bluuebunny/crossref_metadata_2025 huggingface dataset:
A Genomic Strategy To Refine Prognosis and Predict Response to Therapy in Chronic Lymphocytic Leukemia.
Authors: Friedman Daphne R. , Weinberg J. Brice , Potti Anil , Volkheimer Alicia D. , Bond Karen M. , Chen Youwei , Jiang Ning , Moore Joseph O. , Gockerman Jon P. , Diehl Louis F. , Decastro Carlos M. , Nevins Joseph R.
Published: October 2019

Chronic Lymphocytic Leukemia (CLL) is notable for variation in aggressiveness of disease. Many patients with low-risk disease at diagnosis can be followed expectantly, while others rapidly require therapy. While a number of factors aid in determining prognosis, they have no role in predicting response to chemotherapy. We collected clinical data from a cohort of 220 patients with CLL followed at Duke University and the Durham VA Medical Centers. We assessed factors such as stage, leukocyte doubling time, IgVH mutational status, CD38 and ZAP-70 expression, cytogenetics, and time to treatment. We isolated RNA from purified CD5+, CD19+ leukemia cell samples from low and intermediate risk CLL patients, assessed the RNA expression with Affymetrix U133 Plus 2.0 GeneChips, and analyzed differential gene expression using previously developed methods of Bayesian binary regression. In a group of seventy-nine CLL patients who either eventually required (n = 45) or did not (n = 34) require therapy, CD38 status, ZAP70 expression, and cytogenetics were not statistically different (p > 0.5) while IgVH mutational status and leukocyte doubling time were different (p < 0.003). Of the patients treated, 69% were treated with chlorambucil, 51% with fludarabine, 31% with cyclophosphamide, and 49% with rituximab. Using genomic expression data, we developed a 100 gene expression signature that correlated with the need for eventual therapy. The accuracy of this signature was 94% using the leave-one-out cross validation method. Further validation using published data is currently being conducted, and data will be presented. Genes associated with the need for treatment include cell cycle regulators (such as E2F2 and CDK6), inhibitors of apoptosis (such as BAG1), and apolipoprotein B. In addition, using techniques described previously (Potti A et al, Nature Medicine, 2006, 12(11):1294), gene expression data coupled with either in vitro drug sensitivity in the NCI-60 panel of cell lines or in vivo drug response data were used to generate genomic models predictive of sensitivity to drugs commonly used in CLL including fludarabine, cyclophosphamide, and rituximab with accuracies on leave-one-out cross validation of 93%, 96%, and 79% respectively. These predictive models are currently being applied to gene expression data from leukemia samples of CLL patients to predict response to therapy (data to be presented). Thus, a genomic approach can be used to determine which CLL patients will require treatment. Importantly, models of chemosensitivity may predict response to therapy, and thus may ultimately help target the appropriate treatment for each patient.
Mechanistic Paradigm for Combination Signal Transduction Inhibitor Therapy in Zap70+ CLL Involves Bicompartmental Functions of EZH2.
Authors: Hartman Amy D. , Wilson-Weekes Annique , Ruffin Theresa , Hincher Katie J. , Cripe Larry D. , Boswell H. Scott
Published: October 2019

We have characterized a tonically active B cell receptor signaling module of zap70+ve CLL cells that contributes to a high-risk disease phenotype associated with rapid tumor doubling time and cellular bcl-2 overexpression. In CLL cells, syk directs tyrosine phosphorylation of certain adaptors including cbl and crkL and this is also accompanied by tyrosine phosphorylation of the interdomain-B loop of zap70 to affect its own interaction with adaptors and vav1. Vav1 along with zap70 allows JNK activation and the formation of heightened c-jun/AP-1 involved in CLL cell bcl-2 transcription. However, CLL pathogenesis is equally dependent upon ezh2/PRC2-mediated 5′-methylation of CpG islands in several tumor suppressor genes such as PTPRO (a cell-intrinsic syk inhibitor) and DAPK1. In addition, ezh2 functions not only in the nucleus for gene repression, but in cytoplasm for stabilization/perpetuation of a zap70-vav1 signal complex upstream of JNK1 in lymphocytes (IH Su et al., Cell121:425, 2005). We investigated the possible participation of ezh2 in events relevant to the outcome of syk inhibition as a primary therapy for CLL. We used R406/R788, a potent, selective syk inhibitor now in clinical trial (Rigel Pharmaceuticals). We found that R406 treatment strongly inhibited CLL viable cell recovery from culture, and this was associated with inhibition of syk (PY525/catalytic domain) phosphorylation as well as loss of zap70 PY319 (interdomain) phosphorylation with an IC50 of 1 uM R406, associated with downregulation of phospho-c-jun (S73) and of bcl-2. Failure of R406 to inhibit lyn autophosphorylation suggests strongly that syk is the upstream kinase for zap70 in this regulatory pathway. A survey of ezh2 expression showed content of ezh2 correlated with p(319)-zap70 intensity in different CLL cells. R406 caused release of syk substrate cbl from the JNK1 signalosome of R406-treated cells, but ezh2 remained tightly bound to vav1-zap70-JNK1 as demonstrated by immunoprecipitation. To overcome the bi-compartmental functions of ezh2, we used HDAC inhibitor, SBHA (suberoyl hydroxamic acid); a class of compound reported to deplete ezh2 from leukemia cells (W Fiskus et al. Mol Canc. Ther.5: 3096, 2006). In 3 different experiments, SBHA alone, 1 uM, had little inhibitory activity for CLL cell viable cell recovery, however, when combined with R406 (also at 1uM) SBHA caused massive cell death (>80%) only attainable with R406 alone at 2.5X dose. Mechanistic aspects of the reversal of tumor suppressor gene repression and of inactivation of the JNK1 signalosome by SBHA are under scrutiny to fully explain its cooperativity with R406, focused especially on the duality of roles performed by ezh2, and the depletion of ezh2 affected by HDAC inhibition.
Recombinant Activated Factor VII (rFVIIa) in the Treatment of Non-Hemophilia Patients: Physician Underreporting of Thromboembolic Adverse Events.
Authors: Hsia Cyrus C. , Zurawska Joanna H. , Tong Michael Z.Y. , Eckert Kathleen , McAlister Vivian C. , Chin-Yee Ian H.
Published: October 2019

Background: Recombinant activated factor VII (rFVIIa) is a potent hemostatic agent with a short half-life of 2.6 hours and is approved for various hereditary and acquired bleeding disorders. Its “off-label” utilization has increased and is the focus of a number of ongoing clinical trials. One concern is that its beneficial hemostatic effect may be offset by the potential for thromboembolic adverse events (TAEs). Despite its short half-life, rFVIIa likely produces a sustained hemostatic effect and the potential for thromboembolic events may also be prolonged. A recent review of adverse events reported to the FDA suggested that this risk was substantial. The duration that this risk may be present is unclear. Physicians may only consider adverse events to be causally related in the immediate few hours after administration of rFVIIa leading to underreporting TAEs. We conducted a retrospective review of all off-label uses of rFVIIa comparing physician reporting of TAEs to documented TAEs on chart review.

Methods: Chart review of all non-hemophilia adult patients who had received rFVIIa at our tertiary care centre was completed. Following all off-label administration, questionnaires were routinely sent to all treating physicians to report outcomes and adverse reactions with the use of rFVIIa. We included all non-hemophilia adult patients at our tertiary care centre who have received rFVIIa. Exclusion criteria were age < 18 and patients with hemophilia or other bleeding disorders for which rFVIIa use is approved. TAEs identified during chart review were compared to physician reporting on questionnaires.

Results: A total of 71 patients, 32 females and 39 males, satisfied study criteria during the period January 1, 2003 to June 30, 2007. Mean age was 54 (range 18–84). This was a high-risk population with 34 (47.9%) deaths. Mean duration of follow-up was 30 days (within 1 day to 312 days). Total doses of rFVIIa administered ranged from 2.4–19.2mg (mean 8.2mg) given in 1 to 4 divided doses. 11 arterial and 5 venous TAEs identified on chart review were deemed possibly or probably related. These occurred on average 25 days (within 2 day to 89 days) after exposure to rFVIIa. In total, 50 physician questionnaires (70%) were completed. There were no TAEs reported to the transfusion safety officer at our institution by the treating physician(s) in these questionnaires.

Conclusions: Clinically important thromboembolic events due to recombinant activated factor VII are under-reported in this high-risk population. This review was not designed to determine if rFVIIa alters the rate of TAEs but emphasizes the need for careful follow-up, both immediate as well as longterm, of patients who have hemorrhage or are at risk of hemorrhage. Physicians treating patients with rFVIIa should be vigilant for potential TAEs beyond the immediate treatment period.
Single Dose Administration of rFVIIa and NN1731 in Two Hemophilia A Dogs.
Authors: Ezban Mirella , Frost Lone , Viuff Dorthe , Møss Judi , Kloos Mark , Dillow Aaron , Merricks Elizabeth , Raymer Robin , Nichols Timothy C.
Published: October 2019

Introduction: The objective of this pilot study was to evaluate and compare the pharmacokinetic and pharmacodynamic (PK/PD) profile of rFVIIa and NN1731 in two hemophilia A dogs. In addition, it was the aim to evaluate the use of TEG for monitoring rFVIIa/NN1731 activity after in vivo administration and to compare with ex vivo spiking data from a previous study. NN1731 is a new rFVIIa analoge with enhanced activity (Allen et al. Arterioscler. Thromb. Vasc. Biol.2007;27:683–689). In hemophilia patients as well as hemophilia dogs the clot formation is impaired and reflected in coagulation assays such as thromboelastography (TEG) and APTT. The choice of hemophilia dogs is based upon the knowledge that the pharmacokinetics of human coagulations factors (FVIII, FIX and rFVIIa) as well as the effective dose is similar to that in humans. In normal dogs, it is not possible to evaluate the effect of these procoagulant proteins in coagulation assays as no impaired clotting is observed.

Methods: rFVIIa and NN1731 (280 μg/kg IV) were administered to two hemophilia dogs on separate days and plasma samples collected at different time points. FVIIa activity was measured by the FVIIa clot assay and values were used for pharmacokinetic assessment. The same pharmacokinetic models, a non-compartmental method and a two compartment model, respectively, were used as was the case in the First Human Dosing (FHD) trial of NN1731 (NN1731–1639). Analysis of PD markers in dogs included: APTT, PT and whole blood thromboelastography analysis, recently developed for use in hemophilia dogs.

Results: Based on the FVIIa activity profile in the two dogs it was observed that the values obtained at the first time point (C5 min), were higher after treatment with NN1731 than after rFVIIa. All activity based assays including TEG demonstrated that NN1731 was cleared faster than rFVIIa., FVIIa activity (FVIIa clot assay), showed a rapid initial distribution and/or elimination of FVIIa activity (t1/2α:0.3 h) followed by a less rapid elimination phase (t½β:3.5 h). Similar profile and values were obtained for NN1731 in the FHD dose study (J. Møss et al, ISTH, 2007)

Conclusions: This study indicates that in hemophilia A dogs, NN1731 and rFVIIa have distinct PK profiles and very similar to what is observed in man. All activity assays show the same qualitative profile, the FVIIa clot assay being the most sensitive assay. The TEG data obtained in vivo are in accordance with the values obtained after in vitro spiking. The data support the use of hemophilia dogs for evaluating the pharmacokinetic and pharmacodynamic profiles of FVIIa related proteins.
Correlation between Two ADAMTS-13 Activity Assays Based on Different Principles.
Authors: Geiter Sabine , Graf Markus , Vetr Helga
Published: October 2019

Defects in ADAMTS-13, the von Willebrand Factor (vWF) cleaving protease, are thought to be the main cause for the microvascular thrombotic disorder TTP (thrombotic thrombocytopenic purpura) that is in more than 90% of cases fatal if not treated early and appropriately. Usually this disease is clinically diagnosed, but in recent years the need for rapid and reliable diagnostic tests for ADAMTS-13 levels has increased. We present here the comparison of two commercially available assays for quantification of ADAMTS-13 activity both suitable for routine analysis but based on different principles. The two assays differ in their test principle and the readout system as follows: Assay 1 is a fluorogenic assay using a FRETS-vWF73 substrate and a kinetic measurement (TECHNOZYM®ADAMTS-13 ELISA); with this assay ADAMTS13 Antigen can also be determined in a second step. Assay 2 is a chromogenic assay and detects the cleaved vWF73 substrate by a specific monoclonal antibody (TECHNOZYM®ADAMTS-13 Activity ELISA). Citrated plasma of normal donors (n=7), of pooled normal plasma (n=15) and of TTP patients (n=14) were tested in both assays. Results are reported in both assays as percentage of normal activity. The standard for both assays is prepared from a pool of 100 normal donors and defined as 100%. The samples comprised a range from 0.2% up to 107% activity. The overall correlation coefficient between the two different activity assays was 0.96. 5 samples were found to have less than 5% activity in both assays. These results show that data obtained by the new TECHNOZYM®ADAMTS-13 Activity ELISA correlate very well with the fluorogenic assay (TECHNOZYM®ADAMTS-13 ELISA) in spite of the fact that these assays are based on very different principles.
Age-Associated Difference in Gene Expression of Pediatric Myelo-Monocytic and MLL-Rearranged AML.
Authors: Jo Aoi , Tsukimoto Ichiro , Ishii Eiichi , Asou Norio , Igarashi Takashi , Hayashi Yasuhide , Ichikawa Hitoshi
Published: October 2019

Acute myelogenous leukemia (AML) is a heterogeneous disease with a variety of genetic alterations including the MLL gene rearrangement. Microarray-based gene expression profiling has been applied to diagnose AML patients and to explore their underlying molecular pathology. In this study, we focused on pediatric myelo-monocytic leukemia and analyzed 40 pediatric AML patients with FAB M4 and M5 subtypes [excluding inv(16) and t(8;21) cases] using Affymetrix HG-U133plus2.0 microarray. Patients consisted of 14 infants (<1 year old) and 26 children (>=1 year old), and 23 patients possessed MLL gene rearrangements. Unsupervised analysis revealed that 10 of the 14 infants display very distinct gene expression and large set of genes were over-expressed among the infants. Among these highly expressed genes, ZFHX1B, ARHGAP26 (GRAF), and FOXO3A (AF6) were included. With the use of these distinctively expressed genes, we were able to divide the 40 pediatric patients into three subgroups: 10 infants with very high expression as Group A, 2 infants and 10 children with medium expression as Group B, and 2 infants and 16 children with low expression as Group C. The average age of Group A was 0.3 year old, Group B was 3.1 years old, and Group C was 6.6 years old. All subgroups included MLL gene rearrangements as well as normal and other complex karyotypes. FAB subtypes were equally distributed among each subgroup. The comparison of the outcomes among the subgroups showed a striking difference where Group C presented extremely poor outcome (3-year EFS 28%). Genes specifically over-expressed among Group C included poor prognostic factors such as WT1 and KIT. In addition, we found that gene expression signature of MLL rearrangement was different among the subgroups as well. For example, EVI1, SOCS2 and MEF2C were up-regulated in MLL-rearranged AML of Group C but not in the others. HOXA9 and HOXA10 were regulated independently of MLL-rearrangement in pediatric myelo-monocytic AML. These results suggest that age is an important factor contributing to the biology of pediatric myelo-monocytic AML and sub-grouping procedures can provide proper stratification so that the poor prognostic subgroup can be distinguished to receive suitable therapy.
MicroRNA Expression Profiles in Acute Myeloid Leukemia with Common Translocations.
Authors: Li Zejuan , Lu Jun , Sun Miao , Mi Shuangli , Zhang Hao , Luo Roger T. , Qian Zhijian , Neilly Mary Beth , Wang Yungui , Jin Jie , Zhang Yanming , Bohlander Stefan K. , Larson Richard , LeBeau Michelle , Thirman Michael J. , Golub Todd R. , Rowley Janet D. , Chen Jianjun
Published: October 2019

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. It is estimated that 13,410 cases will be diagnosed and 8,990 will die of AML in the United States in 2007 (http://seer.cancer.gov). AML is a genetically diverse hematopoietic malignancy with variable response to treatment. Expression profiling of protein-coding genes using DNA microarray in AML has resulted in inconsistent data from different laboratories. Therefore, further validation of these observations in large cohorts and in independent studies is definitely required before clinical application becomes feasible. Recently, Golub and colleagues described a new, bead-based flow cytometric microRNA (miRNAs, miRs) expression profiling method that could successfully classify tumors. MiRNAs are endogenous ∼22 nucleotide non-coding RNAs, which can function as oncogenes and tumor suppressors. To provide new insights into the complex genetic alterations in leukemogenesis and to identify novel markers for diagnosis and treatment of AML, we performed a genome-wide analysis of miRNA expression profiles using the bead-based method on 54 AML samples with common translocations including t(15;17), t(8;21), inv(16), and 11q23 rearrangement, along with normal controls. In both unsupervised and supervised hierarchical cluster analyses, we observed that t(15;17) samples grouped together as one cluster, as do the 11q23 rearrangement samples. Interestingly, t(8;21) and inv(16), both CBF (core-binding factor) AMLs, grouped together as a unique cluster. Forty-one miRNAs exhibited significantly differential expression between different subtypes of AMLs, and/or between AMLs and normal controls. Notably, expression signature of a minimal number of two, three, and seven miRNAs could be used for class prediction of CBF, t(15;17), and 11q23 rearrangement AMLs, respectively, with an overall diagnostic accuracy of 94–96%. We further showed that overexpression of the two discriminatory miRNAs in CBF AML is associated with epigenetic regulation, rather than DNA copy number amplification. Moreover, several important target genes of these discriminatory miRNAs have also been validated. We are currently exploring the role of these discriminatory miRNAs and their critical target genes in the development of AML using in vitro and in vivo models. This work will enhance our understanding of the biological role of these miRNAs and their targets in leukemogenesis.
Deregulation of RARγ and RARγ-Specific miRNAs in AML.
Authors: Barrett Angela N. , Gupta Rajeev , Glasow Annegret , Grimwade David , von Lindern Marieke , Waxman Samuel , Mills Ken , Burnett Alan , Enver Tariq , Zelent Arthur
Published: October 2019

All-trans-retinoic acid (ATRA) plays important regulatory roles in hematopoiesis and is successfully used in differentiation therapy of acute promyelocytic leukemia (APL). Although ATRA effectively inhibits growth and stimulates myelomonocytic differentiation of myeloid progenitors, it is equally potent in causing expansion of multipotent hematopoietic stem cells. Results of studies utilizing mice that lack expression of a specific RAR and/or RAR subtype specific retinoids, as well as work addressing the molecular pathogenesis of acute promyelocytic leukemia (APL), indicate that the effects of ATRA on hematopoietic stem cells and myeloid progenitors are differentially mediated via the RARγ and RARα, respectively. Using qPCR we have now shown that RARγ is expressed in human cord blood derived stem cells but not in more mature myeloid progenitors or myelomonocytic cells. This change in the RARγ expression levels is paralleled by a reciprocal change in expression of RARγ specific miRNA, which we have identified and validated using multiple experimental strategies, including RARγ 3′UTR based reporter assays. RARγ is also expressed in blasts derived from non-APL AML patients (over 80% of cases examined) and AML cell lines, but is not expressed in ATRA responsive APL cell lines. The expression of RARγ miRNA, on the other hand, is markedly decreased in AML blasts when compared to the levels detected in cord blood derived CD34+ myeloid progenitor cells. Taken together our results suggest that finely tuned and miRNA mediated down-regulation of RARγ expression in the myelomonocytic lineage provides a switch from pro-proliferation to RARα mediated pro-differentiation effects of ATRA. We predict that use of a RARα specific agonist, possibly in conjunction with a strategy that negatively targets RARγ (as with RARγ selective antagonist or siRNA), would be most effective in retinoid based differentiation therapy of non APL-AML.
Truncations in CCND1 mRNA 3′ UTR Alters microRNA Regulation in Mantle Cell Lymphoma.
Authors: Chen Robert W. , Bemis Lynne , Amato Carol , Diane Birks , Han Myint , William Robinson A.
Published: October 2019

Mantle Cell Lymphoma (MCL) represents only 5–10% of all non-Hodgkins lymphomas, making it an uncommon but difficult form of lymphoma to treat. It has a poor prognosis among the B cell lymphomas with median survival of three years. The genetic hallmark of MCL is the t(11,14) translocation causing amplification of cyclin D1 (CCND1), a known cell cycle regulator which is overexpressed in many other cancers. MicroRNAs (miRNA) are a new class of abundant small RNAs that play important regulatory roles at the post transcriptional level. They act by binding to the 3′ untranslated region (UTR) of mRNAs and block either their translation or initiate their degradation. Recent reports have shown truncations in the CCND1 3′ UTR occur in MCL and indicate a worse prognosis. We hypothesized that truncations in 3′ UTR of CCND1 alter it’s regulation by microRNAs. Based on bioinformatics, we identified microRNA 16 with putative docking sites in the 3′UTR of CCND1. Mir-16 has been implicated as a cell cycle regulator. We identified 2 cell lines (Jeko-1 and Z138) with truncations in CCND1 3′ UTR and demonstrated increased CCND1 mRNA expression by qRT-PCR, increased protein expression by western blot, and higher proliferative potential by cell cycle. We prepared a reporter construct by ligating the full length 3′ UTR of CCND1 to GFP. We then co-transfected this construct with mimics of mir-16 into a cancer cell line and demonstrated downregulation of CCND1 protein expression by flow cytometry. In the MCL cell line Granta-519 with non-truncated CCND1, transfection with mimics of mir-16 deminstrated decreased expression of CCND1 mRNA. These studies suggest that the overexpression of CCDN1 In MCL may result from altered regulation of gene expression from loss of a miRNA regulatory site and may give new clues into the patho-biology of this disease and insights into possible new therapies.
The Risk of Thrombotic Events in Neonates Treated with Recombinant Factor VIIa.
Authors: Puetz John , Darling Ginger , Brabec Petr , Blatny Jan , Mathew Prasad
Published: October 2019

Background: In recent years, recombinant factor VIIa (rFVIIa) has been used in non-hemophilia bleeding situations (factor VII deficiency, trauma, liver disease, uremia, surgical bleeding, platelet disorders, and intracranial hemorrhage) for achievement of hemostasis. Although, the risk of thrombosis in hemophilia patients with inhibitors receiving rFVIIa is quite low, its use in other clinical situations has been complicated by some reports of thrombotic events. Recently, rFVIIa has been used to treat coagulopathic and/or bleeding neonates with good success. However, the prevalence of thrombotic events in these neonates is completely unknown. This study was initiated to determine the risk of thrombotic events associated with rFVIIa use in neonates.

Methods: We reviewed all published literature in neonates receiving rFVIIa. In addition, we reviewed all data submitted to the SeveN Bleep Registry, a database developed by the scientific standardization subcommittee on pediatric and neonatal hemostasis of the International Society on Thrombosis and Haemostasis (ISTH) to record all uses of rFVIIa in pediatric non-hemophilic patients. As the baseline prevalence of thrombosis for bleeding and/or coagulopathic neonates is also unknown, we also reviewed the records of 100 consecutive neonates from a single institution who received fresh frozen plasma (FFP) alone to treat their coagulopathy and/or bleeding.

Results: A total of 98 non-hemophilic neonates received rFVIIa. The majority of these neonates received rFVIIa only after failing to achieve hemostasis with standard care (FFP, cryoprecipitate, platelet transfusions). Of those receiving rFVIIa, 7 had a thrombotic event reported. In the control group that received FFP alone, 7 neonates also suffered a thrombotic event. Although the risk of thrombosis in these two groups is similar, neonates receiving rFVIIa tended to have indwelling line related thrombosis, while those receiving FFP tended to have strokes or myocardial insults. Overall the prevalence of thrombotic events in bleeding and/or coagulopathic neonates appears to be 7%, whether or not they received rFVIIa.

Conclusions: In this study, the overall prevalence of thrombotic events was similar in the rFVIIa and FFP group. As data for this study was collected in a retrospective manor, and thereby subject to publication and submission bias, a more accurate determination of the prevalence of thrombosis in neonates will require a prospective study.
Thrombelastographic Whole Blood Coagulation Parameters Demonstrate Improved Clot Strength Induced by Tranexamic Acid during Cardiopulmonary Bypass.
Authors: Sørensen Benny , Christensen Thomas D. , Jepsen Mogens Harrits , Folkersen Lars , Severinsen Inge , Hjortdal Vibeke E. , Ingerslev Jørgen
Published: October 2019

Background: Tranexamic acid reduces the fibrinolytic activity, and may hereby reduce bleeding associated with surgery. However, to date no studies have been published that monitor and display the effect ex vivo. We hypothesized that tranexamic acid improves clot strength and overall coagulation capacity following cardiopulmonary bypass, and the aim of this study was to assess this effect ex vivo using thrombelastography.

Methods: Patients (N=8) undergoing elective coronary artery bypass grafting using cardiopulmonary bypass were randomly assigned to receive either preoperatively 1 gram of tranexamic acid or no tranexamic acid (control group). Blood specimens were collected before and immediately after cardiopulmonary bypass, and whole blood coagulation analysis was performed on a ROTEG® thrombelastograph employing activation with a reaction mixture of tissue factor (Innovin®, final dilution 1:50000 ∼0.15 pM) and single chain recombinant tissue plasminogen activator (4 nM). The coagulation signal was processed to obtain dynamic parameters of clot formation such as maximum velocity (MaxVel), time to maximum velocity (t, MaxVel), minimum velocity (MinVel), and time to minimum velocity (t, MinVel). Overall haemostatic capacity was evaluated using a calculated parameter denoted overall coagulation quality (OCQ) = (maxvel/t, MaxVel)*(t, MinVel - t, MaxVel)).

Results: The integrated mean difference in time (seconds (sec)) to minimal velocity was consistently positive in the tranexamic acid group compared to the control group (1139 sec (95% CI: 463 to 1667) vs −1474 sec (95% CI: −2454 to −588). The overall coagulation quality was compromised in both groups, but less in the tranexamic acid group (−6.98 (95% CI: −25.68 to 11.71) vs −15.31 (95% CI: −37.28 to 6.66)).

Conclusion: Ex-vivo monitoring of the effect of tranexamic acid during cardiopulmonary bypass is feasible, and patients receiving tranexamic acid produce a significantly more lysis resistant clot and a less compromised overall coagulation quality as compared to control patients.
Functional Platelet Counting in Patients with Renal, Haematological and Cardiological Disease Using Blood Collecting Bottles Coated with Collagen, Ristocetin and Arachidonic Acid.
Authors: Smith Graham , Sheridan-Smith Loida , Smith Margaret A. , Pambakian Sam , Campbell Jim
Published: October 2019

Platelet function is a vital factor in preventing bleeding irrespective of circulating platelet number. As a result, patients often have their surgery cancelled due to taking Aspirin or related medications. Similarly, patients bleed in the ITU setting when their counts and humoral coagulation are normal. This raises the concept of exhausted platelets which is often difficult to prove. The availability of a bedside technique which enumerates patients’ "functional" platelets, which is reliable and reproducible, represents a major step forward in determining whether these patients require platelet transfusion. Helena Laboratories, Beaumont, Texas, US have produced blood collection bottles which are coated with platelet agonists equine collagen (C), risocetin (R) or arachidonic acid (AA) in addition to 3.2mg sodium citrate. Our group is the first in Europe to use these bottles in association with point of care testing instrumentation (Horiba ABX Pentra, Chicksands, Beds, England). Samples were taken from normal, renal dialysis, cardiac catheterisation, haematological and ITU patients into K2EDTA and the above bottles and treated identically prior to counting. Samples taken into K2EDTA served as baseline controls whilst free platelets in the synchronously taken agonist samples represented non functional platelets (the normal functional ones having aggregated onto the inside wall of the tube). Data on 54 normal subjects showed that, when using C, only 6.2+/−3.1% of platelets were non functional (NF). Similar figures were found for R and AA. In dialysis patients with eGFR 4–25mls/min (n=32), the R NF% was 58.07+/−17.59%. In a similar group of renal patients on dialysis and Aspirin (n=12), the R NF% was 60.55+/−21.86% which was not statistically different from the non Aspirin group (p=0.955). In cardiac patients, all of whom were on Aspirin, C NF% was 35.23+/−23.12%, AA NF% 51.94+/−23.5% and R NF% 51.53+/−19.04%. C vs AA showed a significant p-value of 0.003and C vs R p=0.002. AA vs R was not significant at p=0.94. These data show that ristocetin is a surprisingly sensitive method of detecting non functional platelets in renal and Aspirin users. Classically, ristocetin is not thought of being a detector of Aspirin effect although data from Sloand et al. JASN (1997) 8(5),799 shows that ristocetin may detect renal effect on platelet GpIb-IX (CD42b). These data demonstrate the potential value of ristocetin aggregation ex vivo in determining the need for platelet transfusion and may provide clinicians with an improved and more logical rationale for such an expensive therapy.
Treatment of Established Graft-Versus-Host Disease by PG490-88.
Authors: Chen Benny J. , Liu Congxiao , Chao Nelson J.
Published: October 2019

Graft-versus-host disease (GVHD) is a major complication following hematopoietic cell transplantation. While prophylaxis of GVHD has improved, treatment for GVHD remains unsatisfactory. PG490–88, a semi-synthetic derivative of a novel compound purified from a Chinese herb, is very effective in prevention of both acute and chronic GVHD. In this study, we tested whether PG490–88 is effective in treating established GVHD. The study was performed in a clinically relevant MHC-matched and minor mismatched model (B10.D2 to BALB/c). BALB/c (H2d, Mls-2a, Mls-3a) mice were lethally irradiated (8.5 Gy) and transplanted with 1x107 bone marrow cells and 1x108 spleen cells obtained from B10D.2 (H2d, Mls-2b, Mls-3b) mice. PG490–88 was administered intraperitoneally (i.p.) daily at a dose of 0.0535 mg/kg/day starting on day +15 post transplantation when the clinical signs of GVHD were first observed. Therefore, all animals in both vehicle control and PG490–88-treated groups developed GVHD before the treatment was started. After treatment, there was no difference in the severity of clinical GVHD between the treated and the control groups. However, PG490–88-treated mice survived significantly longer than the mice in the vehicle control group (Figure, median survival time: 50 vs. 65 days, P<0.0001). These data suggest that PG490–88 as a single agent is active in the therapy of established GVHD. Further preclinical studies will determine optimal schedules and combinations that could be utilized in clinical studies.

Figure Figure
The Molecular Characteristics in CDR3 of TCR Vα and Vβ Genes Associated with cGVHD in Patients after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Wu Xiuli , Li Yangqiu , Zhu Kanger , Du Xin , Chen Shaohua , Yang Lijian , Geng Suxia , Lu Yuhong , Chen Jie
Published: October 2019

Successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) requires reconstitution of normal T-cell immunity. Chronic graft-versus-host disease (cGVHD) is one of the major complications following allo-HSCT. The poor reconstitution of T-cell immunity (including the reconstitution of recent thymic output function and T-cell receptor (TCR) repertoire) was associated with cGVHD. In the previous study, we found that cGVHD predicted low TCR rearrangement excision circles (TRECs) levels and slow naïve T-cell recovery. Because GVHD displayed as clonal proliferation of special T-cells clones, which was triggered by donor T cells to recognize the host’s allogene antigen, in the present study we analyzed the TCR Vα and Vβ repertoire and cloanlity in patients with cGVHD, in order to find the special T-cell clones associated with cGVHD and evaluate the molecular characteristics of the CDR3 of TCR Vα and Vβ repertoire of GVHD-associated T-cell clones. Peripheral blood mononuclear cells (PBMNCs) were obtained from 5 leukemia patients with cGVHD after allo-HSCT. The expression and cloanlity analysis of TCR Vα and Vβ repertoire were detected by RT-PCR and genescan technique. Six donors served as controls. Almost all of TCR Vα and Vβ repertoire with polyclonal pattern were identified in normal controls. However, the skew expression pattern of TCR Vα and Vβ repertoire could be detected in patients with cGVHD even more than 4 year after allo-HSCT. Among 29 Vα and 24 Vβ subfamilies, there were only 4∼12 Vα and 4∼11 Vβ subfamilies expressed in patients with cGVHD. Oligoclonal or monoclonal expanded T cells were identified in TCR Vα 2, 3, 6, 10, 12, 14, 15, 25, 26 and TCR Vβ 1, 3, 7∼9, 13, 17, 19, 20 subfamilies respectively. The CDR3 sequences were further analyzed and all the sequences were blasted by internet (http://www.ncbi.nlm.nih.gov) and confirmed that it belonged to specific TCR Vα or Vβ gene rearrangement. The lengths of CDR3 were ranged from 12 to 15 amino acids. The molecular characteristics of the CDR3 of TCR Vα and Vβ genes rearrangement were TCRVα 3 (new name: Vα 17*01)-N-Jα 48*01-Cα (motif: CATEVDFGNEKLIF), TCRVα 2 (new name: Vα 12–2*01)-N-Jα 20*01-Cα (motif: CAVNLNDYKLIF), TCRVβ 1 (new name: Vβ 9*01)-N-Dβ 2*01-N-Jβ 2–1*01-Cβ 2 (motif: CASSDPPETYNEQFF), TCRVβ 7 (new name: Vβ 4–3*01)-N-Dβ 1*01-Jβ 1–1*01-Cβ 1 (motif: CASSHESGNTEAFF). Some TCR subfamily genes shared similarity in CDR3 amino acid motif. The role of specific sequences of CDR3 of TCR Vα and Vβ repertoire and T-cell clones will be confirmed in vivo by animal models.
Factors Predicting Long Term Survival after Reduced Intensity Allogeneic Stem Cell Transplantation in Acute Myeloid Leukemia.
Authors: Craddock Charles F. , Tauro Sudhir , Gregory Joanne E. , Buckley Laura , Ward Janice , Kottaridis Panos , Thelouli Eleni , Thomson Kirsty , Milligan Donald , Das-Gupta Emma , Liakpopoulou Effie , Snowden John , Yin John , Peniket Andrew , Cook Mark , MacKinnon Stephen , Russell Nigel H.
Published: October 2019

Allogeneic stem cell transplantation (SCT) performed using a reduced intensity conditioning (RIC) regimen represents an important new treatment modality in older patients with high risk acute myeloid leukaemia (AML) whose outlook with conventional chemotherapy would be poor. However assessment of its role has been hampered by the absence of long term follow-up data. Furthermore factors determining survival after RIC SCT have not been rigorously defined. In order to examine the anti-leukemic activity of RIC allografts in more detail we have examined the outcome of 170 patients with AML transplanted using a uniform conditioning regimen over a ten year period. Long term follow-up data (maximum 119 months) was collected on patients transplanted using a conditioning regimen consisting of fludarabine (30 mg/m2 x 5 days), melphalan 140 mg/m2 x 1 day and alemtuzemab (10 mg x 5 days). The median age of transplanted patients was 54 years (range 18–71). 88 patients were in CR1 at the time of transplant, 63 CR2/3 and 19 had relapsed or refractory disease. Cytogenetic information was available on 149 patients and of these 33 patients had adverse risk and 116 intermediate risk cytogenetics by MRC criteria. 83 transplants were performed using an HLA identical sibling donor and 87 using a volunteer unrelated donor. The 100 day transplant related mortality was 9%. 29% of patients developed Grade II-IV acute GVHD and 22% chronic GVHD. The 3 year overall survival (OS) for the whole group was 48% and 3 year disease free survival (DFS) 45%. 20 patients remain in remission more than five years post-transplant. Survival was significantly influenced by status at transplant (p=0.01), patient age (p= 0.01) and presentation cytogenetics (p=0.05) as determined by multivariate Cox proportional hazards regression. The 3 yr OS for patients transplanted in CR1 or CR2 was 51% and 52% respectively compared to 13% for patients with relapsed/refractory disease. Three year OS for patients with intermediate risk cytogenetics was 52% compared with 34% for adverse risk patients. Three year OS for patients under 60 years was 51% compared with 36% for older patients. This study demonstrates the ability of RIC allografts to deliver encouraging long term disease free survival rates in high risk AML. Pre-transplant characteristics can be used to predict outcome after a RIC allograft and older patients with active disease at the time of transplant or adverse cytogenetics require novel strategies in order to improve long term survival.
Identifying NK-Cell Donors for Cell Therapy Based on Functional Phenotype.
Authors: Hurton Lenka , Siddik R. Iram , Singh Harjeet , Olivares Simon , Rabinovich Brian , Hildebrand William , Lee Dean , Champlin Richard , Shpall Elizabeth , Fernandez-Vina Marcelo , Cooper Laurence
Published: October 2019

Donor natural killer (NK) cells after haploidentical hematopoietic stem-cell transplantation (HSCT) and infusion of haploidentical NK-cells have demonstrated a therapeutic effect. NK alloreactivity resulting from appropriate Killer cell Ig-like receptor (KIR)-ligand disparity in human-leukocyte-antigen (HLA)-haplotype mismatched HSCT has resulted in improved engraftment and decreased incidence of leukemia relapse. Yet, not all patient-donor pairs benefit for an allogeneic NK-cell effect. To identify NK-cell donors with a suitable KIR-ligand mismatch, we have developed a functional assay to measure NK-cell killing through KIR-ligand interactions. NK-cell lysis of target cells is blocked by inhibitory KIR that recognize classical HLA class I allotypes and HLA mismatches of an altered allelic repertoire, as in haploidentical HSCT, leading to KIR-ligand mismatch and alloreactive NK cell-mediated target killing (Figure 1A). A cytotoxicity assay was developed based on the NK-cell target HLAnull 721.221 cells, and a panel of targets with enforced expression of HLA genes recognized by KIR. After the killing assay was optimized for high throughput and sensitivity, we used the panel of targets to determine whether bulk populations of donor NK cells could be predicted to kill based on KIR and HLA typing. The results demonstrate patterns of target-cell lysis for the KIR repertoires corresponding, for some donors, with predicted donor-versus-recipient NK-cell alloreactivity (Figure 1B). A relative inhibition of HLA+ target-cell lysis of >30% was associated with binding of KIR to introduced HLA class I molecules. The benefit of this assay to transplant physicians is a tool to actually measure phenotype (lysis), rather than relying on predictive models based on genotype. This assay will be combined with typing data to help identify donors with NK-cell killing function for recipients of haploidentical HSCT and infusion of haploidentical NK cells.

Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box). Figure 1. (A) Schematic of alloreactivity generated between NK cells that are KIR-ligand mismatched with targets. (B) Observed lysis of 721.221 cells, with enforced expression of HLA class I, by KIR-typed donar(box).
DCEP (Dexamethasone, Cyclophosphamide, Etoposide, Cisplatin) Followed by High-Dose G-CSF Is a Highly Efficient and Safe Regimen for Stem Cell Mobilization for Multiple Myeloma.
Authors: Gonsky Jason P. , Lendvai Nikoletta , Donato Michele L. , Rowley Scott D. , Pecora Andrew L. , Feldman Tatyana , Goy Andre , Goldberg Stuart , Siegel David S.
Published: October 2019

High dose chemotherapy with autologous stem cell rescue remains a standard therapy for multiple myeloma patients who can tolerate it. A mobilizing regimen for multiple myeloma should ideally allow for a high yield of CD34+ cells, provide anti-myeloma activity, be well tolerated, and have predictable kinetics regarding initiation of collection of stem cells. Higher numbers of infused autologous CD34+ cells allow for more rapid engraftment and lower incidence of transplant-related morbidity and mortality. The goal for patients with myeloma is to harvest enough CD34+ cells to provide at least two autologous transplants. Previous mobilization regimens utilized G-CSF alone or high-dose cyclophosphamide with G-CSF. However, high-dose cyclophosphamide (4–7g/m2) has only modest efficacy against myeloma and is associated with significant morbidity and up to 1–2% treatment-related mortality. DCEP (dexamethasone, cyclophosphamide, etoposide, cisplatin) is a well established regimen with good efficacy as salvage treatment for myeloma. Additionally, the use of DCEP with G-CSF for mobilization in myeloma has previously been reported to provide an average yield of approximately 6x106 CD34+ cells. We report our experience with DCEP and high-dose G-CSF in mobilizing 88 multiple myeloma patients since 2006. Our regimen consisted of 40 mg dexamethasone IV over 15 minutes x 4 days, cyclophosphamide 500 mg/m2, etoposide 40 mg/m2 (capped at 75 mg), and cisplatin 15 mg/m2 (capped at 25 mg), all continuous IV infusions over 24h x 4 days, with G-CSF starting 24–48h after completion of chemotherapy, administered SQ at 5 mcg/kg x 6 days followed by 10 mcg/kg daily until pheresis is completed. Over 80% of our patients were ready to initiate collection on day 14. Our goal for collection is 10–12x106 CD34+ cells to allow for two or three transplants using at least 4x106 CD34+ cells per transplant. Yields were excellent with a mean yield of 27x106 CD34+ cells, with a range of 7.3–130.5x106 CD34+ cells. 37/88 (42%) of patients required only one day of pheresis, with a mean yield of 34x106 CD34+ cells. 38/88 (43%) of patients required two days of pheresis. Only 15% of patients required more than two days of pheresis. Only 3 patients yielded fewer than 10x106 CD34+ cells (3%), and none yielded fewer than 5x106 CD34+ cells. In conclusion, this regimen is highly efficacious, offers excellent stem cell yields and predictable collection kinetics, can be administered on an outpatient basis, and is safe and well tolerated.
An Essential Role for ras in LPS Mediated Amplification of TREM-1 Induced Neutrophil Activation.
Authors: Radsak Markus P. , Haselmayer Philipp , Schild Hansjoerg
Published: October 2019

The triggering receptor expressed on myeloid cells 1 (TREM-1) is constitutively expressed on polymorphonuclear neutrophils (PMN), monocytes and macrophage subsets and has been recently identified as an important player in the innate inflammatory response to microbial infections and sepsis. Expression of TREM-1 and activation of PMN and monocytes is regulated in concert with microbial products via the recognition of Toll-like receptor (TLR) ligands such as bacterial lipopolysaccaride (LPS). However, it is currently unclear how the amplification of the inflammatory responses by TREM-1 and TLR agonists is mediated on an intracellular level. To this end, we were interested to identify signalling events leading to the synergistic activation of PMN upon TREM-1 and TLR ligation. Using pharmacologic inhibitors and western blot analyses we demonstrate that PI3 kinase, PLC-g and p38 MAP kinase are essential for the initiation of the respirator burst as an important and immediate effector mechanism of human PMN. Furthermore, the phosphorylation of PKB/Akt and MAP kinase ERK show a biphasic pattern upon TREM-1/TLR4 coligation indicating distinct activation mechanisms for these receptors. Interestingly, the TLR4, but not TREM-1 mediated respiratory burst was dependent on the activation of ras leading downstream to an augmented calcium flow and subsequently synergistic burst activity. Taken together, we provide a new mechanism how TREM-1 and TLR interact in PMN mediating enhanced activation. These results shed a new light on our understanding how the innate inflammatory responses are regulated and might contribute to the development of future concept for the treatment of a dysregulated immune response in severe inflammatory conditions such as sepsis.