bluuebunny/crossref_metadata_2025_split huggingface dataset:
Distinct Leukemogenic Activity and Imatinib Responsiveness of a BCR-PFGFRα Fusion Tyrosine Kinase.
Authors: Gavrilescu L. Cristina , Cross Nicholas C.P. , Van Etten Richard A. 
Published: October 2019

Abstract
Chromosomal rearrangements of the platelet-derived growth factor receptor alpha (PDGFRA) gene on 4q12 have been described in a subset of patients with idiopathic hypereosinophilic syndrome/chronic eosinophilic leukemia (IHES/CEL). The most common, found in 12–17% of CEL patients, is the FIP1L1-PDGFRA fusion, resulting from a cytogenetically invisible deletion on 4q12 (Cools et al., NEJM2003; 348:1201; Griffin et al., PNAS2003; 100:7830). FIP1L1-PDGFRA+ CEL patients have an excellent response to low-dose imatinib therapy, often accompanied by complete molecular remission (Pardanani et al., Leuk. Res.2006; 30:965). Additional fusion partners of PDGFRA include KIF5B and CDK5RAP2 in CEL, and BCR in atypical chronic myeloid leukemia with eosinophilia and t(4;22)(q12;q11) (Baxter et al., Hum. Mol. Genet.2002; 11:1391). However, the oncogenic activity and imatinib responsiveness of these other activated PDGFRA alleles are unknown. In this study, we assessed the imatinib sensitivity of BCR-PDGFRα and FIP1L1-PDGFRα in hematopoietic cell lines, and compared their leukemogenic activity in a mouse retroviral bone marrow transduction/transplantation model. Like FIP1L1-PDGFRα, BCR-PDGFRα transformed Ba/F3 cells to become independent of IL-3 for survival and growth. In the absence of IL-3, FIP1L1-PDGFRα-expressing Ba/F3 cells were 30-fold more sensitive to imatinib than BCR-PDGFRα (IC50 = 3 nM vs. 90 nM) for inhibition of proliferation and induction of apoptosis. A FIP1L1-PDGFRα N659D mutant (Cools et al., Cancer Cell2003; 3:459) was relatively resistant to imatinib (IC50 = 200 nM), while the corresponding BCR-PDGFRα N659D mutant displayed increased imatinib resistance (IC50 > 2 μM). Inhibition of cellular proliferation correlated with decreased tyrosyl phosphorylation of the respective fusion kinase and of the downstream substrate PLCγ1. In leukemogenesis experiments, recipients of bone marrow transduced with retrovirus expressing either FIP1L1-PDGFRα or BCR-PDGFRα developed fatal myeloproliferative disease, with similar median and overall survival. This was accompanied by significant eosinophilia, relative to the disease induced by BCR-ABL in this model system (Li et al., Blood2001; 97:1442). Treatment of recipients of FIP1L1-PDGFRA- or BCR-PDGFRA-transduced marrow with imatinib (125 mg/kg/day by oral gavage for 20 days) suppressed leukocytosis and prolonged their survival. These results suggest that distinct signaling pathways activated by leukemogenic PDGFRα fusions in hematopoietic progenitors induce eosinophila in vivo. However, different fusion partners of PDGFRα can significantly influence the sensitivity of the fusion kinase to imatinib treatment.
A Microelectronic DNA Chip Detects the V617F JAK-2 Allelic Ratio in Myeloproliferative Disorders.
Authors: Di Ianni Mauro , Moretti Lorenzo , Del Papa Beatrice , Ciurnelli Raffaella , De Ioanni Mariangela , Falzetti Franca , Tabilio Antonio 
Published: October 2019

Abstract
A single activating mutation in Janus Kinase (JAK)-2 is detected in 65%–97% patients with polycythemia vera (PV), in 23%–57% with essential thrombocytemia and in 35%–57% with idiopathic myelofibrosis. Recent studies have demonstrated the heterozygote population contains clonal and polyclonal granulopoiesis; clonality correlates with the Jak2V617F allelic ratio. We developed a microchip diagnostic platform (NMW NanoChip Molecular Biology Workstation, manufactured by Nanogen Inc, San Diego, CA) to identify the JAK-2 mutation in granulocyte-derived DNA and determine the allelic ratio. The system enables electronic deposition of biotinylated amplicons to selected pads. Twenty-three DNA samples from patients with PV were analysed by direct sequencing, AS-PCR and microelectronic chip. Sequencing results: a fragment of 345 genomic DNA was amplified by PCR under standard condition. 21/23 samples showed mutated JAK-2, 18/21 presented wild-type and mutated alleles; 3/21 had only the mutated allele. 21/23 samples showed mutated JAK-2. Microelectronic chip results: 21/23 samples showed mutated JAK-2, with a frequency of 91%. All samples were genotyped: at the first analysis 3 were identified as homozygotes; 18 were identified as heterozygotes. The allelic ratio in the 3 homozygotes shows residual wild-type allele due, in our view, to residual polyclonal granulopoiesis which the microchip detects. The allelic ratio of the 18 designated heterozygotes showed different T/G percentages, suggesting different disease stages. Combining microchip results with clonality assays could distinguish true heterozygotes. Overall genotyping was performed in 100% of positive cases. Sensitivity assay (on serial dilution of mutated HEL vs unmutated K562 cell lines) showed the electronic microchip detects the JAK-2 mutation in at least 1% of a total cell mixture. Our system is a fast, reliable and sensitive method alternative to DNA sequencing, AS-PCR or dHPLC. It overcomes the relative low sensitivity of direct sequencing and at the same time provides genotyping data even when sequencing fails or results are hard to interpret. This electronic microchip system is as sensitive as dHPLC but does not require an extra sequencing step for genotyping. Specific advantages over AS-PCR include automated calculation of the allelic ratio and consequently the genotype without any reduction in sensitivity.
Altered Expression and Signalling of Bone Morphogenetic Protein Receptor 1A (BMPR-1A) and Enhanced Hematopoietic Progenitor Cell Growth in Long-Term Stromal Culture Assays from Two Patients with a Myeloproliferative Syndrome and Pulmonary Hypertension.
Authors: Hildebrandt Martin , Bommert Kurt , Rautenberg Kirstin , Ludwig Wolf-Dieter 
Published: October 2019

Abstract
The rare association of pulmonary hypertension and myeloproliferative syndromes (MPS) has been described previously (Garcia-Manero G et al., Am J Hematol1999; 60(2): 130-5, Dingli D et al., Chest2001, 120(3): 801-8), but the mechanisms contributing to this potentially hazardous condition await further elucidation. Bone morphogenetic protein receptors (BMPR) modulate the size of the hematopoetic niche (Zhang J et al., Nature2003, 425(6960): 836-41), with an inhibitory effect on myeloproliferation. In the pulmonary vascular bed, a decreased or deficient expression of BMPR has been shown to result in the proliferation of vascular smooth-muscle cells, asymmetric neointimal hyperplasia in small pulmonary arteries and subsequent pulmonary hypertension (Lane KB et al., Nat Genet2000, 26(1):81-4; Du L et al, N Engl J Med2003, 348(6): 500-9). We hypothesized that, in patients suffering from MPS and pulmonary hypertension, changes in the expression of BMPR and subsequent signalling molecules Angiopoietin-1 (Ang-1) and its receptor, TIE-2, may occur not only in the lung, but also in the bone marrow and correspond with enhanced myeloproliferation in vitro.
Bone marrow stromal cells were cultured frompatients with pulmonary hypertension and MPS (n=2),patients with MPS and no evidence of pulmonary hypertension (n=3) andhealthy controls (n=3).The cultured cells were subjected to Western Blot analysis for the expression of BMP receptors BMPR-1A and BMPR-2, Angiopoietin-1 and TIE-2. Furthermore, a modified long-term culture initiating cell (LTC-IC) assay was established using the cultured bone marrow stromal cells as layers for autologous and allogeneic progenitor cell assays.
The two patients suffering from both, MPS and pulmonary hypertension, showed a diminished expression of BMPR-1A and an enhanced expression of Ang-1 and TIE-2 in cultured stromal cells when compared to patients with MPS alone and to healthy controls. In one of the two patients, a three- to fourfold increase in the number of long-term culture-initiating cells after seeding of CD34-positive cells and of bone marrow mononuclear cells from healthy donors and from the other patients included was observed in modified LTC-IC assays.
Our observations argue in favour of changes in BMP receptor expression and signalling with impact not only on pulmonary hypertension as described before, but also on the emergence of a myeloproliferative state.
Enhancement of Hematopoietic Stem Cell Engraftment and Graft vs. Leukemia Effect Induced by Matched and Haploidentically Mismatched Allograft Using Multi-Donor Stem Cell Transplantation (MDT).
Authors: Shapira Michael Y. , Or Reuven , Resnick Igor B. , Samuel Simcha , Gesundheit Benjamin , Slavin Shimon 
Published: October 2019

Abstract
The aim of allogeneic stem cell transplantation (SCT) is to combine tumor cytoreduction with optimal doses of chemoradiotherapy and to replace host with donor immunohematopoietic cells. Induction of host-vs.-graft tolerance by engraftment of donor stem cells, enables durable engraftment of donor lymphocytes, with subsequent induction of graft vs. leukemia effect (GVL) expected to eliminate all residual chemoradio-resistant malignant cells of host origin by alloreactive donor cells. Relapse shortly post SCT indicates tumor refractoriness and carries poor prognosis.
Multi-donor stem cell transplantation (MDT) is an innovative transplantation strategy aiming at both increasing the number of donor stem cells infused to the recipient to improve the chance of engraftment of at least one of the donors, thus avoiding the need for re-transplantation in case of rejection, while in parallel improving the displacement of residual host cells and GVL effect by stimulation of alloreactive donor T cells as bystander effect to the rejection of 1 donor by the other donor in order.
We describe 4 patients that were treated with MDT following early post allogeneic transplant relapse (n=3, resistant AML) or rejection (n=1). In order to reduce the risk for the patients, and with the assumption that MDT will stimulate anti host immune response leading to engraftment and GVL, we chose to combine transplantation from 2 donors (including the 1st donor in the patients transplanted due to relapse and both parents in the patient transplanted due rejection). The patients received a non myeloablative conditioning treatment. All patients engrafted (2 with the 2nd donor, 1 with 1st donor and 1 from his mismatched mother) with 100% donor chimera. Time to engraftment was short - median 11d (range 10–12). CR was achieved with the appearance of GVHD probably due to stimulation of donor vs. donor (and therefore against host alloantigens). No relapse was documented during follow up. Two of the recipients are currently alive with follow-up ranging 1.5–25m.
Conclusions: The use of MDT induced engraftment and GVL in patients with chemo-immuno-resistant leukemia. In view of the profound anti host activating effect of MDT, we plan to assess its therapeutic potential on engraftment facilitation when reduced intensity conditioning is used in patients in need for SCT lacking an HLA matched related or unrelated donor.
ZAP-70 Expression and Stem Cell Transplantation Results in Patients with CLL.
Authors: Villamor Neus , Salamero Olga , Esteve Jordi , Carreras Joaquim , Moreno Carol , Rovira Montserrat , Aymerich Marta , Bosch Francesc , Carreras Enric , Colomer Dolors , Campo Elías , Montserrat Emili 
Published: October 2019

Abstract
The absence of mutations in the VH gene (UM-VH) and the expression of ZAP-70 by leukemic cells are closely related adverse prognostic factors in CLL. Whether ZAP-70 should substitute VH gene analysis as prognostic predictor in patients with CLL is a matter of debate. We and others have shown that allogeneic but not autologous stem-cell transplantation may overcome the negative impact of the UM-VH on the outcome of patients with CLL. Herein we report on transplantation results based on ZAP-70 expression in leukemic cells. Thirty-seven patients (24 M/13 F; median age: 48 years, range: 29-63) received either an autologous (auto-SCT) (n=22) or an allogeneic (allo-SCT) (n=15) transplant. ZAP-70 was assessed by flow cytometry (19 cases), lymph node immunohistochemistry (10 cases) or both (8 cases). No discrepancies were observed in those cases in which both peripheral blood and lymph nodes were analyzed. Thirty-one patients were ZAP-70 positive and 6 ZAP-70 negative. ZAP-70 was positive in all UM-VH (n=24) and in 4 of 8 mutated VH patients. After a median follow-up of 62 months (range: 4 – 142), 5-year survival is 86% (66% to 100%) for allo-SCT and 50% (28% to 72%) for auto-SCT (p=NS), whereas 5-year disease-free survival (DFS) is 80% (54% to 100%) for allo-SCT and 37% (15% to 59%) for auto-SCT (p=0.03). Seventeen patients have relapsed: 16 of 31 of those being ZAP-70 positive and 1 of 6 of those ZAP-70 negative. In ZAP-70 positive patients the cumulative relapse rate at 5 years is 10% (2% to 65%) in the allo-SCT group and 53% (35% to 81%) in the auto-SCT group (p=0.04) (figure 1a). In addition, ZAP-70 positive patients submitted to allo-SCT have a trend for a longer DFS (76% at 5 years; 46% to 100%) than those receiving an auto-SCT (39% at 5 years; 15% to 63%) (p=0.07) (figure 1b). In summary, among transplanted patients those with ZAP-70 positive CLL have a better relapse rate and DFS if submitted to allo-SCT.
Figure Figure
WT1-Specific CD8+ T Lymphocytes May Participate in the Elimination of Acute Lymphoblastic Leukemia Following Allogeneic Stem Cell Transplantation.
Authors: Rezvani Katayoun , Yong Agnes , Mielke Stephan , Savani Bipin N. , Price David A. , Gostick Emma , Douek Daniel C. , Barrett A. John 
Published: October 2019

Abstract
There is clinical evidence that a graft-versus-leukemia (GVL) effect occurs following allogeneic stem cell transplantation for acute lymphoblastic leukemia (ALL). However, the potency of this GVL effect is often associated with unwanted graft-versus-host-disease (GVHD) and disease relapse remains a major contributor to treatment failure. Wilms’ tumor gene 1 (WT1) is overexpressed in 70–90% of cases of ALL and has been identified as a convenient minimal residual disease (MRD) marker. WT1 is an attractive immunotherapeutic target in ALL because peptides derived from WT1 can induce CD8+ T-cell responses, and being non-allelic, WT1 would be unlikely to provoke GVHD. We investigated whether CD8+ T-cells directed against an HLA-A*0201 restricted epitope of WT1 (WT126) occur in ALL patients during the early phase of immune reconstitution post-SCT (days 30–180). We analyzed CD8+ T-cell responses against WT1 in 10 HLA-A*0201+ ALL SCT recipients and their respective donors using WT1/HLA-A*0201 tetrameric complexes and flow cytometry for intracellular IFN-gamma. We studied the kinetics WT1-specific CD8+ T-cell responses in consecutive samples obtained post-SCT. CD8+ T-cells recognizing WT1 were detected ex vivo in samples from 5 of 10 ALL patients post-SCT but not in patients pre-SCT. WT1-tetramer+ CD8+ T cells had a predominantly effector memory phenotype (CD45RO+CD27−CD57+). WT1 gene expression in pre-SCT and donor samples was assayed by quantitative real-time PCR (RQ-PCR). WT1 expression in PBMC from healthy donors was significantly lower than in patients (median 0, range 0–66 ×10−4 WT1/ABL compared to patients, median 12, range 0–2275 ×10−4 WT1/ABL) (P < 0.01). There was a strong correlation between the emergence of WT1-specific CD8+ T cells and a reduction in WT1 gene expression (P < 0.001) (as depicted below) suggesting direct anti-ALL activity post-SCT. Disappearance of WT1-specific CD8+ T-cells from the blood coincided with reappearance of WT1 gene transcripts, consistent with a molecular relapse, further supporting the direct involvement of WT1-specific CD8+ T-cells in the GVL response. These results provide evidence for the first time of spontaneous T-cell reactivity against a leukemia antigen in ALL patients. Our results support the immunogenicity of WT1 in ALL patients post-SCT and a potential application for WT1 peptides in post-transplant immunotherapy of ALL.
Figure Figure
Cytotoxic T Lymphocyte Responses to PR1 Peptide in Chronic Myeloid Leukemia Patients Inversely Correlate with Proteinase 3 and Elastase Expression but Donor PR1 Responses Determine Survival after Stem Cell Transplantation.
Authors: Yong Agnes S.M. , Rezvani Katayoun , Savani Bipin N. , Eniafe Rhoda , Mielke Stephan , Goldman John M. , Barrett A. John 
Published: October 2019

Abstract
The primary granule proteins (PGP) neutrophil elastase (ELA2) and proteinase 3 (PR3) both contain the nonapeptide PR1 which can induce cytotoxic T lymphocyte (CTL) responses in chronic myeloid leukemia (CML). The relative contribution of PR3 and ELA2 to PR1 expression is not known. We previously found that higher levels of PR3 and ELA2 gene expression in CD34+ progenitor cells were associated with longer survival in CML patients. Eradication of leukemia depends on the elimination of leukemic stem cells which are thought to reside within the CD34+ progenitor cell pool. We therefore studied PGP expression and T cell response to PR1 in 23 CML patients and their HLA-identical family donors prior to T-cell depleted allogeneic stem cell transplantation (SCT) with T-cell add-back on day 45–100 post-SCT. CD8+ T cells and CD34+ progenitor cells were purified from mononuclear cells (MNC) of cryopreserved leukapheresis products from HLA-A*0201+ CML patients. Following reverse transcription of RNA from MNC and CD34+ cells, ELA2 and PR3 gene expression was measured using real-time quantitative polymerase chain reaction (RQ-PCR). To assess PR1-CTL responses, T2 cells were loaded with PR1 peptide (VLQELNVTV) at 0.1, 1, and 10μM for 2h, irradiated and subsequently co-cultured with CD8+ T cells for 3h. PR1-CTL response was measured as interferon-γ mRNA expression (relative to CD8) obtained by RQ-PCR. PR1-specific CTL responses were detected in 5/23 CML patients and 6/16 HLA-identical donors. In CML patients, pre-SCT expression of both PR3 and ELA2 in MNC was strongly correlated with the expression in CD34+ cells (p=0.009 and p=0.0006 respectively). There was an inverse relationship between PR1-CTL response in CML patients pre-SCT and PR3 or ELA2 expression (p=0.02 and p=0.01 respectively). This data suggest that both PR3 and ELA2 expression in CD34+ cells and their progeny are a potential source of PR1. However, high expression of these proteins may result in selective deletion of PR1 T cell clones. The presence of PR1-responses in HLA-identical donors was associated with an improved overall survival post-SCT. However, there was no impact of PR1-response in either CML patients pre-SCT or donors, on time to achieve molecular remission post-SCT but a greater proportion of patients whose donors did not have a PR1-response succumbed to fatal graft-versus-host disease. These findings support post-transplant vaccination strategies with PR1 peptide to eradicate minimal residual disease but in patients who are not eligible for SCT, the lower PR1 response in the presence of high PGP expression suggests that there is a risk that vaccines given to patients with minimal residual disease could induce tolerance.
Figure Figure
Anti-Leukemic CTL Generated by RNA Loaded Cord Blood-Derived Dendritic Cells Control Leukaemia in a NOD-SCID Mouse Model.
Authors: Hsu Andy K.W. , Kerr Beverley M. , Lock Richard B. , Hart Derek N.J. , Rice Alison M. 
Published: October 2019

Abstract
Acute Lymphocytic Leukaemia (ALL) patients who relapse after transplantation have few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect may improve their survival. We have developed a method for generating cord blood derived dendritic cells (CBDC) and transfecting them with high efficiency (>90% eGFP positivity) and viability (>90%) (BBMT 2006 12:855–867). We postulate that CTL generated from total ALL RNA-loaded CBDC may be exploited in therapy and therefore tested their ability to control the kinetics of leukaemic growth in a NOD-SCID mouse model of human ALL.
CTL induced using total RNA from the pre-B ALL Nalm-6 cell line lysed 49±11% of Nalm-6 cells compared to 7%±1% of autologous and 2.5%±1.3% of allogeneic mononuclear cells (p=0.03 and p=0.022 respectively). Using total RNA from patient derived ALL xenograft, we generated anti-leukemic CTL cultures comprising a mixed population of 69.9±2% CD4, 12.9±2% CD8 and 17.6±1% NK cells. These CTL lysed 63±6% of ALL xenograft targets and 22±7% of autologous MNC (p=0.04) and also lysed autologous survivin mRNA-transfected DC (16±5.4%). Preliminary results suggest that some of the ALL xenograft CTL also recognize the cell surface protein, HM1.24, which is specifically up-regulated in metastatic tumor cells and chemoresistant cancer cells. This suggests that the total ALL RNA generated CTL comprise a polyclonal population, which may prove efficacious in vivo. To test the in vivo efficacy of these anti-leukemic CTL, we established a NOD-SCID mouse model of ALL using patient samples. By varying the inocula of ALL cells, we established a predictable and reproducible model of emerging relapse.
Administration of anti-leukaemic CTL 2, 3, 4 and 5 weeks post ALL inoculation, in increasing doses (low - 2.5×106 cells/mouse, medium - 10×106/mouse and high - 20×106/mouse), altered the kinetics of ALL growth in vivo (p=0.00032, see figure). By 8 weeks post transplant, mice given ALL alone had 3.6% human CD45+CD19+ leukemic cells in the blood, whereas recipients of the highest CTL dose had only 0.66% CD45+CD19+ leukemic cells in the blood (p=0.05). After 11 weeks the disease load was the same in mice who received ALL alone and those who received ALL and 4 doses of 20 × 106 CTL. These results suggest that anti-leukemic CTL generated by RNA-loaded CBDC can control leukaemia in a NOD-SCID mouse model and that disease control may be enhanced by antigen-specific restimulation of the anti-leukaemic CTL and/or more refined administration. These results represent an important step forward in the development of clinical immunotherapy protocols for the treatment of ALL and its potential application after cord blood transplantation.
Anti-leukaemic CTL infusions alter the kinetics of ALL engraftment Anti-leukaemic CTL infusions alter the kinetics of ALL engraftment