bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Production Rate Predicts the Response to Prednisone Therapy in Patients with Idiopathic Thrombocytopenic Purpura.
Authors: Houwerzijl Ewout , Louwes Henk , Sluiter Wim , Smit Jan , Vellenga Edo , de Wolf Joost
Published: October 2019

The thrombocytopenia in ITP is predominantly caused by autoantibodies that recognize antigens on platelets and megakaryocytes resulting in accelerated platelet destruction and a decreased platelet production rate (PPR). ITP patients with a predominantly suppressed PPR might respond differently to therapy than patients with predominantly peripheral platelet destruction. Tests and/or patient characteristics that can make a distinction between a reduced platelet half life and a reduced PPR could therefore influence diagnostic and therapeutic strategy in ITP. Therefore, in the present study the predictive value of clinical and platelet kinetic parameter for treatment outcome in idiopathic thrombocytopenic purpura (ITP) was investigated. Seventy-five patients with severe ITP (platelets ≤20 × 109/L) were studied. Median age was 46 (range 16–89) years and 34 (45%) patients were males. Median platelet count was 8 (1–20) × 109/L. The mean platelet life was 1 (0.1–6.5) days, and the PPR 160 (2–4670) × 109/day (normal 223 (100–355) × 109/day, p = 0.7). PPR was decreased (<100 × 109/day), normal (100–355 × 109/day) and increased (>355 × 109/day) in 33%, 48%, and 19% of the patients, respectively. All patients started with prednisone at diagnosis (1 mg/kg/day). Initial complete and partial response (CR/PR) was 84% and a durable CR/PR (defined as CR/PR for ≥6 months without treatment) was attained in 44% of the patients. Apart from a higher proportion of patients with a decreased PPR in the group that responded to prednisone therapy (p=0.03), there were no significant differences regarding clinical and platelet kinetic parameters between responders and nonresponders. A durable CR/PR was noticed in 64% of the patients with a decreased PPR (median follow-up of 81 months (18–92)), compared to 34% of the patients with normal or increased PPR (median follow-up 141 (10–284) months (HR: 0.47 [95% CI (0.24–0.92)], p = 0.03). Splenectomy was performed in 32% of the patients with decreased PPR and in 62% of patients with normal or increased PPR (p = 0.03). In addition, patients with a decreased PPR showed significantly less splenic sequestration and a significantly longer mean platelet life than patients with a normal or increased PPR, which underscores that in these patients peripheral destruction of platelets contributes relatively less to thrombocytopenia than suppressed platelet production. Thirty-nine of 42 nonresponders to prednisone underwent splenectomy. Durable CR/PR postsplenectomy was reached in 74%. There were no significant differences with regard to patient characteristics between responders and nonresponders to splenectomy. In conclusion, ITP patients with suppressed PPR have a significant higher durable CR/PR rate to prednisone therapy and are less frequently exposed to splenectomy, than those with a normal or increased PPR.
Constitutive Activation of the Canonical NF-κB Signaling Pathway and Expanded Populations of Splenic Marginal Zone B Cells Characterize Em-BCL10 Transgenic Mice.
Authors: Li Zhaoyang , Xue Liquan , Shin Dong-Mi , Qi Chen-Feng , Zhang Quangeng , Xu Wu , Morris Stephan W. , Morse Herbert , Wang Hongsheng
Published: October 2019

The BCL10 gene was isolated from the breakpoint region of the t(1;14)(p22;q32) chromosomal translocation, a recurrent chromosomal abnormality in mucosa-associated lymphoid tissue (MALT)-type lymphomas. The translocation results in constitutive over-expression and nuclear location of the BCL10 protein. To understand the physiological and pathogenic roles played by BCL10 in B-cell biology, a transgenic (TG) mouse model was developed with a human BCL10 gene driven by Em. The TG was expressed in thymic but not peripheral T cells and in the spleen; lymph nodes were negative. Although the lymphoid compartments of the TG mice were grossly normal, histologic studies showed the splenic marginal zone (MZ) to be significantly expanded and, in older mice, to compress the white pulp. In addition, about 10% of animals developed B-cell neoplasms by 18 months of age. By FACS analyses, the number of MZ B cells was increased nearly 2-fold, and both MZ and follicular (FOL) B cells exhibited a marked down-regulation of CD23 expression. In the peritoneum of TG mice, the B1a B-cell population was markedly reduced while the B1b subset was increased over 2-fold. Studies of purified MZ and FOL B cells from normal and TG mice showed that MZ B cells from TG mice had a survival advantage in culture. Studies of the canonical NF-κB signaling pathway that lies downstream from BCL10 showed that it was constitutively activated with high levels of nuclear p50 and p65 protein identified by Western blotting. Quantitative real-time RT-PCR analyses of purified splenic B cells from normal and TG mice for expression of 384 genes identified 44 with significant differences in expression. Almost half of these genes were known NF-κB targets and included genes known to influence B-cell survival (BAFF, IL-10) or associated with MZ lymphomas (S100A9). These results indicate that constitutive expression of BCL10 in B cells is not sufficient to induce transformation alone and that as-yet-unknown secondary mutations are required. Nonetheless, expression of BCL10 clearly resulted in constitutive activation of the canonical NF-κB signaling pathway and a prominent expansion of the MZ B-cell population, thereby setting the stage for later development of lymphomas.
Bendamustine Is Safe and Effective in Patients with Rituximab-Refractory, Indolent B-Cell Non-Hodgkin Lymphoma.
Authors: Kahl Brad , Bartlett Nancy L. , Leonard John P. , Ganjoo Kristen , Williams Michael E. , Czuczman Myron S. , Robinson K. Sue , Joyce Robin , van der Jagt Richard H. , Cheson Bruce D.
Published: October 2019

Background: Bendamustine (Treanda®) is a purine analog/alkylator hybrid with single-agent activity in patients with relapsed/refractory non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), multiple myeloma, and breast cancer. The objective of this phase 3 multicenter study was to evaluate the efficacy and safety of single-agent bendamustine HCl in patients with relapsed, rituximab-refractory NHL.

Methods: Eligible patients had previously treated, rituximab-refractory, indolent-histology NHL. Rituximab-refractory was defined as no response or progression within 6 months of

first dose of rituximab induction, completion of rituximab maintenance therapy or progression before the next scheduled rituximab dose, or completion of a combination of rituximab and chemotherapy.

Patients received bendamustine 120 mg/m2 intravenously over 60 minutes on days 1 and 2 every 21 days for 6 cycles (2 additional cycles at investigator discretion; max 8). Tumor response was determined by modified International Working Group Response Criteria for NHL. The primary endpoint was overall response rate (ORR) with secondary endpoints of response duration (RD) and progression-free survival (PFS).

Results: Results for the first 38 of 100 enrolled patients are presented. The median age was 60 years, 63% were male, and 63% had stage III/IV disease. Histologies included follicular (53%), CLL/small lymphocytic lymphoma (26%), and marginal zone (21%) lymphoma. Patients had received a median of 3 prior courses of treatment (range 1–10) and 2 prior rituximab-containing courses (range 1–6). Prior alkylator or purine analog-based treatment was administered to 79% and 37% of patients, respectively. Eighteen (47%) patients were considered to be chemoresistant. Four (11%) patients received prior radioimmunotherapy. The ORR in the primary analysis population was 84%, including 29% complete responses (CR), 3% CR unconfirmed, and 53% partial responses. The median RD was 9.3 months and the median PFS was 9.7 months. The primary hematologic toxicity was reversible myelosuppression; grade 3/4 hematologic side effects included leukopenia (60%), neutropenia (60%), thrombocytopenia (24%), lymphocytopenia (95%), and anemia (5%). Febrile neutropenia was not observed. Common nonhematologic adverse events (grades 1/2, 3, 4) were nausea (68%, 5%, 0%), vomiting (42%, 0%, 0%), and fatigue (42%, 13%, 3%).

Conclusions: In this multicenter study, single-agent bendamustine was well tolerated and produced a high rate (84%) of durable responses. It is the first chemotherapeutic agent to demonstrate significant clinical activity in rituximab-refractory indolent lymphoma. These results compare favorably with prior reports of radioimmunotherapy in this patient population. An updated analysis of all 100 patients will be available for the annual meeting.
Expression of the Human Concentrative Nucleotide Transporter (hCNT1) Gene Correlates with Clinical Response in Patients Affected by Waldenstrom’s Macroglobulinemia (WM), Undergoing a Combination Treatment with Cladribine (2-CdA) and Rituximab.
Authors: Laszlo Daniele , Rabascio Cristina , Andreola Giovanna , Saronni Luca , Calabrese Liliana , Rigacci Luigi , Fabbri Alberto , Pinto Antonello , Frigeri Ferdinando , Billio Atto , Pinotti Graziella , Sammassimo Simona , Martinelli Giovanni
Published: October 2019

The aim of this prospective multicenter trial was to evaluate the efficacy of the nucleoside analogue 2-CdA in combination with Rituximab and to identify possible genetic factors that could influence the clinical response of the combination. From December 2003 to November 2006, 29 pretreated/newly diagnosed pts affected by WM were enrolled in the study. Pts characteristics included: sex (M/F) 9/19, median age 64 (range 36–75 yrs), a median IgM level before treatment of 2567mg/dL; 16 pts were newly diagnosed. The combination therapy was Rituximab at standard schedule (375 mg/mq) on day 1 followed by 2-CdA 0.1 mg/kg (sc injection) for 5 consecutive days. Each cycle was administered monthly for a total of 4 cycles. Clinical responses were evaluated two months after the end of treatment, according to Response Criteria from the 3rd International Workshop on WM. Expression analysis of the genes involved in the activity of 2-CdA (deoxycytidine kinase, deoxyguanosine kinase, 5′-nucleotidase, ribonucleotide reductase 1 and 2, human equilibrative nucleotide transporter and hCNT1) was performed on bone marrow cells, at baseline in 22 pts. The therapy was well tolerated, except in three patients who discontinued Rituximab due to cardiac toxicity during the first or second infusion. The treatment was delayed in 3 pts because of haematologic toxicity (G3 neutropenia) and in 2 of them the 2-CdA dose was reduced after 2 cycles. The only non-haematological complications observed were late respiratory infections which occurred in 3 pts. At a median follow-up of 21 (13–41) months we observed 17 (59%) CR/PR, 7 (24%) MR, 1 (3%) SD and 4 (14%) PD/NR. Pharmacogenomic analysis showed a statistically significant lower expression of the hCNT1 gene in pts who failed the treatment and who achieved a MR or SD than in those who achieved a CR or a PR (p=0.014) suggesting a possible relationship between reduced hCNT1 levels and a diminished clinical activity of 2-CdA. No significant difference was detected in the expression of the other genes analyzed. Our results suggest that the combination of 2-CDA and Rituximab is safe and active in WM pts; the use of pharmacogenomic analysis might contribute to a better selection of the patients who are more likely to respond to such a combination treatment.
Persistent Monoclonality after Histological Remission in Gastric Mucosa Associated Lymphoid Tissue (MALT) Lymphoma Treated with Chemotherapy and/or Surgery: Influence of t(11;18)(q21;q21).
Authors: Salar Antonio , Santon Almudena , Garcia-Cosio Monica , Bellosillo Beatriz , Rodriguez Patricia , Cristobal Eva , Serrano Sergio , Besses Carlos , Abraira Victor , Montalban Carlos
Published: October 2019

Background: The molecular response in patients with gastric MALT lymphoma achieving histological remission after chemotherapy with or without surgery is not well established.

Patients and Methods: 19 patients with gastric MALT lymphoma achieving complete histological remission after treatment with chemotherapy and/or surgery was studied retrospectively. IgVH gene rearrangements were studied by PCR in gastric biopsies obtained at diagnosis and during follow-up. Presence of t(11;18)(q21;q21) was studied by FISH or PCR. Sequencing analysis of PCR products of patients with t(11;18)(q21;q21) was performed.

Results: t(11;18)(q21;q21) was present in 3 patients (15.7%) at diagnosis. Median follow-up was 73 months (13–240). In patients with complete histological remission, monoclonality was demonstrated in 11 out of 19 (58%) and maintained monoclonality in 8 out of 10 (80%) who had sequential studies. All 3 t(11;18)(q21;q21) positive patients had maintained monoclonality and sequencing analyses revealed the presence of the same mutated IgVH alleles in diagnostic and in follow-up samples. In contrast, in 2 cases without t(11;18)(q21;q21) the monoclonal rearrangements corresponded to unrelated alleles. Persistent monoclonality was detected after a median of 49 months (6–134) and did not condition histological relapses, either in negative or positive t(11;18)(q21;q21) patients.

Conclusions: More than half of patients with gastric MALT lymphoma achieving histological response after chemotherapy and/or surgery have long term persistent monoclonality. t(11;18)(q21;q21) determines long term persistence of the initial lymphoma population. However, the presence of maintained monoclonality did not determine relapse.
Cytosolic Phospholipase A2α (cPLA2α) Functions at the Nexus of Bidirectional Integrin Signaling in Platelets.
Authors: Prevost Nicolas , Mitsios John V. , Shimizu Takao , Shattil Sanford J.
Published: October 2019

cPLA2 catalyzes the release of arachidonic acid from the sn-2 position of platelet membrane glycerophospholipids, leading via cyclooxygenase to the production of thromboxane A2 (TXA2), a platelet agonist. One function of TXA2 and other platelet agonists is to activate the fibrinogen-binding function of integrin αIIbβ3 through a series of biochemical steps known as inside-out signaling. While some platelet agonists stimulate cPLA2 activity and TXA2 production during inside-out signaling, others appear to require fibrinogen binding to αIIbβ3 and subsequent outside-in signaling through the integrin to elicit TXA2 production. However, the precise relationship between cPLA2 and αIIbβ3 is unclear. Here we have discovered that cPLA2α, the principal cPLA2 isoform in platelets, interacts with αIIbβ3 and participates in both inside-out and outside-in integrin signaling. A pool of cPLA2α was constitutively associated with αIIbβ3 in human and murine platelets, as determined by co-immunoprecipitation with antibodies to cPLA2α or αIIbβ3. Although the mode of interaction of cPLA2α with αIIbβ3 is unknown, vimentin, an intermediate filament protein known to bind to cPLA2α and integrin β tails, co-immunoprecipitated with both cPLA2 and αIIbβ3 from platelets. Selective induction of fibrinogen binding to platelets by MnCl2 caused a detectable increase in cPLA2 activity in both whole platelet extracts and αIIbβ3 immunoprecipitates. However, this response was not observed in platelets from mice deficient in cPLA2α or in human platelets pre-treated with pyrrophenone, a selective cPLA2α inhibitor. Integrin-dependent activation of cPLA2α had functional consequences. First, thromboxane A2 produced in response to fibrinogen binding to αIIbβ3 was required for the recruitment of activated protein kinase Cβ to the αIIbβ3 signaling complex, an event known to be required for cytoskeletal reorganization and full platelet spreading on fibrinogen. Second, platelets from cPLA2α-deficient mice exhibited reduced spreading on fibrinogen compared to platelets from wild-type littermate controls (P < 0.05). Similarly, human platelets pre-treated with either pyrrophenone or a thromboxane A2 receptor antagonist spread poorly on fibrinogen compared to vehicle-treated control platelets (P < 0.05). Third, cPLA2α-deficient platelets stimulated by the GP VI collagen receptor agonist, CRP, exhibited decreased binding of soluble fibrinogen (P < 0.05). These studies establish that cPLA2α can function in association with αIIbβ3 to produce a pool of TXA2 that promotes integrin-dependent activation of protein kinase Cβ, cytoskeletal reorganization and platelet spreading. Given the previous demonstration of prolonged bleeding times and protection from thromboembolism in cPLA2α-deficient mice, these results imply that normal platelet function requires cPLA2α activation both upstream and downstream of αIIbβ3.
Development of RAD51 as Biomarker for Sensitivity to the Histone Deacetylase (HDAC) Inhibitor PCI-24781 in Lymphoma.
Authors: Balasubramanian Sriram , Adimoolam Shanthi , Sirisawad Mint , Evens Andrew M. , Gordon Leo I. , Miller Richard , Buggy Joseph J.
Published: October 2019

Histone deacetylase inhibitors are currently under investigation in the clinic for a variety of malignancies, and one was recently approved for treating cutaneous T-cell lymphoma. We demonstrate here that a novel, orally bioavailable HDAC inhibitor currently in clinical trials, PCI-24781, demonstrates very good activity against lymphoid tumor lines in vitro as well as against primary tumor cells from lymphoma patients cultured ex vivo. Further, we demonstrate the potential of predicting sensitivity to PCI-24781 in lymphoma by measuring expression levels of RAD51, a protein essential for DNA repair by homologous recombination (HR) following exposure to DNA damaging agents. We have recently shown that RAD51 is downregulated by PCI-24781 treatment in solid tumor lines, and this leads to the inhibition of homologous recombination, absence of RAD51 foci and ultimately to apoptosis. However, little is known about the role of RAD51 in lymphoma. In this work, we show that RAD51 is expressed in human lymphoma by immunohistochemical staining of tissue microarrays (TMA) containing over 90 primary tumor sections (50 follicular lymphomas and 40 DLBCL). We found that a majority of these (65%) express significant levels of RAD51. Further, we measured RAD51 protein and mRNA levels in 8 non-Hodgkins lymphoma cell lines, including those derived from B- and T-cell lymphomas, before and after a 24 hour exposure to PCI-24781 at physiologically relevant concentrations. We found that there was a strong correlation between the percentage of apoptotic cells following PCI-24781 treatment as assayed using Annexin V/propidium iodide and the decrease in the level of RAD51 expression, with each of the five cell lines with >70% apoptosis following drug treatment also showing >4 fold decrease in RAD51 mRNA. These cell lines also had the highest pretreatment levels of RAD51. Thus, both the initial level of RAD51 in tumors as well as the fold decrease after treatment are predictive for PCI-24781 activity, and will be used as biomarkers in clinical studies to identify patients most likely to respond to PCI-24781.
Tipifarnib Is Cytotoxic and a Drug Transporter Inhibiting Chemosensitizer in Hodgkin Lymphoma Tumor Cells.
Authors: Boll Boris , Lang Anna , Langendorf Peer , Hansen Hinrich P. , von Strandmann Elke P. , Engert Andreas , von Tresckow Bastian
Published: October 2019

INTRODUCTION: Patients with Hodgkin lymphoma (HL) still suffer from late toxicities and insufficient treatment of relapses. New therapies should therefore be established. Tipifarnib is a farnesyl transferase inhibitor with an excellent toxicity profile and clinical activity in hematologic malignancies. Recently, tipifarnib has also been shown to potentiate the cytotoxicity of anthracyclines in leukemia cells via the inhibition of the multidrug resistance transporter P-glycoprotein. To date, nothing is known about a functional role of drug resistance transporters or the effects of tipifarnib in HL.

METHODS: To test the anti-tumor effects of tipifarnib in HL cell lines, tipifarnib was evaluated in the XTT cytotoxicity assay and the Annexin V binding assay. The combination efficacy of tipifarnib with the anthracycline doxorubicine was monitored using the Chou and Talalay combination index method. Flow cytometry was applied to assess the effects of tipifarnib on drug transporter mediated anthracycline efflux.

RESULTS: First, tipifarnib displayed high single agent toxicity in HL cell lines with an average IC50 of < 0.1 μM. Second, the combination of tipifarnib and doxorubicine was highly synergistic at clinically relevant concentrations (1–3 μM and 0.02–0.2 μM, respectively). Third, measurement of residual doxorubicine after incubation with doxorubicine and tipifarnib indicated a strong inhibition of doxorubicine efflux by tipifarnib suggesting a mechanism for the synergy of the two drugs.

CONCLUSIONS: Since tipifarnib displays high activity in a panel of HL cell lines, the clinical evaluation of tipifarnib in relapsed/refractory patients as single agent is warranted. Interestingly, tipifarnib exhibits a dual targeting mechanism in HL cells: Potent cytotoxicity as single agent and drug transporter dependent chemosensitization leading to a strong synergy with doxorubicine. The synergistic combinations of tipifarnib with doxorubicine correspond to plasma levels of the two drugs in cancer patients. Doxorubicine is one of the most effective but also most toxic drugs in the standard HL polychemotherapies whereas tipifarnib has a very favourable toxicity profile. Consequently, tipifarnib should be evaluated as a combination drug for HL polychemotherapy to save on doxorubicine dose and to lower acute and long-term toxicities. Tipifarnib might also be examined as a chemosensitizer for the combination therapy of patients with refractory disease.
Synergy of the Purine-Scaffold HSP90 Inhibitor, PU-H71, with Doxorubicin in Non-Hodgkin’s Lymphoma Cell Lines.
Authors: Varticovski Lyuba , Wright Mollie H. , Caldas-Lopes Eloisi , Chiosis Gabriela , Robles Ana I.
Published: October 2019

For over 30 years CHOP (cyclophosphamide-doxorubicin-vincristine-prednisolone) has been the standard therapy for Diffuse Large B-cell Lymphoma (DLBCL). Although DLBCL is one of the most chemotherapy-responsive human malignancies, less than 50% of newly diagnosed patients are cured with the conventional anthracycline-based chemotherapy. Novel strategies are sought out to improve the survival of these patients. Heat Shock protein 90 (HSP90) is a molecular chaperone critical for correct conformational folding of many cellular proteins. Many of those proteins are signal-transducing regulators of cell growth, DNA damage response, and survival. Cancer cells are particularly dependent on these regulators for maintenance of the transformed phenotype. Thus, HSP90 is an attractive target for cancer therapy. HSP90 inhibitors block the ATP-binding pocket and inhibit an essential ATPase activity, altering the function of the HSP90 complex and leading to destabilization and eventual degradation of mis-folded proteins. Inhibitors of HSP90 have shown promise alone or in combination with chemotherapy or radiation in preclinical studies, and are currently undergoing PhaseI/II clinical trials. Future clinical trials on their efficacy will likely explore if any additional benefit is apparent from their combination with conventional therapies. We recently demonstrated that schedule of administration of 17DMAG following Doxorubicin (DOX) ia critical for in vitro sensitivity of DLBCL cells. We examined whether the newly developed purine-scaffold HSP90 inhibitor, PU-H71, could similarly enhance the toxicity of cells to DOX when applied 24 hours after exposure to DOX. We found that sequential addition of PU-H71 to DOX resulted in reduction of DOX IC50, from 400 nM to 75 nM. To evaluate the role of schedule of drug exposure as a determinant of the overall response, we analyzed the interaction of DOX and PU-H71 on cell survival. We applied standard criteria for synergy: the median effect/Combination Index (CI) method using an equimolar (1:1) ratio of DOX:PU-H71. Addition of PU-H71 to cells pretreated with DOX for 24 hours was consistently synergistic over the entire range of drug doses, with CI = 0.75 at Fa = 0.5. Although addition of PU-H71 prior to DOX was synergistic with CI = 0.7 at Fa = 0.5, this was only evident for higher doses. Only additive-to-antagonistic effects were observed at lower drug concentrations (Fa < 0.3). These results raise the question on whether synergy between PU-H71 and DOX will be observed at the concentrations achievable clinically. Conclusions: our observations provide a rationale for further preclinical testing and stress the need to consider schedule of exposure as a critical determinant of the overall response when designing clinical trials that combine HSP90 inhibitors with DNA damaging agents.
The Proteasome Inhibitor BSc2118 Causes Growth Inhibition, Cell Cycle Arrest and Cyclin D1 Degradation in Mantle Cell Lymphoma Cell Lines.
Authors: Jakob Christian , Sterz Jan , von Metzler Ivana , Kuckelkorn Ulrike , Braun Hannes A. , Rötzer Susanne , Kaiser Martin , Heider Ulrike , Rademacher Jessica , Kloetzel Peter M. , Sezer Orhan
Published: October 2019

The ubiquitin-proteasome complex has recently been identified as a novel therapeutic target, particularly in hematological malignancies. An increased proteasome activity has been described in certain types of malignant cells including mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has shown in vitro activity in MCL cell lines and also clinical efficacy in patients with refractory or relapsed MCL. We previously described a novel tripeptide compound, BSc2118 which inhibits all three proteolytic activities of the 20S proteasome and has anti-tumor activity in melanoma and multiple myeloma (Cancer Res2006; 66: 7598–7605). We investigated the anti-tumor effects of BSc2118 in the MCL cell lines HBL-2, JeKo-1 and Granta-519 and studied its effects on cell cycle progression and the expression of the cell cycle regulatory proteins p21, p27 and cyclin D1. Furthermore the inhibition of intracellular proteasome and NF-kappa B activity was analyzed in MCL cells. In MCL cell lines HBL-2, JeKo-1 and Granta-519, BSc2118 caused a time- and dose-dependent growth inhibition, an induction of apoptosis and a dose-dependent inhibition of intracellular proteasome activity. After 24 and 48 hours of incubation with 40 – 260 nM BSc2118 we found a time- and dose-dependent cell cycle arrest in G2/M phase in all three MCL cell lines. Furthermore we could demonstrate a dose-dependent stabilization of p21 and a degradation of cyclin D1 in western blot analysis. No significant changes could be seen in p27 expression under proteasome inhibition with BSc2118. After TNF-alpha stimulation we found a low NF-kappa B activity also in untreated MCL cells, which could be partially inhibited by preincubation with BSc2118. In this study we show the effects of the novel proteasome inhibitor BSc2118 on cell cycle and growth inhibition in MCL cells lines and furthermore demonstrate that inhibition of proteasome activity, cyclin D1 degradation and p21 stabilization are crucial mechanisms of action for this compound in MCL. Since recent trials have shown a clinical efficacy of proteasome inhibition in MCL, our preclinical data suggest to consider BSc2118 as a novel agent in drug development against MCL.
Identification of Pleiotrophin as a Novel Growth Factor for Hematopoietic Stem and Progenitor Cells.
Authors: Himburg Heather , Muramoto Garrett , Meadows Sarah , Salter Alice , Zi Ningyi , Chao Nelson J. , Chute John P.
Published: October 2019

Hematopoietic stem and progenitor cells have been demonstrated to reside in association with bone marrow sinusoidal endothelial cells in vivo but the mechanisms through which the BM vascular niche regulates hematopoiesis remains incompletely defined. In an attempt to identify candidate soluble growth factors for hematopoietic stem cells (HSCs) which are produced by endothelial cells (Ecs), we have performed comparative genome-wide expression analysis of a primary human brain-derived Ecs which we have previously demonstrated to support a 10-fold expansion of human SCID-repopulating cells (SRCs) in non-contact cultures. This analysis revealed that pleiotrophin (PTN), a heparin binding growth factor, was 32-fold overexpressed in HUBECs that supported human HSC expansion as compared to non-supportive EC lines. Pleiotrophin has anti-apoptotic, mitogenic and transforming activities, suggesting a role in tumorigenesis. No role for pleiotrophin in hematopoiesis has been described. In order to determine if PTN contributes to the demonstrated HSC-supportive activity of HUBECs, the activity of PTN was blocked by adminstration of a neutralizing anti-PTN mAb to the coculture model. Three doses of PTN neutralizing antibody (5, 25, and 50 ug/ml) were added to non-contact cocultures of HUBECs with human cord blood CD34+CD38-lin- HSCs. The anti-PTN treatment caused a decrease in the numbers of phenotypic HSCs in culture in a dose dependent manner. Relative to control HUBEC cultures, the % and expansion of CD34(+) progenitors decreased signficantly at the two higher doses of anti-PTN (p<0.05, respectively). Additionally, CD34(+)CD38(−) lin(−) progenitors were decreased by 33% at the highest dose of anti-PTN compared to control HUBEC cultures (p<0.05). Transplantation studies of anti-PTN treated versus control HUBEC-cultured progeny into NOD/SCID mice are ongoing to confirm the central role of PTN in the expansion of human HSCs in culture. In a correlative study, the addition of recombinant PTN to cultures of human HSCs with thrombopoietin, SCF and flt-3 ligand was shown to significantly increase CD34+ cell content and CD34+CD38- cell content compared to cytokines alone, again in a dose-dependent manner. Taken together these results suggest that PTN, a growth factor with no previously ascribed hematopoietic activity, is a soluble growth factor for human hematopoietic progenitor cells. Since blockade of PTN activity significantly inhibits the soluble activity of HUBECs to support human HSC proliferation and expansion, PTN is also a candidate growth factor for further study in the context of understanding the function of the BM vascular niche.
Low-Molecular-Weight Heparin Reduces the Cost of Venous Thromboembolism Treatment Compared with Unfractionated Heparin in US Hospitals.
Authors: Merli Geno J. , Ferrufino Cheryl P. , Lin Jay , Hussein Mohamed , Battleman David
Published: October 2019

Background: The current healthcare and economic burden of venous thromboembolism (VTE) in US hospitals is significant. In patients with confirmed VTE, evidence-based guidelines recommend treatment for a minimum of 5 days with either low-molecular-weight heparin (LMWH) or unfractionated heparin (UFH). However, the relative total hospital direct medical costs and VTE-related readmission rates of these regimens for VTE treatment in large real-world populations are, as yet, unresolved.

Methods: This retrospective cohort analysis of discharge and billing records from the Premier Perspective™ database included discharges of patients ≥18 years old and with a primary diagnosis of VTE from January 2003 through June 2005. Discharges with prior VTE during the 12-month period prior to the index hospitalization or a pre-existing contraindication to anticoagulant therapy were excluded from the analyses. Total hospital direct medical costs associated with VTE treatment (including drug costs, hospital costs, and professional costs) were collected and compared for UFH and LMWH. Furthermore, VTE-related readmission rates at days 30 and 90 post-discharge were compared for each of these agents. Total direct medical costs (US $) were compared using generalized linear models (SAS 9.1 PROC GENMOD), adjusting for patient and hospital characteristics. Logistic regression was used to compare the likelihood of readmission within 30 and 90 days.

Results: A total of 38,664 discharges surveyed met the inclusion criteria, 20,577 (53%) receiving LMWH and 18,087 (47%) receiving UFH. The two groups were broadly similar in clinical and demographic characteristics, although mean length of stay was 1.1 days longer in the UFH group (5.7 days [SD=2.9] vs. 4.6 days [SD=2.9] for LMWH, P<0.001). After adjustment for covariates, the mean total direct hospital costs were $3,618 for UFH and $3,068 for LMWH (difference $550, P<0.0001). LMWH was associated with reduced cost in most categories, although anticoagulation therapy costs were higher for LMWH ($242 versus $41 for UFH, P<0.0001). LMWH was associated with lower rates of VTE-related readmission at both 30 days (11.2% vs 12.1%; odds ratio [OR] 0.89, 95% confidence interval [CI] 0.84–0.96; P=0.001) and 90 days (13.1% vs 13.8%; OR 0.91, 95% CI 0.85–0.96; P<0.001).

Conclusion: In a large, real-world population of patients from across the United States, LMWH is associated with reduced total direct medical costs for the acute treatment of VTE when compared to UFH. This reduction occurs despite higher drug-related costs for LMWH. In addition, patients receiving LMWH are less likely to be readmitted to hospital within 90 days with recurrent VTE.
The N-Glycans Modulate the Binding of ADAMTS13 with von Willebrand Factor.
Authors: Zhou Wenhua , Tsai Han-Mou
Published: October 2019

ADAMTS13, a circulating multidomain protease consisting a metalloprotease (MP), a disintegrin, a thrombospondin type 1 repeat [TSR], a cysteine rich region, a spacer, 7 additional TSR and 2 CUB domains, is critical for maintaining the homeostasis of von Willebrand factor (VWF). Severe deficiency of the enzyme can lead to VWF-platelet thrombosis in the microcirculation characteristic of thrombotic thrombocytopenic purpura (TTP). Sequence analysis predicts that ADAMTS13 contains 2 and 6 likely N-glycosylation sites respectively at or near the MP and spacer domains, and 2 possible sites in the CUB domains. To investigate the N-glycosylation status and the role of N-glycans in the function of ADAMTS13, we digested plasma derived ADAMTS13 and recombinant ADAMTS13 or its truncated variants with peptide-N-glycosidase F (PNGase F) or endoglycosidase H. SDS PAGE analysis showed that digestion with PNGase F, but not with endo H, decreased the molecular weights of plasma derived ADAMTS13 as well as recombinant ADAMTS13 proteins, including AD7 (residues 1-1427, full-length), AD5 (residues 1-745, MP-TSR#1), AD2 (residues 1-448, MP-TSR#1), AD8 (residues 556-748, spacer-TSR#2) and AD13 (residues 1016-1427, TSR#8-CUB#2), indicating that the MP, spacer and CUB domains were all N-glycosylated as predicted, and contained complex type carbohydrate chains. Enzyme linked immunoassay showed that removal of the N-glycans did not affect the binding of proteases to immobilized VWF multimers or VWF73 peptide. Removal of the N-glycans modestly affected the protease-TTP IgG binding, increasing the Kd by 190% for AD7 and by 50% for AD5. Expression experiments in Lec1 mutant Chinese hamster ovary (CHO) cells with defective GlcNAc-TI activity due to mutations in the Mgat1 gene showed that the resulting high-mannose type AD7 and AD5 variant proteases were secreted normally, showed no difference in VWF cleaving and TTP IgG binding activities, but exhibited increased binding to VWF multimers or VWF73 peptide. In summary, ADAMTS13 are N-glycosylated with complex type oligosaccharides at the MP, spacer and CUB domains. Intracellular conversion from high mannose to complex type glycans modulates the binding of the protease to VWF, suggesting that this process may help prevent depletion of the protease under conditions of high VWF levels in the circulation.
A Phase I/II Trial on Melphalan, Prednisone, Thalidomide and Defribotide Combination in Relapsed/Refractory Multiple Myeloma Patients.
Authors: Palumbo Antonio , Larocca Alessandra , Rus Cecilia , Gay Francesca , Rossi Davide , Pregno Patrizia , Falco Patrizia , Avonto Ilaria , Magarotto Valeria , Cavallo Federica , Iacobelli Massimo , Gaidano Gianluca , Mitsiades Constantine , Richardson Paul G. , Anderson Kenneth C. , Boccadoro Mario
Published: October 2019

Defibrotide (DF) showed antithrombotic properties and remarkable activity in Multiple Myeloma (MM), without significant anticoagulant effects and bleeding risk. DF may abrogate tumor cells interaction with marrow stromal cells and enhance sensitivity to chemotherapy, thus improving activities of Melphalan, Prednisone and Thalidomide, while protecting against thrombotic state. We designed a multicenter phase I/II trial to define efficacy and safety of Melphalan, Prednisone, Thalidomide and DF (MPTD) in relapsed/refractory MM. The MPTD treatment consisted of 6 35-days cycles of oral melphalan (0.25 mg/Kg day 1–4), prednisone (1.5 mg/kg day 1–4), thalidomide (50–100 mg/day continuously), DF at 3 dose levels (17 mg/Kg iv or 2.4 g po D 1–4, 1.6 g po D 5–35; 34 mg/Kg iv or 4.8 g po D 1–4, 3.2 g po D 5–35; 51 mg/Kg iv or 7.2 g po D 1–4, 4.8 g po D 5–35), every 35 days, without deep vein thrombosis (DVT) prophylaxis. Safety was assessed by defining dose-limiting toxicity (DLT) and maximum tolerated dose (MTD). DLT was defined as the occurrence of febrile neutropenia, G4 neutropenia >1 week, any other G4 hematologic toxicity, or any >G3 non-hematologic toxicity. MTD was the dose level prior to that resulting in DLT. Efficacy was evaluated according to EBMT/IBMTR criteria. Twenty-four patients were enrolled between March and November 2006 and 19 patients completed at least 1 MPTD (median age 69, excluding primary refractory and/or patients receiving anticoagulation) and were evaluated for toxicity and response. Fourty-two percent of patients achieved at least partial response (PR) after a median of 3 cycles (including 16% very good PR), without significant differences among DF dose. The MTD was not reached. DLTs observed were not considered related to DF and included: G3 ileus (1st dose level) and acute myocardial infarction (AMI) in the 2nd. Toxicities ≥G3 consisted of neutropenia 47%, thrombocytopenia 10%, anemia 21%, whereas <5% of patients experienced non-hematological toxicities ≥G3. No DVTs or significant bleeding were detected. Treatment discontinuation occurred in 3 patients for adverse events: AMI (additional anticoagulation required), ileus (because of the diagnosis of amyloidosis AL and disease progression), and persistent G4 neutropenia (heavily pre-treated patients). In this phase I/II study we confirm the efficacy and feasibility of MPTD in the setting of advanced myeloma patients, interestingly a protective role of DF against thrombosis is also suggested. Pharmacokinetic studies and analysis of surrogates are ongoing. Updated data will be presented at the meeting.
Bortezomib in Combination with Bendamustine and Prednisone in the Treatment of Patients with Refractory/Relapsed Multiple Myeloma.
Authors: Poenisch Wolfram , Bourgeois Malvina , Wang Song-Yau , Heyn Simone , Jaekel Nadja , Braunert Leanthe , Rohrberg Robert , Hurtz Hans Juergen , Hoffmann Franz Albert , Schwarzer Andreas , Becker Cornelia , Al Ali Haifa , Niederwieser Dietger W.
Published: October 2019

Introduction: Bortezomib is a novel proteasome inhibitor that has shown important clinical efficacy either as a single agent or in combination with other cytostatic agents in relapsed/refractory multiple myeloma (MM). The combinated treatment of bortezomib with bendamustine and prednisone (BPV) was assessed to determine the efficacy and toxicity of this regiment in patients with advanced MM.

Methods: Between January 2005 and July 2007, 46 patients (median age 63; range 31–77 years) with relapsed or refractory MM (29 patients stage III a, 17 patients III b) were treated with bendamustine 60 (−80) mg/qm on day 1 and 2, bortezomib 1,3 mg/qm on day 1, 4, 8 and 11, and prednisone 100 mg on day 1, 2, 4, 8 and 11. Cycles were repeated every 21 days until maximum response or progressive disease. The time from first diagnosis ranged from 1 to 183 (median 36) months. The duration of the last remission before beginning the BPV-therapy was 6 (range 0–36) months. Previous therapy lines (median 2, range 1–6) included 18 × thalidomide, 10 × autologous PBSCT, and 9 × autologous/allogeneic PBSCT. 16 patients were refractory to the last treatment. 22 patients had preexistent severe thrombocytopenia, leukocytopenia or anemia (WHO grade 3 or 4). Response was assessed using EBMT criteria modified to include near complete remission (nCR) and very good partial remission (VGPR).

Results: 36 patients (78%) responded after at least one cycle of chemotherapy with 2 CR, 5 nCR, 6 VGPR, 15 PR and 8 MR. 4 patients had stable disease and 6 patients had a progress. With a median follow up of 13 months, EFS, and OS at twelve months for patients without severe haematological toxicities due to previous treatments (n=24) were 46% and 79%, respectively. Outcome for these patients was significantly better compared to patients with severe haematological toxicities (grade 3 or 4, n=22) where EFS, and OS were 10% and 22%, respectively (p<0,01). The median number of the BPV-treatment was 2 (1–7) cycles. 20 of 36 responding patients showed a rapid decrease of the myeloma protein and reached the best response after the first cycle and 12 after the second cycle. The regimen was well-tolerated with few significant side effects reported. New cytopenias occured infrequently (four patients had a thrombocytopenia grade 3, and two patients had a grade 4 thrombocytopenia). 1 patient had a moderate new polyneuropathy (grade 2).

Summary: These results indicate that the combination of bortezomib, bendamustine and prednisone is effective and well tolerated in a heavily pretreated population of patients with relapsed or refractory MM.
Coagulation-Related Effect of Bortezomib Treatment in Patients with Relapsed or Refractory Multiple Myeloma.
Authors: Zangari Maurizio , Guerrero Jose , Cavallo Federica , Burns Michael J. , Prasad Keshava , Adcock Dot , Marlar Richard , Tricot Guido , Fink Louis
Published: October 2019

Proteasomes inhibitors have been recently identified as potential agents, which can influence processes leading to thrombosis. We have previously reported a reduced incidence of deep vein thrombosis (DVT) in 69 patients enrolled in Total Therapy III and treated with (B), dexamethasone, thalidomide, cisplatin and doxorubicin with prophylactic anticoagulation. Patients with relapse MM, not previously exposed to B with normal baseline coagulation parameters (PT, PTT fibrinogen and platelet count) were eligible for the study. Except for the two PCR-based genotyping assays Factor V Leiden and Prothrombin Gene, which were assessed only at enrollment, in table one are listed coagulation factors serially assessed at baseline and within 1 hour after the first dose of B on day 1 and 4 of the first treatment cycle. Platelet Aggregation, with epinephrine, collagen, arachidonic acid, ADP and P- selectin by flow cytometry, were obtained at same time points.

Results: Median values of coagulation factors pre and post B infusion are shown in table 1. Platelet count on platelet rich plasma (PRP) decreased after each B treatment (204.1±69.2 × 103 platelets/μL versus 160.8±50.3 × 103 platelets/μL). Platelet aggregation in PRP induced by 10 μM ADP was reduced by 20% and 29% in post-B on day one and four with p-values of 0.033 and 0.009 rispectively. Ristocetin induced platelet agglutination and epinephrine-induced aggregation were both negatively affected by B administration, p=0.0077 and p=0.034. Platelet aggregations elicited by collagen and arachidonic were not affected by B P-selectin expression on platelet surface was overall decrease with each agonist after B administration however no statistical differences were observed. In conclusion this pilot study has shown that even a short in vivo B exposure can significantly impaired platelet number and function and could explain clinical observations.

Table 1 Test Day 1 Pre-dose Median Day 1 Post-dose Median Day 4 Pre-dose Median Day 4 Post-dose Median PT (sec) 13.4 13.85 13.55 14.4 PTT (sec) 17.35 26.2 26.95 25.5 Fibrinogen (mg/dL) 385 360 386 341.5 Protein C (70%–160%) 114 102 120 115 Protein S (65–140%) 89 85 92 92.5 Antithrombin II (190–127%) 98 97.5 100.5 102 APC Ration 2.345 2.33 2.3 2.22 T.T. (<20s) 18.2 17.9 17.7 19.45 D-Dimer (ug/mL) 0.56 0.5 0.5 0.59 Homocysteine (umol/L) 8.77 8.99 8.69 8.13
Ex Vivo Expansion of Cord Blood NK Cell Have In Vivo Efficacy Against Leukemia.
Authors: Xing Dongxia , Fang Wendy , Decker William K. , Li Sufang , Robinson Simon N. , Yang Hong , Steiner David , Thomas Michael , Champlin Richard E. , McMannis John D. , Shpall Elizabeth J. , Zweidler-Mckay Patrick A.
Published: October 2019

Introduction Allogeneic stem cell transplantation has demonstrated the ability to prevent leukemic relapse via an immune-mediated graft-vs.-leukemia effect. Natural killer (NK) cells have been shown to comprise a significant component of this anti-leukemia effect and have been reported to enhance engraftment and reduce graft-vs.-host disease. Ex-vivo expansion of peripheral blood-derived NK cells has been demonstrated. Cord blood (CB) is a promising alternate source of NK cells with enhanced cytokine/antigen-responsiveness, proliferation and increasing availability. We studied expansion conditions for the generation of CB NK cells for clinical use, and evaluated them in vivo in NOD-SCID/IL2Rgnull mice engrafted with human leukemia.

Methods Two expansion protocols were compared:

CD56+ NK cells were isolated and cultured with irradiated CD56− feeder cells in RPMI-1640 supplemented with 100ng/mL IL-2 for 21 days. CB mononuclear cells were depleted of CD3+ cells prior to culture.

In vivo NK cytolytic activity was measured via chromium release assays on leukemia cell lines (K562, Nalm6) and fresh patient leukemia blasts. Functionality was confirmed in vivo as well by injecting 106 K562 cells or 2.5 ×104 Nalm6 cells into NOD-SCID/IL2Rgnull mice. 2 ×106 CB-expanded NK cells were administered on day +1, and an additional 5×106 CB NK cells were administered on day +7. NK killing of leukemic targets was then confirmed by flow cytometry.

Results Feeder cells mediated >30-fold expansion of CB NK cells, generating a nearly pure population that contained 97% CD3−CD56+ cells. Expanded CB NK could lyse >90% of K562 targets at an E:T ratio of 10:1, as well as >40% of patient leukemia blast. In CD3− depleted cultures, similar expansions were observed, however only 55% of these cells were CD3−CD56+ with the remainder being 10% CD3+CD56+ NK/T and 35% CD3+ T lymphocytes. In the NOD-SCID~/IL2Rgnull model, ex vivo expanded CB NK cells demonstrated the ability to reduce the leukemia burden of both AML (K562) and ALL (Nalm6) cells >50%.

Conclusions We have demonstrated the feasibility of expanding CB NK ex vivo to clinically-relevant doses with minimal manipulation. These expanded CB NK cells demonstrated cytotoxic activity in vitro and in vivo against a variety of human leukemic cell lines and patient leukemia blasts. These results provide rationale for immunotherapy of leukemia with CB-derived NK cells.