bluuebunny/crossref_metadata_2025 huggingface dataset:
Disparities of Minor Histocompatibility Antigens in Unrelated Allo-HCT.
Authors: Markiewicz Miroslaw , Siekiera Urszula , Wojnar Jerzy , Wylezol Iwona , Giebel Sebastian , Dzierzak Monika , Karolczyk Agnieszka , Holowiecki Jerzy
Published: October 2019

Disparities of minor Histocompatibility antigens (mHag) have been considered as an important immunogenetic factor influencing immune responses following transplantation despite fully matched HLA of donor and recipient. The aim of our study was to answer whether mHag incompatibility may influence the outcome of allo-HCT. DNA samples from 92 unrelated donor/recipient pairs completely matched in HLA-A*,B*,C*,DRB1*,DQB1* alleles were collected in order to define minor Histocompatibility HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, HwA-9, HwA-10, UGT2B17, HY genotypes with use of Dynal AllSet kits by PCR-SSP method. Unrelated allo-HCTs were performed in the Department of Hematology and BMT in Katowice, Poland, with use the same standard operating procedure from January 2004 until December 2006. Host versus Graft or Graft versus Host direction of immune responses in donor/recipient pairs was analyzed with use of the minor Histocompatibility Database of Leiden University Medical Center. We observed the impact of mHAg mismatches upon chronic GVHD, incidence of leukemia relapse and graft failure. Patients transplanted from donors with mHAg’s incompatibility in GVH direction had higher probability of chronic GVHD in comparison to other pairs (54% vs 31%, p=0.07). This impact was most prominent for mismatched mHAgs with broad tissue distribution (62% vs 36%, p=0.04), especially HY (66% vs 38%, p=0.02). Opposite influence of HY mismatches in GVH direction was observed upon the probability of relapse, which was lower in the HY-mismatched than in HY-matched group (6% vs 23%, p=0.046). Two mHAgs with tissue distribution restricted to hematopoietic cells mismatched in HVG direction were associated with higher incidence of graft failure than in matched pairs: HA-1 (38% vs 12%, p=0.09) and HA-2 (50% vs 13%, p=0.09). Minor Histocompatibility typing of completely HLA matched patients helps to identify individuals who are at higher risk of cGVHD, leukemia relapse and graft failure, and thus supports precise matching of donor/recipient pairs for unrelated allo-HCT.
High CD49d Protein Expression Predicts Short Overall Survival and Early Progression in Chronic Lymphocytic Leukemia Patients.
Authors: Gattei Valter , Bulian Pietro , Del-Principe Maria Ilaria , Zucchetto Antonella , Maurillo Luca , Buccisano Francesco , Bomben Riccardo , Dal-Bo Michele , Luciano Fabrizio , Rossi Francesca M. , Degan Massimo , Amadori Sergio , Del-Poeta Giovanni
Published: October 2019

B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease with highly variable clinical courses which can be at least in part foreseen by investigating the expression of known prognosticators, including the IGHV gene mutational status or CD38/ZAP-70 expression. By analysing the coordinated expression of several surface antigens in a series of CLL with known clinical courses, we identified the simultaneous over-expression of CD38 and CD49d as part of the signature characterizing the subgroup with the worst outcome. The aim of the present study was to investigate the relationship between CD49d and other well-established biologic prognosticators (CD38 and ZAP-70 expression, IGHV gene mutational status), or markers of tumor burden (Rai clinical stage, beta2-M, sCD23), and to define the independent prognostic impact of CD49d in predicting overall survival (OS) and disease progression (evaluated as time-to-treatment, TTT) in CLL patients. The study includes samples from 303 patients affected by CLL according to the current diagnostic criteria (median age: 63.5 years, range 32–97). The entire cohort of patients was utilized to investigate the impact of CD49d and other prognosticators (CD38, ZAP-70, IGHV gene mutational status) on OS. TTT information and additional laboratory parameters (beta2-M, sCD23) were available for 232/303 patients, whose therapies were established according to NCI-WG criteria. CD49d expression was determined by three-color flow cytometry combining anti-CD49d, anti-CD19 and anti-CD5 mAbs; its expression was demonstrated to be stable over-time and the 30% of positive CD5+CD19+CLL cells was chosen as cut-off to discriminate CD49dlow from CD49dhigh cases. CD49d, whose expression was strongly associated with that of CD38 (p=2.2×10exp-16) and ZAP-70 (p=2.6×10exp-5), or with IGHV gene status (p=1.1×10exp-6), was independent predictor for OS (HR=4.39; p=0.0081) along with IGHV status (HR=5.54; p=0.0005) or, if this parameter was omitted, with ZAP-70 (HR=2.90; p=0.0092). CD49d also effectively predicted TTT and refined the prognostic relevance of all the investigated prognosticators. Notably, a CD49dhigh phenotype, while not changing the outcome of good prognosis (ZAP-70low, mutated-IGHV) CLL, was necessary to correctly predict the bad clinical courses of ZAP-70high (HR=3.12; p=0.023) or unmutated-IGHV (HR=2.95; p=0.002) cases. These findings support the introduction of CD49d detection in routine prognostic assessment of CLL patients, and suggest both pathogenetic and therapeutic implications for CD49d expression in CLL, e.g. envisioning the use of a humanized anti-CD49d monoclonal antibody, currently employed in multiple sclerosis (Natalizumab), for selcted CLL patients.
Clinical Efficacy of Lenalidomide in Fludarabine-Refractory Chronic Lymphocytic Leukemia Patients.
Authors: Chanan-Khan Asher A. , Czuczman Myron S. , Padmanabhan Swaminathan , Keating Michael J. , O’Brien Susan M. , Wierda William G. , Ferrajoli Alessandra
Published: October 2019

Introduction: Fludarabine treatment has been shown to be beneficial for patients with Chronic Lymphocytic Leukemia (CLL), and fludarabine-based combinations may even further improve outcomes in patients with CLL. However, most CLL patients eventually become fludarabine refractory, a state which is associated with a relatively short survival. Treatment of fludarabine-refractory patients is challenging, with a median survival of about 10 months. Recently, 2 phase II clinical trials (Chanan-Khan et al. JCO 2006 and Ferrajoli et al. ASH 2006) demonstrated the clinical efficacy of lenalidomide, an immunomodulatory agent, in relapsed/refractory CLL patients. We conducted a subset analysis to examine the efficacy of lenalidomide in patients who are fludarabine refractory.

Methods: All patients enrolled on the 2 phase II single agent lenalidomide clinical trials were evaluated and patients with fludarabine-refractory disease (progressed while on or within 6 months of fludarabine-based therapy) were assessed for clinical efficacy of lenalidomide. Lenalidomide was given orally either at 10 mg daily for 28 days followed by 5 mg increments every 28 days to a maximum dose of 25 mg or given at 25 mg on days 1–21 of each 28-day cycle. Response was assessed using the NCI-WG 1996 criteria.

Results: A total of 80 patients were collectively enrolled in these clinical studies. Among these, 29 were identified to have fludarabine-refractory disease. Important clinical characteristics of these patients are reported in Table 1. The overall response rate in fludarabine-refractory patients was 34.5% (10/29). Complete remission was observed in 2 (6.8%) patients.

Conclusion: Lenalidomide is a novel agent with immunomodulating properties demonstrating clinical efficacy in relapsed or refractory CLL patients. Interestingly, clinical responses to single agent lenalidomide were noted despite refractoriness to fludarabine (a subset of CLL patients with poor survival and limited therapeutic options). This observation of the clinical benefit of lenalidomide independent of responsiveness to prior fludarabine is encouraging and warrants further evaluation.

Table 1 Ferrajoli et al. Chanan-Khan et al. Fludarabine-refractory (N=12) Fludarabine-refractory (N=17) ORR, overall response rate; PFS, progression-free survival; OS, overall survival. Median age, years (range) 62 (51–82) 68 (53–75) Sex, female/male 4/8 4/13 Median no. prior therapies (range) 4 (3–15) 4 (1–10) Median beta microglobulin (range) 5 (3–10) 5 (2–10) Advance Rai Stage (III/IV), n (%) 7 (58.3) 13 (76.4) ORR, n (%) 3 (25.0) 7 (41.2) Median PFS, months 12 14.9 Median OS, months All alive (range, 7–19) 23
AVE5026, a New Anticoagulant for the Prevention of Venous Thromboembolism in Total Knee Replacement Surgery - TREK: A Dose Ranging Study.
Authors: Lassen Michael R. , Dahl Ola E. , Mismetti Patrick , Destrée Dominique
Published: October 2019

Introduction: AVE5026 is a new extractive heparinoid, with predominantly anti-Xa activity (anti-Xa/anti-IIa ratio >30). TREK (a multicenter, randomized, double-blind, double-dummy, parallel group, dose response study of subcutaneous AVE5026 with an enoxaparin calibrator arm in the prevention of venous thromboembolism in patients undergoing elective total knee replacement surgery) evaluated the efficacy and safety of AVE5026 for primary prophylaxis of venous thromboembolism (VTE)

Methods: TREK was a stratified (pre-operative injection/no pre-operative injection) dose-ranging study. Patients were randomized to receive subcutaneously and once daily AVE5026 (5, 10, 20, 40 or 60mg) or enoxaparin 40 mg for up to 10 days after surgery. Depending of the stratum, the first dose was administered 11-13h before or 7–9h after surgery. A mandatory bilateral venography was performed 5–11 days after surgery. The primary efficacy endpoint was the composite outcome of

any deep vein thrombosis (DVT) identified on mandatory venography; symptomatic VTE and VTE-related deaths. The primary safety endpoint was major bleeding.

Results: Of 678 treated patients, 464 were eligible for the primary efficacy analysis with evaluable venous thromboembolism (VTE) assessment. The primary efficacy endpoint rates were at 40.0%, 44.1%, 15.6%, 13.6% and 5.3% for the AVE5026 doses of 5, 10, 20, 40 and 60 mg, respectively demonstrating a highly significant dose response (p<0.0001). The rate in the enoxaparin calibrator arm was 35.8%. A significant dose response was also observed for proximal deep vein thrombosis (DVT) with rates decreasing from 7.7% to 0% (p=0.0002). A statistically significant dose response was found for major bleeding with rates increasing from 0% to 3.4% (p=0.02) and any bleeding ranging from 3.8% to 20.5% (p= 0.0003) for the AVE 5026 dosing arms, compared with 0% and 5.0%, respectively, in the enoxaparin group.

Conclusions: In TREK, AVE5026, a novel anticoagulant, showed a significant dose response in prevention of venous thromboembolic events and a reverse dose-response for bleeding. These data suggest that AVE5026 may be a valuable addition to the armamentarium of antithrombotic agents and support further clinical development.
Eradication of Minimal Residual Disease with Alemtuzumab in Chronic Lymphocytic Leukemia Is Associated with Prolonged Survival and Is an Appropriate Theraputic Endpoint for Relapsed CLL.
Authors: Sayala Hazem A. , Moreton Paul , Kennedy Ben , Lucas Guy , Leach Michael , Rassam Saad M.B. , Haynes Andrew , Tighe Jane , Oscier David G. , Fegan Christopher , Rawstron Andy C. , Hillmen Peter
Published: October 2019

Eradication of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is emerging as a desirable therapeutic end point predicting for better outcome. The monoclonal antibody alemtuzumab (Mabcampath) is approved for patients with fludarabine refractory CLL. We previously published 91 patients with relapsed CLL (74 men and 17 women, median age 58 years [range, 32 to 75 years]; 44 fludarabine-refractory) who received a median of 9 weeks (range 1 to 16) of alemtuzumab, 30mg 3x a week after dose escalation, between 1996 and 2003. 84 patients had i.v. alemtuzumab and 7 received it subcutaneously. Responses to alemtuzumab according to NCI-WG criteria were complete remission (CR) in 32 patients (36%), partial remission (PR) in 17 (19%) and no response (NR) in 42 (46%). Detectable CLL to a level of less than one CLL cell in 10,000 leucocytes, assessed by four-color MRD flow cytometry, was eradicated from the blood and marrow in 18 patients (20%). 8 of these 18 patients were fludarabine refractory. We report here the results of long term follow up of this cohort of patients after a median follow up of 77 months (range 5 to 123 months). Median survival was significantly longer in patients achieving MRD negative responses compared with those with detectable CLL at the end of therapy. The median survival for all 18 MRD negative responders has not been reached but was 87 months for the 8 fludarabine-refractory patients achieving MRD negativity. Overall survival for the 18 patients with MRD-negative remissions was 66% at 72 months (see Figure). MRD positive CR patients had a median survival of 56 months, MRD positive PR patients a median survival of 42 months and non-responders a median survival of 14 months. The median treatment-free interval prior to alemtuzumab for the 18 MRD negative patients was 8 months (range 4 to 35). Excluding planned stem cell transplantation performed in CR, the median time to next treatment for the 18 MRD negative patients was 114 months and 72% (13/18) have required no further therapy. Therefore alemtuzumab can induce MRD negative remissions in CLL resulting in a clear survival advantage with 66% of MRD negative patients alive 6 years after alemtuzumab. The markedly increased treatment-free survival and excellent survival for MRD negative patients strongly suggests that achieving an MRD negative remission is an appropriate therapeutic end-point in relapsed CLL.

Figure Figure
AT7519, a Potent CDK Inhibitor, Is Active in Leukemia Models and Primary CLL Patient Samples.
Authors: Squires Matthew S. , Feltell Ruth E. , Lock Victoria , Smith Donna M. , Lewis Jon E. , Higgins Jacqueline , Yule Murray , Thompson Neil T. , Cooke Laurence , Croce Kimiko D. , Qi Wenqing , Lyons John F. , Mahadevan Daruka
Published: October 2019

Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II. The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. Many leukemic cell lines, which are dependent upon the expression of short half-life transcripts such as Mcl-1 for survival, undergo apoptosis following transcriptional inhibition and the cell cycle effects of such inhibitors are masked. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 in leukemia cell lines (HL60 and MOLT-4). AT7519 causes rapid induction of apoptosis (within 6 hours of initial exposure) in the absence of cell cycle arrest. This induction of apoptosis occurs in parallel with a reduction in the levels of anti-apoptotic proteins such as Mcl-1. In HL60 xenograft models anti-tumour efficacy is observed following 2 cycles of daily dosing of 15 or 10mg/kg for 5 days followed by 2 days off treatment. We observe a 50% cure rate (4/8 mice) at the 15mg/kg dose and a 30% cure rate at the 10mg/kg dose level 40 days following dosing. Pharmacodynamic biomarker studies demonstrate that a greater extent and duration of Mcl-1 knockdown and apoptosis induction are associated with efficacious doses. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. Therefore, we also characterised the transcriptional effects of AT7519 on CLL cells isolated from patients. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–300nM. These effects were correlated with the cytogenetic background of individual patients and the data supports further clinical investigation of AT7519 in B-Cell lymphoproliferative disorders where survival proteins play a pivotal role.
VWF Concentrates - Does Size Really Matter - Von Willebrand Factor Function under Physiological Flow Conditions.
Authors: Schwartz Bruce A. , Fuchs Birte , Fisseau C. , Kessler Craig M. , Kannicht Christoph
Published: October 2019

Introduction: The Von Willebrand Factor (VWF), is a very large glycoprotein present in human plasma essential for normal thrombus formation at sites of vascular injury. Blood coagulation is initiated by exposure of vessel wall components, e.g. collagen, followed by platelet tethering, activation and adhesion leading to the formation of a stable clot. All steps of blood coagulation occur in flowing blood under various conditions depending on vessel size. It is important to assess the function of a VWF concentrate under physiologic conditions. Under static or low shear conditions, platelets can bind directly to collagen without assistance of VWF, while the VWF is essential for mediation of platelet adhesion under high flow occurring in the arterial circulation. It has been assumed that multimer size plays an important role in this binding and in subsequent platelet adhesion. In this study we evaluated the binding of VWF of different multimeric structures to collagen and determined VWF-mediated platelet binding under low to high shear rates in a flow chamber model.

Methods: A flow-chamber coated with human collagen was developed to mimic physiological flow conditions. A high purity VWF/factor VIII (FVIII) concentrate (Wilate®) and two other VWF/FVIII concentrates were tested at shear rates of 400 1700 and 4000 s−1 reproducing shear rates occurring in veins, small arteries and capillaries. Collagen-bound VWF was characterized by antigen determination (VWF:Ag) and multimer (MM) analysis. Binding of labeled platelets was visualized by a fluorescence microscope and surface coverage was quantified.

Results: All VWF MMs independent of MM size were found to bind to collagen even under high shear rates. The amount of collagen-bound VWF:Ag and VWF-mediated platelet adhesion at 1700 s−1 differed significantly between the tested concentrates, when equal VWF:Ag amounts were applied.

Conclusion: Binding of VWF to collagen does not depend on VWF MM size in this model even when measured under high shear rates. The differences in collagen-bound VWF:Ag and VWF-mediated platelet binding do not seem to depend on the VWF MM distribution of the concentrates. Other structural features than VWF MM size may likely be caused by the differences seen in the binding levels of the different concentrates and should be further explored. Possible differences between VWF activity assays performed under static or flow conditions and their appropriate use for VWD diagnosis and quantification of in-vivo activity need to be further investigated.
Microparticle Levels Are High in Patients with Hemophilia A and Can Be Further Increased by DDAVP Administration.
Authors: Artoni Andrea , Santagostino Elena , Mancuso Maria E. , Lecchi Anna , Mannucci Pier M.
Published: October 2019

Microparticles (MP) are circulating submicroscopic cell fragments expressing procoagulant phospholipids and haemostatic proteins as tissue factor. Several evidences support a pro-haemostatic role of MP and suggest that the axis VWF-platelet GpIb is critical in their generation. The aim of this study was to measure the levels of platelet-derived MP (PMP) and tissue factor-expressing MP (TFMP) in patients with hemophilia A to evaluate whether or not a correlation with the phenotype exists. The levels of PMP and TFMP were also measured after DDAVP administration in a subset of patients with mild hemophilia A to assess whether MP levels change in response to DDAVP. FVIII:C, VWF:Ag, PMP and TFMP were measured in plasma 30 min, 1, 2, 4, 8 and 24 h after DDAVP 0.3 μg/kg, subcutaneously. PMP and TFMP were analysed in the pellet of platelet poor plasma by flow cytometry and defined as Annexin V and GpIIb/IIIa or Tissue Factor positive events falling in a gate defined by 1 μm beads. MP were enumerated adding a known number of 7 μm beads to each test tube. PMP and TFMP were measured in 50 patients with severe (FVIII:C<1%) and in 37 with mild (FVIII:C 7–34%) hemophilia A after a wash-out period >72 h, and in 20 male controls. Levels of both PMP and TFMP were lower in controls than in all hemophiliacs (p<0.05), severe hemophiliacs (p<0.05) and mild hemophiliacs (p<0.05). No correlation was found between levels of MP and FVIII:C in mild hemophiliacs. A significant increase in the levels of PMP and TFMP (p<0.05 at all time points vs baseline) was observed in 9 patients after DDAVP administration; peak levels were achieved after 2–8 h. Correlations were found between the increase of VWF:Ag and FVIII:C levels and the peak levels of MP (r=0.62 and r=0.7, respectively). This study showing higher MP levels in hemophiliacs than in controls suggests a role of MP in this patient population. Furthermore, increased MP levels in response to DDAVP administration support the hypothesis that VWF of ultralarge size released by the endothelium interacts with platelets generating MP.

All hemophiliacs (#87) Severe (#50) Mild (#37) After DDAVP (#9) Controls (#20) Median PMP/ml (range) 139 (25–709) 150 (12–586) 128 (28–709) 463 (179–3442) 57 (31–288) Median TFMP/ml (range) 185 (36–859) 169 (36–718) 188 (93–859) 240 (91–1571) 143 (55–314)
Prolonged In-Vivo Half-Life of FVIIa by Fusion to Albumin.
Authors: Schulte Stefan , Weimer Thomas , Wormsbaecher Wilfried , Kronthaler Ulrich , Groener Albrecht , Lang Wiegand , Liebing Uwe
Published: October 2019

For the treatment of hemophilia patients with inhibitors, recombinant Factor VIIa (rFVIIa) is available as a therapeutic option to control bleeding episodes with a good balance of safety and efficacy. The short in-vivo half-life of approximately 2.5 h requires multiple injections, which is inconvenient for treaters and patients. Here we describe the generation of a half-life extended recombinant FVIIa molecule based on genetic fusion of FVIIa to human albumin. In this fusion protein the design of the linker sequence is important to optimize the effect of the albumin moiety on FVII activity. The recombinant FVII-albumin fusion protein (rVII-FP) was expressed in mammalian cells and upon activation displayed a FVII activity comparable to wild type rFVIIa. Pharmacokinetic studies in rats and rabbits demonstrated that the half-life of the activated recombinant FVII albumin fusion protein (rVIIa-FP) was 6 to 9 fold extended compared to wild type rFVIIa. The in-vitro and in-vivo efficacy was evaluated and found comparable to commercially available rFVIIa (NovoSeven®). The results of this study demonstrate that it is feasible to improve the attributes of a rVIIa molecule by extending its half life, while retaining a molecule with very similar hemostatic properties to the wild type factor.
Investigating Mature B-Cell Lymphomas by MicroRNA Profiling.
Authors: Basso Katia , Morozov Pavel , Kitagawa Yukiko , Maute Roy L. , Rosolen Angelo , Chen Chang-Zheng , Califano Andrea , Dalla-Favera Riccardo
Published: October 2019

In order to understand the role of microRNA (miRNAs) in mature B cell function and lymphomagenesis, we analyzed the miRNA expression profile of B cells at different stages of differentiation by cloning of short RNA and by array-based miRNA expression studies. We generated libraries representative of miRNAs expressed in normal germinal center (GC), naïve and memory B cells isolated from human tonsils and in a Burkitt lymphoma cell line (Ramos). Short RNAs were gel purified and linked to adaptor oligonucleotides, reverse transcribed, PCR amplified, cloned into an expression vector, and subjected to sequencing. Candidate miRNAs have been aligned to the human genome to retrieve their potential precursor sequences, whose folding characteristics have been analyzed in order to identify bona fide precursors. The cloned and structurally validated miRNA precursors have been matched to the public miRNA database (miRBase v10.0) to detect previously identified miRNA sequences. The results show that 201 (109 previously known and 92 newly discovered) mature miRNAs were cloned from normal B cells and Ramos cell line. Overall, this analysis led to the discovery of 92 not previously reported miRNAs and allowed us to identify the complete miRNA expression profile of different stages of B cell development. The cloning data show a good degree of concordance with the array-based analysis leading to the identification of a distinct miRNA expression profile for each normal B cell population. Naïve and memory B cells appear to share a large fraction of their miRNA profile while centroblasts display a clearly unique pattern of miRNA expression. We performed a comprehensive analysis of miRNA expression profiles of GC-derived lymphomas (Burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma) using the same microarray platform including approximately 500 miRNA sequences. Each tumor type shows a distinct profile that separates them from their normal counterpart. Interestingly, initial results indicate that a set of miRNAs is expressed in normal GC cells, but not in lymphoma samples and cell lines, suggesting that structural and/or functional alterations of miRNAs occur during lymphomagenesis.
Platelet Pathology in Carriers of X-Linked GATA-1 Macrothrombocyopenia.
Authors: White James G.
Published: October 2019

Mutations in the X-linked hematopoietic transcription factor, GATA-1, have been associated with dyserythropoietic anemia, congenital erythropoietic porphyria and macrothrombocytopenia. Earlier we described the platelet pathology in 3 male members of a previously reported (Mehaffey M.G. et all: Blood. 2001; 98:2681–2688) family with the GATA-1 G208S mutation and serious bleeding problems. All were found to have giant hypogranular platelets with frequent tubular inclusions, parallel associated double membrane sheets connected to channels of the surface connected open canalicular system (OCS), platelets in platelets, and platelets attached to platelets. Their platelets spread normally on formvar grids, but the ability to bind von Willebrand factor (vWF) was half that of normal controls and limited to small multimers rather than the cargo net of large multimers bound by normal platelets. The present study has focused on platelets from four possible female carriers of the GATA-1 mutation in this family. All four of the women have had mild bleeding symptoms, close to normal platelet counts, slight increases in MPV, but no platelet abnormalities on peripheral blood smears. Thin sections of their platelets examined in the electron microscope revealed features consistent with the pathology observed in male family members. Most of their platelets were normal-sized, discoid cells containing the usual complement of alpha and delta storage organelles and channels of the dense tubular system and OCS. However, a significant member of giant platelets containing the usual frequency of alpha and delta granules and hypogranular and agranular giant platelets were observed. The frequency of the macrothrombocytes varied in each of the four women studied, but were present in all. The ability of their platelets to bind multimers of vWF, in contrast to male family members, did not differ from normal controls. Near normal as well as normal platelet counts and the ability of their platelets to bind vWF multimers may protect them from the serious bleeding problems of males with the X-linked GATA-1 G208S mutation. Our findings indicate that obligate female carriers of the GATA-1 gene can be detected by examination of their platelets in the electron microscope.
Risk Prediction of Heparin Induced Thrombocytopenia and Thrombosis in the Tertiary Care Setting.
Authors: Jooma Nuruddin , Kabir Azad , Allison Paul M. , Bracey Arthur
Published: October 2019

Background: Heparin induced thrombocytopenia (HIT) is a challenging diagnosis; especially post cardiac surgery and patients with cardiac support devices. The diagnosis is based on clinicopathologic findings including pertinent clinical history, and heavy reliance on laboratory testing (Heparin PF-4 ELISA) to detect antibodies immunologically or by functional means (serotonin release assay - SRA). Experience with routine measurement of SRA in combination with HPF4 in this setting is limited.

Aim: To determine the clinical and laboratory findings most predictive of thrombosis in patients with HIT.

Patients and Methods: This is a retrospective analysis where we reviewed data on patients who had a positive heparin antibody thrombocytopenia from 1/1/06 to 5/1/07 at the Texas Heart Institute in Houston. We evaluated their diagnosis, O.D., SRA, Doppler ultrasound and platelet count.

Results: Our study found 52 (12.5%) out of 416 patients with positive heparin antibody. Thirty-two patients (62%) with positive heparin antibody had undergone a cardiac procedure within last 30 days. Sixteen (31%) out of the 52 patients with positive heparin antibody had thrombosis confirmed by Doppler/Ultrasound study. Of those 16 patients, 12 (80%) had undergone a cardiac procedure and 10 (63%) had their platelets drop below 75,000. Mean O.D. in patients with thrombosis was 1.10 versus 0.82 for patients with no thrombosis (p=0.14). Serotonin Release Assay (SRA) was considered positive if the serotonin release was > or = 20%. It was positive in only 5 (10%) patients, however, 3 out of those 5 patients had thrombosis. Mean O.D. for patients with positive SRA were 1.88 as compared to O.D. of 0.81 (P<0.001) in patients without positive SRA. All 5 patients had undergone a cardiac procedure.

Conclusion: Our study found almost one third of patients with positive HPF4 antibody developed thrombosis. Patients with thrombosis were more likely to have a positive SRA, higher O.D. and more likely to have undergone a cardiac procedure. Prospective studies are planned to further delineate the relationship between O.D., SRA and thrombosis in the setting of cardiac procedures.
Exploring Genetic Basis of GVHD by Whole-Genome Association Studies in a Large Series from the Japan Marrow Donation Program (JMDP).
Authors: Nannya Yasuhito , Onizuka Makoto , Kashiwase Koichi , Sanada Masashi , Akatsuka Yoshiki , Satake Masahiro , Chiba Shigeru , Kurokawa Mineo , Yamamoto Ken , Saji Hiroo , Maruya Etsuko , Inoko Hidetoshi , Morishima Yasuo , Kodera Yoshihisa , Ogawa Seishi
Published: October 2019

Allogeneic stem cell transplantation (allo-HSCT) has been established as one of the most effective therapeutic options for hematopoietic malignancies. Its major therapeutic benefits are obtained from allo-immunity directed against patients’ tumor cells (graft-versus-tumor effects, GVT). However, the same kind of allo-immune reactions can also be directed to normal host tissues resulting in graft-versus-host disease (GVHD) and subsequently offset the potential benefit from GVT. Because both reactions are initiated by donor T-cells that recognize minor histocompatibility antigens (mHA) that are incompatible between donor and recipient, the knowledge of these mHA is of particular importance to develop a new measure to prevent GVHD while harnessing the antitumor potential of allo-HSCT. On the other hand, the incompatibility of mHAs, as well as other predispositions to development of GVHD, is basically determined genetically, and thus, are plausible targets of genetic association studies, which have recently been enabled with the advent of large-scale genotyping technology. In the current study, we explored genetic basis of GVHD by whole genome association studies, in which relevant SNPs to GVHD development were explored by genotyping a large cohort of GVHD-positive and negative transplantations performed through the Japan Marrow Donor Program (JMDP). Among 7,800 transplants genotyped for HLA types, we selected 1856 transplants (3712 donors/recipients) for large scale genotyping of more than 500,000 SNPs using Affymetrix® GeneChip® 500K arrays, according to the following criteria:

HLA A, B, C, DR, DQ loci were completely matched at the DNA level Short term MTX (stMTX) + CyA or stMTX + FK506 was employed as GVHD prophylaxis, and Genome DNA from both donor and recipient and record of clinical course was available.

Based on the genotyping data, possible association was tested for each SNP in donors and recipients with regard to the development of GVHD. Association of genotype incompatibilities was also investigated for major HLA subtypes to identify the mHA loci that are relevant to GVHD pathogenesis. In the intermediate analysis, a number of candidate polymorphisms in donors and recipients have been identified together with possible mHA loci.
Selective Depletion of Alloreactive T Cells and Study of Anti-Tumor Activity of Specific T Cell Clones in Patients with Leukemia and Renal Carcinoma.
Authors: Matejkova Eva , Buresova Ivana , Vidlakova Petra , Kyjovska Drahomira , Vitetta Ellen S. , Michalek Jaroslav
Published: October 2019

A major challenge in the field of hematopoietic stem cell transplantation (HSCT) is to prevent the alloreactivity of donor T-cells which leads to acute graft-versus-host disease (GVHD) while preserving a graft-versus-tumor (GVT) effect. Selective depletion using anti-CD25 immunotoxin (IT) can eliminate harmful alloreactive T-cells while preserving other donor T-cells with antileukemic/antitumor reactivity. We have used irradiated peripheral blood mononuclear cells (PBMC) from cancer patients and healthy donor PBMC as responder cells in primary mixed leukocyte reaction (MLR).To prepare GVL/GVT-specific T-cells, alloreactive T-cells in primary MLR were depleted with anti-CD25 IT. The remaining T-cells had insignificant alloreactivity in secondary MLR. Allodepleted donor cells were then repeatedly stimulated using purified leukemia/tumor cells from the same cancer patient. Leukemia/tumor-reactive donor T-cells were purified by immunomagnetic separation on the basis of INF-g production. 23 MLRs (12 with acute myeloid leukemia cells, 3 with acute lymphocytic leukemia cells and 8 with renal carcinoma cells) were performed. Selective depletion of alloreactive donor T-cells with anti-CD25 IT led to more than 2log depletion (99.2–100%, median 99.7%). Graft-versus-leukemia (GVL) effect of donor T-cells was well preserved (4.7% of donor T cells were GVL-reactive) while the graft-versus-host (GVH) reactivation of donor cells was negligible even after repeated stimulation with patient’s non-leukemic PBMC. In the case of renal cell carcinoma, GVT effect was less dominant (1.8% of donor T cells were GVT-reactive clones) and GVH reactivation appeared in 2.4% of donor T cells. In conclusion, it is possible to selectively deplete donor alloreactive T-cells with anti-CD25 IT. In case of patients with leukemia, a strong GVL reactivity was noticed in allodepleted donor T cells while in case of renal cell carcinoma the GVT effect was less dominant and associated with mild GVH-reactivation.
Anti-Murine Thymocyte Globulin Prevents Acute Graft-Versus-Host Disease and Induces Foxp3+ T Cells in Target Organs.
Authors: Ruzek Melanie C. , Waire James S. , Weber William , Williams John M. , Richards Susan M. , Garman Richard D.
Published: October 2019

Rabbit anti-human thymocyte globulins (ATG) (Thymoglobulin®) are widely used for treatment and prevention of solid organ transplant rejection. However, more recently these therapies have been shown to also reduce the severity of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. However, dose and schedule of ATG administration in the clinic has not been well defined, and thus, the studies reported here were undertaken to explore optimal dosing and timing regimens as well as possible mechanisms of action of anti-thymocyte globulin in an in vivo murine model of GVHD. We demonstrate that a murine version of ATG, rabbit anti-murine thymocyte globulin (mATG), completely inhibits the development of acute GVHD in a model of allosplenocyte transfer into immunodeficient recipient mice (C57BL/6→BALB/c RAG-2−/ −). This protection is observed even when mATG administration is delayed for up to three days following allosplenocyte transfer. Administration of mATG six or more days following induction of GVHD still ameliorates disease in up to 50% of the animals, depending on the timing of mATG treatment. Murine ATG also remains completely protective down to doses of 1mg/kg if administered at the time of allosplenocyte transfer. Although T cell depletion is still observed at this low, but efficacious dose of mATG, we also find significant increases in Foxp3+ CD4+ regulatory T cells in the spleen (30 fold over control) as well as increased Foxp3+ expression in liver and intestines (3 fold over control). These results demonstrate a potent protective effect of murine ATG in this model of acute GVHD and suggest that the induction of regulatory T cells may participate in the protective effects observed.
Chimerism May Prolong Islet Allograft Acceptance through the Preservation of CD4+CD25+FoxP3+ Treg in Diabetic NOD Mice.
Authors: Bozulic Larry D. , Huang Yiming , Xu Hong , Atay Safinur , Ildstad Suzanne T.
Published: October 2019

In the past several years, novel insights into the role of costimulatory molecules in generating adaptive immune responses have led to the development of new approaches for tolerance induction. We previously demonstrated that preconditioning of diabetes-prone NOD mice with anti-CD8 and combined with blockade of CD40L (anti-CD154) allowed chimerism to be established with 550 cGy TBI. Here, we investigated whether combined costimulatory blockade of CD40L, inducible co-stimulator (ICOS) and OX40L (CD134L) would enhance bone marrow chimerism and promote islet allograft survival in diabetic NOD mice. We also determined whether CD4+/CD25+/FoxP3+ regulatory T-cells (Treg) play an important role in maintenance of tolerance. Spontaneously diabetic NOD mice (blood glucose levels >350 mg/dL) were treated with anti-CD8 antibody (day -3). After 500 cGy TBI (day 0), NOD mice were transplanted with 30 × 106 B10.BR bone marrow cells (day 0) followed by transplantation of 300–400 B10.BR islets under the renal capsule and administered anti-CD154, anti-OX40L, and anti-ICOS mAbs IP (day 0, 1, 2, 3). Chimerism and peripheral blood CD4+/CD25+/FoxP3+ Treg cell expression were quantitated by flow cytometry. Urine and blood glucose levels were monitored. Nonmyeloablatively conditioned mice transplanted with islet allografts and infused with bone marrow (BM) demonstrated acceptance and continued function of islet allografts, with hyperglycemic reversal ≥ 45 (A) and 95 (B) days (Figure 1). Recipients of islet allografts and BM (∼5–12 days), islet allografts and antibody treatment (∼20 days) or islet allografts alone (∼5 days) rejected their grafts. Blood glucose returned to pretreatment hyperglycemic levels in the rejected islet allograft recipient (C) treated in the same manner as the acceptors (A and B) as well as in the control recipients. Donor chimerism at one month post-BMT was 58.5% (A), 89.7% (B) and 0% (C), respectively. Although CD4+/CD25+/FoxP3+ Treg expression was similar between the islet allograft acceptor and rejector recipients, the ratio of CD4+/CD25+/FoxP3+ Treg to CD4+/CD25+/FoxP3− T-effector cells was 2-fold greater in the islet acceptors (3.6 vs. 1.8) (Figure 2). Taken together, our data suggest that prolongation of donor-specific chimerism is associated with tolerance to islet allografts and that CD4+/CD25+/FoxP3+ Treg may be important in maintaining peripheral tolerance. Studies are underway to determine if CD4+/CD25+/FoxP3+ Treg may prevent intra-islet infiltration by autoimmune effector cells, thereby maintaining tolerance to islet allografts.

Figure 1 Figure 1. Figure 2 Figure 2.
Differential Impact of Host Derived Interleukin-18 on Regulatory and Conventional T Cell Expansion Following Murine Allogeneic Hematopoietic Cell Transplantation.
Authors: Zeiser Robert , Zambricki Elizabeth A. , Leveson-Gower Dennis B. , Beilhack Andreas , Kambham Neeraja , Negrin Robert S.
Published: October 2019

Allografting as a curative approach for many hematological malignancies is hampered by the occurrence of acute graft-versus-host disease (aGvHD). Interleukin (IL)-18 stimulates T helper 1 (Th1) and Th2-mediated immune responses and has been shown to modulate aGvHD. It is still unknown whether increased IL-18 levels during aGvHD are of host or donor origin and how the absence of IL-18 impacts migration and expansion of conventional CD4+CD25− (Tconv) and CD4+CD25+ regulatory (Treg) T cells in vivo. By utilizing IL-18 gene deficient donor versus recipient animals we found that the major cytokine production during the early phase of aGVHD induction was recipient derived, while donor hematopoietic cells contributed significantly less. By generating IL-18−/ − luciferase transgenic mice we were able to investigate the impact of IL-18 on Tconv and Treg expansion and trafficking with in vivo bioluminescence imaging. While migration to secondary lymphoid organs was not significantly impacted by the absence of host IL-18, Tconv but not Treg expansion increased significantly. Absence of host IL-18 production translated into lower IFN-γ levels in the early phase after transplantation. We conclude that host derived IL-18 is a major factor for IFN-γ production that may have a protective effect on CD4+ mediated aGvHD, but is non-essential for Treg expansion in an allogeneic environment.
Soluble CD44 Is Increased in Haematopoietic Stem Cell Grafts of Patients with Acute Myeloid Leukemia, Multiple Myeloma and Non-Hodgkin’s Lymphoma.
Authors: Krause Daniela S. , Stowell Christopher P.
Published: October 2019

Autologous haematopoietic stem cell transplantation (autoHSCT) after high dose chemotherapy is a widely used form of therapy for patients with leukemia, lymphoma and multiple myeloma. Malignant cells remaining in the graft, however, may reengraft leading to relapse of the original disease. CD44, a transmembrane glycoprotein, which is overexpressed in many malignancies, including most haematological malignancies, and which plays a role in the engraftment of leukemia-initiating cells, is shed from the surface of malignant cells. In order to establish whether there is a correlation between the amount of CD44 shed from the surface of the malignant cells with the number of contaminating cells remaining in the graft, the engraftment potential of the malignant cell population, or clinical outcome after autoHSCT, we measured soluble CD44 (sCD44) in 86 haematopoietic stem cell grafts from patients with haematological malignancies by enzyme-linked immunosorbent assay (ELISA) and compared the results to sCD44 levels in the HSC grafts from 33 healthy donours. The levels of sCD44 were almost 2fold higher in the grafts of patients with acute myeloid leukemia (AML) (mean: 30,015 ng/ml ± 8524), multiple myeloma (mean: 27,564 ng/ml ± 7,801) and diffuse large B-cell Non-Hodgkin-Lymphomas (mean: 22,539 ng/ml ± 8,382) than in healthy HSC donour grafts (mean: 13,035 ng/ml ± 10,075). As expected, levels of sCD44 were highest in auto HSCT grafts of patients with AML. The majority of patients who achieved a complete remission after autoHSCT had relatively low sCD44 values (mean: 22,610 ng/l ± 7439). Levels of sCD44 were greatest in patients who experienced progressive disease (mean: 28,918 ng/ml ± 10,431), and somewhat less elevated in patients with relapsed disease after auto HSCT (mean: 26,171 ng/ml ± 7616). In addition, patients who had levels of sCD44 <25,500 ng/ml had increased survival (33% at 120 months after autoHSCT versus 18% in patients with sCD44 levels of >25,500 ng/ml). These results argue that the levels of soluble CD44 in an HSC graft could serve as a biomarker for the engraftment potential of contaminating malignant cells in the graft, and may correlate with clinical outcome after autoHSCT. Further, these results raise the possibility that CD44 blocking strategies may be beneficial in autoHSCT.
In Vivo Growth Advantage of Cells Expressing Acquired CSF3R Mutations in Patients with Severe Congenital Neutropenia.
Authors: Germeshausen Manuela , Ballmaier Matthias , Welte Karl
Published: October 2019

Congenital neutropenia (CN) is a heterogeneous group of disorders, characterized by an absolute neutrophil count (ANC) of less than 500 cells per mm3 from birth on and a predisposition of the patients to recurrent and partial life threatening bacterial and fungal infections as a consequence thereof. CN is associated with an exclusive deficiency in neutrophil development. It usually presents early in infancy during the first year of life, and its infectious complications include cellulitis, perirectal abscesses, pneumonitis, peritonitis, stomatitis, and meningitis due to the extremely low ANC (< 200 cells mm 3). The numbers of circulating monocytes and eosinophils are often increased. However, there is a cumulative incidence of 21% for a malignant transformation in CN after 10 years on G-CSF. Point mutations in the gene for the G-CSF receptor CSF3R have been implicated in the progression of CN to leukemia: In 42% out of 219 CN patients tested so far we could demonstrate acquired nonsense mutations in the CSF3R gene. However, in the subpopulation of patients who have developed leukemia 78% are revealing CSF3R mutations. To clarify the involvement of CSF3R mutations in the process of malignant transformation it is important to know at which stage of hematopoietic development these mutations emerge. Therefore we analyzed single cells of flow cytometrically sorted subpopulations of hematopoietic progenitors and mature hematopoietic cells from bone marrow and peripheral blood of 4 CN patients for the existence of CSF3R mutations. The different cell populations reflect the different lineages of hematopoietic development (CD33+, CD34+, CD14+, CD3+, CD19+, CD16+). Two patients characterized by the occurrence of multiple mutations were screened before any signs of malignant transformation and two patients were analyzed at time of secondary AML. Mutated cells were found in all analyzed populations including lymphocytes. However, in the CD34+ cells, eosinphil granulocytes and in the lymphocytic cells the frequency was much lower than in the G-CSFR expressing monocytes and neutrophil granulocytes. These results suggest an in vivo growth advantage of cells expressing a truncated G-CSFR confirming in vitro results with transfected cell lines and underlines the significance of CSF3R mutations in leukemogenesis in CN patients.