, Bcl-2, Bcl-xL and XIAP play key roles in TRAIL resistance, we did not find any change in these factors in the BB-1-treated cells. Instead, we found that BB-1 increased DR5 expression. Subsequent activation of caspase−8, −9 and −3 was observed in a time-dependent manner in the sequentially treated cells, but not in cells treated with BB-1 or TRAIL alone. More importantly, BB1 did not induce DR5 expression or enhance TRAIL sensitivity in PBMCs. Thus, BB-1 augmented TRAIL-induced apoptosis by up-regulating DR5 expression without any cytotoxic activity on normal PBMCs. These results suggest that combined treatment with BB-1 and TRAIL can be a new strategy for cancer immunotherapy against TRAIL-resistant cells.
Proteome Changes Induced by Rituximab: Significance for Monoclonal Antibody Therapy of B-Cell Lymphoproliferative Disorders.
Authors: Everton Kathryn L. , Abbott David R. , Crockett David K. , Elenitoba-Johnson Kojo S.J. , Lim Megan S.
Published: October 2019

Rituximab is a human chimeric monoclonal antibody that targets the CD20 receptor uniquely expressed on normal and malignant B-cells. While it is particularly useful for follicular lymphoma, Rituximab has recently been used for other types of B-cell proliferative disorders. Four major mechanisms have been proposed to account for the activity of Rituximab in B-cell lymphoproliferative disorders: 1) apoptosis and proliferation inhibition via intracellular signaling, 2) increased sensitivity to other chemotherapeutic drugs, 3) complement-dependent cytotoxicity (CDC), and 4) antibody-dependent cellular cytotoxicity (ADCC). A combination of all four mechanisms has also been proposed as the mechanism of action. However, the intracellular events that occur in B-cells in response to Rituximab are largely unknown.

We have employed a global quantitative proteomic profiling approach to study the effects of Rituximab on a t(14;18)+ B cell lymphoma (SUDHL-4). A decrease in viability of 31.3 ± 2.9% was observed after treatment with 10μg/mL of Rituximab for 48 hours. We used isotope-coded affinity tagging (ICAT™) in combination with microcapillary liquid chromatography and tandem mass spectrometry (μLC-MS/MS) to quantitatively determine the proteomic differences between equal amounts of untreated and treated total cell lystates. Protein lysates were labeled with either heavy or light cleavable ICAT™ reagents, digested with trypsin, and purified using avidin affinity chromatography. The labeled peptides were then fractionated using off-line strong cation exchange (SCX) and each fraction was subjected to μLC-MS/MS analysis. Several proteins previously unknown to be expressed in B-cells were identified using this approach. Of the 123 positively identified proteins, 59 proteins were differentially expressed by greater than or less than 1.5 fold (25 upregulated, 34 downregulated). Analysis of differentially expressed proteins identified several functional groups, including integral membrane proteins (Integrin alpha-7 precursor), transcription factors (Zinc finger proteins 445 and 20), proteins involved in migration and adhesion (Astrotactin1), proteins associated with degradation and turnover (26S proteasome non-ATPase regulatory subunit 9), proteins affecting calcium-induced signaling (Tumor-associated calcium signal transducer 1 precursor), proteins associated with lipid rafts (Gemin7), and components of the phosphoinositol (PI4K-alpha) and NF-κB pathways (IKK-E). Our studies reveal the proteomic consequences of Rituximab administration to neoplastic B-cells, and provide novel insights into the mechanism of the drug’s efficacy in the treatment of susceptible B-cell lymphoproliferative disorders.
Syngeneic Hematopoietic Stem Cell Transplantation in Acute Leukaemia. A Report of the Acute Leukaemia Working Party of the EBMT.
Authors: Fouillard Loic , Labopin Myriam , Gluckman Eliane , Gratwohl Alois , Frassoni Francesco , Gorin Norbert Claude , Rocha Vanderson
Published: October 2019

Syngeneic haematopoietic stem cell transplantation (HSCT) is a rare situation and is usually characterised by a high relapse rate because of the absence of graft versus leukaemia effect.

We report results of syngeneic HSCT reported to the EBMT registry from 1975 to 2003. One hundred and 24 acute myeloid leukaemia (AML) and 104 acute lymphoblastic leukaemia (ALL) were reported comprising 150 adults and 78 children, 133 males and 95 females. The number of patients in first complete remission (CR1) was 137, comprising 93 AML (72 adults and 21 children) and 44 ALL (33 adults and 11 children). The number of patients in second complete remission (CR2) was 52 comprising 12 AML (9 adults and 3 children) and 40 ALL (11 adults and 29 children). The number of patients in more advanced disease (AD) was 39 comprising 19 AML (16 adults and 3 children) and 20 ALL (9 adults and 11 children). Total body irradiation was given to 36% of patients. Prophylaxis of graft versus host disease was given to 10% of patients. Source of stem cells was bone marrow for 81% of patients, peripheral blood for 18% and both for 1%.

Outcome at 5 years showed for adult patients with AML in CR1 (n=72) a leukaemia free survival (LFS) of 56+/−7%, a relapse incidence (RI) of 37+/−7% and a non relapse mortality (NMR) of 11+/−5%. For adult patients with ALL in CR1 (n=33), LFS was 60+/−10%, RI 38+/−10% and NRM 3+/−3%.

Outcome at 5 years showed for children with AML in CR1 (n=21) a LFS of 61+/−11%, a RI of 39+/−11% and a NMR of 0%. For children with ALL in CR2 (n=29), LFS was 46+/−10%, RI 52+/−10% and NRM 5+/−5%. Acute graft versus host disease (GVHD) was diagnosed in 12% of patients, 7% grade 1 and 5% grade ≥ 2. Chronic GVHD was observed in 2% of patients.

These retrospective study indicates that syngeneic HSCT can lead to a high LFS in patients with acute leukaemia in CR1 and that GVHD is not a rare event. A graft versus leukemia effect is highly probable in syngeneic HSCT.
Results of Intensive Chemotherapy in 998 Patients Aged 65 Years or Older with Acute Myeloid Leukemia or High-Risk Myelodysplastic Syndrome - Predictive Prognostic Models for Outcome.
Authors: Jabbour Elias , Kantarjian Hagop , O’Brien Susan , Cortes Jorge , Giles Francis , Faderl Stefan , Garcia-Manero Guillermo , Wierda William , Pierce Sherry , Shan Jianqin , Estey Elihu
Published: October 2019

Background. Elderly patients (age ≥ 65 years) with acute myeloid leukemia (AML) have a poor prognosis. AML-type therapy results are often derived from studies in younger patients and may not apply to elderly AML. Many investigators and oncologists advocate, at times, only supportive care or frontline single agents, phase I–II studies, low intensity regimens, or “targeted” therapies. Baseline expectations for outcomes of elderly AML with “standard” AML-type therapy are not well defined.

Study Aims. To develop prognostic models for complete response (CR), induction (8-week) mortality, and survival rates in elderly AML, which define expectations with standard AML type therapy.

Patients and Methods. 998 patients age ≥ 65 years with AML or high-risk myelodysplastic syndrome (≥ 10% blasts) treated with intensive chemotherapy between 1980 and 2004 were analyzed. Univariate and multivariate analyses of prognostic factors used standard methods.

Results. The overall CR rate was 45% and induction mortality 29%. Multivariate analyses identified consistent independent poor prognostic factors for CR, 8-week mortality, and survival. These included age ≥ 75 years, unfavorable karyotypes (often complex), poor performance (3–4 ECOG), longer duration of antecedent hematologic disorder, treatment outside the laminar airflow room, and abnormal organ functions. Patients could be divided into: 1) a favorable group (about 20% of patients) with expected CR rates above 60%, induction mortality rates below 10%, and 1-year survival rates above 50%; 2) an intermediate group (about 50% of patients) with expected CR rates of 50%, induction mortality rates of 30%, and 1-year survival rates of 30%; and 3) an unfavorable risk group (about 25% to 30% of patients) with expected CR rates of less than 20%, induction mortality rates above 50%, and 1-year survival rates of less than 10%.

Conclusions. Prognostic models were developed for elderly patients with AML, which may assist in therapeutic and investigational decisions.
Proteasome Inhibitors Restore to Normal the Decreased Levels of Protein Expression and Nucleolar Localization of Various Mutant Ribosomal S19 Proteins Identified in DBA Patients.
Authors: Cretien Aurore , Proust Alexis , Gazda Hanna , Meerpohl Jorg , Niemeyer Charlotte Marie , Delaunay Jean , Tchernia Gil , Narla Mohandas , Da Costa Lydie
Published: October 2019

Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. We have previously shown that several RPS19 mutant proteins (V15F, InsAG36, W33stop, Y48stop, R56stop, M75stop, R94stop, 274del31, InsG238, G127Q and L131P) exhibit decreased levels of protein expression and do not localize to the nucleolus like the wild type protein in transfected Cos-7 cells. In contrast, other mutants (W52C, T55M, R56Q, R62W, 24Del18, G120S) exhibit normal levels of protein expression and normal nucleolar localization. We hypothesized that decreased levels of expression of mutant proteins such as V15F, G127Q, and L131P may be due to proteosomal degradation. In order to validate our hypothesis, we analyzed the effects of two proteasome inhibitors (MG132 and lactacystin) on mutant RPS19 protein expression levels and their subcellular localization. Following treatment with proteosome inhibitors, the mutant proteins with missense mutations (V15F, G127Q and L131P) were expressed at levels similar to that of wild type protein and localized in the nucleolus. Similarly, proteasome inhibitors also restored the expression levels and normal subcellular localization to RPS19 with non-sense mutations (InsG238, R94stop, and 274del31) that resulted in the translation of RPS19 protein with at least 80 aminoacids. In marked contrast, proteosome inhibitors failed to restore the expression levels of RPS19 with the non-sense mutants that led to synthesis of shortened proteins (InsAG36, W33stop, Y48stop, R56stop, M75stop). Even in the presence of proteosome inhibitors we noted a dramatic decrease in the levels of expression of these mutant proteins and proteins expressed were localized in the cytoplasm. Our findings imply an important role for proteosomal degradation pathway in regulating the expression levels of RPS19. They further suggest that proteasome inhibitors could be considered as a potential treatment for some steroid resistant DBA affected individuals with specific RPS19 mutations.
High-Dose Mitoxantrone-Based Induction Therapy in Newly-Diagnosed AML. Results of 171 Patients Treated at NYMC between 1991–2003.
Authors: Seiter Karen , Liu Delong , Feldman Eric , Shi Qiuhu , Baskind Paul , Arshad Muhammad , Qureshi Anila , Ahmed Tauseef
Published: October 2019

We developed a high dose mitoxantrone-based induction regimen based on the steep dose-response curve for mitoxantrone. Currently we report long-term results of the treatment of patients with newly-diagnosed AML with a single high dose of mitoxantrone combined with once daily cytarabine. One-hundred sixty-five patients treated on four studies of high-dose mitoxantrone-based induction therapy are included.

Study 1: Phase II study, age <60.

Induction: mitoxantrone 80 mg/m2 x 1, ara-C 3 gm/m2/d x 5, VP-16 150 mg/m2/d qod x 3. Consolidation: 5 mos ara-C x 4d with mitoxantrone 20 mg/m2 months 3 + 5, and with VP-16 150 mg/m2 x 2 months 4 + 6.

Study 2: randomized study of high vs standard dose mitoxantrone with ara-C in pts ≥ 60 yrs. Pts did not receive consolidation. Only pts with high dose mitoxantrone are included here.

Study 3: Same as study 1 except pts received ATRA 45 mg/m2 x 3 doses. Pts < 60 received consolidation as in study 1. Pts ≥ 60 did not receive consolidation.

Study 4: Pts ≥ 60 yrs, same as study 2 + temozolomide postremission therapy. In each study, pts with prior antecedent hematologic disorder (AHD) or secondary AML were included. Median age: 59 (21–88); M/F: 91/80; cytogenetics(SWOG): good:13%, intermediate:50%, poor:26%, indeterminate:5%, IM:6%; Prior AHD: 32% of pts. The median follow up time is 65.9 months (95% CI: 55.7–86.2 months). The overall complete remission rate was 64%, with responses in 78% of patients less than 60 years of age and 52% of patients 60 years of age or older. The median duration of response is 21.2 months and 8.0 months, and overall survival is 15.4 months and 7.6 months, respectively. For a subset of patients who would be eligible for most US trials, the complete remission rate was 84% in younger patients and 60% in older patients. The median duration of response was 39.0 months and 8.2 months, and the median overall survival was 19.4 months and 7.6 months, respectively. Prospective analysis of secondary cytogenetic abnormalities showed such changes in 9 patients. Three of these were at the time of relapse, 3 had spontaneous resolution of these abnormalities, and 3 had MDS that evolved to AML. Two of the latter patients had Neurofibromatosis Type-1. Asymptomatic decreases in ejection fraction were common, however only 2 patients developed clinically significant cardiac dysfunction. The efficacy of these regimens compared favorably to results reported with standard “3+7” regimens. Use of a once-daily cytarabine regimen resulted in almost no neurotoxicity and allowed for administration of consolidation in the outpatient setting.
Pharmacological Profile of DZ-697b, a Novel Anti-Platelet Agent -Selective Inhibitor of vWF- and Collagen-Induced Platelet Aggregation.
Authors: Ogihara Yoshiyasu , Muramatsu Sumie , Kaneda Yuki , Iijima Takako , Shibutani Tomoko , Shibano Toshiro
Published: October 2019

Introduction: Bleeding risk accompanied with anti-platelet drugs is an ultimate dilemma in the treatment of thrombosis patient. Under high shear condition of blood flow, vWF- and collagen-induced signaling pathways are likely to trigger the platelet adhesion to the injured endothelium, which leads to the activation of platelets and arterial thrombus formation. Thus, the recent studies suggest that the selective inhibitor of these pathways is a new target of anti-platelet drugs with lower bleeding risk. We report here a pharmacological profile of DZ-697b, which selectively inhibits platelet aggregation evoked by ristocetin and collagen in vitro and ex vivo.

Materials and methods: Human volunteers blood was processed platelet rich plasma (PRP) or washed platelets. PRP aggregation was induced by ristocetin and collagen. To reveal the selectivity, effect of DZ-697b on U46619 (TXA2 analogue), ADP, thrombin and TRAP induced aggregation in the washed platelets were examined. In guinea pigs and cynomolgus monkeys, effects of DZ-697b given orally were also examined on ex vivo PRP aggregation induced by collagen. To investigate the underlying mechanisms of DZ-697b, changes in phosphorylation of FcR γ chain, a common signaling pathway of both vWF- and collagen-induced platelet aggregation, were studied.

Results: DZ-697b potently inhibited both ristocetin- and collagen-induced human PRP aggregation, the IC50 being 0.74 μM and 0.55 μM, respectively. In contrast, DZ-697b even at 50 μM did not show any influences on U46619, ADP, thrombin and TRAP induced platelet aggregation. DZ-697b did not affect ovine COX-1 and COX-2 activities at up to 300 μM. The bioavailability of this compound was more than 80% in monkeys. Oral administration of DZ-697b at 1–3 mg/kg significantly and persistently inhibited collagen induced PRP aggregation in monkeys and guinea pigs. Application of ristocetin, vWF, and collagen significantly increased the intensity of phosphorylation of FcR γ chain in washed platelets, which were inhibited by DZ-697b.

Conclusion: DZ-697b is an orally active compound which selectively inhibits ristocetin- and collagen-induced platelet aggregation and seems to be promising as novel anti-platelet drug.
Biochemical and Pharmacologic Profiling of a Novel Dual Acting Antithrombin Agent with Antiprotease and Antiplatelet Actions. Hematologic Implications.
Authors: Fareed Jawed , Hoppensteadt D. , Haque W. , Diakur J. , Jeske W. , Florian-Kujawski M. , Maddineni J. , Baltasar F. , Neville B. , Walenga J. M.
Published: October 2019

Background: The pathogenesis of thrombosis involves both cellular and humoral processes. Most antithrombotic drugs exhibit either anti-protease or anti-platelet effects. A combination of anti-protease and anti-platelet drugs provides better efficacy in the management of thrombotic disorders. A series of synthetic low molecular weight serine protease inhibitors with varying anti-platelet effects (Medicure Inc.) are being assessed for antithrombotic properties.

Materials and Methods: This investigation reports on a compound with low antithrombin/high anti-platelet activity MC 45301 (A) and a compound with high antithrombin/low anti-platelet activity MC 45308 (B) activity in in-vitro and in-vivo settings used to profile antithrombotic drugs.

Results: A exhibited strong anti-platelet actions as measured using ADP as an agonist (IC50=1.1 g/ml), whereas B had a higher IC50 (9.4 g/ml). In the antithrombin titration assay A (>100 μg/ml) showed a relatively higher IC50 than B (45 μg/ml). In the global anticoagulant assays, A exhibited somewhat weaker effects than B. In the Xa generation assay, both compounds exhibited similar effects. However, in the thrombin generation assays B exhibited stronger effects. In whole blood assays both compounds produced anticoagulant and anti-platelet effects. Intravenous administration of these compounds to rabbits over a dose range of 50–500 g/kg produced strong dose dependent antithrombotic actions. In comparison to direct antithrombin agents such as argatroban, at a comparable dose, B produced identical antithrombotic actions, which were disproportional to the systemic anticoagulant effects. A produced modest antithrombotic actions with minimal ex vivo clotting effects. This data is highly suggestive that compounds with dual targets are able to produce stronger antithrombotic actions relative to monotherapeutic agents. Additional studies in arterial thrombosis may provide newer insights into the antithrombotic actions of compounds with dual sites of action. Moreover, these agents may be more effective in thrombotic conditions where both platelets and the coagulation system are involved.
A Thrombin Inhibitor, but Not a Factor Xa Inhibitor, DU-176b, Aggravates Tissue Factor-Induced Hypercoagulation in Rats.
Authors: Morishima Yoshiyuki , Fukuda Toshio , Tsuji Naoki , Honda Yuko , Matsumoto Chikako , Furugohri Taketoshi , Shibano Toshiro
Published: October 2019

There is rising concern about recurrent thrombotic events after cessation of anticoagulant therapies. Withdrawal of heparins and a direct thrombin inhibitor is reported to be associated with evidence of rebound coagulation phenomenon in patients with coronary artery diseases (Ref 1). Previously we have shown that low-dose administration of a direct thrombin inhibitor, melagatran, worsens consumption of platelet induced by tissue factor (TF) injection (Ref 2).

Objective: To determine whether cessation of melagatran, but not a factor Xa (FXa) inhibitor, DU-176b, aggravates TF-induced hypercoagulation in a rat in vivo model.

Methods: Under halothane anesthesia, melagatran (2 mg/kg, i.v. bolus) and DU-176b (0.3 mg/kg, i.v. bolus) were injected into the jugular vein of male Wistar rats. Five min, 2, 4, 8, and 16 hr after dosing, hypercoagulation was induced by injection of 2.8 U/kg TF into the femoral vein of rats anesthetized with thiopental. Blood samples were collected 10 min after TF injection. Platelet numbers and TAT concentrations were measured.

Results: Both melagatran and DU-176b inhibited platelet consumption and thrombin-antithrombin complex (TAT) generation when hypercoagulation was induced 5 min after the drug administration. This indicated that these compounds effectively inhibited hypercoagulation at this time point. Melagatran, however, 2 and 4 hr after dosing enhanced platelet consumption and TAT generation, indicating that the thrombin inhibitor aggravated hypercoagulation at these time points. Much later (8 and 16 hr), the effects of melagatran towards inhibition or enhancement disappeared. In contrast, DU-176b did not show any exacerbation of TF-induced hypercoagulation.

Conclusion: Cessation of a direct thrombin inhibitor, melagatran, is associated with aggravation of coagulation status. This phenomenon may be implicated in rebound coagulation activation observed in clinical settings. On the other hand, the FXa inhibitor, DU-176b, may be beneficial because of a low risk of recurrent activation of coagulation pathway after cessation.
Prasugrel (CS-747, LY640315) Hydrochloride, a Novel Thienopyridine Prodrug, Shows Potent Antiplatelet and Antithrombotic Effects with Rapid Onset of Action in Rats.
Authors: Niitsu Yoichi , Ogawa Taketoshi , Sugidachi Atsuhiro , Jakubowski Joseph A. , Yokouchi Yuki , Kakusaka Mayumi , Hasegawa Michihiro , Asai Fumitoshi
Published: October 2019

The thienopyridine antiplatelet prodrug clopidogrel bisulfate (C) is widely used for atherothrombotic diseases. However, several reports have described a limitation of C, namely, poor platelet inhibition in many patients following oral dosing. Prasugrel (hydrochloride), currently under investigation in a Phase 3 trial in acute coronary syndrome patients undergoing percutaneous coronary intervention, is a novel thienopyridine antiplatelet prodrug. In recent clinical studies, prasugrel has shown more consistent platelet inhibition than C. To date, several preclinical studies have demonstrated that the prasugrel base formulation has more potent antiplatelet and antithrombotic effects than C. The effects of prasugrel hydrochloride (P), which has a higher solubility than the base formulation, have not been reported. The present studies examined the effects of P on platelet aggregation, arterial thrombosis, and bleeding time in rats. Single oral administration of P (0.3 to 3 mg/kg) resulted in inhibition of platelet aggregation (IPA) induced by ADP in a dose- and time-related manner with an ED50 value of 1.1 mg/kg at 4 hr postdose. After dosing of 3 and 10 mg/kg P, statistically significant IPA was observed at 30 and 15 min, and the time required to achieve 50% IPA was approximately 50 and 23 min, respectively (Figure). These results indicate that P has potent antiplatelet effects with an extremely fast onset of action. Multiple P dosing also resulted in potent IPA, with an ED50 value of 0.45 mg/kg/day (p.o.) at 4 hr after the last dose. Our previous report indicated an ED50 value of C at 4 hr after single oral dosing of 16 mg/kg (Br. J. Pharmacol. 129, 1439, 2000), demonstrating about 10-times greater potency of P compared to C. Potent dose-related antithrombotic effects of P were further confirmed in an arterial thrombosis model in rats. P (0.1–1 mg/kg, p.o.) inhibited thrombus formation in the arterial thrombosis induced by extravascular application of FeCl3 solution. All these results taken together indicate that prasugrel hydrochloride is a novel, potent and fast acting thienopyridine prodrug, potentially providing superior efficacy in the treatment of atherothrombotic disorders.

Figure Figure
Contribution of Nucleic Acid Testing of Plasma Source Material to the Viral Safety of Plasma Derived Coagulation Factors: The French Experience.
Authors: Visse Catherine , Flan Benoit
Published: October 2019

Viral testing of donations and plasma pools is one of the important elements that guarantee the safety of plasma-derived coagulation factors towards the risk of transmitting pathogenic agents. In the last decade, virus testing has benefited from developments in Nucleic Acid Testing (NAT), which has permitted its implementation initially for the testing of plasma pools and more recently at the donation level. Validated NAT techniques significantly increase the safety margin of plasma-derived products by reducing the initial viral load, which could challenge the efficacy of viral inactivation or elimination during the manufacturing processes. NAT for major blood borne viruses such as HIV and HCV allows the detection of the virus during the window period where the donor may escape from serological detection and the viral load can be very high. Implementation of HCV-NAT on plasma pools in France, has allowed detection of 7 HCV RNA positive/anti-HCV negative donations amongst 8.6 million donations tested (period 1997 – 2001) corresponding to an incidence rate of 1/1 230 000 donations, correlating with the extremely low residual HCV transfusion risk in France. NAT has also been voluntarily applied to increase the safety of coagulation factors with regards to non-enveloped viruses, mostly Parvovirus B19 (B19V) and hepatitis A virus (HAV). These viruses have been transmitted through inactivated coagulation factors because they are more resistant to viral inactivation treatments (e.g. Solvent/Detergent treatment) than enveloped viruses. Very high viremia are observed in B19V infection and testing B19 DNA in manufacturing plasma pools with a threshold concentration of 104 IU/ml allowed a dramatic reduction in the initial viral load (reduction factor ~ 6 – 7 log10). After nearly 10-years experience in France, which is the longest experience reported, the mean incidence rate is 1 in 7 710 donations, with epidemics detected in 1997 and 2005. Annual incidence profiles confirm a seasonal evolution of this virus with an increased incidence in the spring period. The contribution of B19 NAT to the increased safety of coagulation factors is supported by pharmacovigilance data in France, with no reports of B19V transmission associated with a coagulation factor since NATs implementation. NAT represents a significant breakthrough in the viral testing of plasma for fractionation. Automated systems in place can readily be adapted to emerging pathogens as shown, in Northern American countries, with the rapid implementation of West Nile Virus PCR testing in the transfusion setting. NAT represents a state of the art development in the manufacture of increasingly safe plasma-derived products.
Histology of Lymphomas with t(3;14)(q27;q32) Correlates with the Molecular Anatomy of the Translocation.
Authors: Ruminy Philippe , Jardin Fabrice , Picquenot Jean-Michel , Gaulard Philippe , Parmentier Françoise , Buchonnet Gérard , Maisonneuve Claire , Tilly Hervé , Bastard Christian
Published: October 2019

The t(3;14)(q27;q32) is the most common translocation involving BCL6 in non Hodgkin B-cell lymphoma. Although this translocation has been predominantly associated with diffuse large B-cell lymphoma (DLBCL), recent studies have shown that it can also be found in follicular lymphomas (FL), where it is often associated with a large cell component. To further investigate the relationship that might exist between this translocation and the phenotype of the tumor, we studied 34 B-cell lymphomas with t(3;14)(q27;q32) and diffuse or follicular growth patterns. As the t(3;14)(q27;q32), involving telomeric ends of both chromosomes 3 and 14, is often difficult to detect by conventional cytogenetic, it should be emphasised that only t(3;14) positive FL or DLBCL where the translocation could be validated either by molecular technics (cloning of the breakpoint, amplification of specific IGH-BCL6 fusion transcripts or fluorescence in situ hybridization) were retained in this study. Noteworthy, none of the samples were found to have a t(14;18), indicating that the two most frequent translocations in B-cell NHL, both seen as early transforming events, are usually exclusive. This observation is in agreement with the hypothesis that BCL2 and BCL6 deregulations probably constitute two different lymphomagenesis pathways. Our data also indicate that most t(3;14) positive DLBCL share a CD10−/BCL6+/MUM1+ phenotype (10/16, 62%) and can be classified in the non-germinal centre subgroup (12/16, 75%)%), while a majority of t(3;14) positive FL are CD10− and BCL6+ (10/12, 83%). Moreover, we identified the IGH switch region (S) involved in the translocation for 32 cases, either by cloning the genomic breakpoint (27 cases), and/or by amplifying the IGH-BCL6 fusion transcripts expressed by the tumors (26 cases). For 17/19 (89%) DLBCL or FL with DLBCL, the translocation involved the Sμ, while for 9/13 (70%) FL with no diffuse areas of proliferation the t(3;14) involved a Sγ. These results indicate that the histology of the tumor and the structure of the translocated allele are correlated (p=0.0016), suggesting that the substituted regulatory regions that comes to deregulate BCL6 after a t(3;14) may affect the presentation of the disease.
Conditional Survival of Patients with Diffuse Large B-Cell Lymphoma.
Authors: Moller Michael B. , Pedersen Niels T. , Christensen Bjarne E.
Published: October 2019

Purpose: Prognosis of lymphoma patients is usually estimated at the time of diagnosis, and the estimates are guided by the International Prognostic Index (IPI). However, survival rates calculated at the time of diagnosis may not be predictive of outcome for patients who have survived 1 or more years. For these patients conditional survival is more informative as only those patients who have already survived a period of time after treatment are considered. Conditional survival data have not been reported for lymphoma patients and the impact of the IPI on conditional survival is not known.

Patients and Methods: Conditional survival was estimated for 1,209 patients with diffuse large B-cell lymphoma from the population-based LYFO-registry of the Danish Lymphoma Group. The patients were diagnosed between 1983 and 1998 with a median follow-up time of 7.8 years, and all patients were treated with curative intent. The Kaplan-Meier method was used to estimate conditional survival at 0–5 years after diagnosis.

Results: The probability of surviving 5 years increased with each year survived for the first 3 years after diagnosis (from 43% to 70%) after which the increase was minimal (Figure 1). The median survival increased from 38 months for all patients to between 108 and 120 months conditioned on survival for at least 3 to 5 years. The distribution of patients in the IPI risk groups changed significantly over time. However this effect was only seen in the first 5 years. At diagnosis, 28% of the patients were allocated to the high-intermediate and high-risk groups, but only 11% of patients who survived 5 years were from these risk groups (P < .0001). Risk group allocation did not change the following 5 years (P = .7747). At the time of diagnosis overall survival differed greatly between the IPI risk groups (P < .0001; figure 2). However, 5 years after diagnosis the rates were similar (P = .0586). Furthermore, the prognostic impact of the individual clinical variables of the IPI (age, stage, performance score, LDH, and extranodal disease) was also significant at diagnosis, but 2 years after diagnosis only age had prognostic impact. Multivariate analysis (including the IPI and the individual IPI variables) of patients who survived ≥3 years identified only age as a prognostic factor (relative risk 4.1; P < .001).

Conclusion: For patients with diffuse large B-cell lymphoma who have survived more than 1 year after diagnosis, the conditional survival probability provides more accurate prognostic information than the conventional survival rate estimated from the time of diagnosis. Furthermore, age is the most important factor for long-term survival, whereas the remaining IPI factors are not informative.

Figure Figure Figure Figure
Lack of BIC and microRNA miR-155 Expression in Primary Cases of Burkitt Lymphoma.
Authors: Kluiver Joost , Haralambieva Eugenia , de Jong Debora , Blokzijl Tjasso , Jacobs Susan , Kroesen Bart-Jan , Poppema Sibrand , van den Berg Anke
Published: October 2019

We previously demonstrated a high expression of primary-microRNA BIC (pri-miRNA-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, a high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral blood samples. In this study we extended our series of BL cases and cell lines for BIC expression by RNA in-situ hybridization (ISH) and quantitative (q)RT-PCR. Both BIC RNA-ISH and qRT-PCR revealed no or only low levels of BIC in 25 BL tissues, including 7 Epstein-Barr virus (EBV) positive cases, compared to HL and normal controls. In agreement with these findings, Northern blotting revealed absence of miR-155 in BL tissues. EBV negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.
CD19-Negative Diffuse Large B-Cell Lymphoma Shows High Serum LDH Level and Poor Prognosis.
Authors: Kimura Miho , Yamaguchi Motoko , Ueno Satoshi , Ogawa Shoko , Miyazaki Kana , Oka Kouji , Kobayashi Tohru , Shiku Hiroshi
Published: October 2019

CD19 is one of the most representative B-cell markers, and is widely used in diagnostic immunophenotyping in diffuse large B-cell lymphoma (DLBCL). However, it is known that the frequency of CD19 expression in DLBCL is less than that of CD20, and the clinical significance of CD19 expression has not yet been thoroughly examined. To clarify the clinical behavior of CD19-negative (CD19−) DLBCL, we have compared the clinical features, immunophenotype, and prognosis in relation to CD19 expression. The diagnosis of DLBCL was made according to the WHO Classification. We examined CD19 expression by means of immunohistochemistry using frozen sections with a monoclonal antibody, Leu12 (Becton Dickinson). Between 1987 and 2002, 227 cases of de novo DLBCL were examined the expression of CD19 in our laboratory. Anthracycline-containing chemotherapies were selected as the first-line treatment in 192 cases (85%). None was treated with rituximab. CD19 was expressed in 205 cases (90%), and 226 cases (99%) were positive for CD20. In 22 cases of CD19-negative (CD19−) DLBCL, the median age was 63 (39–79), and the male/female ratio was 11/11. According to CD19 expression, our DLBCL cases showed the following clinical features: male/female (CD19+ DLBCL 111/94, CD19− DLBCL 11/11: NS), age>60 (70%, 55%: NS), PS>1 (20%, 36%: P=0.07), sLDH>1xN (44%, 73%: P=0.01), extranodal involvement>1 site (12%, 18%: NS), stage III/IV (41%, 54%: NS), B symptom present (29%, 45%: NS). CD19− DLBCL expressed BCL2 protein less frequently than CD19+ DLBCL (P=0.03). The expression of CD5, CD10, CD21, BCL6, and MUM1 did not show a significant difference between CD19+ DLBCL and CD19− DLBCL. CD19− DLBCL showed significantly worse survival than CD19+ DLBCL (P=0.04, log-rank test). These findings suggest that the loss of CD19 expression in DLBCL is associated with high serum LDH level and poor prognosis. Simultaneous examination of CD19 and CD20 in diagnosis of DLBCL is recommended.

Overall survival for patients with CD19+ DLBCL and with CD19− DLBCL. Overall survival for patients with CD19+ DLBCL and with CD19− DLBCL.
A Mid-Treatment FDG-Positron Emission Tomography (PET) Scan Is Highly Predictive of Subsequent Treatment Failure in Patients with Diffuse Large B-Cell Lymphoma (DLBCL).
Authors: Ng Ashley P. , Lee Michael , Hogg Annette , Seymour John F. , Janusewicz Henry , Prince Miles , MacManus Michael , Hicks Rod , Wirth Andrew
Published: October 2019

AIMS Computerised Tomography (CT) scanning is the primary modality for assessment of therapeutic response in DLBCL. We evaluated whether functional imaging utilising FDG-PET may provide additional prognostic information in response assessment prior to completion of chemotherapy for patients with DLBCL.

METHODS We performed a retrospective, single-centre study of patients with DLBCL who received anthracycline-based chemotherapy +/− radiotherapy (RT) and had PET prior to completion of therapy.

RESULTS From 1996–2004 there were 45 eligible patients. Median age was 59 years (range 26–82) with disease stage I–II (n=25) or III–IV (n=20). Median IPI was 2[IPI 0–2(n=27), IPI 3–4(n=13)] in 40 evaluable patients. Therapy included full-course CHOP/CHOP-like therapy (n=26); HyperCVAD (n=6) and limited (3–4 cycles) CHOP/CHOP-like chemotherapy with RT (n=13). Rituximab was used concurrently in 18 patients. Planned RT was administered in 23 patients. 13 (29%) patients were PET positive after a median of 3 chemotherapy cycles (range 1–5). Of these, 7 (54%) progressed a median of 7.2 months following completion of therapy. Of the 6 progression-free, 4 demonstrated residual low-grade activity at sites of prior bone involvement (median follow-up 50.8 months), while 2 patients (stage I and IV disease) had limited follow-up (<8 months). Of 32 patients who were PET-negative, only 3 have progressed (median 7.7 months follow-up); 2 with stage IV (IPI 1 and 2 respectively) and 1 with stage I disease. Overall the Positive predictive value (PPV) of a positive-interim PET was 58.3% [Hazard Ratio of 6.67 (95% C.I. 2.6 – 46.3)] and Negative PV(NPV) was 90.9% (see figure 1). Excluding patients with Stage IE bone lymphoma (see Figure 2), the PPV of positive-interim PET was 88%(77–98%) and NPV was 91%(82–100%). The median number of chemotherapy cycles after which PET was performed was equivalent according to interim PET status (3 cycles) as was median baseline IPI [IPI 2(PET-positive), IPI 1.5(PET-negative); p=0.17].

CONCLUSIONS Patients with DLBCL receiving chemotherapy who have persisting PET-positivity during their therapy have a greatly increased risk of treatment failure and should be considered for treatment intensification. Bone lymphoma may represent a distinct entity with a good prognosis despite some minor persisting PET-positivity prior to completion of therapy.

Patients in remission Patients in remission Patients in Remission excluding Stage IE Bone DLBCL Patients in Remission excluding Stage IE Bone DLBCL
Allogeneic Stem Cell Transplantation with Peripheral Blood Stem Cells Mobilized by Pegylated-G-CSF.
Authors: Hill Geoff , Morris Edward S. , Fuery Maddona , Hutchins Cheryl , Butler Jason , Grigg Andrew , Roberts Andrew , Bradstock Ken , Szer Jeffrey , Kennedy Glen , Morton James , Durrant Simon
Published: October 2019

The mobilization of stem cells with pegylated-G-CSF (peg-G-CSF) modulates regulatory T cell and NKT cell function, separating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects in animal models. We have initiated a phase I/II study to analyse the feasibility of mobilizing stem cells from sibling donors with peg-G-CSF and their ability to restore hematopoiesis in HLA matched transplant recipients who have received myeloablative conditioning. Results were compared to a cohort of donors mobilized with standard G-CSF at 10ug/kg/day (n=19). The administration of 6mg of peg-G-CSF (n=6) resulted in suboptimal stem cell mobilization with a peak peripheral blood CD34+ count of 29 ± 4/uL. Apheresis 4 days after peg-G-CSF administration yielded 2.7 ± 0.3 x106 CD34+ cells/kg recipient ideal body weight and all patients required a second collection on day 5 to yield a total of 4.0 ± 0.5 x106 CD34+ cells/kg recipient weight. Following escalation of the dose to 12mg (n=9), the peak CD34+ count was 109 ± 13/uL and all donors collected sufficient stem cells for transplantation in a single apheresis (9.8 ± 1.7 x106 CD34+ cells/kg recipient weight). The 6mg dose of peg-G-CSF was significantly inferior to standard G-CSF for stem cell mobilization (P<0.01) while the 12mg dose was at least equivalent (P=0.07).

Bone pain was similar between the 6mg and 12mg cohorts and to that seen with standard G-CSF. However, in addition to the expected rises in serum ALP and LDH, transient rises in hepatic transaminases were noted 5 to 12 days after peg-G-CSF administration in 7 of 9 donors receiving the 12mg dose. One donor developed NCI grade 3 hepatic toxicity and splenomegaly. After allogeneic transplantation of peg-G-CSF mobilized grafts (Cy/TBI conditioning in 13 of 14 recipients), median neutrophil and platelet engraftment occurred on days 18 and 14 respectively and was identical to that seen with grafts mobilized by standard G-CSF. With a median follow up of 165 days (range 55–532), the incidence of grade II-IV and grade III/IV acute GVHD is 50% and 21% respectively. No patients have relapsed to date and overall survival is 86%. The mobilization of stem cells with peg-G-CSF in normal donors is feasible and 12mg appears the optimal dose. Further data are required to more closely analyse the effect of peg-G-CSF on donor liver function and the ability of stem cell grafts to separate GVHD and GVL effects.

Figure Figure