bluuebunny/crossref_metadata_2025_split huggingface dataset:
BCR/ABL Kinase Disrupts Formation of Mismatch Repair Complex To Induce Genomic Instability.
Authors: Stoklosa Tomasz , Slupianek Artur , Basak Grzegorz , Skorski Tomasz 
Published: April 2020

Abstract
Among different genotoxic agents, BCR/ABL cells are more resistant to N-methyl-N’-nitro-N-nitrosoguanine (MNNG), which methylates 06 position of guanine. 06 -MeG bases pair with either T or C during replication giving rise to mismatches. Mismatch repair (MMR) proteins are responsible for detecting and removing misincorporated nucleotides, which escaped proofreading activity of DNA polymerases. MMR proteins assembled on the mismatch can signal to repair or apoptosis. Defects in expression of MMR genes leads to drug resistance and mutator phenotype, observed in several different solid tumors. The role of MMR system in drug resistance and/or genomic instability of leukemic cells remains poorly understood. Parental cells and BCR/ABL expressing clones were incubated with MNNG for 4 weeks resulting in their MNNG-resistant derivatives, which may accumulate mutations in their genomic DNA resulting from methylating activity of the drug. To investigate the mutation rate and phenotype, ouabain-resistance test was employed. Cells become resistant to ouabain, a glycoside that inhibits ATP1A1 subunit of Na/K ATP-ase, when mutations arise in the gene fragment encoding this subunit. The clonogenic assay revealed over 5 times more ouabain resistant colonies in MNNG-resistant BCR/ABL-positive cells than in parental counterparts. To investigate the type of mutations, a fragment of Na/K ATP-ase gene encoding the ATP1A1 subunit was sequenced from MNNG-resistant BCR/ABL-positive and parental cells. The dominating mutation in BCR/ABL MNNG-resistant cells was C to T, while A to G mutations were prevalent in parental cells. In order to check the status of MMR proteins, Western blotting and immunofluorescence studies were performed. Expression of MMR proteins in BCR/ABL transformed cells was similar to parental cells, however immunofluorescence visualized dramatic changes after DNA damage in the nuclear co-localization of MMR proteins in BCR/ABL-transformed cells (CML patient cells and leukemic cell lines) in comparison to normal cells. Co-localization of MSH2 and MSH6 proteins, forming a heterodimer homologous to bacterial MutS, remained similar in parental and leukemia cells upon MNNG treatment. However, co-localization of MLH1 and PMS2 proteins, which form a heterodimer homologous to bacterial MutL, was detected in non-transformed cells, but not in BCR/ABL leukemia cells. In addition, MLH1 and MSH2 co-localized in normal, but not BCR/ABL-positive cells after MNNG treatment. The defects in interaction of MMR proteins in leukemia cells were reversed by inhibition of BCR/ABL kinase by STI571. Thus, the assembly of MMR proteins on mismatched bases and subsequent signaling to repair and/or apoptosis may be impaired in BCR/ABL leukemia cells. These results suggests a novel mechanism of regulation of DNA damage response proteins by oncogenic tyrosine kinase that can lead to genomic instability and drug resistance of leukemic cells.
ATR-Chk1 Axis Is Activated, but the Function of Chk1 Is Disrupted in BCR/ABL Leukemia Cells Responding to DNA Damage.
Authors: Nieborowska Margaret , Stoklosa Tomasz , Datta Mandrita , Czechowska Agnieszka , Rink Lori , Blasiak Janusz , Skorski Tomasz 
Published: April 2020

Abstract
BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assays. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. BCR/ABL leukemia cells displayed higher activity of ATR kinase, which is stimulated by replication-dependent DSBs. ATR phosphorylates histone H2AX on serine 139 (γ-H2AX) on megabase-length fragments near DSB sites. In accordance with PFGE and comet assay, BCR/ABL-positive leukemia cells accumulated more γ-H2AX protein and γ-H2AX nuclear foci than normal counterparts during 6h after genotoxic treatment. γ-H2AX started to disappear in BCR/ABL cells, while continued to increase in parental cells. Altogether, these results support the hypothesis that higher kinase activity of ATR in BCR/ABL leukemia cells results from their response to elevated levels of drug-induced replication-dependent DSBs, and that more efficient repair is associated with better survival. More abundant expression and prolonged phosphorylation on serine 345 of the Chk1 kinase, an important ATR kinase downstream effector and cell cycle regulator, was detected in leukemia cell lines and CML patient cells after genotoxic treatment. Interestingly, inhibition of ATR kinase by caffeine, but not Chk1 kinase by indolocarbazole inhibitor SB-218078 sensitized BCR/ABL leukemia cells to mitomycin C. In conclusion, although ATR - Chk1 axis appeared strongly activated in BCR/ABL leukemia cells responding to DNA cross-linking agents causing numerous replication-dependent DSBs, its function in drug resistance may be disassembled downstream of the Chk1 kinase.
Chronic Myeloid Leukemia (CML) Cells Express Tumor Associated Antigens Eliciting Specific CD8+ T Cell Responses Despite of Deficient Expression of Costimulatory Molecules.
Authors: Greiner Jochen , Li Li , Giannopoulos Krzysztof , Brunner Christian , Döhner Konstanze , Döhner Hartmut , Schmitt Michael 
Published: April 2020

Abstract
Specific immunotherapies for CML patients targeting T cell antigens might eliminate residual CML cells after therapy with imatinib or chemotherapy and might enhance a specific graft versus leukemia effect after allogeneic stem cell transplantation without aggravating the graft versus host disease. However, for effective specific immunotherapies in CML, extended studies on the expression, the function and the immunology of leukemia-associated antigens (LAAs) and on the LAA presentation by leukemia cells are required.
Here, we investigated on expression and functional aspects of tumor/leukemia-associated antigens (TAAs/LAAs) in CML. Several LAAs are expressed and are therefore candidate structures for specific immunotherapies: bcr-abl (100%), G250 (24%), hTERT (53%), MPP11 (91%), NEWREN60 (94%), PRAME (62%), Proteinase3 (71%), RHAMM/CD168 (83%) and WT1 (53%), but not BAGE, MAGE-A1, SSX2 or NY-ESO-1. The expression of LAAs varied according to leukocyte subsets and the expression of RHAMM/CD168, Proteinase3 and PRAME was upregulated in accelerated phase and blast crisis. Quantitative expression of several TAAs/LAAs correlated with the clinical course. In flow cytometry, CD34+ CML progenitor cells typed positive for HLA-molecules, but were deficient for CD40, CD80, CD83 and CD86. This lack of costimulatory molecules might constitute a tumor escape mechanism. However, RHAMM/CD168-R3-specific T cell responses were demonstrated by ELISPOT analysis and specific lysis of R3-peptide pulsed T2 cells in chromium-51 release assays. These CD8+ cells could be phenotyped as CCR7-CD27-CD45RA+ early effector T cells by tetramer staining. In conclusion, several LAAs are expressed in CML and are therefore candidate structures for specific immunotherapies. CML progenitor cells express HLA-molecules, but lack costimulatory molecules. However, as CML patients show RHAMM/CD168-R3-specific early effector T cells, peptide vaccination might be therefore a promising approach to enhance specific immune responses in CML.
Reduced-Intensity Unrelated Cord-Blood Transplantation (RI-UCBT) for Patients with High-Risk Myeloid Malignancies.
Authors: Yuji Koichiro , Miyakoshi Shigesaburo , Takagi Shinsuke , Kato Daisuke , Miura Yuji , Narimatsu Hiroto , Kusumi Eiji , Kishi Yukiko , Matsumura Tomoko , Kobayashi Kazuhiro , Hamaki Tamae , Uchida Naoyuki , Kami Masahiro , Masuoka Kazuhiro , Murashige Naoko , Wake Atsushi , Ueyama Junichi , Yoneyama Akiko , Matsuzaki Michio , Morinaga Shinichi , Muto Yoshitomo , Takaue Yoichi , Taniguchi Shuichi 
Published: April 2020

Abstract
&lt;Background&gt;Allogeneic stem cell transplantation (allo-SCT) is a curative treatment for advanced myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The therapeutic benefits are attributable to myeloablative radiochemotherapy and graft-versus-leukemia effect, whereas severe regimen-related toxicity (RRT) limits the efficacy of allo-HSCT to young patients without co-morbidities. Reduced-intensity stem-cell transplantation (RIST) using a non-myeloablative preparative regimen has been developed to decrease RRT, whilst preserving an adequate antitumor effect. RIST may be a curative treatment for heavily pretreated elderly patients with myeloid malignancies. Umbilical cord blood from unrelated donors has been used as an alternative stem cell source. We report the results of reduced-intensity unrelated cord-blood transplantation (RI-UCBT) in patients with advanced or high-risk myeloid malignancies.
&lt;Patients and Methods&gt;Forty-eight patients (median age, 59 yr; range, 17–70) underwent RI-UCBT with a preparative regimen consisting of fludarabine 125 mg/m2, melphalan 80 mg/m2, and 4 Gy of total body irradiation from 2002 to 2004. Twelve-seven patients were classified as AML not otherwise categorized, 16 as AML with multilineage dysplasia, 4 as RAEB and 1 as RA. The disease status at transplant was 2 CR1, 5 CR2 or CR3, 3 untreated and 38 refractory. The median infused total cell dose was 2.8 (range, 1.8–4.5) x 107/kg. HLA match was 6/6 in 1, 4/5 in 8, and 4/6 in 39 cases. GVHD prophylaxis was composed of cyclosporine (n=25) or tacrolimus alone (n=23).
&lt;Results&gt; Fourty-three patients achieved primary neutrophil engraftment after a median of 20 days. Twelve patients developed grade II–IV acute GVHD and 7 developed chronic GVHD. Fifteen patients achieved CR. Eleven patients experienced relapse. Thirty-one patients died as a result of relapse (n=10), GVHD-associated complications (n=10), or infection (n=11). With a median follow-up of 489 days (range, 192–768), 17 of 48 patients are alive, resulting in a 2-year overall survival rate of 31% (95% CI, 16% to 45%). GVHD prophylaxis influenced outcomes (p&lt;0.05).
&lt;Discussion&gt; These data suggest that RI-UCBT may be an effective option for patients with high-risk myeloid malignancies who lack an HLA-matched donor. RI-UCBT is associated with high TRM, providing a rationale for a larger clinical study, which should be modified to focus on enhancing any GVL effect, minimizing toxicities, and controlling infectious complications.
Figure Figure Figure Figure
Results of Alemtuzumab-Based Reduced-Intensity Allogeneic Transplantation for Advanced Chronic Lymphocytic Leukemia: A BSBMT Study.
Authors: Delgado Julio , Thomson Kirsty , Russell Nigel , Ewing Joanne , Stewart Wendy , Cook Gordon , Devereux Stephen , Lovell Richard , Chopra Rajesh , Mackinnon Stephen , Milligan Donald 
Published: April 2020

Abstract
We report 41 consecutive patients with advanced chronic lymphocytic leukemia (CLL) who underwent allogeneic hematopoietic cell transplantation (HCT) following fludarabine, melphalan and alemtuzumab reduced intensity conditioning. Donors were 24 HLA-matched siblings and 17 unrelated volunteers (4 of them mismatched). Median age at transplant was 54 (range 37–67) years, interval from diagnosis to HCT was 54 (10–164) months, and number of previous chemotherapy regimens was 3 (1–6). Eleven patients were refractory to fludarabine at the time of transplant and 3 others (8%) had it stopped due to immune cytopenias. Eleven patients had failed autologous HCT. At the time of transplant, 7 patients (17%) had chemo-refractory and 34 (83%) chemo-sensitive disease, but only 5 (12%) were in complete remission.
All but 3 patients had initial hematological recovery, but 5 more patients had delayed graft failure that responded to subsequent stem-cell infusions. Median intervals to neutrophil (&gt; 0.5 × 109/l) and platelet (&gt; 20 × 109/l) recovery were 14 (range 9–30) and 11 (range 8–45) days, respectively. Eleven patients (27%) relapsed and received escalated donor lymphocyte infusions, but only 3 of them had a sustained response. Acute and chronic graft-versus-host disease (GVHD) was observed in 17 (41%) and 13 (33%) patients, respectively.
With a median follow-up of 15 (range 0.2–62) months, 17 patients have died, 5 of progressive disease and 12 of transplant-related complications. The 2-year overall survival, progression-free survival and transplant-related mortality are 51% (CI 33%–69%), 45% (27%–62%) and 26% (14%–46%), respectively (Figure 1). In multivariate analysis, fludarabine refractoriness prior to transplant was the only factor to predict a worse progression-free survival in this setting.
In conclusion, the alemtuzumab-based regimen was feasible and effective in patients with CLL with a relatively low rate of GVHD. However, transplant-related mortality remains relatively high as a result of a variety of viral and fungal infections. Ongoing studies are aiming to address the efficacy of reduced doses of alemtuzumab in this group of very immunosupressed patients.
Figure Figure
Activated Protein C Light Chain Mutations Distinguish Exosite Residue Requirements for Anticoagulant Versus Cytoprotective Activities.
Authors: Mosnier Laurent O. , Griffin John H. 
Published: April 2020

Abstract
Activated protein C (APC) can exert two major distinct activities: 1) anticoagulant activity, via inactivation of factors Va and VIIIa, and 2) cytoprotective activity including anti-apoptotic and anti-inflammatory activities mediated by two receptors, Endothelial Protein C Receptor (EPCR) and Protease Activated Receptor-1 (PAR-1). Interest in APC’s receptor-mediated effects is sparked by APC’s reduction of mortality in severe sepsis patients and by the beneficial neuroprotective activities of APC on brain endothelium, on neurons and in rodent ischemic stroke models. Our previous targeted mutagenesis of exosite residues in the protease domain in APC’s heavy chain proved that anticoagulant activity can be almost ablated while leaving anti-apoptotic activity intact, thus showing that some heavy chain exosite residues are required for one activity but not the other. To further characterize APC exosites required for various functional activities, we extended site-directed mutagenesis studies to the C-terminal region of the light chain (residues 142–155) which was indirectly implicated as an anticoagulant exosite but which has not been targeted using mutagenesis. Alanine-scanning mutagenesis was used to probe the requirement for Arg143, Lys146, Arg147, Glu149, Lys150, Lys151 and Arg152 for zymogen activation and for expression of APC’s anticoagulant and cytoprotective activity. Kinetic data for activation of each mutated zymogen by thrombin and by thrombin-thrombomodulin were indistinguishable from wild type protein C results. APC mutants with single Ala substitutions at residues 146, 147, 150 and 151 had significantly reduced anticoagulant activity in APTT assays while substitution at 143 and 152 showed little change relative to wild type APC. Surprisingly, the Ala149-APC exhibited over 3-fold increased anticoagulant activity. No mutation altered the half-life of APC amidolytic activity in plasma, suggesting that APC’s reaction with key serine protease inhibitors was unaltered by these mutations. As determined using staurosporine-induced endothelial cell apoptosis assays, replacement of the various Arg and Lys residues by Ala had little significant effect on APC cytoprotective activity; however, the Glu149 to Ala substitution markedly reduced APC cytoprotective activity. Thus, whereas this latter mutation increased by 3-fold anticoagulant activity, it diminished APC direct effects on cells. In summary, these studies of the C-terminus of the APC light chain emphasize the distinction between exosite residue requirements for APC’s anticoagulant versus cytoprotective mechanisms; furthermore, these results may contribute to the design of safer therapeutic APC variants with reduced risk of bleeding due to reduction in anticoagulant activity but with the normal beneficial direct effects of APC on cells.
Transplantation of HLA-Mismatched Stem Cell Transplantation without T Cell Depletion for the Treatment of Malignant Hematological Disease.
Authors: jun Huang Xiao , Liu Daihong , Liu Kai Yan , ping Xu Lan , Chen Huan , Han Wei , Chen Yu Hong , zhi Wang Jing , yong Gao Zhi , Zhang Yao Chen , Jiang Qian , xia Shi Hong , pei Lu Dao 
Published: April 2020

Abstract
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only therapeutic option for many hematological malignancies. Many patients requiring allo-HSCT do not have a human leukocyte antigen (HLA)-matched donor. For those patients, unrelated bone marrow or cord blood is often used but with many limitations such as time-consuming search and deficient cell numbers. Recently, HLA mismatched family donors were used in allo-HSCT, but which are associated with higher rates of graft rejection and acute graft versus host disease (aGVHD) if T cells are not first depleted. We developed a new technique for HLA mismatched allogeneic HSCT using G-CSF primed bone marrow plus G-CSF-mobilized peripheral blood stem cells without ex vivo T cell depletion. In this study, 176 patients, including 88 with high-risk or advanced leukemia, were transplanted with cells from a HLA-haploidentical family donor with 1–3 mismatched loci. After conditioning, patients received G-CSF-primed bone marrow grafts that had not been depleted ex vivo of T cells, in combination with G-CSF-mobilized peripheral blood stem cells, as well as GVHD prophylaxis. The total infused nucleated cell count was 6x108 to 8x108/kg recipient weight. All patients achieved sustained, full donor-type engraftment. The incidence of grade II–IV aGVHD was 41.5% (73/176), including 21 patients with grade III–IV aGVHD. The development of aGVHD was not associated with the extent of HLA disparity. Chronic GVHD was observed in 81 of 120 evaluable patients (67.5%). Forty-six of the 81 patients had limited-stage disease and 35 had extensive cGVHD. Fifty-two patients died among whom 9 died of recurrent disease and 43 of transplant-related complications. One hundred and twenty-four of the 176 patients survived, and 112 remained disease free at the time of a median follow-up of 22 months (4 to 59 months). The 2-year probabilities of disease-free survival were 74.8% and 69.3% for standard- and high-risk patients, respectively. In summary, we have established a new regimen to use bone marrow from haploidentical family donors without ex vivo T cell depletion, in combination with G-PBSCs, as a source of stem cells even in cases of HLA mismatched transplantation. This method showed us good effect even similar as that of HLA-matched allo-HSCT. It will be very useful for patients with hematological malignancies especially much more patients in China who are the only child in the family.
Peripheral Blood Stem Cell (PBSC) Mobilization in Patients with Multiple Myeloma: Low Dose Versus Intermediate Dose Cyclophosphamide.
Authors: Hiwase Devendra K. , Schwarer Anthony P. , Mckendrick Joe J. , Bollard Geraldine M. , Hiwase Smita D. , Michael Bailey M. 
Published: April 2020

Abstract
Aim: Autologous peripheral blood stem cell transplant (PBSCT) is standard of care for multiple myeloma patients younger than 65 years of age. However there is no agreement on mobilization regimen for PBSC collection. We have compared efficacy and toxicity of low dose & intermediate dose of cyclophosphamide for stem cell mobilization.
Methods: Eighty-seven patients with multiple myeloma had stem cell mobilization: Intermediate dose cyclophosphamide (3 to 4 gm/m2) [ID-CY] was used between Jan 1995 to Jun 2002 and low dose cyclophosphamide (1 to 2 gm/m2)[LD-CY] was used between 2002 and 2004.
Mobilization chemotherapy: LD-CY or ID-CY was infused along with hydration, antiemetics and mesna. Stem cell collection was planned between day 10 to day 14 after chemotherapy and G-CSF 10 ug/kg (5 to 20 ug/kg) was started 4 days prior to stem cell collection. PBSC collected by processing 7 litres of blood by Fenwal CS3000 (Baxter) or Cobe Spectra.
Statistical Analysis: The Mann-Whitney U and Kruskal-Waliis test were used for comparison between treatment groups. Chi-Square and Fisher exact test were used for comparisons of categorical variables
Result: There were 87 patients of multiple myeloma, 61 in LD-CY group and 26 in ID-CY group. There was no statistically significant difference between two groups for basic demographic, disease parameters & prior duration of treatment. The median G-CSF/kg dose was similar in both groups.
Comparison of Stem cell collection and toxicity: LD-CY Vs ID-CY CD34 Yield LD-CY ID-CY P Number of Patients 61 26 Total CD34/kg Median (Range) 5.17 (0.23 to 17.3) 7.71 (0.08 to 26.4) 0.018 Total CD34/kg/L Median (Range) 0.62 (0 to 2.47) 1.07 (0.01 to 3.77) 0.010 CD34/kg on 1st day of leukapheresis 2.33 (0.03 to 15.8) 5.11 (0.04 to 15.9) 0.001 number of patients collected &gt; 2 x 10^6/kg 54/61 (88%) 24/26(92%) Number of Leukapheresis Median (Range) 2 (1 to 5) 2 (1 to 3) 0.001 Number of patients had febrile neutropenia 8/61(13%) 10/26 (38%) 0.0085 Number of patients required antibiotics 8/61 (13%) 10/26(38%) 0.0085 Number of patients requiring hospitalisation 10/61(16.39%) 11/26 (42.30%) 0.010
Conclusion: There was statistically significant higher stem cell yield with ID-CY as compared to LD-CY, however at the cost of increased toxicity. In LD-CY group 88% patients had &gt;2 x106/kg stem cells collected which is sufficient for single autograft. We conclude that LD-CY mobilisation chemotherapy is adequate for patients who are planned for single autograft with minimal toxicity.