bluuebunny/crossref_metadata_2025 huggingface dataset:
2-Phenylacetylenesulfonamide Upregulates Noxa and Induces p53-Independent Apoptosis of CLL Cells.
Authors: Steele Andrew J. , Prentice Archie , Cwynarski Kate , Hoffbrand A. Victor , Wickremasinghe R. Gitendra
Published: April 2020

The classical pathway of p53-mediated apoptosis involves transcriptional upregulation of pro-apoptotic proteins including Puma and Noxa. However, recent studies have identified a novel non-transcriptional mechanism mediated by direct binding of p53 to Bcl-2 or Bcl-XL at the mitochondrial surface and consequent neutralization of their anti-apoptotic function (Chipuk and Green, Cell Cycle, 2004; 3: 429–431). Induction of apoptosis of chronic lymphocytic leukaemia (CLL) cells by cytotoxic agents is strongly dependent on a functional p53 system and genetic changes which compromise p53 function result in drug resistance. We are therefore attempting to identify novel therapeutic agents which induce selective apoptosis of CLL cells by mechanisms independent of p53. Here we present data suggesting that 2-phenylacetylenesulfonamide (PAS; trivial name pifithrin μ) is a potential therapeutic agent for CLL. PAS was toxic to CLL cells, with a median LC50 of 10.72 μM (SD 3.8 μM n=20) as measured by a dye reduction cytotoxicity assay. Killing of CLL cells was by apoptosis, documented by the ability of 10–20 μM PAS to induce cytochrome c release from mitochondria (Fn2) to the cytosol (Fn1; Figure 1, upper), conformational change of Bax to a pro-apoptotic conformation, translocation of Bax from the cytosol to mitochondria (Figure 1, lower) and cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP). PAS toxicity was p53-independent, since p53-deleted CLL isolates were susceptible to killing. Furthermore, PAS was toxic towards leukemic cell lines with either inducible (Daudi), non-functional (Raji) or deleted (K562) p53. PAS treatment of CLL cells resulted in upregulation of Noxa and the subsequent displacement by Noxa of the pro-apoptotic Bim long (L) and extra-long (EL) isoforms from Bcl-2, as determined by western blot analysis of Bcl-2 immunoprecipitates (Figure 2). These observations contrast with those of Strom et al (Nat Chem Biol, 2006; 2: 474–479), who reported that PAS caused the displacement of p53 from mitochondria to the cytosol and thereby protected the cells from p53-dependent apoptosis. These differences may result from ectopic viral expression of p53 in the latter study whereas this protein was under normal cellular regulation in CLL cells. Normal T lymphocytes were relatively resistant to PAS, with IC50 values four times greater than those observed for CLL cells. In conclusion, our data suggest that PAS represents a novel class of agent which is toxic towards CLL cells via a mechanism involving p53-independent upregulation of Noxa and the subsequent unleashing of pro-apoptotic Bim from anti-apoptotic Bcl-2. This p53-independent toxicity may be of particular value in the treatment of drug-resistant CLL patients with compromised p53 pathways.

Figure 1. Figure 1. Figure 2. Figure 2.
Non-Relapse Mortality Associated with Autologous Stem Cell Transplant (ASCT).
Authors: Stotler Christy , Kalaycio Matt , Dean Robert M. , Sobecks Ronald , Copelan Edward A. , Andresen Steven , Pohlman Brad , Sweetenham John William , Rybicki Lisa , Brown Stacey , Bell Kim , Cherni Kelly , Bernhard Laura , Ferraro Christina , Bolwell Brian J.
Published: April 2020

Advances in supportive care have reduced early treatment related mortality with ASCT to approximately 1%. Registry data has shown that relapse is the cause of treatment failure in approximately 80% of patients. However, non-relapse mortality (NRM), over time, affects 20% of transplant recipients. Delayed NRM is poorly studied. To characterize the factors associated with NRM, we reviewed 1573 consecutive autologous transplants performed at the Cleveland Clinic from 1/1992 through 12/2005. This analysis included only adult patients (pts) receiving peripheral stem cells, busulfan based preparative regimens, single transplants, and diagnoses of NHL, HD and MM (n = 856). The median age was 49, 62% were male, and 30% received prior radiation therapy. The most common number of prior chemotherapy regimens was 2 (48%); the primary diagnosis was NHL (67%), HD (18%), MM (15%); and 90% had sensitive disease at the time of transplant. 471 (55%) are alive and 385 (45%) have died. Relapse was the most common cause of death, occurring in 303 (79%) patients. Non-relapse mortality occurred in 82 patients (21% of deaths). The most common cause of NRM was pulmonary toxicity, occurring in 26 patients, followed by secondary malignancy in 19 pts, infection (12 pts), cardiac toxicity (7 pts), other organ failure (7 pts), and other causes (11 pts). Patients who died from secondary malignancy were significantly more likely to have received prior radiation therapy (p = 0.004), to require more days of pheresis to collect stem cells (p<0.001) and had a longer time to platelet engraftment than did other patients (p<0.001). 16/19 deaths due to secondary malignancy were from hematologic malignancies. Causes of death in the “other” category included accidents (n=3), homicide (n=1) and suicide (n=1). Liver toxicity was the most common organ affected in the “other organ failure” category. 46% of patients with NRM due to pulmonary toxicity had a prior exposure to radiation therapy, as did 43% of those with cardiac toxicity. The time to development of NRM is shown below:

Figure Figure

The substantial majority of NRM due to pulmonary toxicity, other organ failure or infection occurs within one year post-transplant. This suggests a need for more intensive surveillance of recipients of autologous transplantation for 12 months, possibly with regular visits to the transplanting institution. We have adopted more stringent follow up with promising early results at reducing 1 year NRM. The median time of NRM from secondary malignancy is 3.5 years, which suggests that surveillance of blood counts should continue well beyond one year post-transplant. Whether more aggressive surveillance will reduce NRM associated with ASCT requires further study.
Induction of GLUT1 during Erythropoiesis Triggers a Switch from Glucose to DHA Uptake in Vitamin C-Defective Mammals.
Authors: Montel-Hagen Amelie , Kinet Sandrina , Manel Nicolas , Mongellaz Cedric , Prohaska Rainer , Battini Jean-Luc , Delaunay Jean , Sitbon Marc , Taylor Naomi
Published: April 2020

Glucose provides a key supply of energy and carbon and its transport is achieved via multimembrane-spanning glucose transporters (GLUTs). The human erythrocyte is the cell type expressing the highest level of the GLUT1 glucose transporter, harboring greater than 200,000 molecules per cell. We now demonstrate that GLUT1 transcripts increase by 3-logs during erythropoiesis and high GLUT1 surface expression is observed following passage through the basophilic erythroblast stage. Paradoxically though, glucose transport significantly decreases. As GLUT1 also transports L-dehydroascorbic acid (DHA), the oxidized form of ascorbic acid (AA), transport of this molecule was assessed and indeed, it increases dramatically during erythropoiesis. The switch from glucose to DHA transport is coupled to the physical association of GLUT1 with stomatin, an integral erythrocyte membrane protein. We find that stomatin inversely regulates the relative transports of glucose and DHA by GLUT1. Moreover, in a patient with overhydrated hereditary stomatocytosis, a rare genetic disorder of red cell membrane permeability wherein stomatin is absent, glucose uptake is significantly higher but there is a concomitant 50% reduction in DHA transport. Intriguingly, erythrocyte-specific DHA transport is not conserved amongst all mammalian species; we did not detect GLUT1 on mature murine erythrocytes where DHA uptake is minimal. Notably though, humans differ from the vast majority of the greater than 5,000 mammalian species in that they are unable to synthesize ascorbic acid from glucose. This trait is shared only with other higher primates, guinea pigs and and fruit bats. We have determined that erythrocyte GLUT1 expression and associated DHA transport are specific features of these diverse ascorbic acid-deficient mammals. Within the primate order, defective ascorbic acid synthesis is due to an inactivation of the L-gulonolactone oxidase (GLO) enzyme at the Haplorrhini-Strespsirrhini split. In accord with the data presented above, we find that erythrocyte GLUT1 expression and associated DHA transport are characteristic of erythrocytes from primates within the Haplorrhini suborder but not Strepsirrhini lemurs. Thus, the erythrocyte-specific co-expression of GLUT1 and stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize the essential ascorbic acid metabolite.
Clinical and Haematologic Features of 300 Patients Affected by Hereditary Spherocytosis as a Function of the Type of the Membrane Protein Defect.
Authors: Barcellini Wilma , Mariani Mariagabriella , Vercellati Cristina , Marcello Anna P. , Fermo Elisa , Pedotti Paola , Boschetti Carla , Zanella Alberto
Published: April 2020

Hereditary Spherocytosis (HS) is caused by defects of red cell membrane proteins (spectrin, ankyrin, band 3 and band 4.2). The aim of this study is to analyse a large database of 300 HS patients grouped according to SDS-PAGE, 1) to ascertain whether the clinical/haematological features and response to splenectomy are related to the type of molecular defect, and 2) to compare the sensitivity of the most common laboratory screening tests for HS in the various subsets of patients. Three hundred consecutive patients were investigated; 41 patients had been splenectomised before the time of the study, and 21 thereafter. In not splenectomised subjects, anaemia was severe in 7% of cases, mild to moderate in 52% and compensated in the remaining cases. The median number of spherocytes at the peripheral blood smear examination was 7%. The most frequent protein abnormalities revealed by SDS-PAGE were band 3 (54%) and spectrin and/or ankyrin (34%) deficiency. The membrane protein defect was undetectable in 3% of splenectomised versus 11% of not splenectomised patients. In patients evaluated by SDS-PAGE before and after splenectomy surgery allowed the identification of the defect (one band 3 and seven spectrin/ankyrin deficiency) in all the 8 previously unclassified cases. No significant differences were observed among clinical and haematological parameters of patients grouped according to the type of biochemical defect, although the degree of anaemia, haemolysis markers and median spherocyte number were higher in spectrin deficient than in the other groups of patients. Splenectomy was clinically effective in correcting both anaemia and haemolysis, but splenectomised spectrin/ankyrin deficient patients showed after splenectomy a slightly lower median rise in haemoglobin, and a higher reticulocyte number and unconjugated bilirubin level than band 3 deficient patients. The red cell osmotic fragility tests’ sensitivity varies greatly and ranged from 48 to 95%. The sensitivity was similar in patients with band 3 and spectrin/ankyrin deficiency and also in patients without detectable membrane defect. Furthermore, the sensitivity of all the methods investigated increased in splenectomised cases. AGLT displayed the highest sensitivity, and the association of AGLT and NaCl test on incubated blood reached a sensitivity of 99%, enabling the diagnosis of the atypical HS cases, such as those with rare or no spherocytes in blood smears, normal MCHC and reticulocyte counts.
Bacterial Omptins Proteolytically Inactivate Tissue Factor Pathway Inhibitor (TFPI).
Authors: Yun Thomas H. , Cott Jessica , Tapping Richard I. , Slauch James M. , Morrissey James H.
Published: April 2020

Activation of the clotting system is an effector function of the immune response, resulting in extravascular fibrin deposition whose purpose appears to be to limit the spread of invasive microorganisms. Activation of the clotting cascade may also modulate inflammatory reactions. Some bacteria are known to counteract the host coagulant response by modulating specific components of the clotting system, thereby promoting fibrinolysis or inhibiting coagulation activation. Pla, a member of the omptin family of Gram-negative outer membrane proteases, is a known virulence factor for the plague agent, Yersinia pestis. Pla’s ability to activate plasminogen by limited proteolysis is thought to facilitate the organism’s escape from fibrin meshworks, promoting its dissemination through extravascular tissues. We now show that Pla, as well as its homologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium, abrogate the function of the endogenous anticoagulant, tissue factor pathway inhibitor (TFPI). Using gene deletions, we found that bacterial inactivation of TFPI required the expression of these omptins. Consistent with prior studies of other omptin substrates, cleavage of TFPI by the Salmonella omptin was demonstrable only in rough LPS backgrounds. Western blotting experiments showed that proteolysis of TFPI correlated with loss of TFPI anticoagulant activity in clotting assays. The specificity of the omptin/TFPI interaction is supported by the finding that both common and distinct TFPI cleavage fragments are generated by the omptins in these three bacterial species. Furthermore, TFPI inactivation proceeds even in plasma where an abundance of competing protein substrates would be expected to render TFPI proteolysis almost non-existent if the reaction were not specific. We hypothesize that the sensitivity of TFPI to inactivation by bacterial omptins is a novel host-pathogen interaction that potentiates the procoagulant immune response to bacterial infection. With the heavy bacterial loads present in the circulation during the late stages of severe septicemia, this interaction may contribute to the development of disseminated intravascular coagulation and end organ failure.
Thrombin-Releasable Protein Z-Dependent Protease Inhibitor Identified in Platelets and Is Likely Due to Uptake from Plasma.
Authors: Han Xin , Nguyen Martin H. , Fernandez Michael , Broze George J. , Bueso-Ramos Carlos E.
Published: April 2020

Protein Z (PZ) is a vitamin K-dependent protein, whereas PZ-dependent protease inhibitor (ZPI) is a member of the serine protease inhibitor superfamily. ZPI rapidly inhibits factor Xa in the presence of PZ, calcium, and phospholipids and inhibits factor XIa in a PZ-independent fashion (Blood2000; 96:3049–3055). PZ circulates as a complex with ZPI in plasma and deletion of either the PZ or the ZPI gene is associated with the prothrombotic phenotype in mice. In addition, W303X or R67X nonsense mutations in the ZPI gene are reportedly associated with deep venous thrombosis in certain human populations. Western blot analysis of platelets stimulated with thrombin (0–200 mU/mL) showed they contained and released ZPI (approximately 200 ng/109 platelets) with the same molecular weight as plasma ZPI (72 kDa). The majority of the ZPI was released within 1 min. by 25 mU/mL thrombin. PZ was not detected in platelets by western blot analysis. Immunohistochemical staining using a monoclonal anti-ZPI antibody demonstrated a cytoplasmic fine granular staining pattern in maturing megakaryocytes in bone marrow aspirates and in circulating platelets, suggesting that ZPI may be stored in alpha granules. ZPI mRNA, however, was not detected by reverse transcriptase polymerase chain reaction (RT-PCR) in platelets or bone marrow aspirates, but was detected in human liver cDNA. RT-PCR for platelet factor 4 and glyceraldehyde 3-phosphate dehydrogenase mRNA showed amplified products with expected sizes. In conclusion, thrombin-releasable ZPI, but not PZ, is present in platelets and is most likely derived from the uptake of ZPI from plasma. ZPI released from activated platelets may play a role in the regulation of local coagulation at a site of injury.
Genomic, Proteomic and Functional Screens Identify PAI-1 as an Important Factor Influencing Mesenchymal Stromal Cell Survival Post-Transplantation In Vivo.
Authors: Copland Ian B. , Cuerquis Jessica , Wang Eugenia , Galipeau Jacques
Published: April 2020

MSCs have robust reparative properties through their ability to limit apoptosis, enhance angiogenesis and direct positive tissue remodelling. However, a major limitation on the widespread application of MSC transplantation is that transplanted cells have low survival rates in vivo. Thus, enhancing MSCs survival post-transplantation is critical for the successful implementation of cellular therapy. One explanation for their low survival is that MSCs are often transplanted into ischemic tissue - such as infarcted myocardium - where there is poor blood supply and low oxygen tension. How these factors influence MSC survival is still unclear, therefore the objective of this project was to better understand how hypoxia under low nutrient conditions affects the behaviour and survival of MSCs. To answer this question, we performed the following series of experiments:

Isolate and immunophenotype murine MSCs. Assess impact of in vitro ischemic conditions (hypoxia and serum deprivation) on MSCs by microarray analysis and ITRAQ proteomics for secreted proteins. Confirm changes of specific factors in vitro. Isolate and immunophenotype MSCs from knock-out mice for specific factors. Determine whether knock-out MSCs have altered survival in vitro and in vivo.

As confirmed by PCR and Western blotting, microarray and proteomic screens, identified plasminogen activator inhibitor-1 (PAI-1) as one factor consistently upregulated in hypoxia (3% oxygen) treated MSCs. In vitro migration studies demonstrated that PAI-1 blocks migration of MSCs towards a chemotactic gradient, while isolation of PAI-1 knock-out MSCs, revealed an enhanced survival of PAI-1null MSCs over wild-type MSCs. In vivo subcutaneous transplantation of beta-galactosidase engineered wild-type and PAI-1null MSCs, recapitulated our in vitro results, such that 14 days post-implantation PAI-1null MSCs had a five fold increase in survival compared to wild-type MSCs. In conclusion, PAI-1 is a multifunctional protein. As an inhibitor, PAI-1 acts as ‘bait’ regulating the conversion of plasminogen to plasmin thus functioning as a major control point in the regulation of systemic fibrinolysis and local growth factor availability. In addition, PAI-1 can also act as a ligand for vitronectin to influence cellular migration/adhesion. Up-regulation of PAI-1 by MSCs after transplantation impacts MSC survival. Determining how to block PAI-1 following transplantation of MSCs is currently under investigation.
AML1-ETO9a Correlated with C-KIT Overexpression/Mutation and Indicated Poor Disease Outcome in t(8;21) Acute Myeloid Leukemia.
Authors: Liang Yang , Jiao Bo , Wu Chuan-Feng , Xiong Shu-Min , Gu Long-Jun , Chen Zhu , Chen Sai-Juan
Published: April 2020

AML1-ETO fusion gene is generated from chromosome translocation t(8;21) in acute myeloid leukemia (AML). It is recently reported that its spliced variant AML1-ETO9a induced leukemia rapidly in murine model. Here we detected AML1-ETO9a expression in 58 of 71 (81.7%) patients with t(8;21) AML by using qualitative RT-PCR. Quantitative RT-PCR revealed significantly higher C-KIT levels in the AML1-ETO9a positive group than in the negative group (P=0.0001). Among 29 cases harbored C-KIT mutations, 28 (96.6%) expressed AML1-ETO9a. Clinically, patients expressing AML1-ETO9a exhibited significantly elevated white blood cell counts and a short overall survival time (P=0.0455 and 0.0039, respectively). Morphologically, although there is no difference of leukemic blasts percentage between AML1-ETO9a positive and negative cases (P=0.1169), the latter possessed more aberrant myelocytes (P=0.0462). Taken together, AML1-ETO9a correlated with C-KIT overexpression/mutation and indicated poor disease outcome in t(8;21) AML.
Thromboprophylaxis for Catheter-Related Thrombosis in Patients with Cancer: A Systematic Review.
Authors: Carrier Marc , Tay Jason , Fergusson Dean , Well Philip S.
Published: April 2020

Background Patients with malignancy have an overall 4-fold increased relative risk of developing a venous thrombosis. Central venous catheters are commonly used in patients with cancer (chemotherapy, blood products, parenteral nutrition, etc) and further increase the absolute risk of thrombosis in this population. Thromboprophylaxis to prevent catheter-related thrombosis in patients with cancer is controversial. Both low-molecular-weight-heparin (LMWH) and low dose warfarin have been studied in adults. We sought to summarize and quantify the clinical effects of LMWH and low dose warfarin to prevent catheter-related thrombosis in patients with cancer.

Methods A systematic literature search strategy was used to identify potential trials on MEDLINE (1950 to January week 4 2007), EMBASE (1980 to 2007 week 5), the Cochrane Register of Controlled Trials (1st quarter 2007) and all EBM Reviews (4th quarter of 2006) using an OVID interface. RCTs were evaluated using a previously validated scale (Jadad scale) and assessed for allocation concealment. The primary outcome measure was symptomatic catheter-related thrombosis. Secondary outcome measures included: clinically significant catheter-related thrombosis, death and bleeding episodes. Relative risk (RR) was used as the primary measurement with 95% confidence intervals (CIs). Pooled measurements were calculated for randomized clinical trials using a random-effects model.

Results In total, 510 records were identified and 16 records (14 articles and 2 abstracts) were deemed potentially eligible. 6 articles and one abstract met our inclusion criteria; 3 investigated LMWH and 4 low dose warfarin. Three studies were double-blinded and 2 reported on allocation concealment. The pooled RR of symptomatic catheter-related thrombosis for LMWH thromboprophylaxis was 0.43 (95% CI: 0.12–1.56). The pooled RR of symptomatic catheter-related thrombosis for low-dose warfarin thromboprophylaxis was 0.82 (95% CI: 0.46–1.47). All RCTs also reported the proportion of clinically significant catheter-related thrombosis. The pooled RR was 0.42 (95% CI: 0.12–1.42) and 0.74 (95% CI: 0.41–1.32) for LMWH and warfarin respectively. Very few studies reported any adverse events (death, major and minor bleeding).

Conclusions Based on the available data, we cannot recommend routine thromboprophylaxis for patients with cancer receiving a central venous catheter. Further studies with a greater number of enrolled patients are required before we can draw firmer conclusions regarding its utility.
The Impact of Histological Subtypes on Outcome in Patients with Mantle Cell Lymphoma Treated with or without Autologous Stem Cell Transplant.
Authors: Zulfiqar Muhammad I. , Weisenburger Dennis D. , Loberiza Fausto R. , Vose Julie M. , Bierman Philip J. , Bociek R. Gregory , Armitage James O.
Published: April 2020

Mantle cell lymphoma (MCL) accounts for 7% of all non-Hodgkin’s lymphomas, with median overall survival in most series of 3–4 years. MCL has been classified into three histological subtypes which include diffuse MCL, nodular MCL, and blastic MCL. A relatively small number of studies have examined the prognostic importance of histology in MCL. The aim of this study was to determine if the progression free survival (PFS) and overall survival (OS) rates in mantle cell lymphoma differ among histological subtypes. A total of 102 patients with MCL, treated by the Nebraska Lymphoma Study Group between January 1986 and June 2006, with a median age of 60 years (range 32–89 years) were available for study. Patients were treated with HyperCVAD or a CHOP like regimen with or without rituximab and autologous hematopoetic stem cell transplant (ASCT). All cases were confirmed using cyclin D1 staining. Regardless of treatment, our study failed to show a significant difference in PFS (p=0.26) or OS (P=0.06) among histological subtypes. There was a trend for better survival in patients with nodular MCL. However, in patients receiving ASCT, there was a significantly higher PFS (P=0.0001) and OS (p=0.0005) compared to patients not receiving ASCT. The 3 year PFS for patients receiving HyperCVAD followed by ASCT was 64% compared to the 3 year PFS for HyperCVAD alone of 0 (p=0.008). In conclusion, we failed to show association between histological subtypes of MCL with outcomes regardless of treatment. However, the use of ASCT improved survival.
Risk Factors Analyses for Outcomes after Unrelated Cord Blood Transplantation for Children with Myelodysplastic Syndromes. An Analysis on Behalf of EWOG-MDS and Eurocord Groups.
Authors: Moreno-Madureira Adrienne B. , Teira Pierre , Locatelli Franco , Piccardi Alessandra , Woolfrey Ann , Chan Ka-Wah , Socié Gérard , Vora Ajay J. , Bertrand Yves , Bonfim Carmem , Garnier Federico , Ionescu Irina , Gluckman Eliane , Niemeyer Charlotte M. , Rocha Vanderson
Published: April 2020

Myelodysplastic syndromes (MDS) are very rare diseases in children and hematopoietic stem cell transplantation remains the only curative treatment. This is a retrospective registry-based study of 42 children with MDS (refractory cytopenia n=21, refractory anemia with excess of blasts n=15, and refractory anemia with excess of blasts in transformation n=6), who underwent unrelated cord blood transplantation (UCBT). Both databases from Eurocord and EWOG-MDS group were used. Diagnosis of MDS was established by each reporting center based on the WHO classification modified for pediatric patients. Median age was 7.4 years, median cell dose infused was 4.2 × 107/Kg and median follow-up was 39 months. All patients received one cord blood unit, with 0 or 1 HLA disparity in 22, and 2 or more in 20. Twenty-five children received a Busulfan-based conditioning regimen. The association of Cyclosporine A and prednisone was used in 69% of patients. The 60-day cumulative incidence (CI) of engraftment was 69±7%, in a median time of 25 days. In multivariable analysis, ABO match (Hazard Ratio=2.94, 95% Confidence Interval=1.23–6.66, p=0.01) and refractory cytopenia (HR=2.38, 95% Confidence interval=1.01–5.55, p=0.05) were favorable factors associated with neutrophil recovery. Thirteen patients developed acute GVHD grades II–IV. Moreover, 16 children had skin, 5 liver, 6 gastrointestinal involvements (stages 2–4). The 100-day CI of aGVHD was 29±7%. Of 20 evaluable patients, 10 developed chronic GVHD and the 1-year CI of cGVHD was 24±7%. The 2-y CI for treatment-related mortality was 56±8%. In univariable analysis, recipient’s weight was the only statistical significant factor associated with TRM (47% for children weighting less than < 38Kg versus 80% for remainders, p=0.05). In addition, there was a trend of higher TRM for patients who received CBU with 2 or more HLA disparities (p=0.09). The 2-y overall survival and disease-free survival were 37±8% and 32±8% respectively. In multivariable analysis, OS improved when recipient’s weight at UCBT was lower than 38kg (HR= 2.53, 95% confidence interval=1.16–5.55; p=0.02). In conclusion, since hematopoietic stem cell transplantation is the only treatment with curative potential, UCBT is a reasonable option for children without an identical sibling donor. Moreover, outcomes might be improved with better donor and patient’s selection.
GPVI Mutation and Absent Platelet Collagen Activation in a Patient with a Bleeding Disorder.
Authors: Freson Kathleen , Hermans Cedric , Thys Chantal , Van Geet Christel
Published: April 2020

The physiological relevance of the collagen GPVI receptor was known prior to its recognition as a platelet membrane receptor since several patients lacking GPVI as a consequence of autoantibody inhibition present with a mild bleeding diathesis. Remarkably, patients with a proven GPVI gene mutation could not yet been identified. We here describe a female patient with ecchymoses, epistaxis, several posttraumatic and postsurgery bleeding complications since her childhood and menorraghia. Coagulation and vWF studies were normal. The patient has a normal platelet count and electron microscopy showed no structural platelet abnormalities. All standard platelet aggregations were normal except for an absent response to Horm collagen, convulxin and the collagen-related peptide even at high concentrations of these agonists. Platelet ATP secretion was normal for ADP stimulation but absent for collagen. The mechanism of this dysfunction was further analyzed. Flow cytometric analysis of the different platelet membrane receptors showed a normal expression of integrin alphaIIb/beta3, integrin alpha2 and GPIbalpha but a completely absent membrane expression of GPVI. In contrast, immunoblot analysis of total platelet lysates showed a 50% reduced expression of both GPVI and its binding partner Fcgamma. Molecular analysis of the immunoreceptor GPVI/Fcgamma revealed no abnormalities in Fcgamma gene, except for the presence of Fcgamma mRNA with exon 2 skipping which could also be detected in control platelets. The patient was heterozygous for the known SKTQH/PEALN functional GPVI haplotype (Joutsi-Korhonen et al, Blood, 2003) but importantly also carried a heterozygous out-of-frame 16 bp deletion in exon 3 of GPVI gene. Subcloning and sequencing of the platelet GPVI mRNA resulted in only 25% of clones without the exon 3 deletion. The normal GPVI allele carried in addition to the SKTQH haplotype also 3 SNPs in the 3′UTR of the GPVI gene. The GPVI downstream intracellular signaling and collagen adhesion studies for this patient are ongoing. This study presents the first patient with a genetic GPVI defect with in contrast to patients with an autoantibody against GPVI a normal platelet count and ultrastructural morphology.
Long-Term Follow-Up of BEACOPPescalated Chemotherapy in Patients with Advanced-Stage Hodgkin Lymphoma on Behalf of the German Hodgkin Study Group.
Authors: Andreas Engert , Franklin Jeremy , Diehl Volker
Published: April 2020

The HD9 trial of the German Hodgkin Study Group (GHSG) compared two different doses (baseline and escalated) of the novel chemotherapy regimen BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone) in patients with advanced-stage Hodgkin lymphoma (HL). The previous analysis with a median follow-up of 5 years showed improved tumor control (FFTF) and overall survival (OS) for BEACOPPescalated. Since BEACOPPescalated had been associated with more toxicity as compared with ABVD we report the results of long-term follow-up of 1196 patients enrolled and randomized in that study. The median follow-up was 112 months; a total of 370 centres contributed. Patients received one of three chemotherapy regimens: 8 cycles of COPP (cyclophosphamide, vincristine, procarbazine, and prednisone) alternating with ABVD (doxorubicin, bleomycin, vinblastin, and dacarbazine), 8 cycles of standard-dose BEACOPP or 8 cycles of escalated-dose BEACOPP. At 10 years, FFTF rates were 64%, 70% and 82%, OS rates were 75%, 80%, and 86 for COPP-ABVD (arm A), BEACOPPbaseline (arm B), and BEACOPPescalated, respectively (p < 0,001). Importantly, BEACOPPescalated was also significantly better than BEACOPPbaseline in terms of FFTF (p < 0.0001) and OS (p = 0.0053). Death due to HL occurred in 11.5%, 8.1% and 2.8% in arms A, B, and C, respectively. 74 second malignancies were documented, including secondary acute myeloid leukaemias (1,7,14), Non-Hodgkin lymphoma (7,8,5), and solid tumors (7,16,9) in arms A, B, and C respectively. The corresponding overall secondary malignancy rates were 6.7%, 8.9% and 6.8%. Importantly, the risk of secondary AML (sAML), although increased in this study after BEACOPPescalated, amounts to 0.9% in our follow-up study with BEACOPPescalated (HD12) in 1502 advanced-stage HL patients randomized and four years median follow-up. Although the higher rate of secondary AML after BEACOPPescalated in HD9 most likely occurred by chance, interestingly, 70% of patients in this group had additional radiotherapy whereas only 39% were radiated in HD12. Taken together, the 10 years follow-up of BEACOPPescalated chemotherapy demonstrates a stabilized significant improvement in long-term FFTF and OS for advanced-stage HL. Although for formal prove the results of ongoing prospective randomized comparisons with 8 cycles of ABVD might be required, these results clearly challenge ABVD as standard of care for this patient population.
FDG-PET for Assessment of Residual Tissue after Completion of Chemotherapy in Hodgkin Lymphoma - Report on the 2nd Interim Analysis of the PET Investigation in the Trial HD15 of the GHSG.
Authors: Diehl Volker , Kobe Carsten , Haverkamp Heinz , Dietlein Markus , Engert Andreas
Published: April 2020

Introduction The prospectively randomized HD15 multicenter trial of the German Hodgkin Study Group (GHSG) included advanced-stage Hodgkin lymphoma patients comparing 8 cycles of BEACOPPescalated with 6 cycles or 8 cycles time-condensed BEACOPPbaseline. One other main study endpoint was the prognostic value of 18F-fluorodesoxyglucose (FDG) positron emission tomography (PET) following chemotherapy. The aim was to specify the negative predictive value of PET (NPV) in patients with residual tumour mass after chemotherapy.

Methods Entry criteria for the PET question were partial remission (PR) after the end of chemotherapy with at least one involved nodal site measuring more than 2.5 cm in diameter by computed tomography (CT). Exclusion criteria included diabetes, elevated blood sugar levels and skeletal involvement with risk of instability. Calculations were restricted to those cases with either progressive disease (PD) or relapse within 12 months after PET or at least 12 months of follow-up. A total of 275 patients were eligible for this analysis. The assessment was based on those patients confirmed by an expert panel as being PET-negative. CT verification was performed to identify false positive PET findings. The NPV was defined as the proportion of patients without progression or relapse within 12 months.

Results 9/216 patients with PET-negative residues and 9/59 patients with PET-positive residues had PD or relapse within one year of follow-up. The NPV was 0.958% (95% CI 0.931 – 0.985%). In 244/245 cases with PET-negative residual masses, no irradiation was given. In the 62/66 cases with PET-positive residues, additional radiotherapy was performed. Progression/relapse rates were significantly different between those patients with residual mass being PET-negative or PET-positive (p=0.0053). PET-negative patients, who were assessed as partial response by CT, had a prognosis similar to those in complete remission. There was no significant difference in the progression free survival in this trial and the prior GHSG trials HD12 (arms pooled) and HD9 (arm C) for advanced-stage HL (p=0.266). Importantly, the proportion of patients receiving radiotherapy decreased from 70% (HD9-C) to 39% (HD12) and 12% (HD15).

Discussion The high NPV of PET suggests that radiotherapy following 6 or 8 cycles of BEACOPP might be restricted to those patients who are PET-positive after chemotherapy.
FDG-PET Guided Consolidative Radiotherapy in Patients with Advanced Stage Hodgkin Lymphoma with Residual Abnormalities on Post Chemotherapy CT Scan.
Authors: Savage Kerry J. , Connors Joseph M. , Wilson Don , Klasa Richard , Shenkier Tamara , Hoskins Paul , Voss Nicholas , Sehn Laurie H.
Published: April 2020

Consolidative radiotherapy (RT) is often administered following chemotherapy for advanced stage Hodgkin lymphoma (HL) with bulky disease at diagnosis or for residual abnormalities on post-chemotherapy CT imaging. It is unknown whether RT is necessary for such patients if the residual mass is FDG-PET-negative (PET-neg) following chemotherapy (CHT). Further, it has been previously shown that a post-therapy PET-positive (PET-pos) scan is highly predictive of future relapse; however, it is unclear whether consolidative RT can be used to salvage these cases.

Methods Since July 2005, adult (> 16 y) patients in British Columbia (BC) with advanced stage HL (stage III /IV and/or the presence B symptoms and/or bulky disease (> 10cm)) have been treated with 6 cycles with ABVD followed by a PET scan for further treatment planning if residual abnormalities ≥2 cm were observed on CT imaging. Prior to this, post-treatment PET scans were obtained in some individuals by self-pay. If the PET scan was positive, RT was administered if feasible, otherwise, patients were observed. There were 68 eligible patients, including 7 self-pay patients, that were identified in the BC Lymphoid Cancer Database. 16 patients were excluded (PET not performed (5); palliative tx or refusal (3); composite lymphoma (1); progressive disease during CHT or at the time of PET scan (4); HIV + (1); PET mid-therapy (1); death due to unrelated cause prior to PET scan (n=1).), leaving a total of 52 patients who had a PET scan post-chemotherapy for a residual mass and who had adequate follow-up (median 19 m, 10–59 m). Patient characteristics include: M:F 1.1:1, Median age 29 (17–81); 55% stage II, 46% stage III/IV 44%; 49% bulky disease; 69% B symptoms; IPFP Prognostic score 0 8%; 1 39%; 2 26%; >3 26%.

Results In total, 40/52 (77%) were PET-neg and 12/52 (23%) were PET-pos (SUV mean 4.4, 2.2–6.8). PET-pos patients had an inferior 2 y PFS compared to PET-neg patients (26% vs 91%, p=.001). In the PET-pos group, 10/12 received the planned RT and 5 have relapsed. In the PET-neg group, there was no difference in the 2 y PFS in patients with bulky (n=17) vs non-bulky disease (n=20) (86% vs 94%, p=.35).

Conclusions Advanced stage HL patients who achieve a post-CHT PET-neg status are at a low risk of relapse and do not require additional consolidative RT thus avoiding potential long-term side effects. The majority of patients with bulky disease who have a PET-neg scan following CHT also remain in remission, however, longer follow-up is still needed. Although some patients with a positive PET-scan may be salvaged by consolidative RT, a high proportion ultimately relapse.

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Analysis of Class I and II Histone Deacetylase Fails To Identify a Human Leukemia Specific Expression Profile.
Authors: Garcia-Manero Guillermo , Quesada Andy S. , Maddipoti Shirisha , Kuang Shaoqing , Tong Weigang , Wierda William G. , Bueso-Ramos Carlos M. , Yang Hui
Published: April 2020

Histone deacetylase (HDAC) inhibitors are being developed clinically for the treatment of leukemia. Because HDACs are composed of a large number of different proteins, and substrate specificity may differ among different HDAC inhibitors, it is important to understand if human leukemias are characterized by specific HDAC expression patterns. To study this, we have analyzed using real-time PCR and Western blots, all major class I and II HDACs in normal marrow controls (N=13, including 10 CD19+ B cell specimens), leukemia cell lines (N=25), samples from patients with AML (N=6), MDS (N=12), CLL (N=10) and human samples (N=6) obtained pretreatment and sequentially from patients with leukemia treated with two different phase I clinical trials of HDAC inhibitors: MGCD0103 and vorinostat. In general, normal controls were characterized by low levels of HDACs 1 to 10, although normal CD19+ B cells exhibited a significant increased expression of HDAC1 and 5. In leukemia cell lines, HDAC 1, 2 and 3 were expressed at higher levels than 4 to 10 but there were no differences between leukemia cell lines and normal controls or B cells. HDAC mRNA expression was not modified by cell proliferation or treatment with HDAC inhibitors. No specific HDAC expression profiles were detected in primary human AML or MDS samples. In contrast, CLL primary samples were characterized by an overexpression of HDAC 1,3,5 and 10, although this pattern was not significantly different than that of normal CD19+ B cells. Sequential analysis of human samples obtained from patients treated with two different HDAC inhibitors, vorinostat or MGCD0103 on two different clinical trials, did not affect expression profiles in patients with MDS or AML. Overall, mRNA expression results correlated with protein levels. In summary, our results indicate that AML or MDS are not characterized by a leukemia specific HDAC expression profile but that B cells and B cell leukemia are characterized by a significant overexpression of HDAC 1. This could explain the activity observed with HDAC inhibitors in B cell malignancies and serve as the bases for clinical studies of HDAC inhibitors in CLL.
Novel Function of FGFR1 in Hematopoietic Stem Cell Stress Response.
Authors: Ross Jason T. , Li Linheng
Published: April 2020

The hematopoietic stem cell (HSC) is one of the best characterized tissue-specific stem cells in adult mammals, yet a great deal remains to be learned about the signaling pathways involved in its regulation. Fibroblast growth factors (FGFs), a large family of cytokines, and their cell-surface receptors (FGFRs) are known to play key roles in regulating cell proliferation, differentiation, and migration, both during embryonic development and in the adult. Although HSCs are known to express certain FGF pathway components, the functional significance of this is not well understood. In order to investigate the contribution of FGF signaling to HSC regulation, we generated a conditional Mx1-Cre/FGFR1fx/fx knockout mouse line. Though lacking an overt phenotype within the hematopoietic system under homeostatic conditions, these mice displayed defects in mobilization of HSCs to the peripheral circulation and spleen after myelosuppression with the chemotherapeutic agent 5-fluorouracil (5-FU), as compared to littermate controls. We are currently exploring the mechanism of this defect by directly testing the migratory ability of a population of bone marrow cells containing stem and progenitor cells. Of interest are cells from FGFR1 knockout mice that displayed defective migration toward recombinant chemokine (C-X-C motif) ligand 12 (CXCL12), indicating a link between FGF signaling and CXCL12/CXCR4-mediated chemotaxis, a process with known roles in mobilization. We competitively transplanted LSK/Flk2- cells (a population highly enriched for HSCs) from FGFR1 knockout and control mice into lethally irradiated recipients to test their repopulation ability. Our findings indicate an important role for signaling through FGFR1 in the mobilization of HSCs in mice, knowledge which may enhance clinical mobilization protocols.
A Novel, Small Non-Peptidyl Butyzamide Activates Human Thrombopoietin Receptor and Promotes Megakaryopoiesis.
Authors: Nogami Wataru , Yoshida Hiroshi , Koizumi Kenzo , Yamada Hajime , Abe Kenji , Arimura Akinori , Yamane Noriko , Takahashi Koji , Tanaka Yoshikazu , Yamane Akiko , Takemoto Hiroshi , Ohnishi Yasuyuki , Ikeda Yasuo , Miyakawa Yoshitaka
Published: April 2020

Butyzamide is a novel non-peptidyl molecule which has agonistic activity to the thrombopoietin (TPO) receptor Mpl. Butyzamide promotes the proliferation of murine pro B cell line Ba/F3 expressing human Mpl (hMpl), and induces phosphorylation of JAK2, STAT3, STAT5 and MAPK. Interestingly, butyzamide does not promote the proliferation of Ba/F3 cells expressing murine Mpl (mMpl). To elucidate the mechanisms for this species specificity, we created stable transfectants such as Ba/F3-hMpl(H499L) and Ba/F3-mMpl(L490H) cells. Butyzamide induced the proliferation of Ba/F3-mMpl(L490H) but not Ba/F3-hMpl(H499L) cells, indicating that a histidine residue in the transmembrane domain of human Mpl is critical for butyzamide-induced signaling. Butyzamide induced the colony-forming unit-megakaryocyte and polyploid megakayocytes from human CD34+ hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was orally administered to immunodeficient NOD/Shi-scid/IL-2Rγcnull (NOG) mice transplanted with human fetal liver-derived CD34+ cells, the human platelet count increased by 6.2- and 22.9-fold at the doses of 10 and 50 mg/kg for 20 days, respectively. Butyzamide also increased the number of reticulated human platelets and human matured megakaryocytes in NOG mice. These results indicate that butyzamide is an orally bioavailable Mpl activator and suggest its potential for clinical development as a therapeutic agent in patients with thrombocytopenia.
GM1 Expression of Non-Hodgkins Lymphoma Determines Susceptibility to Rituximab Treatment.
Authors: Bueschenfelde Christian Meyer zum , Feuerstacke Yvonne , Goetze Katharina S. , Schlolze Kathrin , Peschel Christian
Published: April 2020

The monoclonal antibody rituximab directed against the cell surface molecule CD20 of mature B cells has been proven to be successful in the treatment in a variety of B cell malignancies. However, resistance against rituximab occurs and there is no prognostic marker to predict individual response. Rituximab has been shown to induce cell killing via antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and the induction of apoptosis. However, the mechanism that renders rituximab so effective in vivo remains elusive as does the reason for treatment failure in a subgroup of patients. On the molecular level the function of rituximab has been associated with the partitioning of CD20 molecules to lipid microdomains. We now show that the extent of CD20 recruitment to lipid rafts correlates with response to rituximab. In addition, analyzing 11 different Non Hodgkin’s lymphoma cell lines we demonstrate that the expression of the raft associated sphingolipid GM1 on lymphoma cells is associated with the susceptibility to rituximab. Of note, lymphoma cells treated with the sphingolipid synthesis inhibitor PDMP exhibited reduced GM1 expression levels and showed impaired susceptibility to rituximab as compared to non treated lymphoma cells. Furthermore, analyzing the GM1 expression on lymphoma cells of 217 patients with various types of indolent Non-Hodgkin’s lymphoma, we demonstrate significantly different GM1 expression in various Non-Hodgkin’s lymphoma subtypes. Whereas chronic lymphocytic leukemia (CLL) cells have a low GM1 expression, marginal zone lymphoma (MZL) cells exhibit much higher levels. Differences were not only detected between various lymphoma subgroups but also within one lymphoma subtype. Interestingly, whereas CLL cells from patients with high GM1 expression responded to rituximab, patients with low GM1 expressing CLL cells did not. Similar results were observed analyzing lymphoma cells from patients with mantle cell lymphoma (MCL) suggesting that GM1 expression correlates with responsiveness to rituximab in further types of Non-Hodgkin’s lymphoma. Collectively, these data demonstrate the importance of membrane microdomains for the effect of rituximab and may offer a predictive factor for the responsiveness of lymphoma cells to rituximab.