bluuebunny/crossref_metadata_2025 huggingface dataset:
A Randomized Study of Daily Versus Weekly Administration of 2-Chlorodeoxyadenosine in Patients with Hairy Cell Leukemia.
Authors: Zenhausern Reinhard , Leupin Nicolas , Schmitz Shu-Fang Hsu , Solenthaler Max , Tichelli Andre , Heim Dominik , Hess Urs , Leoncini Leda , Bargetzi Mario , Tobler Andreas
Published: October 2019

Background: In hairy cell leukemia (HCL), one cycle of 2-chlorodeoxyadenosine (CDA) is considered a standard treatment. The treatment often results in a high frequency of severe neutropenia and neutropenic fever. Weekly administration of CDA has been reported as effective as daily administration, but with a reduction of febrile episodes and infectious complications. This trial compares the two regimens for efficacy and toxicity.

Design and Methods: HCL patients (pts) were randomized to receive either the standard schedule CDA 0.14 mg/kg/day as a s.c. bolus injection on days 1–5 (Arm A) or the experimental schedule of one weekly dose of CDA 0.14 kg/day as a s.c. bolus injection on five consecutive weeks (Arm B). The primary endpoint was the average of 6 weekly white blood cell count after randomization, analyzed using ANCONA to adjust for baseline count. 100 pts were required to detect a difference between 0.8 G/L and 1.2 G/L with 5% type I errror and 80% power. Secondary endpoints were response rates, other acute hematotoxicity, acute infection rate and fever within 10 weeks, remission duration (RD), event-free survival (EFS) and overall survival. Clinical and laboratory examinations were repeated weekly during the first 6 weeks, then biweekly up to week 10, every 3 months up to 2 years and every 6 months thereafter. Response was assessed at week 10, then every 6 months up to 2 years, and every year thereafter.

Results: 50 pts were enrolled to each arm. There was no significant difference between treatment arms (A vs. B) in the primary endpoint (2.2 G/L vs. 3.0 G/L), the rate of grade 3/4 leucocytopenia (94% vs. 84%) or neutropenia (90% vs. 80%), acute infection of any grade (44% vs. 40%), fever (44% vs. 38%), hospitalization (38% vs. 34%), and blood transfusion (22% vs. 30%). Response rate (CR + PR) at week 10 was 78% with 95% confidence interval (CI) (64%, 88%) vs. 68% with CI (54%, 80%) (1-sided p-value 0.13). Over all follow-up visits the best response rate was improved to 86% in both arms. The median follow-up time was 50.2 months vs. 42.3 months. The median values for RD and EFS have not been reached, and the between-arm differences were not significant. One pt in each arm died during treatment and one additional pt in arm A died during follow-up.

Conclusions: Weekly application of CDA fails to show any advantage in toxicity or efficacy compared to standard approach.
Pattern of Relapse in Localized Gastric Lymphoma Registered within the Prospective German Multicenter Study GIT NHL 01/92.
Authors: Liersch Ruediger , Berdel Wolfgang E. , Willich Normann A. , Reinartz Gabriele , Tiemann Markus , Koch Peter
Published: October 2019

Introduction: In 2001 (Koch et al.,JCO) we published the treatment results of 185 patients (pts) with localized primary gastric lymphoma (PGL) registered within the GIT NHL 01/92 German multicenter study. After a median time of observation of 130 months we present clinical and pathological features of relapsed patients.

Methods: We compared treatment results in PGL after combined surgery and conservative (S/CM) versus conservative management (CM). Radiotherapy (Rx) and chemotherapy were stratified according to histological grading, stage, and the inclusion or omission of surgery as follows: PGL pts with indolent lymphomas in stage I received an extended field (EF) Rx with total abdominal irradiation and in stage II additionally a T-field preceded by 6 cycles of COP. The dose was 30 Gy for EF followed by a boost of 10 Gy on sites of tumor. All pts with aggressive PGL were treated with four (stage I) or six (stage II) cycles of CHOP-14 followed by EF Rx (stage I) or involved field Rx (stage II), dosage of Rx corresponded to Rx for indolent NHL.

Results: Between the years 1992 and 1996, a total of 185 pts were registered. Recurrence of NHL occurred in 17 cases during follow-up. Analysis revealed no specific features of relapsed pts with respect to initial symptoms, clinical features (except stage II2 - more than regional lymph nodes involved) and initial treatment. DLBCL was the most common histological subtype seen in relapsed pts (58.8%). Indolent lymphoma relapsed in 41.2% pts. Interestingly 70% of the DLBCL initially had an additional small-cell component, and 56% relapsed as marginal zone B-cell lymphoma, particularly at primary site independent of the initial treatment.

Conclusion: In this non-randomized study there was no disadvantage for a stomach preserving approach in the context of tumor recurrence. Patients with distant nodal involvement (Stage II2) and DLBCL relapsed more often than pts. with localized stages (Stage I/II1) and indolent lymphomas. Interestingly, aggressive lymphomas, with an initial small-cell component, predominantly relapsed as marginal zone B-cell lymphomas at primary site, while indolent lymphomas relapsed also generalized.
Immunosuppression (IST) Can Be Safely Ceased during Chemotherapy for PTLD in Renal Transplant Patients.
Authors: Mollee Peter , Hourigan Matthew , Johnson David , Jones Mark , Isbel Nikki , Hawley Carmel , Marlton Paula , Gandhi Maher , Campbell Scott , Gill Devinder
Published: October 2019

Aim. The optimal management of IST in renal transplant patients with PTLD is uncertain. As chemotherapy regimens used for PTLD are in themselves immunosuppressive, IST may not be required during this phase of treatment. Subsequent long-term reduction in IST is important to prevent relapse. We examined whether a protocol (instituted in 1994) of ceasing IST during chemotherapy for PTLD and recommencing IST at reduced doses after chemotherapy (calcineurin inhibitor at 50%, prednisolone <6mg daily and no third agent) was associated with deleterious effects on renal graft function.

Methods. We performed a retrospective audit of adult renal transplant patients with PTLD requiring chemotherapy who were managed according to this protocol. Outcomes were compared (matched 1 case to 2 controls) to renal transplant patients without PTLD matched for age, gender, diabetes, graft type (cadaveric or live donor) and year of transplant. Matching was performed blinded to outcome.

Results. 20 cases were identified: median age at PTLD diagnosis 39yrs; 85% male; 17 late onset PTLD; median time to onset of PTLD post-transplant 110 months (range, 5–276). 18 patients received prior triple combination IST (cyclosporine/tacrolimus plus azathioprine/mycophenolate mofetil plus prednisone). All patients received anthracycline-based chemotherapy with additional therapies of rituximab in 6 and radiation therapy in 4. 85% attained complete remission. 4 patients have relapsed. The 5yr OS was 75% with a median follow-up of 67 months from PTLD diagnosis. 6 cases (30%) had died (5 due to PTLD, 1 unknown) compared to 1 control (2.5%). Of the surviving 14 cases, 2 patients had failed renal allografts at 14 and 19yrs post-transplant and recommenced haemodialysis compared to 18 controls; neither case was retransplanted compared to 8 controls. 2 cases had >25% increment in serum creatinine but not requiring dialysis (assumed secondary to chronic allograft nephropathy) compared to 4 controls. 10 cases had normal functional allografts with no significant decrement in renal function. The cumulative rate of renal allograft failure requiring change of treatment to dialysis at 22yrs post-transplant was 34% vs 63% for cases and controls, respectively (p<0.05).

Figure Figure

Conclusions. IST can be safely ceased during chemotherapy for PTLD in renal transplant patients and recommenced at reduced doses after chemotherapy, without deleterious effects on renal graft function.
GS-9219 - A Novel Prodrug of the Acyclic Nucleotide PMEG Demonstrates Significant Therapeutic Activity Against Spontaneous Non-Hodgkin’s Lymphoma as Modeled in Pet Dogs.
Authors: Vail David M. , Thamm Douglas H. , Tumas Daniel B. , Reiser Hans , Ray Adrian S. , Wolfgang Grushenka H.I. , Babusis Darius , Kurzman Ilene D. , Jeraj Robert , Plaza Susan , Anderson Christie , Wessel Mackenzie A. , Robat Cecilia , Lawrence Jessica
Published: October 2019

Non-Hodgkin’s lymphoma (NHL) is the fifth leading cause of cancer deaths and the second fastest growing form of cancer in the United States. While clinical remission can be induced using currently available antineoplastic agents, drug resistant relapse is common. Canine NHL has proven to be a relevant model for preclinical testing of novel therapeutics prior to human trials. GS-9219, a novel double prodrug of the anti-proliferative nucleotide analog 9-(2-phosphonylmethoxyethyl) guanine (PMEG), has been shown to have potent cytotoxic activity in lymphoblasts and leukemia cell lines and was designed to load and functionally target malignant lymphoid cell populations upon intravenous administration. To generate proof-of-concept, activity and safety data in lymphoid malignancies, a dose-finding and activity trial with GS-9219 monotherapy was initiated in pet dogs (N = 38) with naturally occurring chemotherapy naïve or refractory NHL. Dose cohorts of 3 to 6 dogs were treated using several schedules including daily treatments for 5 consecutive days out of every 21 days, once weekly, once biweekly and once every 3 weeks. Adverse events, when observed, were manageable and reversible, dose- and regimen-dependent and included predominantly Grade 1 to 2 transient neutropenia, gastrointestinal signs, lethargy, dermatopathy and proteinuria. Major antitumor responses were noted in 79% of dogs treated with GS-9219 monotherapy (61% complete and 18% partial responses) as measured by tumor size (RECIST criteria). Responses were similar across the different treatment regimens evaluated. Importantly, responses were noted in both previously untreated dogs (N=16) and dogs with chemotherapy-refractory NHL (N=22), implying a role both in induction therapy and in the rescue of drug resistant disease. B and T cell phenotypes were both found to respond to GS-9219 therapy; however, first remission durations were significantly longer in dogs with B cell NHL. The overall median first remission duration of 4.5 months observed compares favorably to other monotherapies reported in dogs with NHL. Furthermore, in dogs retreated with GS-9219 after recrudescence of their NHL, 78% achieved a second remission. A subset of nine dogs were further evaluated with 18F-fluorothymidine (FLT) PET/CT imaging prior to and after their first treatment with GS-9219; 8 of 9 displayed initially high FLT uptake (mean standardized uptake values [SUV] of 81.55) prior to treatment which decreased significantly after GS-9219 monotherapy (SUV of 13.39; p < 0.05). Given these very promising results in this relevant preclinical model, a Phase I study of GS-9219 in humans with NHL has been initiated.
Marked Therapeutic Efficacy of a Novel Polyethyleneglycol-SN38 Conjugate, EZN-2208, in Xenograft Models of Non-Hodgkin’s Lymphoma.
Authors: Sapra Puja , Mehlig Mary , Malaby Jennifer , Kraft Patricia , Longley Clifford , Zhao Hong , Greenberger Lee M. , Horak Ivan D.
Published: October 2019

Background: The clinical utility of SN38 (10-hydroxy-7-ethyl-camptothecin), the active moiety of CPT-11 (Camptosar®), has been severely compromised due to its poor solubility. EZN-2208 is a novel, water-soluble, polyethyleneglycol (PEG)-SN38 conjugate generated by linking SN38 with a multi-arm high molecular weight PEG, 40k 4-arm-PEG, via a glycine linker. Here, we compare the efficacy of EZN-2208 and CPT-11 in the treatment of animals bearing non-Hodgkin’s lymphomas.

Methods: The in vitro cytotoxicity of EZN-2208 and CPT-11 were tested in human lymphoma (Raji, Daudi, DoHH2) cancer cell lines using the tetrazolium assay. The pharmacokinetics and maximum tolerated dose (MTD) of EZN-2208 and CPT-11 were evaluated in tumor-free severe combined immunodeficient (SCID) mice and the therapeutic efficacy was evaluated in xenograft models of non-Hodgkin’s lymphoma (Raji and Daudi).

Results: In vitro, the IC50 of EZN-2208 ranged from 2–20nM and the in vitro potency of EZN-2208 was 30 to 50-fold more than CPT-11. The MTDs of EZN-2208 and CPT-11 in SCID mice were 30 and 60 mg/kg, respectively, when injected as a single dose. When administered in a multiple dose regimen (q2d x 5), the MTDs of EZN-2208 and CPT-11 were 10- and 40 mg/kg respectively. The pharmacokinetic profile of EZN-2208 was biphasic showing a rapid distribution phase (t1/2a =0.6h) and a slow elimination phase (t1/2b =19.3h for conjugate and 14.2h for SN38). In the Raji xenograft model, treatment with a single MTD of EZN-2208 resulted in a 500% improvement in life span (ILS) and 50% cures of animals compared with 19% ILS observed for mice treated with a single MTD of CPT-11. Multiple dose treatment of EZN-2208 resulted in 90% cures of animals compared with 63% ILS and no cures observed for the CPT-11 group. In the Daudi xenograft model, a single injection of EZN-2208 given as early treatment resulted in cures of 100% of animals in contrast to 66% ILS and no cures observed for CPT-11 group. When treatment was delayed, a single MTD of EZN-2208 caused 90% cures of animals, whereas CPT-11 treatment was completely ineffective.

Conclusions: EZN-2208 demonstrated excellent efficacy in preclinical models of non-Hodgkin’s lymphoma. Ongoing Phase I studies will determine the optimal dose and schedule of EZN-2208.
Notch Signaling Pathway in Hematopoietic Stem Cells Is Activated through Interactive Communication with Mesenchymal Stem Cells.
Authors: Kikuchi Yuji , Kume Akihiro , Urabe Masashi , Mizukami Hiroaki , Suzuki Takahiro , Ozaki Katsutoshi , Nagai Tadashi , Ozawa Keiya
Published: October 2019

Mesenchymal stem cells (MSCs), which are key elements of hematopoietic microenvironment in bone marrow, are known to play a critical role in supporting hematopoiesis. A variety of hematopoietic growth factors are produced from MSCs, and cell-to-cell contact is also believed to be crucial in the interaction between hematopoietic stem cells (HSCs) and MSCs. However, the molecular mechanisms of hematopoiesis-supporting ability of MSCs are still unclear. In particular, there is little information regarding the effects of HSCs on MSC function. In the present study, we investigated the cellular and molecular events in the interactive communication between HSCs and MSCs using a differentiation-inducible MSC model; i.e. parent C3H10T1/2 cells and 10T1/2-derived cell lines, A54 preadipocytes and M1601 myoblasts. These cells were co-cultured with murine HSCs (Lin-Sca1+) isolated from bone marrow. There was 9-fold increase in the number of hematopoietic progenitors after co-culture with A54 preadipocytes, whereas there was no increase when co-cultured with parent 10T1/2 or M1601 cells. More intriguingly, cobblestone areas were observed only when HSCs were co-cultured with A54 cells. Quantitative RT-PCR showed that A54 cells express significantly higher levels of SCF, SDF-1, and angiopoietin-1 (Ang-1) compared with parent 10T1/2 cells and M1601 cells, although these cytokines were not up-regulated when co-cultured with HSCs. To search for the genes involved in the interaction between HSCs and MSCs, we compared gene expression profiles before and after the co-culture by using a microarray analysis. Among the candidate genes with up-regulation after the co-culture, we paid attention to the Notch system, because Notch ligands are considered to play an important role in nurturing HSCs within the hematopoietic microenvironment. As a result, the expression of Notch ligands, Jagged1 and Dll3, increased in A54 cells after the coculture with HSCs. On the other hand, the expression of Notch1 and Hes-1 also increased in HSCs upon co-culture with A54 cells. These data were confirmed by quantitative RT-PCR. Moreover, when HSCs were co-cultured with A54 cells without cell-to-cell contact using Transwell permeable supports, there was neither increase in the number of progenitors in the upper wells, nor the up-regulation of Notch ligands in A54 cells in the lower wells. These findings support the idea that HSCs act on MSCs to induce the expression of Notch ligands via direct cell-to-cell contact and that the Notch ligands derived from MSCs act on HSCs in turn to activate Notch signaling pathway, possibly leading to the cobblestone formation with the maintenance of immature state of HSCs. The Notch system may be one of the critical elements in the interactive communication between HSCs and MSCs.
Prospective In Vivo Identification of Osteogenic Stem/Progenitor Cells from Bone Marrow-Derived Lin−/Sca-1+/CD45− Cells.
Authors: Wang Zhuo , Jung Junghun , Kucia Magdalena , Song Junhui , Shiozawa Yusuke , Ratajczak Mariusz Z. , Krebsbach Paul H. , Taichman Russell S.
Published: October 2019

We previously developed an in vivo prospective assay for identification of non-cultured cells with MSC potential. Using this assay we identified a population of cells that were slowly cycling and of low density that were capable of multilineage differentiation both in vitro and in vivo (Z. Wang et al, Stem Cells. 2006 24(6):1573). Further characterization of these cells suggested that they resemble a homogenous population of rare Lin−/Sca-1+/CD45− cells that have the morphology and express several markers of undifferentiated embryonic-like stem cells. In vitro the Lin−/Sca-1+/CD45− cells may differentiate into cells from all three germ-layers (M. Kucia et al, Leukemia. 2007 21(2):297). To determine the in vivo fate of this population, we transplanted 500 or 5,000 Lin−/Sca-1+/CD45− cells from a GFP mouse into SCID mice in each group (n=3) immediately after cell sorting to evaluate tissue generation in vivo. At 4 weeks the regenerative potential of these populations was evaluated by micro-CT and histology, and cells were tracked by gross examination of the harvested tissues by fluorescent microscopy. The results showed that a large number of GFP+ cells are located in the implants, indicating that the transplanted cells maintain the ability to contribute to the generation of new tissue. Bone-like tissue was observed in the Lin−/Sca-1+/CD45− group with as low as 500-cells/implant, while 5,000 Lin−/Sca-1+/CD45− cells generated significantly larger mineralized tissue volume, which was confirmed by micro-CT. Lin−/Sca-1+/CD45+ cell only implantation did not form any mineralized tissue, however, while mixed with 2x106 whole bone morrow cells, positive mineralized tissue occurred. Whole bone marrow mixture also improve bone formation in Lin−/Sca-1+/CD45− cell implants compared the actual bone volumes measured by micro-CT. This study demonstrates that non-cultured BM-derived Lin−/Sca-1+/CD45− cells exhibit the capacity to form bone in vivo with as low as 500 cells/implant. Whole bone marrow mixtures can enhance the bone formation, presumably through the interaction of other populations cells. Based on these findings, it is proposed that non-cultured BM-derived Lin−/Sca-1+/CD45− cells are enriched osteogenic cells that can be applied to bone regeneration in vivo.
Artificial Hematopoietic Stem Cell Niche Sustains Growth and Differentiation of Human ES-Derived Early Hematopoietic Progenitors.
Authors: Esmaeli-Azad Babak , Srivastava Anand S. , Frederico Cybele , Martinez Geraldo , Yasukawa Satoshi , Carrier Ewa
Published: October 2019

Using a novel Microplate Biomaterial Microarray (MBM™) technology, we have created an artificial hematopoietic stem cell niche that can sustain growth and differentiation of human embryonic stem cells-derived (hES) early hematopoietic progenitors. This hydrogel based ex-vivo niche allows uploading of human embryonal stem cells, human mesenchymal stem cells (MSC), genes (bcl-2 preventing apoptosis and HoxB4 enhancing hematopoiesis) and extracellular matrices to support growth and differentiation of human ES cells. These experiments were done using NIH-approved hES cell lines H1 and H9. Serum-free, feeder-free culture conditions were established and early hematopoietic progenitors grown using SCF, TPO, VEGF and IL-3 with high efficiency. At day 3–5 dual CD34+/CD31+ progenitors were identified, while on day 7–8 CD34+ hematopoietic progenitors were isolated, which formed typical hematopoietic colonies. These progenitors expressed genes related to early hematopoiesis, such as TAL1/SCL, FLT1, GATA2, GATA1, EPOR and TPOR. The early dual endothelio-hematopoietic progenitor (hemangioblast) expressed PECAM-1 and CD34 and showed typical blast-like morphology. Based on mathematical simulations, various micro-niches were designed to establish optimal differentiation conditions for this progenitor using IL-3, IL-6, TPO, EPO, VEGF, SFC, Flt-3 ligand and various extracellular matrices. Specific micro-niches were created for generation of CFU-E, BFU-E, CFU-GM, CFU-GEMM, CFU-M, CFU-G, and CFU-MK progenitors from human ES-derived hemangioblast. Kinetic uploading of TPO, EPO, SCF and VEGF created a niche-sustaining growth of ES-derived hemangioblast with high efficiency and low apoptosis rate. These niches used pulse -delivery of anti-apoptotic bcl-2 gene and hematopoiesis-enhancing Hoxb4 gene. The model of artifical niche sustaing growth and differentiation of human ES-derived hemangioblast was established. In the future, this system will allow optimized and upscaled generation of early hematopoietic progenitors from human ES cells, as a first step towards clinical applications of human embryonic stem cells.

Figure Figure
Elevated Red Cell Microparticles (RMP) as Risk Factor for Thrombosis in Patients with Renal Failure.
Authors: Fontana Vincenzo , Jy Wenche , Horstman Lawrence , Pamela Dudkiewicz , Carina Fernandez , Bidot Carlos , Ahn Ahn S.
Published: October 2019

BACKGROUND: Cell-derived microparticles (MP) are microvesicles released during activation or apoptosis from endothelial cells (EMP), platelets (PMP), leukocytes (LMP) and red cells (RMP). Their roles in hemostasis and inflammation are increasingly coming to light. Chronic and acute renal failure (RF) is associated with increased risk of bleeding as well as thrombosis but the pathogenesis is unclear. Platelet hyperaggregation, elevated clotting factors, deficiency in natural anticoagulants, impaired fibrinolysis and hyperhomocysteinemia have all been implicated in thrombosis in RF. Recently high levels of EMP, PMP and LMP have been reported in RF but their clinical significance is obscure. We investigated these MP as well as RMP in patients with RF.

METHODS: Twenty-seven patients (14M/13F, mean age 53yr) with RF were investigated. Incidence of thrombosis was assessed. Healthy controls were n=109. Flow cytometry was used to assay LMP by anti CD45, PMP by anti CD41, RMP by anti-glycophorin, and EMP by CD31+/CD41−.

RESULTS: The RF patients were divided into groups with thrombosis (TB) and without (NTB). Nine patients (33%) had thrombosis, most often venous. When MP were compared between controls and all RF patients, EMP (p=0.0004) and RMP (P=0.01) were elevated in RF. However, PMP and LMP in RF did not differ from controls (FIG. 1). On the other hand, when we compared MP between TB and NTB, only RMP was significantly greater in TB (mean 2064/uL) than NTB (mean 972/uL), p=0.001. Mean levels of EMP, PMP and LMP did not differ significantly between TB and NTB in RF (FIG. 2).

CONCLUSION / DISCUSSION: First, we confirm elevated EMP in RF compared to normal controls, and also report for the first time elevated RMP in RF. This indicates activation of endothelium and and red cells. Elevation of procoagulant MP likely contribute to risk of thrombosis in RF. Interestingly, PMP and LMP were not significantly increased in RF. Secondly, in comparing MP between the two groups of RF patients, only RMP, not other MP, were significantly higher in TB than NTB. The potential role of RMP in hemostasis and thrombosis is unknown, but the present study demonstrates a unique association of elevated RMP with thrombosis. Specifically, only RMP discriminate the TB from the NTB group in RF. This may suggest a role of RMP in thrombosis. RMP carry procoagulant phosphatidylserine (PS). Expression of PS was increased in RBC of patients with RF. The mechanism of increased RMP in RF is not known. Increased membrane fragility and decreased deformability of RBC seen in uremic patients, together with enhanced membrane lipid peroxidation from oxidative stress would contribute to increased hemolysis and RMP in RF. Thus, the present findings should further stimulate investigation into the causes and significance of RMP in RF and other TB disorders.

Figure 1 Figure 1. Figure 2 Figure 2.
Epistatic Interaction between Fibrinogen alpha and gamma Genes and Factor V Leiden in Children with Venous Thrombosis: Results from a Family-Based Association Study.
Authors: Nowak-Gottl Ulrike , Weiler Hartmut , Seehafer Tanja , Thedieck Sabine , Stoll Monika
Published: October 2019

Background: Fibrinogen, the precursor of fibrin, is an essential component of the hemostatic system. A previous large case-control study showed that genetic variation in the fibrinogen gamma gene (FGG) increased the risk for VT in adults. Here we investigated the association of haplotypes comprising the fibrinogen alpha (FGA) and gamma (FGG) genes, carriership of the Factor V Leiden mutation and risk for VT in a large family-based study sample for pediatric VT.

Methods: We genotyped 188 pediatric VT families for seven single nucleotide polymorphisms (SNPs) (rs6050, rs2070016, rs2070014 and rs2070011, rs1049636, rs2066861, rs2066860) as well as the G1691A Factor V Leiden (FV) polymorphism. Association was assessed using the Transmission Disequilibrium Test (TDT) and corrected for multiple testing using permutation testing as implemented in HAPLOVIEW. Interaction between FV and FGA and FGG, respectively, was assessed by TDT in families stratified for presence or absence of the FV mutation in the affected child.

Results: rs6050, rs2070016, rs2070014 and rs2070011 located in the FGA gene are in tight linkage disequilibrium (LD) and define 5 common haplotypes (HT) and are linked with the neighboring FGG gene (q= 0.91). rs1049636, rs2066861, rs2066860 located in FGG are in tight LD and define 4 HTs. HTs in both, FGA and FGG are significantly overtransmitted from parents to affected offspring (FGA: HT1 (AACT), HT frequency 0.32, T:U 62: 32, p=0.0025; FGG: HT2 (ATC), HT frequency 0.32, T:U 60:32, p=0.0035). When stratifying for FV status, it became apparent that the association between FGA and FGG and VT was more pronounced in FV-negative families (FGA, HT1, T:U 55:24, p=0.0006; FGG, HT2, T:U 55:24, p=0.0005), while absent in FV-positive families.

Conclusion: Our results indicate that genetic variation in FGA and FGG are risk factors for VT in children, and further that an epistatic interaction between FGA/FGG and FV Leiden influences the risk of FGG and FGA on pediatric VT. Our study highlights the complex nature of VT and the necessity to evaluate gene-gene interactions in association studies of complex, polygenic diseases.
Rates of Guideline-Recommended Venous Thromboembolism Prophylaxis in Surgical Patients among US Hospitals: A Real-World Analysis.
Authors: Amin Alpesh N. , Lin Jay , Yang Guiping , Stemkowski Stephen
Published: October 2019

Background: In spite of the wide availability of evidence-based guidelines for the thromboprophylaxis of venous thromboembolism (VTE), the burden of this syndrome in the US remains high. The improvement of VTE prophylaxis use is a major healthcare goal for the US Joint Commission. We therefore assessed the real-world rate of appropriate thromboprophylaxis in at-risk, hospitalized surgical patients, in accordance with the 7th American College of Chest Physicians guidelines, in hospitals across the US.

Methods: Premier’s Perspective™ inpatient data were used to assess the rate of VTE prophylaxis in discharges undergoing surgical procedures. Discharges aged ≥ 40 years, with a length of stay ≥ 6 days, and without contraindications for anticoagulation were included in the analysis. Appropriate thromboprophylaxis was determined by comparing the daily use of anticoagulants and compression devices, dosage, and duration of prophylaxis with the 7th ACCP recommendations for each surgical procedure. A number of other factors, including geographic region, hospital type and size, and admission source were also investigated for potential influence on the levels of appropriate thromboprophylaxis.

Results: A total of 116,915 surgical discharges from 1,574,320 discharges between January 2002 and December 2006 met the inclusion criteria and were included in this study. VTE prophylaxis rates varied among surgical groups, with an overall mean of only 14.4% of discharges receiving appropriate prophylaxis. Appropriate prophylaxis rates were 68.0% in orthopedic surgery (n = 2,404), 14.9% in vascular surgery (n = 54,170), 13.9% in general surgery (n = 39,072), 12.3% in laproscopic surgery (n = 14,905), 6.5% in gynecological surgery (n = 610), 5.2% in urological surgery (n = 2,640), and 4.0% in neurological surgery (n = 3,114).

Conclusion: There is a significant gap in adherence to the ACCP guidelines in surgical patients, with on average approximately 85% of surgical patients not receiving evidence-based VTE thromboprophylaxis. Although the rates of appropriate prophylaxis for orthopedic surgery patients were considerably higher than in other surgical categories, one-third of these patients still did not receive appropriate prophylaxis. As performance measures evaluating hospital’s VTE thromboprophylaxis use are rolled out across the US, it is important for individual hospitals to increase their awareness and use of the ACCP recommendations for VTE thromboprophylaxis.
Extended-Duration Enoxaparin for Venous Thromboembolism Prophylaxis in Acutely Ill Medical Patients: An Evaluation of the EXCLAIM Study Based on a Recently Recommended Composite Efficacy Endpoint.
Authors: Schellong Sebastian M. , Hull Russell D. , Tapson Victor F. , Monreal Manuel , Samama Meyer-Michel , Turpie Alexander G.G. , Yusen Roger D.
Published: October 2019

Introduction The EXCLAIM study has shown that in acutely ill medical patients who have received a standard 10-day period of prophylaxis with enoxaparin, extended-duration prophylaxis with enoxaparin for 4 weeks reduces the relative risk of the primary composite endpoint of asymptomatic proximal deep-vein thrombosis (DVT), symptomatic DVT or pulmonary embolism (PE), and fatal PE by 44% compared with placebo (2.8% vs 4.9%; RR 0.56; 95% CI 0.39–0.80; p=0.0011). We assessed the consistency of these results using the composite efficacy endpoint recently proposed for venous thromboembolism (VTE) prophylaxis studies by the European Committee for Medicinal Products for Human Use (CHMP) which, in addition, takes all-cause mortality into account.

Methods Patients enrolled in the EXCLAIM study had a recent reduced mobility (≤3 days) due to a medical illness, were aged 40 years or older, and had anticipated level 1 (total bed rest or sedentary without bathroom privileges) or level 2 (level 1 with bathroom privileges) reduced mobility with further risk factors. Eligible patients received enoxaparin 40mg SC once-daily for 10±4 days. Patients were then double-blind randomized and received either enoxaparin 40mg SC once-daily or placebo for a further 28±4 days. Asymptomatic DVT were diagnosed by bilateral compression ultrasound after completion of the randomized treatment period. Suspected cases of symptomatic DVT or PE were confirmed by objective tests. Fatal PE were confirmed by autopsy where possible. Data were analyzed using the CHMP-recommended composite efficacy endpoint of proximal DVT, adjudicated PE and all-cause mortality.

Results Eligible patients enrolled at 370 sites in 20 countries received standard-duration prophylaxis with enoxaparin. Of these, 4114 patients were double-blind randomized and 4040 received extended-duration prophylaxis with enoxaparin (n=2013) and placebo (n=2027), respectively. Extended-duration enoxaparin reduced the relative risk of the CHMP composite endpoint by 26% compared with placebo (5.8% vs 7.9%; RR 0.74; 95% CI 0.58–0.95; p=0.018).

Conclusion The significant reduction in the incidence of the primary efficacy endpoint associated with the use of extended-duration enoxaparin compared with placebo, following standard duration enoxaparin, was consistent when applying both the pre-defined EXCLAIM and the CHMP-recommended composite endpoints, the latter of which included all-cause mortality in addition to proximal DVT and adjudicated PE.
Dose-Escalated Low Molecular Weight Heparin Provides Effective Anticoagulation for Women with Mechanical Heart Valves during Pregnancy: A Single-Centre Experience.
Authors: Quinn John , Brooks Ruth , Walker Fiona , Peebles Donald , Cohen Hannah
Published: October 2019

Effective anticoagulation for women with mechanical heart valves during pregnancy remains a major challenge. The use of standard dosage low molecular weight heparin (LMWH) is associated with an unacceptable incidence of morbidity and mortality for this patient group. In this prospective audit of anticoagulation management, we report pregnancy outcome for a cohort of women with mechanical heart valves, who received anticoagulation with a higher dosage LMWH regimen. We audited 12 consecutive pregnancies in 10 women (mean age 27.8 (18–41) years) with mechanical heart valves (mitral 4, aortic 2, aortic and mitral 2, systemic tricuspid AV valve 2). Past obstetric history (on warfarin) included 6 fetal losses: 2 miscarriages <12 weeks and 4 terminations including one therapeutic at 22 weeks gestation because of an intracerebral fetal haemorrhage. In 11/12 pregnancies treated with LMWH, warfarin was discontinued at <6 weeks and in 1/12 at 8 weeks. LMWH (dalteparin 7, enoxaparin 5) +/− low-dose aspirin (4) was then started at full therapeutic dosage (subcutaneous 12 hourly) with regular monitoring to maintain anti-Xa levels at 1.0–1.2U/mL (0.8–1.2 in the first 4/12 pregnancies). This necessitated stepwise increases in LMWH with median doses (12 hourly) pre-delivery 11,750IU for dalteparin and 95mg for enoxaparin, representing a median 47.7% increase over initial dosage. 11/12 pregnancies on LMWH resulted in live births (median BW 2.5 kg (range 1.0–3.7) at median gestation 36 (range 26–38 weeks) with 1 IUFD at 37 weeks. One patient had non-fatal valve-related thrombosis at 26 weeks associated with a sub-therapeutic anti-Xa level (0.64 U/ml). Of note, she had a past history of pulmonary embolism and was heterozygous for the G20210A prothrombin gene mutation. There were no other valve-related thrombotic events. Minor bleeding occurred during 3 pregnancies. There were 5 major bleeding episodes in 4 patients: 3 antepartum (1 with placenta praevia) and 1 post-partum haemorrhage plus 1 abdominal wound haematoma. Our data suggest that in this patient group escalated dose LMWH provides effective anticoagulation. These patients require meticulous anticoagulant monitoring and close surveillance for bleeding and thrombotic complications within a multidisciplinary setting, with urgent intervention when indicated.
Comparison of the 2-Year Clinical Outcomes of VTE Prophylaxis Options in Medical Patients at Risk of VTE.
Authors: Deitelzweig Steven B. , Lin Jay , Benner Josh , Becker Russ
Published: October 2019

Background: Hospitalized medical patients are at significant risk of venous thromboembolism (VTE). Although evidence-based guidelines exist which provide recommendations for thromboprophylaxis in hospitalized medical patients, the optimum regimen for prophylaxis is not clear. We have therefore created a model, based on established literature, which examines the 2-year clinical outcomes following no prophylaxis, thromboprophylaxis with unfractionated heparin (UFH), or thromboprophylaxis with low-molecular-weight heparin (LMWH) in medical patients at risk of VTE.

Methods: A decision-analytic model was developed that replicates and extends an existing, published VTE model (McGarry et al. Am J Manag Care.2004;10:632–42) from 30 days to 2 years. Hypothetical cohorts of 10,000 medically ill patients at risk of VTE (according to MEDENOX criteria) were assembled, using a Markov chain model with resampling, to compare the rates of primary VTE events and related outcomes at 90 days and VTE complications and recurrent events at 2 years. Clinical outcomes were estimated from published clinical trial and observational data, and compared between three interventions, namely no prophylaxis, UFH, and the LMWH enoxaparin. Outcomes included in the analysis were clinical or venographically detected primary VTE, major and minor bleeds, asymptomatic or symptomatic heparin-induced thrombocytopenia, and death within the first 90 days, as well as VTE recurrence, post-thrombotic syndrome, pulmonary hypertension, and death within 2 years. Sensitivity and threshold analyses were performed to test the general applicability of the model. The simulation model was run using TreeAge software (Williamstown, USA).

Results: VTE rates and death were the lowest in the enoxaparin prophylaxis cohort, followed by the UFH and no prophylaxis cohorts respectively (Table 1). Adverse events were lowest in the no prophylaxis group, followed by the enoxaparin group and the UFH group (Table 1).

Conclusion: In this Markov model, based on robust data from clinical trials and observational studies, prophylaxis with enoxaparin reduced VTE occurrence and mortality over two years when compared with no prophylaxis or UFH prophylaxis in hospitalized medical patients at risk of VTE. Enoxaparin was also associated with a reduced incidence of adverse events when compared with UFH.

Table 1. Two-year outcomes in simulated cohorts of hospitalized medical patients at risk of VTE Outcome (n) Enoxaparin (n=10,000) Unfractionated heparin (n=10,000) No prophylaxis (n=10,000) VTE at 2 years 683 791 1787 DVT 545 633 1426 PE 138 158 361 Death 1573 1600 1745 Adverse events at 90 days 364 725 314 Minor bleed 285 510 244 Major bleed 65 116 55 Asymptomatic HIT 6 45 7 Symptomatic HIT 8 54 8
Downregulation of Mpl Marks the Transition to Lymphoid-Primed Multipotent Progenitors with Gradual Loss of Granulocyte-Monocyte Potential.
Authors: Luc Sidinh , Anderson Kristina , Kharazi Ms Shabnam , Buza-Vidas Natalija , Boiers Charlotta , Jensen Christina T. , Ma Zhi , Wittman Mrs Lilian , Jacobsen Sten Eirik W.
Published: October 2019

Evidence for a novel route of adult hematopoietic stem cell (HSC) lineage commitment through Lin−Sca-1+Kit+Flt3hi (LSKFlt3hi) lymphoid-primed multipotent progenitors (LMPPs) with granulocyte/monocyte (GM) and lymphoid but little or no megakaryocyte/erythroid (MkE) potential was recently challenged, as LSKFlt3hi cells were reported to possess MkE potential. Herein residual MkE potential segregated almost entirely with LSKFlt3hi cells expressing the thrombopoietin receptor (Thpor), whereas LSKFlt3hiThpor− LMPPs lacked significant MkE potential in vitro and in vivo, but sustained combined GM and lymphoid potentials, and co-expressed GM and lymphoid but not MkE transcriptional lineage programs. Gradually increased transcriptional lymphoid priming in single LMPPs from Rag1GFP mice was shown to occur in the presence of maintained GM lineage priming, but gradually reduced GM lineage potential. These functional and molecular findings reinforce the existence of GM-lymphoid progenitors with dramatically downregulated probability for committing towards MkE fates, and support that lineage restriction occurs through gradual rather than abrupt changes in specific lineage potentials.
Single Amino Acid Substitution (Asp111Ala) in Coagulation Factor Va Causes Subunit Dissociation and Disrupted Calcium and Copper Binding.
Authors: Song Jina , Talbot Kimberley , Hewitt Jeffrey , Camire Rodney M. , MacGillivray Ross T.A. , Pryzdial Ed L.
Published: October 2019

Factor V (FV) is the essential inactive precursor of blood coagulation cofactor Va (FVa). FV is synthesized as a single chain protein that is activated at sites of injury by thrombin to FVa. As a ternary complex with the serine protease factor Xa and an anionic phospholipid (aPL) membrane in the presence of calcium (prothrombinase), FVa functions to accelerate the rate of prothrombin to thrombin conversion, the critical step in hemostasis. FVa is a non-covalent heterodimer composed of a heavy (FVaH) and a light subunit (FVaL). It has been well established that FVa requires calcium to stabilize a functional subunit interaction, although copper is also known to bind. A 1:1 divalent cation:FVa stoichiometry has been reported for both metals. Despite the importance of FV few studies have investigated the specific amino acids and divalent cations that mediate the FVaH-FVaL association. In order to understand the role of divalent cations in maintaining the FVa subunit interaction and consequent function, we investigated the effect of mutating FV Asp111 to Ala (D111A), which was suggested by our previous work, others’ crystallography studies and homology to coagulation factor VIII, to potentially participate in calcium binding. Compared to wild type recombinant FV (wtFV), there was no difference in the kinetics of thrombin-mediated conversion of purified FVD111A to FVaD111A assessed by gel electrophoresis. Binding to small unilamellar aPL-containing vesicles was followed by light scattering and also showed no change. However, the cofactor activity of FVD111A was observed in clotting and purified prothrombinase chromogenic assays to be significantly inhibited (39% and 27% reduction, respectively). When the wtFV or FVD111A was pre-activated by thrombin (i.e. 2-stage assay), markedly greater inhibition was observed (94% and 73% reduction, respectively). The 1-stage/2-stage discrepancy seen here can be explained by spontaneous dissociation of subunits in FVD111A upon activation. Indeed, thrombin activation of FVD111A was shown by light scattering to result in rapid dissociation of subunits even in the presence of excess calcium. To quantitatively investigate the direct effect of the mutation on divalent cation coordination, graphite furnace atomic absorption spectrometry was utilized to measure the amount of bound calcium and copper. As predicted, calcium binding to FVD111A was severely inhibited resulting in a stoichiometry of <0.16:1. Interestingly, the stoichiometry of copper binding was also appreciably reduced to ∼0.5:1. These data demonstrate for the first time that a single point mutation can result in the spontaneous subunit dissociation of purified FVa. The finding that mutation of Asp111 disrupted not only the coordination of calcium but also that of copper, suggests that interdependent metal ion binding sites may contribute to FVa configuration and function.
Pegylated Liposomal Doxorubicin (PLD) in Combination with Bortezomib (B) May Provide Therapeutic Advantage for High-Risk Multiple Myeloma Patients Relapsing within 12 Months of Stem Cell Transplant.
Authors: Kumar Shaji , Blade J. , Miguel J. San , Hajek R. , Nagler A. , Sonneveld P. , Spencer A. , Sutherland H.J. , Robak T. , Mundle S.D. , Zhuang S.H. , Harousseau J.L. , Orlowski R.Z.
Published: October 2019

Background: Patients (Pts) with multiple myeloma (MM) who relapse within 12 months of autologous stem cell transplantation (SCT) have a poor prognosis. As reported by Mahmood et al. (ASCO 2007), of 432 pts who received SCT at the Mayo Clinic between 1994–2005, those with early relapse within 12 months (94/432– 22%) showed poorer median overall survival. Kaplan-Meier estimates of 12-month survival from the date of first relapse were 37% for pts relapsing within 12 months after SCT as compared to 85% for those relapsing after 12 months. In that study, pts had received regimens other than B. In a recent report of a large, phase III study (DOXIL-MMY3001), the combination of PLD+B improved time to progression (TTP) as compared to B alone (Orlowski et al. JCO 2007). The present analysis examined the 12-month post-randomization survival of patients who had early (<12 months) vs. late (≥12 months) relapse following SCT, as well as the effect of PLD+B vs. B alone in pts who had relapsed early.

Methods: This was a retrospective analysis of 646 pts who received intravenous B, 1.3 mg/m2 on days 1,4,8 and 11 of every 21-day cycle ± PLD, 30 mg/m2 on day 4.

Results: 359 pts had previously received transplant, 114 (32%) of whom relapsed within 12 months from SCT. The median age, gender distribution, time from diagnosis trial enrollment, measures of disease burden (M-Protein levels and B2M) and renal function were comparable between the early and late relapse groups. There was no difference in overall response rates [complete + partial response (CR+PR)] or very good PR (VGPR) rates between the two groups, or between treatment arms within each group. There was no significant difference in TTP between early vs. late relapse groups (HR=0.94). 12-month survival from randomization was significantly lower in the early relapse group as compared to late relapse (83% vs. 92% respectively, p=0.009). However, within the early relapse group 12-months post-randomization survival rate was significantly superior following treatment with PLD+B as compared to B alone [52/56 patients (93%) vs. 43/58 patients (74%) respectively, p=0.01]. Correspondingly, TTP was better in this group with PLD+B vs. B alone (276 days vs. 205 days respectively, p=0.13). Overall, the toxicity profiles of the combination and B alone were comparable regardless of early or late relapse following SCT.

Conclusions: The present analysis of the MMY3001 study corroborates the prior Mayo observation of lower survival in MM pts relapsing within 12 months of SCT. Importantly, it demonstrates that PLD+B may provide a therapeutic advantage for high-risk MM pts with early relapse following SCT.
Preferential Proliferation of T Effectors and Tumor Protection by Adoptive Transfer of Primed Anti-Lymphoma Splenocytes into Lymphodepleted Hosts.
Authors: Brody Joshua D. , Goldstein Matthew , Levy Ronald
Published: October 2019

Clinical trials of immunotherapy for lymphoma have demonstrated induction of immunologic anti-tumor responses but only a minority of clinical responses. Possible reasons include tumor-produced factors (e.g. IL10, VEGF, TGFβ) and immune-regulatory mechanisms (e.g. Tregs, immature myeloid cells, tumor-associated macrophages). Immunotherapy in the post-transplant setting could overcome both these obstacles, as it is a state of minimal residual disease and of relative lympho- and myelodepletion. We have previously described a method of CD8 T cell-dependent vaccination against established lymphoma tumors by a combination of chemotherapy and intra-tumoral injection of the TLR9 agonist CpG oligodeoxynucleotides. Herein, we transferred splenocytes from such vaccinated mice into lymphodepleted recipient mice. The transferred tumor-specific T cells proliferate in these “empty” mice at a 30-fold excess over that in normal non-depleted recipients. The lymphodepleted recipients of adoptive transfer were completely protected against challenge with the lymphoma tumor at a 100-fold greater tumor cell dose than that required to kill normal animals. Because the transferred effector lymphocytes may still be contaminated by the same immuno-regulatory elements (e.g. Tregs) present in the donor mice, we asked whether these might proliferate and diminish the effector function of anti-tumor T cells. By tracking the homeostatic proliferation of the transferred cells we observed an apparent preferential proliferative capacity of the tumor-responsive CD8 cells and a relative decreased proliferative capacity of transferred foxP3(+) Treg cells. These data suggest that the post-transplant environment can skew the relative proportion of different T cell subsets, which may be a central mechanism for the dramatic improvement in tumor protection observed.