bluuebunny/crossref_metadata_2025 huggingface dataset:
Comorbidity Indexes and Allogeneic Stem Cell Transplantation (SCT).
Authors: Xhaard Alienor , Porcher Raphael , Chien Jason , de Latour Regis Peffault , Robin Marie , Ribaud Patricia , Martin Paul J. , Socié Gérard
Published: April 2020

Despite recent advances, mortality rates after allogeneic hematopoietic cell transplantation remain high and cannot be accurately predicted. During the last years several reports from the Seattle group suggest the use of comorbidity indexes (CI) to provide valid and reliable scoring of pre-transplant comorbidities that predicted non-relapse mortality (NRM) and survival [Blood 2004 (CCI), 2005 (HCT-CI) & Ann Int Med 2006 (PAM)). However whether such indexes could be applied by other groups, and if one index better predicts NRM than another, is unknown yet. Patients undergoing their first allogeneic SCT at Saint-Louis between April 2004 and December 2005 were considered. A total of 183 patients were first included in the study (median age 31, range [4–64], 41% from unrelated donors, 87% intermediate or high-risk diseases). Survival curves were estimated using Kaplan-Meier estimator, and cumulative incidence curves of NRM, treating relapse as competing event. The likelihood ratio statistics in proportional hazards models was computed for each index, with its associated P-value, as a measure of association between the comorbidity indexes collapsed into risk groups and the outcomes. Discriminative performance of comorbidity indexes was then evaluated by the c-index. Standard errors of c-indexes were computed from that of Somers’ Dxy rank correlation coefficient. P-values for comparison of c-indexes were obtained using a nonparametric bootstrap procedure. Using CCI, 21% of our patients had indexes of 1 or more; median HCT-CI was 1; and median PAM score was 24. The discriminative properties of the 3 CI were rather low in our patient population with c-index of 0.54, 0.49 and 0.57 for the CCI, HCT-CI and PAM indexes, respectively. Comparisons of patients- and transplant-characteristics between our and Seattle group’s cohorts however reveal significant differences with more cord blood HCT and HCT for Fanconi anemia in St Louis. Finally direct comparison of scoring items revealed that, unrelated HCT, renal function, and lung function (PFTs) had however similar impact on NRM and OS in our cohort. Additional analyses including patients transplanted in 2006 are underway.
Therapeutic Drug Monitoring (TDM) of IV Busulfan in Patients with Severe Obesity.
Authors: Hutcherson Donald A. , Sessions Jolynn K. , Mihelic Ronald A. , Sanvidge Katherine , Stricker Stephen R. , Langston Amelia , Flowers Christopher , Lechowicz Mary Jo , Waller Ned , Lonial Sagar
Published: April 2020

It has been previously reported that increases in relapse and graft rejection can be associated with a low busulfan area under the curve (AUC) and hepatic toxicities are associated with a high AUC. This has lead to strategies to adjust busulfan dosing to achieve a target AUC. A retrospective analysis was performed in patients with severe obesity, defined as a Body Mass Index (BMI) greater than 40, to evaluate the reliability of IV busulfan dosed using the package insert dosing strategy to reach a targeted AUC. The study included 11 females and 8 males who received IV busulfan as part of their hematopoietic progenitor cell transplant preparative regimen. There were 11 autologous and 8 allogeneic transplants for lymphoma (10) and leukemia (9). The mean and median ages were 38 and 41 years (range 19–51). The mean and median BMIs for this population were 47.8 and 46.2 (range 40.4–62.7). The mean and median weights were 139.5 kg and 142.5 kg (range 107–192.3). TDM was performed with the first dose of busulfan along with subsequent dosage adjustments to achieve a target AUC of 1150–1350 micromol X min/L. Busulfex package insert dosing recommendations are for 16 doses of 0.8 mg/kg (12.8 mg/kg) using an adjusted ideal body weight (AIBW) for obese patients. AIBW = 0.25 × (actual weight − ideal body weight) + ideal body weight. Using the AIBW dosing, the mean starting dose to actual body weight ratio was 0.48 mg/kg (range 0.40–0.55 mg/kg). Based on the AUC analysis, the predicted mean AUC using the AIBW dosing was 972 (range 702–1356). Six AUCs were below 900, 10 AUCs were above 900 but below 1150, 2 AUCs were within the target range and 1 AUC was greater than 1350. Thirteen of the patients had repeat AUC analyses following a subsequent dose to confirm that the dose adjustments achieved the targeted AUCs. Seven of these patients had further dose adjustments following the repeat AUC analysis. Five patients with a busulfan mean half-life of 235 minutes (range 218–247 minutes) had higher AUCs than the rest of the group. The mean and median AUCs for these 5 patients were 1177 and 1223 (range 997–1357). The remaining 14 patients with a mean busulfan half-life of 177 minutes (range 151–201) had mean and median AUCs of 899 and 903 (range 702–1105). The total therapy delivered ranged from 12.7 to 21.4 mg/kg based on the AIBW. When calculated on actual weight, the range was 6.9 to 11.24 mg/kg.

Conclusion: The AIBW dosing strategy of IV busulfan is inadequate to achieve the targeted AUC of 1150–1350 in severely obese patients. Severely obese patients require dose increases in the majority of cases, while those with delayed clearance could be overdosed if higher doses are administered without TDM. Severely obese patients require TDM and the majority will require dosage adjustments to administer IV busulfan in the targeted therapeutic range.
Reduced Fibrinolysis in the Hepatic Venous Occlusive Disease: Effect of Defibrotide on Plasmin Activity.
Authors: Echart Cinara , Repice Cinzia , Ferro Laura , Anderson Kenneth , Richardson Paul , Falanga Anna , Fareed Jawed , Iacobelli Massimo
Published: April 2020

Introduction: The fibrinolytic activity has been shown to be reduced in many vascular diseases, including hepatic veno-occlusive disease (VOD) after stem cells transplantation (SCT). Defibrotide (DF) is a polydisperse oligonucleotide with antithrombotic, profibrinolytic, anti-ischemic, and anti-adhesive properties. Numerous clinical studies have shown promising results with DF in the treatment of VOD, with minimal toxicity. In laboratory studies, DF has been shown to decrease plasminogen activator inhibitor-1 (PAI-1), increase tissue plasminogen activator (t-PA) levels and increase the overall plasma fibrinolytic activity in patients with VOD. Similar results have been observed in pre-clinical studies with endothelial cells stimulated by lipopolysaccharide. Plasmin is a potent and nonspecific serine protease, generated from the proenzyme plasminogen by plasminogen activators. Plasmin plays a pivotal role in fibrinolysis by virtue of its ability to effectively degrade fibrin clots. The purpose of this study was to investigate whether DF is capable of modulating the activity of plasmin in different in vitro studies.

Method: Plasmin activity was measured in the presence and absence of DF, at different concentrations, by following the cleavage of the chromogenic substrate S-2251. S-2251 contains the preferential cleavage site for plasmin and mimics the fibrin polymer substrate present in fibrin clots. In addition, we evaluated the activity of plasmin generated by t-PA and urokinase-plasminogen activation as well as the plasmin activity in plasma in the presence and absence of DF. Further, plasmin activity generated in fibrin clot plate was measured in the presence and absence of DF. Finally, the inhibition of plasmin by aminocaproic acid was tested in these systems.

Results: DF increased the activity of plasmin, to hydrolyze its substrate, in a dose dependent manner. Similar concentration-dependent effects of DF were observed when plasmin was generated by t-PA or urokinase activation of plasminogen and on plasmin in plasma samples (p<0.001). In contrast, DF had no direct effect in activation of plasminogen to plasmin. We also showed that DF was able to enhance the activity of plasmin to degrade the fibrin clot formed from fibrinogen, plasminogen and thrombin (p<0.001). This effect was concentration dependent and directly correlated with the enzymatic activity of plasmin.

Conclusion: The present study demonstrates that DF is capable of enhancing the activity of plasmin. The stimulation of plasmin by DF was concentration-dependent. These findings can, in part, explain the fibrinolytic activity of DF and support its effect in restoring the fibrinolytic vascular phenotype in conditions such as VOD.
Gemtuzumab Ozogamicin as Part of Reduced-Intensity Conditioning in Patients with Relapsed or Refractory Acute Myeloid Leukemia - Results of a Phase I/II Trial.
Authors: Bornhaeuser Martin , Illmer Thomas , Uwe Platzbecker , Rainer Ordemann , Johannes Schetelig , Markus Schaich , Alexander Kiani , Ulrich Schuler , Gerhard Ehninger
Published: April 2020

Gemtuzumab ozogamicin (GO) has been associated with an increased risk of liver sinusoidal obstruction syndrome (SOS) when applied within three months before or after allogeneic stem cell transplantation. In addition, excess of liver toxicity has been observed when GO was used directly before conventional intensive preparative regimens. We hypothesized that GO might be safe and effective as part of a fludarabine-based reduced-intensity conditioning regimen. 30 patients relapsing after conventional induction chemotherapy (n=17) or after previous transplantation (autologous n=3; allogeneic n=10) have been included in a prospective phase I/II trial. Per protocol the preparative regimen contained 6 mg/sqm and 3 mg/sqm GO on day-21 and day-14 followed by fludarabine 120 mg/sqm, 200 or 800 cGy total-body irradiation (TBI; depending on age and pretreatment) and stem cell infusion during GO-induced aplasia. Five patients who had previously undergone TBI received melphalan 140 mg/sqm. GvHD prophylaxis was performed with tacrolimus (starting day-1) and mycophenolate mofetil (1.5g BID from day 0). Four patients with progressive disease after GO did not proceed to conditioning therapy and went off study. A reduction of marrow blast counts after GO monotherapy was achieved in 15/30 patients (50%). In 11 cases without response to GO, an additional salvage regimen containing anthracyclines and high-dose cytarabine was administered before conditioning therapy and transplantation. 26 patients received G-CSF mobilized peripheral blood progenitor cells from matched-sibling (n=6) or unrelated donors (n=20). Primary engraftment was observed in all cases. With a median follow-up of 20 months (range 6–55) only one patient experienced sinusoidal obstruction syndrome which was reversible after treatment with defibrotide. Grade II-IV acute GvHD occurred in 15 patients (54%). Nine patients are alive in complete remission. Reasons for death in the other patients were relapse/progression (n=10) and GvHD/Infection (n=7). The probability of overall and disease-free survival at two years for patients having received a transplant is 38% and 32%, respectively. Blast reduction after GO was associated with a superior disease-free survival (48%, p=0.1). These data suggest that GO can be integrated into reduced-intensity conditioning therapy before allogeneic stem cell transplantation in patients with relapsed AML. Refractory patients with no response to GO monotherapy have a low chance of cure and should probably receive other combination therapies.
Notch Signaling Induces Megakaryocytic Cell Fate.
Authors: Mercher Thomas , Cornejo Melanie , Sears Christopher , Kindler Thomas , Moore Sandra , Aster Jon , Gilliland D. Gary
Published: April 2020

The Notch pathway regulates a broad range of biological mechanisms including proliferation, border formation and cell fate decisions. In the hematopoietic system, Notch signaling is generally thought to specify T cell lineage fate at the expense of the B cell whereas its role in the myeloid lineage development is unclear. When using heterotypic co-cultures of murine primary hematopoietic stem cells (HSC: Lin-Sca1+Kit+) with OP9 stromal cells, or OP9 cells expressing the Notch ligand Delta1 (OP9-DL1), we unexpectedly observed the development of large cells with cytoplasmic protrusions reminiscent of proplatelet production by megakaryocytes on OP9-DL1 stroma. These cells stained positive for acetylcholinesterase, specific for megakaryocyte, and displayed large polylobated nuclei. Flow cytometric analysis indicated a 10-fold increase in the number of CD41+ cells in OP9-DL1 co-cultures compared to parental OP9 co-cultures. Expression of a constitutively active intra-cellular Notch (ICN) mutant allowed differentiation of HSC into CD41+ cells in parental OP9 co-cultures without DL1 stimulation, whereas expression of a dominant-negative MAML1 (dnMAML1) mutant abrogated this effect in OP9-DL1 co-cultures. In addition, megakaryocyte differentiation in OP9-DL1 co-cultures was blocked by γ-secretase inhibitors treatment and rescued by ectopic expression of ICN. Global gene expression analysis demonstrated engagement of a megakaryopoietic transcriptional program when HSC were co-cultured with OP9-DL1 vs. OP9 stroma or OP9-DL1 stroma treated with γ-secretase inhibitor. Bone marrow transplantation experiments with ICN, resulted in enhanced megakaryopoiesis in vivo with increased MEP numbers and megakaryocyte colony formation. Furthermore, transplantation of bone marrow cells transduced with dnMAML1 significantly impaired megakaryopoiesis in vivo with a 4- to 7-fold decrease in maturing megakaryocytes. These findings demonstrate a positive regulatory role for Notch signaling in specification of megakaryocyte development, and indicate that Notch plays a complex role in cell fate decisions among myeloid progenitors. They suggest the possibility that inhibition of Notch signaling may have therapeutic potential in malignancies of the megakaryocytic lineage. Furthermore, Notch pathway stimulation could be of value in enhancing megakaryocyte growth in clinical contexts associated with severe thrombocytopenia, such as hematopoietic reconstitution following bone marrow transplantation or chemotherapy.
Dyskeratosis Congenita: Low Regimen-Related Toxicity Following Hematopoietic Cell Transplantation (HCT) Using a Reduced Intensity Conditioning Regimen.
Authors: Tolar Jakub , Orchard Paul J. , Miller Jeffrey S. , MacMillan Margaret L. , Wagner John E. , Alter Blanche P. , Baker K.S.
Published: April 2020

Dyskeratosis congenita (DC) is a congenital disorder characterized by very short telomeres. Clinical presentation includes a diagnostic triad (lacey reticular pigmentation, nail dystrophy, and leukoplakia), aplastic anemia (the main cause of premature death), myelodysplastic syndrome/leukemia and solid tumors. Allogeneic HCT is the only curative option for the hematologic complications of DC but has been associated with a high risk of peri-transplant morbidity and early death. Only about fifty HCT for DC have been performed to-date, and the five year survival after related donor HCT has been about 75%, but only approximately 35% when an unrelated donor was used. To improve survival in DC patients by decreasing transplant mortality, we introduced a reduced intensity regimen including cyclophosphamide (50 mg/kg), fludarabine (200 mg/kg), low dose total body irradiation (200 cGy), and (in patients 3 and 4) campath 1H (1 mg/kg). To decrease the risk of graft rejection, grafts were not T-cell depleted. We report outcomes in four consecutive patients, two adults and two children, all of whom engrafted with donor hematopoiesis:

Age (years) Sex Graft and HLA match NC dose (×108/kg) CD34 dose (×108/kg) Follow-up (months) Donor chimerism 24 M URD dUCB 4/6, 4/6 0.55, 0.39 0.5, 0.43 1 (dead*) 83% 29 F REL PBSC 6/6 13.93 4.43 20 (alive) 100% 5 F URD BM 7/8 1.38 1.55 16 (alive) 100% 2 M URD BM 8/8 5.92 2.31 3 (alive) 100%

Legend: M, male; F, female; NC, nucleated cell; URD, unrelated donor; REL, related donor; BM, bone marrow; dUCB, double umbilical cord blood; PBSC, peripheral blood stem cells; *Patient had autologous recovery after the first dUCB and died of sepsis 1 month after the second dUCB; HLA matching is reported for antigen level HLA-A, B and allele level DRB1 for cord blood, and allele level typing for HLA-A, B, C, DRB1 for PBSC or BM. The most recent donor chimerism is reported. To decrease the risk of graft rejection and prevent graft versus host disease (GvHD) patients received cyclosporine and mycophenolate mofetil. Patient 2 developed limited chronic GvHD and patient 4 developed grade III skin acute GvHD. Both were treated successfully with systemic and topical steroids. Our data suggest that this conditioning regimen results in a low rate of transplant related complications without compromising engraftment. Critically, early fatal pulmonary and vascular complications, common in post-transplant courses in DC patients, were not observed. This highlights the need to avoid drugs that are associated with pulmonary toxicity such as busulfan, and to limit radiation to the lung in patients with DC. This new less intensive conditioning regimen appears to result in a low rate of transplant related complications, and yet has adequate immunosuppressive activity to permit engraftment from alternative donors in DC patients.
Systematic Genomic Screen for Tyrosine Kinase Mutations in CLL.
Authors: Brown Jennifer R. , Levine Ross , Raderschall Elke , Thompson Christina , Gilliland D. Gary , Freedman Arnold S.
Published: April 2020

Genomic screens have identified activating somatic mutations in tyrosine kinases in a spectrum of acute and chronic hematologic malignancies. These mutations lead to increased cellular proliferation and/or impairment of apoptosis. Chronic lymphocytic leukemia is a common chronic leukemia of adults that is thought to result at least in part from failure of apoptosis. Recent data suggest that CLL cell turnover is higher than previously thought and may correlate with disease progression, suggesting that somatic mutations that activate signal transduction may contribute to CLL pathogenesis and/or progression. The purpose of this study was to look systematically for activating mutations in protein tyrosine kinases in CLL using high-throughput DNA resequencing. 95 CLL patients were selected to encompass the range of prognostic groups defined by somatic hypermutation of the immunoglobulin heavy chain gene, ZAP-70 status and FISH cytogenetics. DNA was isolated from Ficoll separated peripheral blood mononuclear cells, and germline DNA was obtained from buccal swabs and/or saliva samples. 70 tyrosine kinases were selected for resequencing based on their involvement in lymphocyte biology, known altered gene expression in CLL, or location at a site of chromosomal gain, loss or mutation in CLL. All coding exons of ZAP-70 were included. Nested M13 tailed primers were designed to amplify and sequence the exons encoding the activation loops and JM domains. Bidirectional sequence analysis was performed using Mutation Surveyor version 2.28 (SoftGenetics). Candidate mutations were reamplified and sequenced from the original CLL DNA sample for verification; in parallel, the same exon was amplified and resequenced from the paired germline sample to assess whether non-synonymous mutations not known to be SNPs were present in the germline. A total of 178 exons were sequenced per sample. Analysis of 6.21 megabases of sequence data identified nine novel non-synonymous alterations in seven different genes; at least seven of these nine alterations are present in the germline, with the other two presently undergoing analysis. Although these non-synonymous alleles may have functional relevance to the pathogenesis of CLL, these data indicate that a significant proportion of non-synonymous alleles identified in high-throughput genomic screens are non-synonymous polymorphisms. These findings further suggest that the rate of acquired somatic mutation in the tyrosine kinome in CLL is low, and demonstrate the importance of matched germline DNA to allow assessment of candidate non-synonymous mutations in human cancer samples.
Prognostic Assessment of Three Single Nucleotide Polymorphisms (BCL2–938C>A, MTHFR 677C>T, GNAS1 T393C) in Chronic Lymphocytic Leukemia.
Authors: Zenz Thorsten , Duehrsen Ulrich , Dohner Harald , Siffert Winfried , Stilgenbauer Stephan , Nuckel Holger
Published: April 2020

A number of single nucleotide polymorphisms (SNP) have been implicated to impact on the disease course in chronic lymphocytic leukemia (CLL). Nonetheless, there have been few polymorphisms for which replications of association were done in different studies. We genotyped three recently described SNPs (BCL2 –938C>A, MTHFR 677C>T, GNAS1 T393C) in a large group of CLL patients (n=273) with mature follow up and detailed analysis of molecular genetics. The cohort had a median follow-up of over 150 months and the known poor risk molecular genetics (17p–, 11q– and unmutated VH-status) were associated with poor prognosis. The allele frequencies for all three SNPs were in Hardy Weinberg equilibrium and we could not identify imbalances when we stratified for subgroups (genomic aberrations, VH status, stage according to Binet). Moreover, the polymorphisms did not affect clinical outcome (time to first treatment and overall survival) in univariate or multivariate analysis in the current cohort. Comparing CLL patients with over 100 healthy controls we found no significant differences in allele frequencies or genotype distributions for all three polymorphisms. While there have been reports showing the molecular effects and clinical consequences of the polymorphisms we studied, we did not find SNP genotype-dependent effects in a large cohort of CLL patients. Potential reasons for the discrepancy of the results are numerous. Because of the advancement of genotyping technologies, a large number of SNPs can be evaluated (multiple testing). The sample sizes of the reported association studies are often too small and frequently only one patient cohort is investigated. Moreover, negative results are often not published (publication bias). One major reason for the different results is the different genetic background of the populations due to ethnicity (selection bias). Summarized, this study supports the importance of confirmation of initial findings in an independent data set. However, results of genome-wide association studies will soon be reported with additional novel genes identified and will help making great strides in identifying the genetic determinants of CLL risk.
Screening for Copy Number Alterations and Loss of Heterozygosity in Chronic Lymphocytic Leukemia - A Comparative Study of Four Differently Designed, High Resolution Microarray Platforms.
Authors: Gunnarsson Rebeqa , Staaf Johan , Jansson Mattias , Ottesen Anne Marie , Göransson Hanna , Liljedahl Ulrika , Ralfkiaer Ulrik , Mansouri Mahmoud , Smedby Karin Ekström , Hjalgrim Henrik , Syvänen Ann-Christine , Borg Åke , Isaksson Anders , Jurlander Jesper , Juliusson Gunnar , Rosenquist Richard
Published: April 2020

Screening for copy number alterations (CNA) has improved by applying genome wide microarrays, where SNP-arrays also allow analysis of loss of heterozygosity (LOH). Currently, comparisons of high resolution microarray platforms are few, thus we performed a study to evaluate the power of differently designed microarrays for copy number analysis and LOH. We here analyzed 10 diagnostic chronic lymphocytic leukemia (CLL) samples (five IGVH mutated and five IGVH unmutated) using four different high-resolution platforms: BAC-arrays (32K), oligonucleotide-arrays (185K, Agilent), and two SNP-arrays (250K, Affymetrix and 317K, Illumina). Comparison of copy number data showed that the platforms are concordant in terms of detecting large CNA, including the known recurrent alterations. Mono-allelic and bi-allelic loss of 13q14 (3 and 1 sample, respectively), mono-allelic loss of 11q (1 sample), trisomy 12 (2 samples) and mono-allelic loss of 17p (2 samples) were concordant in all platforms. These aberrations were validated with FISH, which in addition identified subclones with mono-allelic loss of 13q14 in two cases, only detected with the BAC platform, rendering a cut-off for the power of detecting subclones to approximately 25% of investigated cells. As expected, all poor prognostic aberrations were detected in patients carrying unmutated IGHV genes whereas four of five mutated samples were detected with mono-allelic loss of 13q14, as the only recurrent alteration. Furthermore, detection of small CNA were in many cases discordant between platforms. Therefore, we defined alterations identified by at least two platforms and identified 47 losses and 31 gains using this criterion. We are currently validating the presence of a number of these alterations using other techniques. Evaluation of LOH showed concordance for 86 regions between the Illumina and Affymetrix platforms. Of these regions 12 LOH coincided with CNA, leaving the remaining 74 as copy-neutral LOH. In conclusion, all platforms investigated are powerful tools for screening of CNA, however, since non-overlapping CNA were detected by individual platforms, we emphasize the importance of validating findings. Also, there is a cut-off for detecting subclones, here estimated to 25%. Genomic arrays will improve the detection of new recurrent aberrations, which may potentially refine the prognostic hierarchy established by FISH.
Clinical and Outcomes Findings for Thrombotic Thrombocytopenic Purpura among 467 Persons with Severely Versus Not Severely Deficient ADAMTS-13 Levels.
Authors: Bennett Charles L. , Luu Thanh Ha , Zakarija Anaadriana , Kwaan Hau C. , Bandarenko Nicholas , Pandey Dilip K. , Cursio John F. , McKoy June M. , Buffie Charlie , Winters Jeffrey L. , Ortel Thomas L. , Sarode Ravindra , Zheng X. Long , Raife Thomas J. , Matsumoto Masanori , Fujimura Yoshihiro , Rock Gail A.
Published: April 2020

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare disorder that presents with microangiopathic hemolytic anemia and thrombocytopenia, fevers, renal insufficiency and neurologic features. We reviewed clinical, laboratory, and outcome data for TTP cases with severely deficient versus non-severely deficient ADAMTS13 activity levels.

Methods: Mean and median data were from the Surveillance, Epidemiology and Risk Factors for TTP (SERF-TTP) study group for idiopathic TTP cases, the Canadian Apheresis Group (CAG), and five published series (Zheng 2004, Raife 2004, Vesely 2003 (Oklahoma TTP-HUS Registry), Matsumoto 2004 (Japan Referral Center), Bennett 2007).

Results: Compared to TTP cases with near-normal ADAMTS13 activity levels (n= 282), TTP cases with severe ADAMTS13-deficiency (n=185) were more likely to have severe thrombocytopenia, normal renal function and neutralizing ADAMTS13 antibodies. Severe ADAMTS13 deficient TTP cases have better overall survival after therapeutic plasma exchange (TPE) but are more likely to relapse. TTP patients with severe ADAMTS13 deficiency were primarily categorized as idiopathic or ticlopidine-associated, while TTP patients with non-severely deficient ADAMTS13 activity levels were frequently categorized as idiopathic, secondary to drugs (clopidogrel, quinine), stem cell transplantation, or cancer.

Conclusions: Severe ADAMTS13 deficiency is most commonly idiopathic, has better survival following TPE, and a 35–40% spontaneous relapse rate. By contrast, non-ADAMTS13 deficient TTP cases are usually associated with an underlying disorder or external insults. Amongst this cohort, four series have 47–62% survival rates and three series, which contain mostly idiopathic cases, have 83–90% survival rates following TPE. From this, we propose that TTP may occur by three possible mechanism; ADAMTS13-deficient (antibody-mediated), an immunologic mediated pathway independent of ADAMTS13 (i.e. quinine) that is responsive to TPE, and endothelial injury related TTP that is unresponsive to TPE.

Platelet count mean (x10^9/L) Creatinine mean (mg/dl) ADAMTS13 neutralizing antibodies (%) Survival % Relapse % * <15% ADAMTS13 activity cutoff Severe ADAMTS13 Deficiency (<10–15%) SERF-TTP (n=30) 19 1.3 83 97 41 Zheng (n=16) 19 1.6 44 81 38 Bennett (n=26) 15 85 Oklahoma (n=18) 12 1.8 94 81 38 Raife (n=50) * 13 1.2 92 35 Japan (n=34) 35 91 Canada (n=11) 16 2.4 82 Not Severely Deficient ADAMTS13 Activity (> 15%) SERF-TTP (n=22) 57 3.9 35 90 0 Raife (n=57) * 44 2.7 83 9 Canada (n=17) 57 4.1 88 Zheng (n=13) 40 3.0 0 54 Bennett (n=13) 62 Japan (n=66) 9 62 Oklahoma (n=94 ) 23 47 3
Effects of Serotonin on Proplatelet Formation and F-actin Reorganization in Human Megakaryocytes.
Authors: Cheng Yuan Shan , Liu Yuan Sheng , Chan Godfrey ChiFung , Ye Jie Yu , Lau Yu Lung , Yang Mo
Published: April 2020

We previously reported that serotonin (5-HT) is a growth factor for hematopoietic stem cells and megakaryocytic progenitor (Yang et al, Stem Cells, 2007). We further proposed a possible role of serotonin on megakaryocyte differentiation and platelet formation. The effect of serotonin on proplatelet formation and F-actin reorganization in human megakaryocytes (MKs) was investigated in this study. Our results showed that:

There was a stimulating effect of serotonin on proplatelet formation in human bone marrow megakaryocytes. Human BM MK progenitors cultured in serum free medium with either 5-HT (200nM) or TPO (50 ng/ml) had more proplatelet bearing MKs than the control group (5-HT(11.33% ± 4.93) vs. control (6% ± 3.60), P=0.026; TPO (14.66% ± 1.53) vs. control, P=0.043; n=3). The 5-HT treatment group showed more mature and more in the final stage MK cells as compared to TPO group; The effect of serotonin on proplatelet formation in Meg-01 cells were via 5-HT2 receptors. Meg-01 cells strongly expressed 5-HT 2A, 2B, 2C receptors by using western blot method. 5-HT also promoted proplatelet formation in these cells and this effect was reduced by 5-HT2 receptor inhibitor ketanserin (KE); and Serotonin acted on cytoskeleton reorganization in human megakaryocytes via 5-HT2 receptors and ERK1/2 pathway.

Using an immunofluorescence microscope with F-actin specific binder rhodamine-phalloidin staining, the polymerized actin level was lower in the control group (serum free) than the 5-HT group and actin distributed diffusely throughout the cytoplasm. In contrast, polymerization actin level was higher in 5-HT group. Adding ketanserin and ERK1/2 inhibitor PD98059 to 5-HT treatment, the fluorescence intensity was correspondingly reduced (5HT vs. Control, P=0.006; 5-HT vs.5-HT plus KE, P=0.014; n=6). Our data also demonstrated that ERK1/2 was activated in MK cells treated with 5-HT for 30 minutes (21.76% ± 7.42). Our studies showed that serotonin had a stimulating effect on proplatelet formation and F-actin reorganization in human megakaryocytes and this effect involved the 5-HT2 receptors and the activation of ERK1/2 pathway.
Differential Expression of ERG Isoforms in Acute Myeloid and T-Lymphoblastic Leukemia Is Regulated by DNA-Methylation.
Authors: Baldus Claudia D. , Schlee Cornelia , Thibaut Julia , Heesch Sandra , Bohne Arend , Thiel Eckhard , Hofmann Wolf K.
Published: April 2020

The oncogenic ETS transcription factor ERG is involved in various cellular pathways including developmental regulation, proliferation, and differentiation. In hematopoiesis ERG plays a specific role during normal T-cell differentiation showing high expression levels in stem cells and down regulation in the progenitor compartment. In this regard, it is intriguing that aberrant expression of ERG was found in a subset of patients with acute T-lymphoblastic leukemia (T-ALL) and was associated with an inferior outcome. Furthermore, high level ERG expression was of adverse prognostic significance in patients with newly diagnosed acute myeloid leukemia (AML), thus highlighting ERG’s potential role in myeloid as well as T-lineage leukemogenesis. ERG3 (NM_182918) and ERG2 (NM_004449) represent the main isoforms and show abundant expression in myeloid and lymphoid hematopoietic progenitor cells. The expression pattern of specific ERG isoforms in acute leukemias has yet to be investigated. To further elucidate the nature of aberrant ERG expression we have determined the existence and transcriptional regulation of ERG isoforms in pretreatment bone marrow samples of adult T-ALL (n=21) and AML (n=20) patients as well as in normal CD34+ hematopoietic cells of healthy volunteers (n=5). 5′RACE revealed the presence of a new ERG isoform (ERG3Δex12) characterized by expression of exon 5 and absence of exon 12. Expression of ERG3Δex12 was verified by RT-PCR in AML, T-ALL, and CD34+ cells. In addition, real-time RT-PCR showed concomitant expression of the two main isoforms ERG2 and ERG3 in AML and normal CD34+ cells. In contrast, T-ALL patients lacked expression of ERG isoforms harboring exon 4 (ERG2). Promoter analyses of ERG2 and ERG3 revealed the presence of two CpG islands in the ERG2 promoter region, whereas no CpG island was predicted in the ERG3 promoter. Bisulfit conversion of genomic DNA and sequencing of cloned PCR products revealed a significantly higher degree of methylation of CpG island 2 in T-ALL samples (median: 86.4%, range: 16.0 – 98.8%) as compared to AML (median: 38.1%, range: 10.9 – 60.7%; P-value=0.0002 - two sided T-test). As for CpG island 1, CD34+ cells had the lowest rate of methylation in CpG island 2 (median: 7.7%, range: 2.4 – 20.7%). Thus, the differential expression of ERG isoforms is mediated by epigenetic silencing of exon 4 containing transcripts in T-ALL. In conclusion, the identification of the new ERG isoform (ERG3Δex12) suggests the association with different partners as the central exons, including exon 12, guide the interaction with different proteins. Furthermore, the distinct expression of specific ERG transcripts controlled by methylation adds to the complexity of ERG directed downstream pathways in different leukemic subtypes.
The Risk of Intrauterine Growth Restriction (IGR) Is Increased by Risk Determinants of Arterial Thrombosis.
Authors: Gerhardt Andrea , Howe Nadja , Krussel Jan S. , Bender Hans G. , Scharf Rudiger E. , Zotz Rainer B.
Published: April 2020

Background: As previously shown, the risk of cardiovascular disease is higher after a maternal placental syndrome, especially in the presence of fetal compromise. The HPA1-b allele of the ß3 subunit of the essential platelet integrin Alphallbbeta3 is a risk factor for increased platelet thrombogenicity, leading to arterial vascular occlusion also triggered by inflammatory endothelial alterations. We performed a case-control study to assess hereditary risk factor for arterial thrombosis in addition to the known risk determinants for venous thrombosis as risk determinants for fetal IGR.

Materials and Methods: 121 women with severe fetal IGR (defined by birth weight below the 5th- percentile for gestational age and sex) and 300 normal women were evaluated. Women with other reasons of IGR (history of venous thrombosis, fetal loss, and preeclampsia, neonatale immunthrombocytopenia) were excluded. The fetuses were born alive after the 24th week of gestation.

Results: A significant risk association could be shown for the HPA-1b/1b genotype OR 3.1 (95%CI 1.2–8.8), increased levels of lipoprotein (a) (>0.2g/l) OR 1.7 (95%CI 1.0–2.9), increased levels of fibrinogen (>median 284mg/dl) OR 2.8 (95%CI 1.8–4.4) increased levels of FVIII:C (>median 147%) OR 1.7 (95%CI 1.1–2.7), and increased levels of von-Willebrand-factor antigen (vWF-Ag) (>median 129%) OR 2.7 (95%CI 1.1–2.7). The combination of risk determinants led to a further increase in risk (e.g. HPA-1b allele, increased lipoprotein (a) and vWF-Ag OR 14.0) There was no significant risk association for factor V Leiden G1691A OR 1.3 (95% CI 0.7–2.81), and prothrombin G20210A mutation. OR 2.0 (95% CI 0.6–6.5)

Conclusions: Increased platelet thrombogenicity via interaction with an inflammatory vascular process and/or via plasma levels of components of hemostasis/receptor ligands appear to be more important in women with IGR than the established risk determinants for venous thrombosis. Pharmacological control of the observed platelet thrombogenicity in patients at risk may be of clinical relevance. In consequence, it will be of importance to examine whether the critical subgroup of patients can benefit from prevention with specific antiplatelet agents.
Contributions of Phosphatidylserine Exposure on Leukemic Cells to Unregulated Procoagulant Activity in Acute Promyelocytic Leukemia.
Authors: Zhou Jin , Cao Fenglin , Lu Chengfang , Li Jinmei , Hou Jinxiao , Yu Hongjuan , Ma Ming , Liu Shuchuan , Shi Jialan
Published: April 2020

Acute promyelocytic leukemia (APL) causes coagulation, which can worsen following initiation of chemotherapy and is improved or corrected by the differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (As2O3). The relationship of procoagulant activity to phosphatidylserine (PS) exposure on leukemic cells has not been clarified, although prior studies indicate that APL cells expose tissue factor (TF). Procoagulant activity of leukemic cells was measured in a modified prothrombin time in which leukemic cells replaced thromboplastin. The presence of phosphatidylserine (PS) in neutrophils and mononuclear cells (MNC) from healthy donors and APL cells from patients was investigated by flow-cytometry and confocal microscopy. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. Lactadherin was utilized as a probe to track PS exposure and as an agent to block exposed PS. Isolated APL cells exhibited patches that stained with lactadherin, but neutrophils and MNC did not, indicating more PS exposure on APL cells than the other two cell types. Exclusion of propidium iodide by the leukemic cells indicated that PS exposure occurs in the absence of frank apoptosis. Coagulant activity increased approx 20-fold after cells were exposed to 0.1 μM daunorubicin for 24 hr with 70% of apoptosis and decreased by 85% after 24 hr treatment with 1 μM ATRA or As2O3 with partial differentiation. Inhibition of procoagulant activity by ATRA and As2O3 corresponded to decreased PS and decrease activity for all three enzyme complexes. Lactadherin, which blocks PS binding sites, inhibited Xase and prothrombin conversion by their respective APL-assembled activating complexes. APL cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant PS and TF exposure at the outer leaflet of cell membrane. In contrast, less PS on neutrophils and MNC do not support the coagulation.
Epac Is Involved in cAMP-Mediated Exocytosis of Weibel-Palade Bodies in Endothelial Cells through the Activation of the Small GTPase Rap1.
Authors: Voorberg Jan , Bierings Ruben , Fernandez Mar , van Mourik Jan A.
Published: April 2020

Vascular endothelial cells provide a dynamic interface between circulating blood and underlying tissues that is critically involved in maintaining vascular integrity and homeostasis. The endothelium provides a surface for adhesion and subsequent extravasation of leukocytes to sites of inflammation. In addition, vascular endothelial cells are involved in the regulation of vascular tone, contribute to neo-vascularisation and mediate the formation of a platelet plug in the event of vascular damage. Rapid recruitment of bio-active components from intracellular storage pools has been shown to contribute to the critical role of endothelial cells in maintaining vascular homeostasis. A significant number of haemostatic components and inflammatory mediators originate from endothelial cell-specific, cigar-shaped organelles called Weibel-Palade bodies (WPBs) that function as storage vesicles for von Willebrand factor (VWF), a multimeric adhesive glycoprotein crucial for platelet plug formation, the leukocyte receptor P-selectin and a number of bioactive compounds that include the chemoattractants IL-8 and eotaxin-3. Following stimulation with agonists that increase intracellular Ca2+ levels, such as thrombin or histamine, WPBs fuse with the plasma membrane, thereby releasing their content onto the cellular surface or into the circulation. More recently, agonists such as epinephrine and vasopressin that raise intracellular levels of cAMP have been shown to promote the release of WPBs. The physiological importance of this pathway is illustrated by the rise in VWF levels in patients with von Willebrand’s disease and mild hemophilia A following administration of the vasopressin analogue desmopressin (DDAVP) or epinephrine. We have previously shown that cAMP-mediated WPB release is dependent on protein kinase A (PKA) and involves activation of the small GTPase RalA. Here, we have investigated a possible role for another, PKA-independent cAMP-mediated signaling pathway in the regulation of WPB exocytosis, namely the exchange protein activated by cAMP (Epac) and its substrate, the small GTPase Rap1. SiRNA-mediated knockdown of the cAMP-GEF Epac1 results in decreased epinephrine-induced WPB exocytosis. Epinephrine stimulation of endothelial cells leads to activation of the small GTPase Rap1 in a PKA-independent fashion. In addition, siRNA-mediated knockdown of Epac1 completely abolished epinephrine-induced activation of Rap1 indicating that activation of Rap1 in response to epinephrine proceeds via Epac1. We subsequently addressed whether activation of Rap1 is crucial for WPB release. Downregulation of Rap1 activation through expression of Rap1GAP effectively inhibits epinephrine-induced WPB exocytosis. Taken together, these data uncover a new pathway by which endothelial cells can regulate WPB exocytosis in response to agonists that signal through cAMP.
Requirements for Retroviral Targeting of a Suicide Gene to Alloreactive Memory Stem T Cells for Adoptive Immunotherapy of Leukemia.
Authors: Bondanza Attilio , Hambach Lothar , Kaneko Shin , Mastaglio Sara , Nijmeijer Bart , Van Halteren Astrid , Ponzoni Maurilio , Aldrighetti Luca , Toma Salvatore , Radrizzani Marina , Ciceri Fabio , Bordignon Claudio , Goulmy Els , Bonini Chiara
Published: April 2020

In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.
STAT3 Signaling in Donor-Derived CD4+ T-Cells Plays a Critical Role in the Induction of Acute and Chronic GVHD in Murine Models of alloBMT.
Authors: Radojcic Vedran , Pletneva Maria A. , Yen Hung-Rong , Harris Timothy J. , Pardoll Drew M. , Drake Charles G. , Luznik Leo
Published: April 2020

Growing evidence from several murine models suggests that STAT3 signaling in CD4+ T-cells plays a pivotal role in the pathogenesis of autoimmunity. We herein tested the hypothesis that the same pathway plays a critical role in the induction of GVHD in murine models of allogeneic BMT. To test our hypothesis and to compare the differential ability of STAT3 signaling in CD4+ T-cells to mediate GVHD in MHC-mismatched and-matched murine models of alloBMT, CD4-Cre × STAT3flox/flox (STAT3KOCD4+), or corresponding wild-type control mice were used as donors of CD4+ T-cells. In an MHC-mismatched model, groups of lethally irradiated (775 cGy) BALB/c (H-2d) mice received T-cell depleted (TCD) bone marrow (BM) from wild-type C57BL/6 (H-2b) mice alone, TCD B6 BM plus 4 × 105wild-type, or STAT3KOCD4+ T-cells, all on a C57BL/6 background. In repeated experiments we observed significantly decreased clinical signs of GVHD in animals receiving STAT3KOCD4+ in comparison to those receiving wild-type CD4+ T-cells (median GVHD score of 2.25 vs. 5.29; P <.001) but no difference in lethality in chimeras monitored up to day 60 posttransplant. In an MHC-matched, minor histocompatibility mismatched setting, we conducted experiments using the B10.D2 (H-2d) BALB/c (H-2d) model in which GVHD is CD4+ T-cell-mediated and has clinicopathologic consistent with human chronic GVHD. Since the dominant clinical manifestation in this model is cutaneous sclerodermatous GVHD that is associated with in situ dermal mononuclear infiltrates, the use of BALB/c-CD45.1 (CD45.1+) mice as recipients enabled us to examine the fate of skin CD11c+ dendritic cells. After conditioning (775 cGy) recipient mice received B10.D2 (CD45.2+) TCD BM and 9.3 ×106 TCD splenocytes, repleted with 106 wild-type CD8+ and 1.9 ×106 wild-type, or STAT3KOCD4+ T-cells. We reproducibly induced all signs of chronic GVHD in chimeras receiving wild-type CD4+ T-cells, but not in chimeras injected with STAT3KOCD4+ T-cells (median GVHD score of 5.2 vs. 0.0; P <.001). We found that development of cutaneous GVHD was accompanied by prominent dermal infiltration of donor-derived inflammatory monocytes and complete turnover to donor CD11c+ epidermal DC chimerism (88.4±4.3% vs. 0.9±0.6%; P <.001). Interestingly, splenic CD11c+ DC (99.9±0.1% vs. 99.8±0.1%), CD4+ (99.9±0.2% vs. 99.6±0.7%) and CD8+ (99.9±0.2% vs. 99.9±0.2%) T-cell chimerism was nearly completely donor-derived and did not differ between the two sets of described chimeras. In summary, our data suggest that intact STAT3 signaling in CD4+ T-cells is required for clinical manifestations of delayed acute and/or chronic GVHD, but it does not interfere with lymphohematopoietic graft-versus host response and achievement of full donor chimerism. Further exploration of the STAT3 signaling pathway in pathophysiology of delayed acute and/or chronic GVHD is warranted.