bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Functional Significance of Genes Regulated by Structurally Different ABL Kinase Inhibitors.
Authors: Nunoda Kousuke , Tauchi Tetsuzo , Takaku Tomoiku , Sumi Masahiko , Okabe Seiichi , Sashida Goro , Akahane Daigo , Ohyashiki Junko H. , Ohyashiki Kazuma
Published: April 2020

Imatinib is an ABL-specific inhibitor that binds with high affinity to the inactive conformation of the BCR-ABL tyrosine kinase and has been shown to be effective in the treatment of chronic myelogenous leukemia. Dasatinib is an ATP-competitive, dual-spesific SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical stand point, dasatinib is particular attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant CML patients. In the view of the fact that the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on a wild type p210 BCR-ABL expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 hrs, and gene expression data was obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib- responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The genes whose expression was affected by imatinib and dasatinib were categorized into different functional groups based on their biological function, and genes in the cell proliferation and apoptosis categories were examined in detail. Imatinib and dasatinib affected the expression of several cyclin-dependent kinases (CDK2, CDK4, CDK6, CDK8, and CDK9), cell division cycle genes (CDC6, CDC7, CDC25C, and CDC34), and cyclones (cyclin A2, C, D2, D3, E1, E2, F, G1, G2, and H). Imatinib and dasatinib also modulated the expression of apoptosis-related genes including APAF1, BAK1, BCL2, BCL10, MCL1, CASP3, and CASP6). One of the distinct genes which are selectively modulated by dasatinib are CDK2 and CDK8, which had a maximal fold reduction of <8-fold in microarray screen. Immunoblotting confirmed that gene expression changes induced only by dasatinib correlated with changes in protein expression. To assess the functional importance of dasatinib regulated genes, we used RNA interference to determine whether reduction of CDK2 and CDK8 affected the growth inhibition. The siRNA to CDK2 or CDK8 specifically reduced cdk2 or cdk8 in K562 cells. K562 cells pretreated with CDK2 or CDK8 siRNA showed the additive growth inhibition with imatinib but not with dasatinib. These finding demonstrate that the additive/synergistic growth inhibition by imatinib and dasatinib may be mediated by CDK2 and CDK8.
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

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

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

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

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.

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).

 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<0.05).

 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

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 (> 0.5 × 109/l) and platelet (> 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

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

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

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 > 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 >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.
Variability in Measures of Health Outcomes in Sickle Cell Disease (SCD) Patients with Access to Comprehensive Care Centers.
Authors: Shankar Sadhna M. , Abrogast Patrick , Mitchel Ed , Ding Hua , Wang Winfred , Griffin Marie R.
Published: April 2020

The mortality of children less than 5 years of age with SCD has decreased substantially and now is similar to that of children without SCD. However the impact of comprehensive care on health outcomes of SCD has not been fully evaluated. This study compared the medical care utilization and mortality in children and adults with SCD in four regions in the state of Tennessee with variable access to a comprehensive sickle cell center (CSCC). This is a retrospective longitudinal study of a cohort of patients with SCD enrolled in TennCare, Tennessee’s Medicaid managed health care program, from January 1995 to December 2002. Children and adults who had at least one inpatient or two outpatient physician visit claims, at least 30 days apart, listing an International Classification of Diseases, Ninth Revision, Clinical Modification code for SCD (homozygous, SC, sickle beta thalassemia) constituted the cohort. TennCare data linked to Tennessee vital records were used to define the population and identify the outcomes. One of the four large urban counties in Tennessee has a CSCC. The patients were assigned to one of four regions based on the area of residence on the day of their hospitalization or outpatient visit: Area 1a- urban county where CSCC is located, Area 1b- counties surrounding the CSCC area, Area 2: 3 urban counties which include large academic medical centers, Area 3- the remainder of Tennessee counties. Rates of hospitalizations, outpatient and emergency department (ED) visits, and deaths were compared in the four regions of the state. The cohort included 2102 patients with a total of 11,814 person-years (py). 58% of patients resided in area 1a, 9% in area1b, 19% in area 2 and 14% in area 3. 59% were females and 49% were older than 20 years. 14% were younger than 5 years. The following figure shows the rates of hospitalizations, ED and outpatient visits and deaths in 4 regions. Among children less than 5 years of age the rate of hospitalization was highest in area 1a (p<0.001). ED and outpatient visits for children were highest in area 3, (p<0.001). Among adults, 20 years or older, the rates of hospitalization and ED visits were highest in area 3, (p<0.001). The outpatient visits were highest in area1b (p<0.001). There was no significant difference in the death rates in the four regions. Males had significantly higher death rates in all regions (p<0.001). Trend analysis over 8 years showed a significant trend for increased rates of ED and outpatient visits in all regions (p<0.001) and deaths in area 2 (p=0.014). No clear pattern of improved outcomes as measured by hospitalizations, ED and outpatient visits and deaths are identified in relation to access to a CSCC or large academic medical centers. Area of residence may not affect care for SCD. Alternatively, there may be selective distribution of sicker patients in these areas based on their need for care.

Figure Figure
Activation of AKT and Enhanced Resistance to Apoptosis Induced by Surface IgM Ligation in ZAP-70-Positive Chronic Lymphocytic Leukemia B Cells.
Authors: Chen Liguang , Meraz Gabriel A. , Kipps Thomas J.
Published: April 2020

Signaling through the B-cell receptor (BCR) for antigen has been implicated to play a role in the pathogenesis and/or progression of chronic lymphocytic leukemia (CLL). Previously we found that BCR ligation on CLL cells that expressed ZAP-70 induced significantly higher levels of phosphorylation in p72Syk, BLNK, and phospholipase C-gamma than did BCR ligation on CLL cells that lacked expression of this protein tyrosine kinase. We hypothesized that CLL B cells that express ZAP-70 also may have enhanced levels of AKT activation following BCR ligation that is associated with increased resistance to apoptosis. CLL B cells were stimulated via surface IgM ligation and monitored at various time points for phosphorylation of cytoplasmic signaling molecules via immunoblot analyses and for viability by flow cytometry after staining with propidium iodide and DiOC6 to detect changes in mitochondrial membrane potential associated with apoptosis. We found that CLL cells that expressed ZAP-70 experienced significantly higher levels of AKT phosphorylation within 10 minutes following BCR-ligation than did ZAP-70-negative CLL cells. Over time a significantly higher proportion of ZAP-70-negative CLL cells were induced to undergo apoptosis by BCR-ligation than similarly treated CLL cells that were ZAP-70 positive. The differences between these two groups in leukemia cell viability over time after BCR ligation could be abrogated by addition of LY294002, a PI3-K inhibitor, to the CLL cell cultures 30 minutes prior to surface IgM ligation. We conclude that ZAP-70-positive CLL cells are relatively resistant to apoptosis induced by surface IgM ligation under the experimental conditions used, a characteristic that is associated with enhanced phosphorylation of AKT and activation of AKT-dependent pathways following BCR ligation. These studies support a model proposing that BCR ligation induces signaling that results in enhanced growth and/or survival of CLL cells that express ZAP-70 relative to that of leukemia cells that lack expression of this tyrosine kinase. Because expression of ZAP-70 typically is associated with expression of unmutated Ig V genes in CLL, the improved signaling afforded by expression of this tyrosine kinase may account in part for the greater tendency for disease progression observed in patients with CLL cells that use unmutated Ig V genes.
Expression of Specialized Error-Prone DNA Polymerases in Chronic Lymphocytic Leukemia (CLL).
Authors: Crespo Marta , Gine Eva , Muntanola Ana , Villamor Neus , Montserrat Emili , Bosch Francesc
Published: April 2020

Specialized DNA polymerases are low fidelity enzymes required to bypass DNA lesions that are also involved in somatic hypermutation. Deregulation of these enzymes can lead both to an increased mutation rate and to an enhanced translesion synthesis capability. DNA polymerase Kappa (DNA pol Kappa) has been found to promote tumorigenesis and to be overexpressed in some cancers. Expression levels of specialized DNA polymerases Iota, Eta, Zeta and Mu were analyzed in primary B-cells from 12 CLL patients (6 with IgVH mutated genes [M] and 6 with unmutated [UM] genes). Expression of DNA pol Kappa was also assessed in 27 CLL samples (13 M and 14 UM), in B-cell lines derived from pre- and post-germinal center malignancies (NALM-6 [preB-acute lymphoblastic leukemia], JVM-2 [prolymphocytic leukemia], Granta, NCEB and REC [mantle-cell lymphoma] and Daudi [Burkitt’s lymphoma] and in normal B-cells from peripheral blood and tonsil. Expression of DNA polymerases was measured by Quantitative RT-PCR using Ramos (a constitutively hypermutating Burkitt’s lymphoma cell line) as a reference, considering an arbitrary unit of 1 for each gene. Although somatic hypermutation is absent in UM CLL cells, there were no differences in the expression of the DNA polymerases analyzed according to the mutational status. Expression of DNA pols Iota and Zeta was similar in CLL cells and Ramos (mean value: 0.95 and 1.3, respectively), whereas expression of DNA pol Mu was slightly higher (mean 3.24) and DNA pol Eta was underexpressed (mean 0.19). In contrast, among all the hematological malignancies analyzed, significant overexpression of DNA pol Kappa was only found in CLL samples (mean value; 8.43;range: 1.42–19.47). Induction of AID expression by stimulating CLL cells with CD40 ligand and IL-4 did not modulate DNA pol Kappa expression. Finally, expression of DNA pol Kappa in normal B cells from peripheral blood was similar to that in Ramos (0.87) and slightly higher in B cells from tonsil (2.02). In conclusion, no differences in DNA polymerases were observed between M and UM CLL. As previously described in some solid tumors, CLL cells showed an increased expression of DNA pol Kappa, regardless the mutational status of the IgVH genes, suggesting that the expression of this polymerase is not related to the process of somatic hypermutation that occurs in some CLL cells. Taken together, these results support the concept that DNA pol Kappa may behave as an oncogene in CLL and that its regulation may be crucial for maintaining genomic stability.
Thalidomide (T) in Combination with Fludarabine (F) as Initial Therapy for Patients (pts) with Treatment Naïve Chronic Lymphocytic Leukemia (CLL): Preliminary Results of a Phase I/II Clinical Trial.
Authors: Chanan-Khan Asher Alban , Miller Kena C. , Marshall Patricia , Padmanabhan Swaminathan , Brady William , Bernstein Zale P. , Wallace Paul , Czuczman Myron S.
Published: April 2020

Introduction: The importance of VEGF and TNF-a and their interaction with other cytokines in the pathogenesis of CLL has recently been identified. These cytokines in the tumor microenvironment (ME) form a network of growth promoting signal(s) that regulate the CLL cell survival and progression. T is an immunomodulatory agent, reported to exert antitumor activity through down regulation of VEGF, TNF-a and IL-6. We hypothesized that targeting the tumor ME with T will interrupt the growth promoting signals and predispose CLL cells to enhanced proapoptotic activity of F with resultant improvement of clinical responses. In this abstract we report our findings to date of this phase I/II clinical trial.

Patients and Methods: Treatment naïve pts requiring therapy for CLL were eligible for this study. T was started at D1 and continued for 6 months while F (25mg/m2 x 5 D every 4 wks) was given for 6 cycles starting on D7. Three doses of T (100,200,300 mg) were studied in phase I and as no dose response curve or DLT was noted, 200mg of T was selected for the phase II evaluation for concerns of cumulative toxicity of T. Low-dose coumadin (1 or 2 mg) was used for prophylaxis of venous thromboembolism (VTE).

Results: To date 20 pts (15M, 5F; median age 66, range 38–76 yrs) have been enrolled. Of these 11 pts had limited (0-II) stage and 9 pts had advance Rai stage (III/IV) disease. Pts who completed the intended 6 months of T were evaluable for response. Six pts were removed from study for toxicity (2VTE - 1 in the 1st week of therapy prior to F infusion, 2nd during the 4th cyc; 1 Hep C reactivation presumably secondary to F, 1 secondary to early death from advance disease, 1 non-compliance, and 1 early termination for poor tolerance to therapy), without evidence of disease progression. Thirteen pts completed 6 months of therapy and are available for response. Among the evaluable pts 9 (69.2 %) achieved a complete response (CR) and 4(30.7%) were noted to have a partial remission (PR). While all pts who completed 6 months of T achieved a major response, the ORR in the intent-to-treat population was 65% with median duration of response 12+ (range 3 – 18+) months. Fatigue, thrombocytopenia, and anemia were the most common side effects, noted in 75%, 65%, and 55% of the 20 patients, respectively.

Conclusion: T when combined with F results in a higher ORR when compared to F alone based on historical data. The combination FT was well tolerated with manageable toxicity. Completion of this ongoing phase II study will identify the role of this new combination in pts with CLL.
In Vivo Evaluation of Immunomodulating Effects of Lenalidomide (L) on Tumor Cell Microenvironment as a Possible Underlying Mechanism of the Antitumor Effects Observed in Patients (pts) with Chronic Lymphocytic Leukemia (CLL).
Authors: Chanan-Khan Asher Alban , Padmanabhan Swaminathan , Miller Kena C. , Pera Paula , DiMiceli Laurie , Ersing Noreen , Wallace Paul , Rickert Michael , Porter Carl
Published: April 2020

Introduction: L is a more potent analogue of thalidomide with antitumor activity reported in MDS and multiple myeloma. Clinical anti-leukemic activity of L is reported for the first time by our group in pts with CLL. The underlying mechanism of its antitumor activity remains undetermined. We investigated the effect of L on the tumor microenvironment and studied the modulation of soluble cytokines and immune cells (T and NK cells) in pts receiving L.

Patients and Methods: CLL pts enrolled on the clinical study with L were eligible. Pre and post (day 7) samples were obtained for evaluation of changes in serum cytokine and immune cell environment. Malignant cells were also obtained to investigate the in vitro antitumor activity of L prior to initiating treatment on clinical trial.

Results: With Anexin V staining for evaluation of apoptosis induction, in vitro testing of pts samples (n=10) showed only a modest increase in apoptosis at 200mg of L - levels clinically not achievable. Yet same pts treated with L on clinical study showed significant antitumor response, suggesting the mechanism to be possibly related to modulation of the tumor microenvironment. In evaluation of the tumor cytokine microenvironment (n= 10) we noted significant L induced increase in IL-10 (n=6), IL-8 (n=8), IP-10 (n=10), IL-8 (n=8), TNF-alpha (n=4) and decrease in PDGF (n=5) and RANTES (n=5). While evaluation of the immune cell repertoire we observed an absolute increase in T-cell as well as NK-cell after treatment with L.

Conclusion: Our in vitro evaluation does not suggest a direct apoptotic effect of L on the malignant CLL cells and thus support the hypothesis that the anti-leukemic effect noted in the clinical trial (reported separately) is most likely from in vivo modulation of the tumor microenvironment as is demonstrated from changes in the cytokine milieu and the cellular immune response. Collectively these changes may be responsible for the immune modulating properties of L and the resultant anti-CLL activity in pts.
Immune Therapy for Chronic Lymphocytic Leukemia Induces the Antibody Response Against a Novel Tumor-Associated Antigen, the Orphan Tyrosine Kinase Receptor ROR1.
Authors: Fukuda Tetsuya , Rassenti Laura Z. , Kipps Thomas J.
Published: April 2020

Transduction of Chronic lymphocytic leukemia (CLL) CLL cells with replication-defective adenovirus encoding CD154 (Ad-CD154) enhances the capacity of such cells to function as antigen presenting cells. In a clinical trial patients received multiple infusions of autologous CLL cells transduced ex vivo with Ad-CD154 to examine the safety and efficiency of Ad-CD154 gene therapy. Treated patients were found to mount antibody responses against the recombinant adenovirus and cellular immune responses against autologous CLL cells. We examined the antibody response generated in some of the treated patients against CLL cells. Pre and post treatment sera were incubated with CLL cells followed by detection with human anti-IgG antibody via flow cytometry. We found that 3 out of 6 patients examined developed IgG antibodies after therapy that could bind CLL cells. Microarray analyses of CLL samples identified a relatively small number of genes that were differentially expressed in CLL cells compared with normal B cell subsets or neoplastic B cells of other B cell malignancies. These CLL signature genes are candidates TAAs of CLL. One such a CLL signature gene encodes ROR1, a surface receptor tyrosine kinase. Surface expression of ROR1 was on CLL cells, but not on normal lymphocytes. We used two methods to detect the anti-ROR1 activity. One was the flow cytometric analysis using Chinese hamster ovary cell transfected with ROR1 cDNA. Another strategy used an ELISA with plates coated with recombinant ROR1protein. Both methods revealed that patients who developed anti-CLL autoantibodies following gene therapy also developed IgG antibodies reactive with ROR1. As such, ROR1 appears to be a candidate TAA that may be targeted by immune responses induced by Ad-CD154 gene therapy.
RhG-CSF Mobilized and Apheresis-Collected Endothelial Progenitor Cells for Therapeutic Vasculogenesis.
Authors: Korbling Martin , Reuben James M. , Gao Hui , Lee Bang-Ning , Giralt Sergio A. , Khouri Issa F. , Saliba Rima M. , Champlin Richard E. , Estrov Zeev
Published: April 2020

Endothelial progenitor cells (EPCs) have been identified as part of hematopoietic tissue-derived progenitor cells that are mobilized into the peripheral blood (PB) as a result of tissue injury. It therefore seems likely that circulating EPCs have therapeutic potential by aiding in the neovascularization of ischemic tissue. It was the purpose of this study to provide clinical data on the availability of circulating EPCs at steady-state and after rhG-CSF mobilization, and their collection by leukapheresis. Eight healthy donors underwent rhG-CSF (10 μg/kg) treatment over 4 days. Continuous-flow leukapheresis (COBE Spectra, Version 4.7) was performed on the fourth day of rhG-CSF treatment. Blood samples were drawn before starting rhG-CSF treatment, before apheresis, and from the apheresis collection bag. The total cell numbers collected per apheresis were based on processing a median of 17 L [10.7–20.1] blood volume or approximately three times the donor’s total blood volume. Blood and apheresis samples were analyzed by flow cytometry for EPC surface markers CD34, CD133, VEGFR-2, and by forming EPC colonies (CFU-EPC). The Wilcoxon matched-pairs signed-ranks test was used to compare the distributions at various sampling points.

All CD133 subsets were CD14 negative to exclude differentiated monocytes or macrophages. The median steady-state PB concentrations of circulating CD34+CD133+VEGFR-2+ cells was 0.9/μl [0–3], or 1.7/μl [0.9–4] for CD34+133−VEGFR-2+ EPCs. After 4 days of rhG-CSF mobilization treatment the PB CD34+CD133+VEGFR-2+ subset concentration increased by a median of 8-fold [p 0.01], and the CD34+133−VEGFR-2+ subset concentration by a median of 10-fold [p 0.01] over baseline. The median PB concentration of circulating CFU-EPCs increased by 10-fold [p 0.02]. The median absolute number of CD34+CD133+VEGFR-2+ and CD34+CD133-VEGFR-2+ cells collected by leukapheresis per kg of body weight was 0.845 x 106 [0.12–2.55] and 1.54 x 106 [0.06–2.4], respectively. A small population of CD133+34− VEGFR-2+ cells was identified in steady state and mobilized PB, and in the apheresis collect (0.11 x 106/kg [0–0.26]). CD34− progenitor cells expressing CD133 are believed to represent a primitive progenitor cell population containing SCID-repopulating cells. VEGFR-2 coexpression of those immature cells may define a circulating cell population that contributes to vasculogenesis.

Our data suggest that clonogenic circulating EPCs can be mobilized by rhG-CSF and collected by continuous-flow leukapheresis generating large numbers of EPCs, specifically in the range of 60 x 106 EPCs per leukapheresis procedure for a 70 kg individual. Circulating EPCs represent a novel blood cell component that can be clinically used in large quantities, either unmanipulated or EPC-selected, for therapeutic vasculogenesis.
The Induction of Apoptosis by Histone Deacetylase Inhibition in Chronic Lymphocytic Leukemia (CLL) Cells Is Mediated through the TNF-Related Apoptosis-Inducing Ligand (TRAIL) Pathway.
Authors: Szwajcer David , Gibson Spencer B. , Johnston James B.
Published: April 2020

Background and Objectives: Histone Deacetylase Inhibitors (HDIs) have been shown to induce cell cycle arrest, differentiation and apoptosis in a variety of myeloid and lymphoid malignancies while being relatively nontoxic to normal cells. HDIs have also been shown to sensitize CLL cells to TRAIL-induced apoptosis. In this study we investigated the effects of two HDIs, trichostatin (TSA) and valproic acid (VPA), alone and in combination with fludarabine (FLU) on apoptosis in the BJAB lymphoblastoma B-cell line and in primary CLL cells.

Materials and Methods: Cell viability was assessed by the MTT (3-(3,5-dimethylthiazol-2yl)- 2,5 diphenyl tetrazolium bromide) colorimetric assay, following 4 days of continuous drug treatment. Apoptosis was assessed by staining cell suspensions with acridine orange and quantitating apoptotic cell populations using fluorescence microscopy. Protein expression for the TRAIL receptors, DR4 and DR5, was determined by western blotting.

Results: As determined by the MTT assay, the mean concentrations of TSA and VPA to reduce cell viability by 50% (IC50) in CLL cells were 0.23 uM and 650uM, respectively. TSA at 0.5uM and VPA at 2mM induced 60% apoptosis following 48 hours of drug exposure in both BJAB and CLL cells, as measured by acridine orange staining. Addition of DR4:FC, which sequesters TRAIL away from its receptors, decreased TSA- and VPA-induced apoptosis in BJAB and CLL cells by 9 and 20% respectively, indicating activation of the TRAIL apoptotic pathway by these HDIs. In addition, exposure to TSA at a concentration of 1uM for 24 hours, up regulated the expression of DR5 in BJAB cells, but this was not observed with VPA at a concentration of 1.5mM, after up to 48 hours of treatment. Neither HDI influenced DR4 protein expression in these cells. Preliminary results show that VPA potentiated FLU-induced apoptosis by 20% in both BJAB and CLL cells.

Conclusions: Both TSA and VPA induce apoptosis in BJAB and CLL cells by activation of the TRAIL apoptotic pathway. For TSA, this effect may be potentiated by up regulation of DR5 expression. VPA potentiates FLU-induced apoptosis in BJAB and CLL cells, suggesting a therapeutic role for the combination of HDIs and nucleoside analogues in the management of CLL. Ongoing studies are determining whether this effect is specific for CLL cells, as compared to normal lymphocytes, and the optimum scheduling for synergy.
Identification of Candidate Tumor Suppressor Genes Silenced Epigenetically in Mantle Cell Lymphoma.
Authors: Kawamata Norihiko , Saitoh Takayuki , Sakajiri Sakura , Koeffler Phillip H.
Published: April 2020

Many tumor suppressor genes are silenced by epigenetic mechanisms in human cancers, including mantle cell lymphoma (MCL). In this study, we have used a variety of research tools to screen for genes that are epigenetically silenced in MCL. Changes in the global gene expression profile of the MCL cell line, Jeko1, were analyzed after treatment with the combination of the demethylating agent, 5-aza-2′-deoxycytidine, and the histone deacetylase inhibitor, suberoyl anilide bishydroxamide, by DNA microarray technique. By screening over 22,000 genes, we identified 26 candidate tumor suppressor genes, expression of which were enhanced by the treatment, in the MCL line. Basal expression of these 26 genes were low in Jeko1 cells. The treatment enhanced the expression more than 2 folds and the enhancement was also confirmed by real-time PCR. Methylation status of these 26 genes were examined by bisulfite sequencing and/or combined bisulfite and restriction enzyme digestion assay in Jeko1 cells. We found hypermethylation of a CpG island in the middle of the INPP5F gene. We also found the hypermethylation of that region of INPP5F in normal peripheral blood. We also examined expression levels of these 26 genes in normal mantle cells by real-time PCR and found only 11 genes showed high levels of transcription in laser-dissected normal mantle cells. We examined expression of these 11 genes in eight MCL clinical samples by real-time PCR and found that only three genes, INPP5F, DUSP10 and FGD2 showed very low expression levels. We conclude that expression of INPP5F, DUSP10 and FGD2 genes were suppressed in MCL cells although the expression of these genes are high in normal mantle cells. INPP5F is a inositol phosphatase and could be involved in PI3K pathway. DUSP10 is a dual specific phosphatase and could be involved in JNK pathway. FGD2 is a RAS-GAP gene and could be involved in RAS pathway. These three genes may be candidate tumor suppressor genes in MCL and further functional analysis is ongoing.