bluuebunny/crossref_metadata_2025 huggingface dataset:
Peripheral Blood Stem Cell Mobilisation with Pegfilgrastim Versus Filgrastim in Children and Adolescents
Authors: Fritsch Peter , Schwinger Wolfgang , Lackner Herwig , Sovinz Petra , Benesch Martin , Urban Christian E.
Published: April 2020

Background: Several studies have shown that Pegfilgrastim, the long acting agent of rh-GCSF, is as effective as Filgrastim in children undergoing cytotoxic chemotherapy by reducing the duration of neutropenia without increased adverse events as bone pain and headache. Recent studies in adults have shown that Pegfilgrastim is also effective to mobilize CD 34+ stem cells. In contrast to Filgrastim studies showed a reduction of time to reach the peripheral blood CD 34+ cell peak, resulting in earlier performance of leukapheresis. Although the use of Pegfilgrastim in children after conventional chemotherapy is established, only few data have been published yet describing the ability to mobilize CD 34+ stem cells. The aim of the study was to compare the efficacy of Pegfilgrastim versus Filgrastim for CD 34+ stem cell mobilization in children and adolescents.

Methods: Patients admitted after 2006 were stimulated with Pegfilgrastim on day 4 following a chemotherapy course of the according treatment protocol followed by peripheral stem-cell-apheresis. Filgrastim was added when CD 34 + stem cells did not reach 15/μl between day 15 to 20. Patients were compared to historical controls (2002–2005) stimulated with conventional daily Filgrastim.

Results: 3 groups of patients were compared: Group 1 (historical group): 6 patients with Ewing’s Sarcoma undergoing chemotherapy according to the EURO-EWING protocol receiving Filgrastim 10–20 μg/kg (median 11 μg/kg) underwent a median of 3,8 peripheral stem cell collections (PSCC) between day 12–24 (median 15,4). A median of 16,9 CD 34+ stem cells/kg BW were collected. Group 2: 3 Patients with Ewing’s Sarcoma admitted after 2006 undergoing chemotherapy according to the EURO-EWING protocol received only Pegfilgrastim 100–200 μg/kg (median 166 μg/kg). 3 PSCC were performed between day 11–13 (median 12) and a median of 24,3 CD 34+ stem cells/kg BW were collected. Group 3: 5 Patients suffering from recurrent neoplasm [B-NHL (n=1), medulloblastoma (n=1), ependymoma (n=1) and germ cell tumor (n=2)]. Chemotherapy was performed according to relapse protocols followed by Pegfilgrastim 150–200μg/kg (median 188 μg/kg), however 2 patients needed further cytokine stimulation with Filgrastim 10–15μg/kg (median 12,5 μg/kg) combined with stem cell factor. A median of 3,2 PSCC was performed between day 8–30 (median 17). A median of 11,9 CD 34+ stem cells/kg BW were collected.

Discussion: Our data show that stem cell mobilisation with Pegfilgrastim seems to produce earlier CD34+ peaks and better CD34+ yields than Filgrastim in children when performed during primary or without previous long lasting chemotherapy. However patients with previous long lasting chemotherapy might need additional mobilisation therapy with Filgrastim and/or stem cell factor.
HHV6 and EBV Reactivations Associate with Encephalitis in Patients Post Allogeneic Haematopoietic Stem Cell Transplantation
Authors: Jaskula Emilia , Tarnowska Agnieszka , Suchnicki Krzysztof , Lange Andrzej
Published: April 2020

Herpes viruses reactivation is frequent post HSCT and constitutes a major complication when the reactivation becomes clinically symptomatic. 102 patients at several time points (1058 measurements for each Herpes virus, on average 10 measurements per one patient, from one week to one year post transplant) were investigated with the use of quantitative PCR for the presence of DNA viral copies in blood cells. CMV, EBV and HHV6 DNA copies in blood cells were determined with the use of the real-time PCR technique employing an Opticon 2 thermocycler (MJ Research, Watertown, USA). PCR conditions and the sequences of PCR primers and probes were selected from BALF5 region of EBV, US17 region of CMV and U67 region of HHV6 as described by Kimura et al, (1999), Machida et al, (2000) and Locatelli et al, (2000), respectively.

It was found: CMV and EBV DNA copies (above 100 copies/100 000 cells) were detected in 35% and 23% of patients, respectively. HHV6 reactivation was considered as significant at lower number of copies (above 10 copies/100 000 cells) and was found in 41% of HSCT recipients. Reactivations were more common at early stage post HSCT, with a peak between +31 and +60 day, then at later time. This difference was statistical significant for CMV (p=0.021) and similar trends were also seen when EBV and HHV6 reactivation were analyzed. CMV and EBV reactivations were associated with severe post transplant complications what resulted with poorer 5 year survival (30% vs 72% p=0.015 and 35% vs 65% p=0.016) in patients with CMV and EBV reactivation, respectively. This was not seen when HHV6 was considered. The poorest 5 year survival was seen in patients having aGvHD and presenting reactivation of CMV (23% vs 67%, p= 0.023). Patients with hepatitis were more frequently positive for CMV (0.54 vs 0.30, p=0.071) and EBV (0.41 vs 0.20, p=0.091) reactivation as compared to those lacking clinically apparent hepatitis. Full blown post transplant limbic encephalitis was seen in one case only and was associated with the presence of HHV6 DNA copies in blood (10 copies/100 000 cells) and in spinal fluid (5865 copies/200μl). However, in 11 cases some neurological, psychological and/or psychiatric symptoms were seen. Notably, among these encephalitis cases nine, six and three patients had HHV6, EBV and CMV reactivation, respectively. In five patients with encephalitis reactivation of two Herpes viruses were detected. Separate analysis of investigated Herpes viruses showed the statically significant association of encephalitis with HHV6 (0.75 vs 0.37 p=0.008) and EBV (0.54 vs 0.20 p=0.019) reactivation. Encephalitis was diagnosed in 25% and 21 percentages of pts with EBV and HHV6 reactive respectively. Seven out of 11 patients with encephalitis died. It appears that HHV6 and EBV reactivations may associate with encephalitis that affects the survival post HSCT.
Full Donor Engraftment without Chronic GvHD Following HLA-Matched Sibling Donor Haemopoietic Stem Cell Transplantation for Diamond Blackfan Anaemia
Authors: Alamelu Jayanthi , O’Boyle Farah , Killeen Natalie , New Helen , Atra Ayad , Dokal Inderjeet , Davis John , Foroni Letizia , Szydlo Richard M. , Roberts Irene , de la Fuente Josu
Published: April 2020

Long-term survival in Diamond Blackfan Anaemia (DBA) is compromised by transfusional iron overload and/or side effects of high-dose steroids as well as increasing recognition of the increased risks of aplasia and malignancy. Allogeneic haemopoietic stem cell transplantation (HSCT) can cure the haematological manifestations of DBA, but the optimal conditioning protocol is yet to be defined, particularly avoiding irradiation in view of the increased risk of malignancy. Four consecutive children with DBA were conditioned with oral busulfan 14 mg/kg (days-9 to -6), cyclophosphamide 200 mg/kg (days-5 to -2) and horse ALG 45 mg/kg (days-4 to -2) prior to HLA-matched sibling donor HSCT. Graft-versus-host disease (GvHD) prophylaxis was provided with ciclosporin from day-1 for six months to a target range of 100–150 ng/mL and methotrexate 10 mg/m2 on days +4 and +7.

P1: 10 years old boy who was transfusion dependent for two years having lost the response to steroids. In addition, he had deafness, short stature, gastrooesophageal reflux and cleft palate. He received bone marrow (8 × 108 TNC/kg). P2: 10 years old boy who was steroid dependent and had developed diffuse liver steatosis and bone fractures. In addition, he had short stature and undescended testis. He received bone marrow (1.91 × 108 TNC/kg). P3: four year old girl who was transfusion dependent with no associated abnormalities. She received a combination of cord blood (0.83 × 107 TNC/kg) and bone marrow (0.738 × 108 TNC/kg) from a sibling born following pre-implantation genetic diagnosis and HLA typing. P4: six year old boy born at 34 weeks of gestation who was transfusion dependent. He received a combination of cord blood (1.4 × 107 TNC/kg) and bone marrow (2.73 × 108 TNC/kg).

Neutrophil and platelet engraftment were prompt in all 4 patients (neutrophils: day 15–22; platelets day 10–33). Stable complete donor haemopoiesis was confirmed in all four patients using peripheral blood genomic DNA microsatellites markers by genescanning on day +28, 6 months and 1 year post-transplantation. The conditioning protocol was well tolerated by all patients with no life-threatening infections, though one patient had moderate veno-occlusive disease responding to defibrotide followed by gastrointestinal haemorrhage and another suffered reversible ciclosporin-associated posterior leucoencephalopathy and stage IV, steroid-responsive, acute gut GvHD. All four patients are alive with full donor haemopoiesis and normal haematological parameters at 11, 12, 15 and 24 months post-transplantation. Sibling allogeneic HSCT with this non-TBI protocol is a reasonable and feasible option for DBA patients suffering from unacceptable steroid side effects or chronic transfusion therapy.
Allogeneic Stem Cell Transplantation (SCT) in Advanced Chronic Myeloid Leukemia (CML) Patients after Tyrosine Kinase Inhibitor (TKI) Therapy
Authors: le Coutre Philipp D. , Hemmati Philipp , Neuburger Stefan , Terwey Theis , Massenkeil Gero , Daniel Peter , Dörken Bernd , Arnold Renate
Published: April 2020

In contrast to chronic phase CML patients (pts), in whom imatinib is the treatment of choice, in advanced CML pts allogeneic SCT remains the only therapeutic option with a curative potential. In order to adress the impact of pre-transplant parameters such as remission status, duration of disease, baseline therapies and cytogenetic and/or mutation status we analysed advanced CML pts with TKI pre-treatment (imatinib, nilotinib and/or dasatinib) who subsequently received an allogeneic stem cell transplant. A total of 24 pts (15 males, 9 females) was studied (median age 43,8 years, range: 21 – 58). Of these 7 pts were in 1. chronic phase (CP), 10 in 2nd or 3rd CP, 2 in accelerated phase (AP) and 5 in blast crisis (BC). Eleven of the 17 CP pts had either a CHR (complete hematologic remission) or HR at SCT and seven had a major cytogenetic remission (MCyR, including 4 CCyR). Cytogenetics and molecular analysis at SCT showed additional chromosomal abnormalities in 9/21 and point mutations in the BCR-ABL kinase domain in 2/17 pts (E255V & Y253H). All pts received imatinib prior to SCT, in 4 pts a 2nd line TKI therapy (dasatinib or nilotinib) and in one patient a 3rd line TKI therapy (dasatinib) was given. In 8/24 pts additional chemotherapy or irradiation after the last TKI were given prior to conditioning therapy. In 23/24 pts a myeloablative conditioning regimen was given. 15 pts were transplanted from an unrelated and 9 pts from an HLA identical family donor. The median follow-up of all patients is 19.8 months (range: 0 – 71). 12/24 pts died (SCT failure: n= 1, infection: n=5, GvHD: n=3, relapse: n=3). Risk stratification by basline parameters showed a statistically relevant survival advantage for patients with at least a MCyR and no non-TKI therapy prior to conditioning therapy (14 versus 10 pts; 14 versus 5 months; p=0.036; Chi-square test). Likewise, a survival benefit was identified for pts receiving SCT with at least a baseline HR (15 versus 9 pts; 11 versus 5 months; p=0.011; Chi-square test). Conversely, no statistically relevant differences were observed when pts were stratified upon additional chromosomal alterations, donor origin or stage of disease at baseline. In summary our data confirm the feasibility and benefit of allogeneic SCT in advanced CML patients following TKI treatment. Relevant pre SCT risk factors that could be identified as part of this analysis included baseline response and the absence of a non-TKI treatment prior to the conditioning therapy. Further studies are required to optimize the pretransplant treatment of advanced CML patients that are eligible for allo SCT.
Feasibility and Efficacy of BEAM as a Frontline High Dose Chemotherapy Supported by Autologous PBSCT in Elderly Patients (≥60 Years) with DLBCL
Authors: Ivanov Vadim , Coso Diane , Rey Jerome , Aurran Therese , Stoppa Anne-Marie , di Collela Jean-Marc Schiano , Blaise Didier , Bouabdallah Reda
Published: April 2020

Limited data is available concerning feasibility and efficacy of high dose therapy (HDT) supported by autologous PBSCT in elderly patients with non-Hodgkin lymphoma (NHL). In young patients with poor prognostic features intensification supported by PBSCT as a part of first-line treatment suggests survival benefit. It is not clear if the same strategy is applicable to the older patients. The Institute Paoli-Calmettes database was reviewed for all DLBCL patients who received BEAM followed by PBSCT in patients >=60 years old between January 1998 and December 2006 (9 years). All patients were HIV-negative and received BEAM intensification as a part of front-line treatment. All of them were in a complete or partial response after CHOP or R-CHOP induction prior to autograft. Twenty seven auto-transplanted patients were identified (median age 63 y, range 60–68). This cohort was compared with closely matched group of 37 patients of same age range, who received first-line CHOP or R-CHOP regimen without intensification in the same 9-years interval. Only patients in a complete response after first line were included. As frontline autoPBSCT was performed in high-risk patients, the group without HDT was naturally privileged in the terms of Ann-Arbor stage and aaIPI index. There was significant difference in the localised vs disseminated disease (stage I–II: 54% in no-HDT vs 26% in HDT group, p=0.03)) and aaIPI (0–1: 66% in no-HDT vs 37% in HDT, p=0.046) between the two groups. Factors evaluated included treatment-related mortality (TRM), overall survival (OS) and event-free survival (EFS). TRM in the HDT group (1/27 pts (3,7%)) was comparable with previously published data. The estimated 5-year OS was 75,5% (95%CI 52–90 %) for HDT group compared to 79,9% (95%CI 58–92%) in the no-HDT group (p=0,75). There were 8 events (1 TRM and 7 relapses) in the HDT group and 11events (all relapses) in no-HDT (5-year EFS 49,4% vs 64,2%, p=0.45). We conclude that frontline autologous PBSCT with BEAM conditioning can be safely performed in patients aged 60 years or above with DLBCL after CHOP of R-CHOP induction. There was no difference in OS and EFS between cohorts with and without intensification even if the auto-transplantation procedure was reserved for the high risk patients. We conclude that first-line HDT with autologous PBSCT in older patients with high-risk IPI score might improve survival in this group and produce results similar to those in the low-risk group.
The Glycogensynthase Kinase 3b (GSK-3β) Is Involved in Leukemic Transformation
Authors: Bäsecke Jörg , Lehmann Anneke , Bergholz Ulla , McCubrey James , Trümper Lorenz , Stocking Carol , Horn Stefan
Published: April 2020

Purpose: Signal transduction pathways, such as the PI3K/AKT cascade, are frequently activated in acute myeloid leukemia and stimulate the proliferation and survival of leukemic cells. Mutations in upstream genes such as Class III receptor tyrosine kinases are frequent but not exclusive causes of that activation. An important downstream target of PI3K/AKT is the key regulator GSK-3β. It controls anti-apoptotic genes such as NF-kappaB and Cyclin D1, is involved in wnt-pathway activation and drug resistance of leukemic cells. Deregulated signaling by GSK-3β occurs through inhibition by AKT-mediated phosphorylation. We have observed that constitutive phosphorylation of GSK-3β occurred in hematopoietic cells with pro-leukemogenic PI3K-mutations. We wanted to evaluate the relevance of GSK-3β inactivation in the transformation process of hematopoietic cells.

Methods and Results: We used an in vitro factor-independent growth assay, GSK-3b inhibitors (Lithium, BIO) and established a second hit model using retroviral gene transfer of the weak oncogene Bcl XL. Signaling cascades were analysed by western blot. We demonstrate that inactivation of GSK-3b alone was not sufficient to induce factor-independent growth in IL-3 dependent early hematopoietic cells (Ba/F3). Induction of apoptosis upon growth factor withdrawal was reduced, but not prevented, in the presence of GSK-3b inhibitors, leading to a delayed Caspase 3 activation, PARP cleavage and DNA fragmentation. Overexpression of Bcl-XL also did not result in a prevention of apoptosis. GSK-3b inhibition in synergy with Bcl-XL overexpression resulted in the establishement of several growth factor-independent cell lines, which were characterized by the activation of multiple signaling cascades including AKT, MAPK, STAT5, but not STAT3. Also Cyclin D1 was overexpressed in contrast to other cyclines (D2, D3) which are no substrates of GSK-3b.

Conclusions: Our data show that GSK-3β is part of the apoptotic response to growth factor withdrawal and suggest that GSK-3β is causaly involved in the transformation process of hematopoietic cells and seems to have an synergistic role in addition to other pro-leukemic mutations.
Histone Deacetylase 11 (HDAC11) as a Novel Molecular Target in Mantle Cell Lymphoma
Authors: Villagra Alejandro v , Maurin Michelle m , Cheng Fengdong , Wang Hongwei , Dessureault Sophie , Seto Ed , Tao Jianguo , Wright Ken , Pinilla Javier , Sotomayor Eduardo M.
Published: April 2020

Mantle Cell Lymphoma (MCL) is a B-cell Non-Hodgkin’s Lymphoma characterized by the translocation t(11;14)(q13;32) and a consequent Cyclin D1 over-expression. The clinical evolution of MCL is very aggressive, and currently there is no standard of care for this malignancy.

In spite of the known association of Histone Deacetylases (HDACs) and solid malignancy growth, little is known regarding the role of these enzymes in MCL. We recently found newest member of the HDAC family, HDAC11, a molecule with an unknown prior physiological role, is a strong transcriptional repressor of gene expression1. Interestingly, we have found a significant over-expression of HDAC11 in murine as well as in human MCL cell lines. In primary cells from patients with MCL, we observed that higher levels of HDAC11 are associated with a more aggressive disease, while low expressions of HDAC11 are seen in patients with the indolent variant of MCL. Microarray analysis of cells in which HDAC11 expression was silenced revealed significant down regulation of specific genes involved in the control of apoptosis, cell cycle, methylation and B-cell survival. Critical genes regulated by HDAC11 include IL-10 and IL-6, cytokines known to be survival factors for malignant B-cells. In addition, we have unveiled a previously unknown association between HDAC11, cyclin D1, and Cyclin Dependent Kinase Inhibitors (CDKI), molecules that play a central role in the pathogenesis of MCL.
Potential Synergism of Green Tea and Aspirin as a Possible Cause of Bleeding in a Diabetic
Authors: Munro Roger C , Wakeman Lisa J , Al-Ismail Saad
Published: April 2020

A 58 year old lady with Metabolic Syndrome of 10 years duration presented with post-menopausal PV bleeding, haematuria, occasional epistaxis and ecchymoses. Prescribed medication which had remained unchanged for the preceding two years included daily doses (mgms) of Aspirin (150), Atenelol (50), Metformin (500 × 3), Bendroflumethiazide (2.5), Losartan (50) and Simvastatin (20). Intravenous urogram, cystoscopy, cytological examination of urine sediment, hysteroscopy, a cervical scan and endometrial biopsies showed neither evidence of overt pathology nor any physiological indication for the cause of haematuria or PV bleeding. Tests for urinary infection were negative. Apart from the raised blood glucose (9.1: NR 3.3 – 6.0 mmol/L), the biochemistry profile including liver enzymes, coagulation profile and blood count were normal (Platelets = 265 × 109/L). Bleeding episodes were observed after commencement of a daily intake of 7–8 cups of green tea for a period of six months. Green tea intake was self-instigated in response to reported amelioration of risk factors associated with Metabolic Syndrome (reduction in LDL cholesterol and serum triglyceride levels; elevation of protective HDL; potent antioxidant activity; ACE inhibition and promotion of glucose metabolism). Hot water extract of green tea specifically inhibits platelet adhesion and lowers sub-maximal platelet aggregation and prolongs the lag time in a dose-dependent manner. Previous fractionation studies of these hot water extracts, has revealed that the tea catechins (tannins) actively inhibit thromboxane A2 production and that ester-type catechins are more effective than free-type catechins. One of the ester-type catechins, epigallocatechin gallate (EGCG), suppresses thrombin and collagen-induced platelet aggregation completely at a concentration of 0.2 mg/ml. EGCG also inhibits aggregation by a mechanism which differs from that of aspirin by inhibiting platelet activating factor (PAF). The IC50 values of EGCG and aspirin indicate that their potencies are comparable. Bleeding symptoms ceased two weeks after the patient stopped drinking green tea. Our assumption of the causal effect of green tea on anomalous bleeding in this patient needs to be confirmed by structured platelet function tests in both aspirinised and non-aspirinised patients. Since inhibition of cyclo-oxygenase is an additional anti-thrombotic property of aspirin which differs from that of green tea, diabetic patients taking prophylactic low-dose aspirin should continue to do so and potentially beneficial ingestion of green tea should not be considered without consultation with an appropriate health professional in view of its synergistic potential on the effect of aspirin and the associated haemorrhagic risks.
Willebrand in the Netherlands: WiN Study. Background and Study Design
Authors: De Wee Eva M. , Mauser-Bunschoten Eveline P. , Van der Bom Anske G. , Eikenboom Jeroen C.J. , Fijn van Draat Karin J. , De Goede-Bolder Arja , Meijer Karina , Nováková Irena , Plug Iris , Leebeek Frank W.G.
Published: April 2020

The clinical presentation of patients with Willebrand Disease (VWD) is highly variable, and not always predictable upon laboratory measurements. The mechanism of this clinical variability is unknown. Also the impact of VWD on the quality of life is highly variable between patients.

This ongoing study aims to register and investigate all patients in the Netherlands with moderate and severe von Willebrand Disease to assess clinical presentation, treatment and related complications of treatment. One of the aims is to assess which factors influence the bleeding phenotype. Another goal is to investigate the influence of von Willebrand Disease on quality of life.

We have identified 1071 eligible patients. All patients are known at 12 hemophilia treatment centers. Inclusion criteria are moderate or severe VWD defined as; VWF antigen or activity ≤ 30% and/or FVIII:C ≤ 40%. The patients will be sent a questionnaire and a blood sample will be obtained for plasma and DNA.

The characteristics of the patients are the following: median age is 34 years (range 0–86) in males and 42 years (range 1–92) in females. 192 children are included (<18 years). Sixty-one percent is female. In this cohort 59% has type 1, 30% has type 2, 4,5% has type 3, 0.5% is compound heterozygote and 6% is non-specified. Among type 2 we identified 50% 2A, 26% 2B, 10% 2M, 6% 2N, and 8% non-specified. In this cohort 45% has blood group 0, 29% non-0 and in 26% this is unknown. The VWF:Ag, VWF:Act (CB or RCo) and FVIII:C levels are mentioned in table 1.

Conclusion: The WiN study has identified 1071 moderate to severe VWD patients in the Netherlands who will form the basis for further research that will result in more insight into the clinical features and quality of life in VWD.

Table 1:

VWF:Ag VWF:Act* FVIII:C * VWF:CB of VWF:RCo <0.10 IU/mL 91 (9%) 246 (23%) 62 (6%) 0.10 – 0.19 IU/mL 157 (15%) 368 (34%) 63 (6%) 0.20 – 0.29 IU/mL 287 (27%) 332 (31%) 137 (13%) >0.30 IU/mL 527 (49%) 119 (11%) 799 (75%) Missing data 9 (1%) 6 (1%) 10 (1%)
Rituximab for the Treatment of High Titer Acquired Factor VIII Inhibitor with Long Term Follow up
Authors: Sreenivasappa Shylendra B , Telfer Margaret Clare
Published: April 2020

Acquired factor VIII inhibitors are auto antibodies directed against factor VIII (FVIII) and have a reported incidence of 0.2–1.0 per million individuals per year. It is a rare cause of serious bleeding associated with high mortality. Individuals with high titer antibodies (> 100 Bethesda units (BU)), often have difficulty achieving a complete sustained remission although different strategies have been advocated for these patients. Rituximab has been demonstrated to show efficacy in patients with acquired hemophilia. In this study we retrospectively studied three patients with high titer acquired FVIII inhibitors with long term follow up. Two of the patients were resistant to steroids and Intravenous Immunoglobulin, one patient had received steroids and Cyclophosphamide prior to rituximab therapy. Patients ages were 36, 45 and 48 years, two were women and one male. One of the women has a h/o pregnancy about 8 months ago and other two were thought to have idiopathic inhibitors. The patient’s inhibitor titers ranged from 124–800 BU at the time of treatment with Rituximab. All received 4 weekly infusion of rituximab at 375mg/m2. No significant treatment related complication was noted. One patient did not respond to treatment. Two patients had a decline in inhibitor titers. One patient had a gradual drop in titer from 124 BU to 0.00 BU over 18 months. The other responder had a drop in titer from 800 BU to 41 BU in 16 months and then relapsed with significant bleeding and anti factor 8 titers of 700 BU and was retreated with rituximab and had a gradual drop in inhibitor titers to 50 BU over 8 months. The two patients have been followed for 28 and 30 months respectively. The non responder continues to have high tires of antibodies and has been followed for 25 months.

We conclude that in patients with high titer of acquired inhibitors rituximab alone may produce a drop in inhibitor titer but is not sufficient to achieve a complete response. Rituximab in combination with other therapies or novel agents may provide better results,
Altered Immune Responses in Patients with Chuvash Polycythemia
Authors: Niu Xiaomei , Miasnikova Galina Y , Sergueeva Adelina I , Okhotin Daniel J , Nouraie Mehdi , Ammosova Tatiana , Nekhai Sergei , Gordeuk Victor R.
Published: April 2020

Chuvash polycythemia results from a homozygous 598 C>T mutation in exon 3 of the von Hippel-Lindau (VHL) gene. This disrupts the normoxia pathway for degrading hypoxia inducible factor (HIF)-1a and HIF-2a causing altered expression of HIF-1 and HIF-2 regulated genes. As hypoxia induces expression of pro-inflammatory cytokines, we hypothesized that there might be an elevation of Th1 cytokines in the setting of Chuvash polycythemia. Concentrations of Th1 (interleukins-2 and 12, interferon-g, GM-CSF, tumor necrosis factor-a) and Th2 cytokines (interleukins-4, 5, 10 and 13) were measured in plasma using the Bio-Plex multiplex suspension array system. Concentrations of all of these cytokines were elevated in patients with Chuvash polycythemia 598C>T homozygous compared to the control wild type participants. In parallel, peripheral blood concentrations of CD3 positive T-helper cells and CD-4 positive T-helper cells were lower in patients with Chuvash polycythemia compared to controls. The ratios of interferon-g and tumor necrosis factor-a to interleukin-10 did not differ by genotype. Thus, although the upregulated hypoxic response in Chuvash polycythemia is associated with increased plasma products of the Th1 and Th2 pathways, the peripheral blood concentrations of T-helper cells are reduced, perhaps as part of a feed-back mechanism, and the balance between the two pathways may be preserved. Such countervailing responses to unregulated hypoxia sensing may explain why patients with Chuvash polycythemia do not present clinically with immunologic disorders.
Impaired Immune Reconstitution after HLA-Mismatched Transplantation Due to Antigen Specific T-Cell Recognition Restricted by the Disparate Haplotype
Authors: Amir Avital L. , Heiden Pim L.J. , Falkenburg J.H. Frederik , Heemskerk Mirjam H.M.
Published: April 2020

The applicability of HLA matched hematopoietic stem cell transplantation (SCT) is limited by the lack of HLA matched donors. In contrast, an HLA haploidentical donor can be found for almost all patients. Posttransplantation immunodeficiency leading to high morbidity and mortality from viral infections is a major barrier to the broader application of haploidentical SCT. In HLA mismatched SCT profound T cell depletion is necessary to prevent GVHD, but also leads to slower T-cell reconstitution. Vigorous T-cell depletion, however, does not fully explain the increased immunodeficiency. Despite a gradual normalization of absolute T-cell and total lymphocyte number after haploidentical SCT, the immunodeficiency may persist for years. Additionally, an increase in the number of mismatched HLA alleles between patient and donor leads to more profound post transplantation immunodeficiency. We hypothesized that another explanation for the long lasting immunodeficiency is T-cell antigen recognition via the haplotype disparate between patient and donor. T-cells recognizing viral epitopes in the donor non-shared HLA molecule will attack virus infected hematopoietic cells of donor origin but not virus infected non-hematopoietic cells of patient origin. To investigate the role of T-cell disparate haplotype restriction in posttransplantation immunodeficiency, we characterized the immune responses against CMV in two patients at various time points after haploidentical and mismatched SCT. The first patient was HLA-A2 negative and transplanted with SCT of an HLA-A2 positive haploidentical donor. After the SCT he suffered from life threatening CMV reactivations for almost two years. Using tetramers composed of the HLA-A2 molecule and CMV-pp65-NLV epitope, we determined that in this patient CMV specific T-cells restricted to the donor HLA-A2 were present from 6 months after the SCT and remained present during the two year period of recurrent CMV infections. Since the HLA-A2 allele was not shared between patient and donor, these results indicate that T cells restricted by non-shared donor-HLA were unable to protect against infections. The second patient transplanted with an HLA mismatched SCT, suffered from CMV reactivations only in the first 6 months after SCT. Already 3 months after SCT CMV specific T cells restricted to the shared HLA-B8 allele were present in high concentrations, whereas the CMV specific T-cells restricted to the non-shared donor HLA-A1 allele disappeared in time. Our results suggest that for an effective immune response the T cells need to recognize viral epitopes restricted to the HLA shared between patient and donor and that impaired immune reconstitution after haploidentical SCT is, in part, the result of antigen specific T cell responses restricted to the disparate haplotype.
Exploiting Cytokine Secretion to Rapidly Produce Multivirus-Specific T Cells for Adoptive Immunotherapy
Authors: Fujita Yuriko , Leen Ann M. , Sun Jiali , Nakazawa Yozo , Yvon Eric , Heslop Helen E. , Brenner Malcolm K. , Rooney Cliona M.
Published: April 2020

Viral infections remain a major cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT), and conventional small-molecule therapeutics often have modest benefit, high cost and adverse effects. Adoptive transfer of donor-derived, virus-specific T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (AdV) proved feasible and safe after HSCT, and reconstituted immunity to all 3 viruses. Current protocols to generate multivirus-specific T cells are lengthy, taking up to 12 weeks. Since viral infections often occur <30 days after HSCT, more rapid production is required. The IFN-γ capture (Miltenyi) and tetramer methods can select antigenspecific T cells directly from peripheral blood and have been used clinically to prevent infections with AdV and CMV respectively. Tetramer selection is limited to certain HLA class I molecules with known epitope specificities. By contrast, the IFN-γ capture assay can be used to select multi-epitope and multi-antigen, CD4+ and CD8+ T cells from any donor, irrespective of HLA type. We now describe a modified rapid selection method for production and characterization of CD4+ and CD8+ T cells specific for CMV, EBV and AdV in a single infusate using the IFN-γ capture assay. We could manufacture a specific product from small volumes of peripheral blood over a 48 hour time period. These cells are suited for infusion into immunocompromised patients. Activated PBMCs were transduced either with Ad5f35pp65 vector or Ad5f35LMP2 vector and then combined. After overnight culture, IFN-γ-secreting antigen-specific T cells were captured. The number of selected cells ranged from 0.06 ×106 to 0.36 ×106 cells (mean: 0.14 ± 0.06 ×106; n=5) per 20 × 106 PBMCs.Importantly, we demonstrate that the selected cells lack alloreactivity, indicating that the infused cells should be safe in vivo. By expanding a small fraction of the selected product we could characterize the phenotype, specificity and function of the infused product, to allow subsequent comparison with clinical outcome. All products were able to secrete IFN-γ in response to each antigen and kill CMV, EBV and AdV antigen-expressing targets. These results show that we can successfully generate trivirus-specific T cells for infusion within 3 days without sacrificing the product characterization required to correlate product with clinical outcome.
Immunopathology of ATG Prior to Bone Marrow Transplantation in Mouse Model
Authors: Sadeghi Behnam , Hashmi Sulaiman Al , Hassan Zuzana , Rozell Bjorn , Abedi-Valugerdi Manuchehr , Hassan Moustapha
Published: April 2020

Introduction: Polyclonal anti-thymocyte globulin (ATG) is mainly used in hematopoietic stem cell transplantation, as a prevention for graft rejection as well as for GVHD. Immunosuppressive properties of ATG have been considered primarily from the depletion of peripheral lymphocytes. However direct or indirect effects of ATG on other immune components still is controversial. In the present study we evaluated the immunopathologic effects of ATG on immune system in a mouse model.

Methods Ten to fourteen weeks old BALB/c and C57BL/6 mice, were injected intra- peritoneally or intravenously with different doses of ATG (0.2mg/kg -25mg/kg) at three dosage schedules. Considering bone marrow transplantation as day 0, ATG were given at days -10, -7 and -5 or -7, -5 and -1 or -5, -3 and -1. At day 0, mice were killed; spleen (SP), bone marrow (BM), lymph nodes (LN) and thymus were removed and analyzed for T-, B-, DC, NK-, T-reg and myeloid cells.

To evaluate the efficacy of immunosuppressive effect of ATG, a group of BALB/c mice were conditioned using busulfan (Bu) and ATG and compared to a control group of BALB/c conditioned with Bu (80mg/kg) followed by cyclophosphamide (200mg/kg). Both groups transplanted with BM and spleen cells from C57BL/6 and followed for engraftment and/or GVHD.

Results The administration of ATG (0.2- 4.5mg/kg) has resulted in an increase in spleen cellularity while in lymph node and thymus a decreased cellularity was observed. We have found that injecting 4.5mg/kg of ATG at day -7, -5 and -3 significantly decreased T-cell population in spleen and LN compare to Bu-Cy conditioning. The ratio CD4/CD8 increased after ATG treatment showing that CD8 cells are six-fold more sensitive to ATG treatment compared to CD4 lymphocyte. Interestingly T-reg cell population increase after ATG at day 0, however, the increment was negatively correlated with the administration time and positively correlated with the dose.

ATG treatment has resulted in an increase in B-cell population by two- and three- fold in spleen and lymph nodes, respectively. Moreover, 1.2- to 3.5-fold increase in DCs, NK and myeloid cells was observed in SP and LN. Thymus cellularity and cell phenotype was less affected while BM cellularity was not affected by ATG treatment. No differences in the ATG effect on cellularity or cell phenotype between IP and IV route was observed.

Despite the high immunosuppressive effect observed in T-cell population compared to that seen in Bu-Cy conditioning, no chimerism was observed when Cy was substituted with ATG (4.5 – 10 mg/kg).

Conclusion No donor chimerism could be obtained using ATG as a single agent up to 25mg/kg. ATG dose and the administration time are important factors affecting the repopulation of residual T-cells in spleen and lymph node that have to be considered in bone marrow transplantation setting.
The Study on Influence of T Cell Ratios Between Grafts to Recipients on Gvhd in HLA- Haploidentical Allogeneic Peripheral Blood Stem Cells Transplantation
Authors: Liang Hui , Deng Xiao-hui , Weng Xia
Published: April 2020

Objective To study the influence of T cell ratios between grafts to recipients on GVHD in HLA- haploidentical allogeneic peripheral blood stem cells transplantation ( PBSCT ).

Methods:

Animal experiment: We set up rat allo-BMT model. Male SD rats were transplanted with female Wistar rats.Recipient rats of experiment group were transplanted with the CD3+ T cells of female Wistar rats at different graft-receptor T cell ratios, 1:1 (group A1), 2:1 ( group A2), 4:1 ( group A3) respectively. Control group (group B) were infused with grafts consisting of 3×107 bone marrow cells by classical method. Observe the incidence and severity of GVHD of different groups. Clinical trial: 6 cases of HLA-haploidentical allogeneic PBSCT. 4 patients were transplantated with purified allo-CD34+ cells.

Next day, adjusted graft-receptor T cell ratios to 2:1 by infusing donor CD3+ T cells. Another 2 patient were treated with classical allo-PBSCT.

Results

Animal experiment: The severity of GVHD in group A1, A2 were lighter compared with group A3,B. The difference is significant among group A1,A2 and A3. Clinical trial: An ultra-acute severe GVHD occurred in 2 patients who were transplanted with classical method.

They died from interstitial pneumonia secondary to PBSCT. The remaining 4 cases only presented mild aGVHD.Among these 4 cases, 3 patients had DFS (disease free survival) over 3 years, 1 patient died from polyradiculitis on +155d.

Conclusion: On the basis of our animal experiment, the appropriate graft-receptor T cell ratios would be 1:1 or 2:1. If we applied this ratio to clinical transplantation, especially to part HLA-related and HLA-haploidentical transplantation, we could obtained a better GVL effect and a lighter GVHD.
Escalating Costs of Cancer Treatment: The Local Effect of a Global Reality
Authors: Souza Danielle Leão
Published: April 2020

While incidence of cancer has been increasing worldwide, so has the cost of cancer treatment. As newer anti-cancer drugs and diagnostic tools have become available, the results of pharmacoeconomic studies have also become more important in the recent years, in guiding cancer treatment policies and reimbursement decisions.

In Brazil, there will be more than 466,000 new cases of cancer in 2008. In this very populated developing country, health policy makers and administrators of private and public health services are becoming increasingly concerned about the cost of cancer treatment.

This health economic project utilized the database of Unimed, a private health care plan which is the biggest in the southern Brazil. The Unimed health plan has over 45,000 clients & 300,000 users in Cascavel region.

The cost of oncology drugs from 2003 to 2007 at two cancer treatment centers in the region was analyzed. The results showed a progressive increase in the expenses at these centers.

Over the period of 5 years, the claims for oncology drug increased 4 fold in one center, and an astronomical 13 fold in the other center. By 2007 the total cost of oncology drugs exceeded US$ 600,000 & US$ 1 million in these 2 oncology centers. Details of the population treated, average cost per patients etc over this period will be presented in the full poster.

While only about 20% (40 million) of the population of Brazil are covered by such private health plans, the dramatic increase of cancer treatment costs seen within this health plan of a single patient population puts in perspective the concerns and funding pressures facing health plans in Brazil, and perhaps the rest of the world.

There is a need for more robust pharmacoeconomic studies to provide clearer treatment guidance and for formulating healthcare policies, so that patients receive better treatment outcomes. A comprehensive Health Technology Assessment processes may also help to streamline how private and state health plans cover such newer treatments, thereby enabling them to provide optimal level of care and services to their patients.

In conclusion, these local Brazilian data confirms that escalating cost of cancer treatment is also a reality in the developing world. Without the support of strong health economic data, it may become increasingly challenging for cancer patients in Brazil to have access to these newer cancer medications.
Direct Confirmation of Quiescent Property of AML Stem Cells by Showing Markedly Decreased Plating Efficiency in Single Leukemic Stem Cell Culture
Authors: Shin Myung-Geun , Kim Hye-Ran , Kim Chang-Soo , Kim Hyeoung-Joon , Kook Hoon , Kee Seung- Jung , Kim Soo-Hyun , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook
Published: April 2020

Cancer stem cell, recently discovered to exist in colon cancer and brain tumor, is resistant to anti-cancer drugs and radiotherapy, demanding the development of new drugs and treatment strategies targeting tumor stem cells. Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). Various anti-cancer medicines, particularly anti-proliferative agents, have been ineffective in treating LSC due to its slower division process and longer interphase compared to normal stem cell and hematopoietic cell. This study comparatively examined growth and proliferation capacity (plating efficiency) of clonogenic hematopoietic progenitors and LSC from healthy donors and AML patients using single cell sorting and culture system (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA). A total of 384 normal hematopoietic stem cells (CD34+CD38+/CD38−) were obtained from peripheral bloods and cord bloods donated by four donors using single cell sorter, and individual single cells were cultured in 96-well plates with each well containing 100ul of serum media, 100ng/ml of stem cell factor, 100ng/ml of Flt-3, 100ng/ml of thrombopoietin and 50ng/ml of G-CSF for five days. 768 single LSC (CD34+CD38−) and 384 single CD34+CD38+ cells were obtained from three AML patients. Growth and proliferation capacities of normal hematopoietic stem cell and LSC were determined in terms of plating efficiency (number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Plating efficiency of individual normal single hematopoietic stem cells varied between samples. Eighty eight out of 192 single stem cells originated from cord blood grew into more than two cells, yielding plating efficiency of 45.8% and cells from the peripheral blood of two healthy donors 30.2% (58/192). In contrast, single LSC originated from the AML patients showed significantly lower plating efficiency with 14.6% (42/288), 3.6% (7/192) and 8.0% (23/288). These results directly confirmed quiescent and slowly dividing properties of LSC. In addition, plating efficiency of normal hematopoietic stem cells was shown to vary between their originating locations in healthy donors.
In Vitro Differentiation of Human Placenta Derived Mesenchymal Stem Cells into Dopaminergic Cells
Authors: Dongmei He , Li Chen , Yuan Zhang
Published: April 2020

Mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after Parkinson diseases. Compared with other stem cells, MSCs derived from human placenta have significant advantages and greater potential for immediate clinical application. The aim of this study was to investigate whether human placenta mesenchymal stem cells(hpMSCs) could be induced to differentiate into dopaminergic cells in vitro. MSCs from human placenta were isolated by digest and density gradient fractionation. Cell surface glycoproteins of these hpMSCs were analyzed by flow cytometry. These hpMSCs were cultured under conditions promoting differetiation to osteoblasts and adipocytes. Using a cocktail that includes basic fibroblast growth factor(bFGF), all trans retinoic acid(RA), ascorbic acid(AA),3-isobutyl-1-methylxanthine(IBMX), hpMSCs were induced in vitro to become dopamine (DA) neurons. The expression of mRNA of nestin and tyrosine hydroxylase(TH) genes known with significant associated with dopaminergic neuron differentiation from the induced hpMSCs was assayed by RT-PCR. The expression of proteins of nestin, dopamine transporter(DAT), neuronal nuclear antigen(NeuN) and TH was determined by immunofluorescence. The synthesized and secreted DA by induced hpMSCs was determined by ELISA. We found that hpMSCs exhibited fibroblastoid morphology. Flow cytometric analyses showed that the hpMSCs were positive for CD44 and CD29, negative for CD34, CD45, CD106 and HLA-DR. Moreover, they could be induced into adipocytes and osteocytes. When hpMSCs were induced with bFGF, RA, AA, IBMX, the MSCs showed a change of neuronal-like cells in morphology. The induced cells express nestin and TH mRNA. The induced MSCs also express nestin, DAT, NeuN, and TH protein. ELISA showed the induced MSCs synthesized and secreted DA. Our results suggest that MSCs from human placenta have the ability to differentiate into dopaminergic cells in vitro.
The Effect of Mesenchymal Stem Cells as a Cell Therapy in Renal Ischemia
Authors: Hong Dae Sik , Park Seongkyu , Won Jong-Ho , Kim Chan Kyu , Lee Sang Chul , Lee Kyu Taeg , Bae Sang Byung , Park Hee Sook
Published: April 2020

Introduction: The most commonly used therapeutic targets in nephrology are the reduction of injury, the delay of progression, or renal replacement therapy. Many animal and human studies demonstrated the role of stem cells in repair and regenerations of kidney. Mesenchymal stem cells (MSCs) have shown to improve outcome of acute renal injury models. It is controversial whether MSCs can reduce injury following a toxic/ischemic event and delay renal failure in chronic kidney disease. We evaluated the hypothesis that the treatment with MSCs could improve renal function and attenuate injury in chronic renal failure (CRF).

Materials and methods: Sprague-Dawley female rats (8 weeks old, 182.2 ± 7.2g) were underwent modified 5/6 nephrectomy. Rats in the MSC group received an injection of MSCs (1 × 106 cells) via tail vein 1 day after nephrectomy. Blood and urine samples were collected after 7 days and every month thereafter. The kidneys of rats were removed for histologic evaluation after 24-hr urine collection and blood sampling. The Y-chromosome stain using fluorescent in situ hybridization was performed to verify the presence of male MSCs in the kidney of female recipients.

Results: No significant differences in blood urea nitrogen and creatinine concentration were observed between MSC group and untreated CRF group. However, the weight gain and creatinine clearance in the MSC group were greater than those of the CRF group. Proteinuria in the MSC group was less after 4 months. Y chromosome was detected in the kidney of MSC group. Although no significances were observed between two groups, the histologic analysis suggests that MSCs have positive effect against glomerulosclerosis.

Conclusions: These results suggest that MSCs help preserve renal function and attenuate renal injury in CRF.