bluuebunny/crossref_metadata_2025 huggingface dataset:
CEBPA Mutations Predict Favorable Prognosis in Pediatric AML
Authors: Ho Phoenix , Alonzo Todd A. , Gerbing Robert B. , Pollard Jessica A. , Stirewalt Derek , Hurwitz Craig Alan , Heerema Nyla A. , Hirsch Betsy , Raimondi Susana C. , Lange Beverly , Franklin Janet , Radich Jerald , Meshinchi Soheil
Published: April 2020

C/EBPα is a transcription factor that regulates terminal granulocytic differentiation. Although functional mutations in the CEBPA gene have been associated with improved outcome, they have not been well-characterized in pediatric AML. We evaluated the prevalence and prognostic significance of CEBPA mutations in 847 children with de novo AML treated on three consecutive pediatric AML trials (CCG-2941, CCG-2961, and COG-AAML03P1). Clinical characteristics and outcomes for patients with CEBPA mutations were compared to that of patients with wild-type CEBPA. Two types of CEBPA mutations—N-terminal truncating mutations and in-frame bZip domain mutations—were detected in 38/847 (4.5%) patients tested, and 31/38 (82%) patients with mutations harbored both mutation types. Polymorphisms were also identified in 7.8% of patients screened; patients with polymorphisms were analyzed in the CEBPA wild-type subgroup. The presence of functional mutations was correlated with laboratory and clinical characteristics, and clinical outcome. CEBPA mutations were significantly more common in older patients, patients with FAB M1 and M2, and patients with normal cytogenetics. These mutations did not occur in patients with either favorable or unfavorable risk cytogenetics. CEBPA mutations were found almost exclusively in the normal karyotype cohort, and patients with CEBPA mutations accounted for 17% of all patients with normal cytogenetics. Actuarial event-free survival at 5 years from study entry was 69% vs. 38% (p=0.017) with a cumulative incidence of relapse after complete remission of 13% vs. 44% (p=0.007) for those with and without CEBPA mutations. For patients with known diagnostic cytogenetic information, clinical outcome of those with CEBPA mutations was compared to that of those with favorable risk Core Binding Factor (CBF) AML. Patients with CEBPA mutations had an overall survival similar to those with CBF AML (78% vs. 74%, p=0.999). The presence of CEBPA mutations was an independent prognostic factor for improved outcome (hazard ratio 0.24, p=0.047). CEBPA mutations are associated with a lower relapse rate and improved survival in pediatric AML. CEBPA mutation analysis needs to be incorporated into initial screening for risk identification and therapy allocation at diagnosis.
Humoral Immune Responses against the Immature Laminin Receptor Show Prognostic Significance in Patients with Multiple Myeloma.
Authors: Zeis Matthias , Siegel Sandra , Friedrichs Birte , Schmitz Norbert
Published: April 2020

Multiple myeloma (MM) is characterized by a highly variable clinical course. The role of an autologous tumor-specific immune control contributing to the variable length of survival in MM is poorly understood. In the current study, we investigated whether humoral immunity specific for the tumor-associated oncofetal antigen/immature laminin receptor (OFA/iLR) has a prognostic value in MM. In patients with monoclonal gammopathy of unknown significance (MGUS) we found significant levels of anti-OFA/iLR antibodies in 21 of 30 sera (70%) and in 7 of 27 (26%) patients with asymptomatic MM (AMM). In contrast, in sera from patients with progressive MM (n=67) and in normal healthy donors (HD, n=60) no humoral reactivity against OFA/iLR was present. To look on long-term persistence of humoral anti-OFA/iLR immune responses, significant and stable titers of anti-OFA/iLR-specific IgG antibodies in follow-up samples of MGUS patients were detected. IgG subclass analyses showed a predominant IgG1 and IgG3 response suggestive for a Th1-driven immune response in the vast majority of patients with MGUS and to smaller extent in patients with AMM. OFA/iLR-antibodies were capable of recognizing and selectively killing OFA/iLR-expressing myeloma cells in complement-mediated and antibody-dependent cellular cytotoxicity (ADCC) assays. To determine whether the presence of anti-OFA/iLR antibodies influenced progression-free survival (PFS), patients were analyzed according to the presence/absence of a humoral immune response against OFA/iLR protein. In a univariate analysis, the group of patients with reactive sera (n=28) showed a significantly lower probability of disease progression compared to patients with non-reactive sera (n=29). In the patient group with significant anti-OFA/iLR titers only 2 of 28 patients developed disease progression, but 18 of 29 patients with OFA/iLR non-reactive sera (p=0.001). Our data suggest that spontaneous tumor-specific humoral immune responses against OFA/iLR exist in a significant proportion of MM patients and that superior PFS in those patients could reflect autologous immune control.
T Cell Modulation Combined with Intratumoral CpG Cures Lymphoma without the Need for Chemotherapy.
Authors: Houot Roch , Levy R.
Published: April 2020

Introduction: We have previously shown that intratumoral injection of CpG oligodeoxynucleotide plus systemic chemotherapy can induce T cell immunity against lymphoma and serve as a therapeutic vaccine to cure tumors in a murine lymphoma model (Li et al., J Immunol. 2007). CpG is a Toll-like receptor 9 agonist capable of activating tumor-infiltrating dendritic cells and/or tumor B cells to force tumor antigen presentation in situ when injected intratumorally. Here, we demonstrate that antibody-mediated modulation of T cells enhances the efficacy of CpG vaccination, thereby eliminating the need for chemotherapy.

Materials and Methods: Mice were inoculated s.c. with A20 lymphoma tumor cells at two sites (right and left abdomen). Only one site was injected with CpG allowing us to evaluate the systemic anti-tumor response at the distant site. Treatment started when tumors became palpable. CpG was administered intratumorally. T cell modulation was accomplished using systemic (i.p.) administration of monoclonal antibodies against T cell targets:

- Regulatory T cells were depleted using anti-Folate Receptor 4 (FR4) antibody or functionally blocked using anti-GITR antibody, - Effector T cells were stimulated using anti-OX40 antibody (to trigger their costimulatory molecule) or anti-CTLA4 antibody (to block inhibitory signals).

Results: Treatment with intratumoral injection of CpG alone did not cure any mice. Treatment with CpG and a single antibody (anti-OX40, anti-CTLA4, anti-FR4, or anti-GITR) cured 20–30% of mice. Interestingly, some combinations of antibodies (anti-OX40+anti-CTLA4, anti-OX40+anti-FR4, anti-CTLA4+anti-GITR) potentiated T cell modulation and further enhanced the efficacy of CpG vaccination. In particular, the combination of anti-OX40 and anti-CTLA4 appeared to be especially potent when combined with intratumoral CpG. Indeed, this combination (CpG+anti-OX40+anti-CTLA4) induced anti-tumor T cells capable of secreting IFN-γ in response to overnight culture with A20 tumor cells; it cured more than 80% of mice bearing large and systemic lymphoma tumors without the need for chemotherapy (effective therapy required both CD4 and CD8 T cells); finally, this therapy produced high numbers of anti-tumor memory T cells and provided long-lasting immunity against tumor re-challenge.

Conclusions: Our results show that antibody-mediated T cell modulation greatly enhances the therapeutic efficacy of intratumoral vaccination with CpG. Importantly, we show that T cell modulation can be potentiated by appropriate combinations of antibodies against T cell targets. As these reagents (CpG, anti-OX40 and anti-CTLA4 notably) are becoming available for human clinical trials, the combination of intratumoral CpG and immunomodulatory T cell antibodies holds promise for therapeutic vaccination against lymphoma.
Preclinical Analysis of the Combined Activity of SGN-40, Anti-CD40 Monoclonal Antibody, with Rituximab in Non-Hodgkin Lymphoma.
Authors: Lewis Timothy S. , McCormick Renee S. , McEarchern Julie A. , Kissler Kim , Stone Ivan J. , Gerber Hans-Peter , Drachman Jonathan G. , Grewal Iqbal , Law Che-Leung
Published: April 2020

SGN-40 is a humanized monoclonal antibody against CD40, a TNF receptor family member expressed in non-Hodgkin lymphoma (NHL), multiple myeloma, and several carcinomas. SGN-40 is cytotoxic to NHL cell lines via activation of proapoptotic signal transduction pathways and mediates antibody dependent cellular cytotoxicity (ADCC) effector function activity. In this study we examined the anti-tumor activity of SGN-40 in combination with the anti-CD20 antibody, rituximab, in lymphoma cell line models. Cell proliferation data (3H-thymidine incorporation assay) was generated for SGN-40 and rituximab alone and in combination for three NHL cell lines (Ramos, RL, and SU-DHL-4) and combination index (CI) analyses performed. SGN-40 was reproducibly synergistic with rituximab in Ramos cells and additive in the RL and SU-DHL-4 cell lines in this assay. This suggested that different anti-proliferative signaling events are activated by these antibodies, which produce a greater anti-tumor effect when combined. To better understand the combined activity of SGN-40 and rituximab, the signal transduction pathways activated by each antibody were examined. In Ramos cells SGN-40 signaling caused the degradation of pro-survival BCL-6 oncoprotein and upregulation of TAp63α, a proapoptic p53 family member, while only BCL-6 degradation was triggered in the RL and SU-DHL-4 cell lines. In contrast, rituximab signaling degraded BCL-6 protein in only one cell line (SU-DHL-4), and did not upregulate TAp63α expression in any of the cell lines examined. To further define the combined activity of SGN-40 with rituximab the effector function activity of both antibodies were examined in vitro. ADCC assays in the WIL2-S and Raji cell lines both showed a greater percent cell lysis in the presence of both SGN-40 and rituximab compared to either drug alone. Next, the SGN-40 and rituximab were tested in subcutaneous mouse models of NHL to evaluate this combination in vivo. In a Ramos model, SGN-40 and rituximab (dosed at 4.0mg/kg, q4dx4, ip) had significantly greater anti-tumor response when combined compared to the equivalent dose of either antibody alone. The anti-tumor response achieved with dual dosing of SGN-40 and rituximab was greater than the response expected if the combination was additive. Our data suggests that the improved efficacy of SGN-40, rituximab combination therapy in vivo is due to distinct apoptotic signaling pathways activated by these two antibodies in addition to augmented effector function activity. The combination of SGN-40 and rituximab is currently being studied in clinical trials of NHL.
A Promising Therapeutic Implication of a Novel Bcl-2 Family Inhibitor ABT-737 for Adult T-Cell Leukemia/Lymphoma.
Authors: Ishitsuka Kenji , Yotsumoto Fusanori , Katsuya Hiroo , Ushijima Emiko , Miyamoto Shingo , Tamura Kazuo
Published: April 2020

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-cell lymphotrophic virus type I. Conventional chemotherapeutic regimens used against other malignant lymphoma have been administered to ATLL patients, but the treatment outcome of acute and lymphoma type ATLL remains very poor. Therefore, new therapeutic strategies are needed to improve patient outcome. Anti-apoptotic proteins, Bcl-2 and Bcl-XL have been reported to be highly expressed in ATLL cells, and confer resistance to chemotherapy. The aim of this study is to elucidate the therapeutic implication of ABT-737 (Abbott Laboratories, Abbott Park, IL, USA), a potent small-molecule inhibitor of Bcl-2, Bcl-XL and Bcl-w for ATLL. We first examined the direct toxicity of ABT-737 against tumor cell lines in vitro. ABT-737 significantly inhibited growth of ATLL cell lines (MT-1, MT-2 and HuT102) and an acute T-cell leukemia cell line, Jurkat with IC50 values at 72h of 2.5μM, 0.4μM, 0.01μM and 0.75μM, respectively. In contrast, Burkitt lymphoma cell lines, Raji and Ramos were resistant to ABT-737 with IC50 values at 72h of 24μM and 7.0μM, respectively. Importantly, Bcl-2 protein was highly expressed in MT-1, MT-2, HuT102 and Jurkat cells, but not in Raji and Ramos cells. We next examined the mechanism of ABT-737 induced growth inhibition using MT-1 and MT-2 cells. ABT-737 induced apoptosis with cleavage of caspase 9 and caspase 3 without modulating cell cycle distribution. Furthermore, ABT-737 significantly induced apoptosis in fresh tumor cells obtained from ATLL patients. We finally elucidated the potential of ABT-737 to enhance the apoptosis induced by anti-Fas monoclonal antibody (mAb), and cytotoxicity induced by conventional chemotherapeutics. ABT-737 enhanced apoptosis induced by anti-Fas mAb in MT-1 and MT-2 cells. Most importantly, ABT-737 synergistically enhanced the cytotoxicity induced by either of doxorubicin, vincristine or etoposide. These results suggest that ABT-737, used either alone or in combination with other conventional cytotoxic drugs, represents a promising novel targeted approach to overcome drug resistance and improve patient outcome in ATLL.
Blockade of mTOR Signaling Potentiates the Ability of Histone Deacetylase Inhibitor to Induce Growth Arrest and Differentiation of Acute Myelogenous Leukemia Cells.
Authors: Nishioka Chie , Ikezoe Takayuki , Jing Yang , Koeffler H. Phillip , Yokoyama Akihito
Published: April 2020

This study found that MS-275, a novel synthetic benzamide histone deacetylase inhibitor (HDACI), blocked Akt/mTOR signaling in acute myelogenous leukemia (AML) HL60 and acute promyelocytic leukemia (APL) NB4 cells, as assessed by decreased levels of the phosphorylated (p)-Akt, p-p70 ribosomal S6 kinase (p70S6K), and p-S6K by Western blot analysis. Interestingly, further inactivation of mTOR by rapamycin analogue RAD001 (everolimus) significantly enhanced MS-275-mediated growth inhibition and apoptosis of these cells in parallel with enhanced upregulation of p27 kip1 and downregulation of c-Myc. In addition, RAD001 potentiated the ability of MS-275 to induce differentiation of HL60 and NB4 cells, as measured by expression of CD11b cell surface antigens, as well as reduction of nitroblue tetrazolium. Importantly, RAD001 potentiated the ability of MS-275 to induce expression of the myeloid differentiation-related transcription factor CCAAT enhancer binding protein e in these cells in association with enhanced acetylation of histone H3 on its promoter. Furthermore, RAD001 (5 mg/kg) significantly enhanced the effects of MS-275 (10 mg/kg) to inhibit proliferation of HL60 tumor xenografts in nude mice without adverse effects. Taken together, concomitant administration of a HDACI and a mTOR inhibitor may be a promising treatment strategy for the individuals with a subset of human leukemia.
Therapeutic Nucleic Acids: A Potential Source of Resistance to Cancer, Antiviral and Immunosuppressive Therapy.
Authors: Buhmann Raymund , Yang Ting , Schifferer Monica , Obermeier Martin , Jaeger Gundula , Kolb Hans-Jochem
Published: April 2020

According to their unique structural and chemical properties nucleic acids were recognized to provide inherent therapeutic potential beyond sole information storage. In the meantime an increasing number of nucleic acid based drugs achieved FDA approval and entered clinical trails (e.g. “antisense” or “immunosustimulatory CpG” oligodeoxynucletides (ODNs), aptamers, ribozymes, RNA interference or defibrotide). But so far, no or less information is available whether these compounds might

compete with chemically and structurally related drugs, e.g. nucleoside analogues (NA) widely used in cancer or antiviral therapy, or interfere with the intracellular nucleic acid metabolism.

In the present report we provide evidence, that nucleic-acid based drugs antagonize fludarabine, acyclovir or mycophenolate mofetil (MMF). In presence of defibrotide (DF), a polydisperse mixture of single-stranded oligodeoxyribonucleotides (15 to 30 kD) e.g. used for treatment of hepatic veno-occlusive disease and other endothelial disorders, fludarabine treated lymphocytes or myeloid blasts where rescued from apoptosis. According to nucleic acid degradation the resulting metabolite deoxycytidine turned out to be the key substrate competing with fludarabine for phosphorylation by deoxycitidine kinase (dCK) and suggested interference with nucleic acid metabolism rather than direct competition with the drug for cellular uptake. Moreover, in standard drug resistance assays using acyclovir sensitive herpes simplex virus (HSV) strains (V0631508), 4 mM of DF restored viral replication in presence of 50 mM acyclovir. This was confirmed by quantitative PCR of viral DNA. Here, deoxythymidine turned out to be the main competitor for intracellular phosphorylation mediated by virus thymidine kinase. To further extent our findings, that an increase of the extracellular concentration of nucleic acids directly interfere with the intracellular nucleic acid metabolism, mixed lymphocyte reactions (MLRs) were performed, to test whether the immunosuppressive effects of mycophenolate mofetil (MMF) could be reversed. As control cyclosporine A (CsA) was used. Here, addition of singular ribonucleotides almost completely antagonized the T cell inhibitory effects of mycophenolic acid (MPA), respectively its prodrug MMF, but not of CsA. As MPA is known to be a potent, selective, uncompetitive and reversible inhibitor of inosine monophosphate dehydrogenase, a key enzyme for the de novo pathway of guanosine nucleotide synthesis, addition of guanosine to the MLRs was found to be effective and sufficient to reverse the immunosuppressive effects of MPA. We conclude that treatment with nucleic-acid-based drugs interfere with the intracellular nucleic acid metabolism, thus decreasing the efficacy of NAs used for cancer and antiviral therapy or the immunosuppressive therapy using MMF. Prospective clinical trials are required to confirm these in vitro findings.
A Functional Promoter Polymorphism (−8C>G) of the Proteasome Subunit α6 Gene Is Associated with Multiple Myeloma and Poor Outcome Independent of Circulating Proteasome Serum Levels.
Authors: Bachmann Hagen S , Novotny Juergen , Sixt Stephan , Liebisch Peter , Frey Ulrich , Peters Juergen , Duehrsen Ulrich , Siffert Winfried , Nückel Holger
Published: April 2020

Introduction: The proteasome system plays a crucial role in several malignant diseases, especially in multiple myeloma (MM). Recently, the serum 20S circulating proteasome level (CPL) was shown to be an independent prognostic factor in MM.

A single nucleotide polymorphism (SNP) −8C>G in PSMA6, one of seven α-subunit genes of the 20S proteasome, was currently demonstrated to be associated with myocardial infarction. Additionally, it has been shown that PSMA6 expression is genotype-dependently altered e.g. in human B cells, whereas the G allele is associated with a 1.8 fold higher expression. Demonstrating the extensive role of the proteasome system in MM we investigated the role of the novel SNP in our cohort of 116 patients with MM.

Methods: DNA-samples of 116 patients with MM, all treated at the University Hospital Essen, and 125 healthy controls were genotyped for PSMA6 −8C>G. CPL of 70 patients were studied by an anti-20S proteasome enzyme-linked immunoabsorbant assay (ELISA). PSMA6 −8C>G genotypes were correlated with patients’ survival and CPL.

Results: Patients’ genotype distribution (69 CC, 44 CG, 3 GG) and genotype distribution of healthy controls (90 CC, 31 CG, 4 GG) were consistent with Hardy-Weinberg equilibrium. Genotypes were significantly associated with MM in a dominant genetic model (CC vs. CG+GG), with an odds ratio of 1.75 (95% confidence interval (CI): 1.02–3.00, p=0.043). Kaplan-Meier curves revealed a significant association of PSMA6 −8C>G with 5-year survival (p=0.014). Median survival time was 43 months for the GG genotype and 50 months for the CG genotype. It was not reached within follow-up by the CC genotype (CC 5-year survival rate 61.2%). Following hazard ratios (HR) were calculated: CC vs. CG: 2.007, 95%CI 1.11–3.63, p=0.022; CC vs. GG: 2.515, 95%CI 0.58–10.86, p=0.217 and in the dominant genetic model CC vs. CG+GG: 2.038, 95%CI 1.14–3.65, p=0.017. In multivariate analysis the GG/GC genotypes were independent prognostic factors (HR 2.1, p=0.014).

To proove if the detected effect of individual PSMA6 genotypes was dependent or independent from CPL, ELISA experiments were performed. There was no detectable difference in CPL between the genotypes. Mean CPL was 255 ng/mL for CC homozygous and 205 ng/mL for G allele carriers (p=0.718).

Conclusions: These results suggest the PSMA6 −8C>A polymorphism as a survival prognosticator as well as indicator of a high risk group within patients with MM. PSMA6 genotypes were not associated with CPL. Therefore, the SNP is independent of this known prognostic factor and could lead to additional prognostic information for MM patients.
The Novel NFkB Inhibitor V1810 Induces Apoptosis and Cell Cycle Arrest in Multiple Myeloma Cells and Overcomes NFkB Mediated Drug Resistance.
Authors: Meinel Felix , Mandl-Weber Sonja , Baumann Philipp , Leban Johann , Schmidmaier Ralf
Published: April 2020

Multiple Myeloma (MM) is a fatal malignancy characterised by the accumulation and expansion of antibody producing plasma cells in the bone marrow. Evidence is increasing that nuclear factor kappa B (NFκB) is a promising target for new anti-myeloma therapies. In this study, we assessed the in vitro activity of V1810, a novel NFκB inhibitor. V1810 potently induces cell death in all tested MM cell lines (OPM-2, U266, NCI-H929, RPMI-8226) with an IC50 ranging between 5μM to 10μM as well as in primary MM cells from patients. Cell death induced by V1810 clearly shows biological features of apoptosis such as DNA fragmentation and caspase 3 cleavage. In OPM2, U266 and RPMI-8226 cells induction of apoptosis is accompanied by cell cycle arrest. Western blots revealed downregulation of cyclin D1 (U266) or cyclin D2 (OPM2, NCI-H929, RPMI-8226) respectively, but not cyclin D3. Consistent with the downregulation of cyclin D1/2, retinoblastoma protein was found to be hypophosphorylated. Considering that cyclin D1 and D2 are known to be NFκB target genes, this is in line with our finding that V1810 inhibits baseline NFκB activity in MM cells (36% relative reduction). Importantly, V1810 also abrogates NFκB activation induced by genotoxic drugs like melphalan and doxorubicin. Accordingly, V1810 and melphalan synergistically decrease MM cell viability. Taken together, V1810 induces apoptosis and cell cycle arrest in MM cells by inhibition of NFκB and overcomes NFκB mediated drug resistance to melphalan. The maximum tolerable dose (MTD) of V1810 in BalbC mice was 10mg/kg i.v. and plasma concentrations of 9.5μM are achievable in NRMI mice after 5mg/kg V1810 i.v., which corresponds well to the used in vitro concentrations. This study strongly supports the further development of NFkB inhibitors in MM, especially in combination with genotoxic drugs.
Kpm/Lats2 of the Hippo Pathway Is Linked to Chemo-Sensitivity of Leukemic Cells.
Authors: Kawahara Masahiro , Hori Toshiyuki , Chonabayashi Kazuhisa , Oka Tsutomu , Sudol Marius , Uchiyama Takashi
Published: April 2020

The Hippo pathway is now recognized as a major signaling network for development and organ size control in mammals as well as in Drosophila melanogaster. We previously identified human kpm/Lats2, the key serine/threonine kinase in the Hippo pathway, and reported that its overexpression induced cell cycle arrest and apoptosis. Down-regulation of kpm/Lats2 expression has been described in various malignancies. It is of particular importance in hematology that low expression of kpm/Lats2 has been reported to be associated with poor prognosis in acute lymphoblastic leukemia. In the present study, we first measured the expression level of kpm/Lats2 mRNA in various hematological malignancies and found that it was markedly decreased in adult T cell leukemia (ATL) and NK cell leukemia/lymphoma, both of which are known to be highly resistant to conventional chemotherapy. In order to investigate the relationship between down-regulation of kpm/Lats2 expression and chemo-resistance, we made kpm/Lats2-knockdown sublines from KG-1a, an AML-derived cell line, and ED-40515+, an ATL-derived cell line, by using shRNA-expression retrovirus vector targeting kpm/Lats2. Silencing of kpm/Lats2 expression in both leukemic cell lines did not change the rate of cell growth but rendered them resistant to DNA damage-inducing agents such as DOX and ETP. Expression of p21 and PUMA was strongly induced by these agents in control cells, despite defective p53, but was only slightly induced in kpm/Lats2-knockdown cells. DNA damage-induced nuclear accumulation of p73, a member of p53 family, was clearly observed in control cells but hardly detected in kpm/Lats2-knockdown cells. ChIP assay showed that p73 was recruited to the puma gene promoter in control cells but not in kpm/Lats2-knockdown cells after DNA damage stress. The analyses with transient co-expression of Kpm/Lats2, YAP2, and p73 showed that Kpm/Lats2 contributed to the stability of YAP2 and p73, which was dependent on the kinase function of Kpm/Lats2 and YAP2 phosphorylation at serine 127. These results strongly suggest that Kpm/Lats2 is involved in the fate of p73 and the induction of p73-target genes that underlie chemo-sensitivity of leukemic cells.
Latexin: A Potential Tumor Suppressor.
Authors: Liang Ying , Van Zant Gary
Published: April 2020

We recently found that latexin is a negative regulator of the size of the hematopoietic stem cell population in mice. It acts by increasing apoptosis and decreasing cell proliferation. This 29 kD protein bears a strong structural resemblance to tazarotene-induced gene 1 (TIG1), a tumor suppressor down-regulated in a variety of cancers. The structural similarity and close genetic linkage led us to hypothesize that latexin also may have tumor suppressor properties. We found that latexin was down-regulated in a variety of human leukemias and lymphomas as determined by a survey of malignant cell lines and by analysis of CD34+ cells isolated from the blood and marrow of patients diagnosed with these malignancies and presenting with very high white cell counts. Bisulfite sequencing revealed that methylation of CpG dinucleotides in the latexin promoter at least partially accounted for latexin down-regulation. 5-aza-deoxycytidine treatment reinitiated or significantly increased latexin expression in K562, Molt4, CRF-CEM, Jurkat, U937, HL60, KG-1, and Sup B15 cell lines. To test the hypothesis that ectopic latexin expression in tumor cells would inhibit their growth, we developed a retrovirus-based expression vector with which we infected the murine lymphoma cell lines, WEHI231 and A20, neither of which contained significant latexin levels by Western blot. A vector containing GFP, but not latexin, was used to infect control cells. In triplicate experiments, the growth of both cell lines in vitro was inhibited an average 48% by infection with the latexin vector. Western blots revealed that latexin was durably expressed throughout the 2-week culture period at 2- to 4-fold the level expressed in normal T and B cells. As we found in our normal stem cell studies, latexin caused growth inhibition of lymphoma cells by significantly increasing apoptosis by 6-fold, and by suppressing cell proliferation by 2-fold. To test whether tumor inhibition extended to lymphomas in vivo, we injected either control or latexin vector-infected A20 cells subcutaneously in the flanks of BALB/c mice. Three weeks following adoptive transfer of identical numbers of cells, in duplicate experiments the latexin-expressing cells developed tumors only half the volume of those caused by the control cells. These results are consistent with a tumor suppressor role for latexin and suggest that latexin, or molecular mimics thereof, may have clinical efficacy in the treatment of malignancies.
VIDAS D-Dimer in Combination with Clinical Pre-Test Probability to Rule out Pulmonary Embolism. A Systematic Review of the Management Outcome Studies.
Authors: Carrier Marc , Righini Marc , Djurabi Reza Karami , Huisman Menno , Perrier Arnaud , Wells Philip , Wuillemin Walter , Le Gal Gregoire
Published: April 2020

Background: Clinical outcome studies have shown that it is safe to withhold anticoagulant therapy in patients with suspected pulmonary embolism (PE) who have a negative D-dimer result and a low pre-test probability (PTP) either using a PTP model or clinical gestalt.

Purpose: To assess the safety of the combination of a non-high PTP using the Wells or Geneva models with a negative VIDAS© D-dimer result to exclude PE.

Data Source: A systematic literature search strategy was conducted using MEDLINE, EMBASE, the Cochrane Register of Controlled Trials and all EBM Reviews.

Study Selection: Seven studies (6 prospective management studies and 1 randomized controlled trial) reporting failure rates at three months were included in the analysis. Non-high PTP was defined has “unlikely” or “low/intermediate” PTP using either, the Wells’ score, the Geneva, Revised Geneva Score, or gestalt estimation.

Data extraction: Two reviewers independently extracted data onto standardized forms.

Data Synthesis: A total of 5,622 patients with non-high PTP were assessed using the VIDAS© D-dimer. PE was ruled out by a negative VIDAS© D-dimer test in 40% (95% confidence intervals (CI) 38.7 to 41.2%) of patients. The three-month thromboembolic risk in patients left untreated was 0.14% (95% CI 0.05 to 0.4%).

Table 1. Accuracy Indices

Total non-high PTP and negative VIDAS© D-Dimer Wells’ “unlikely” PTP and negative VIDAS© D-dimer Geneva* “low/intermediate” and negative VIDAS© D-dimer Number of patients 5,622 2,017 3,208 Sensitivity (%, 95% CI) 99.7 (99.0– 99.9) 98.7 (96.2– 99.6) 100.0 (99.4–100) Specificity (%, 95% CI) 47.4 (46.0– 48.9) 57.3 (55.0– 59.6) 40.8 (38.9– 42.7) NPV (%, 95% CI) 99.9 (99.6– 100) 99.7 (99.1– 99.9) 100.0 (99.6– 100)

Conclusion: The combination of a non-high PTP with a negative VIDAS© D-dimer result, effectively and safely exclude PE in an important proportion of outpatients with suspected PE.
The Relationship Between D-Dimer Level, Venous Valvular Reflux and Development of the Post-Thrombotic Syndrome after Deep Venous Thrombosis.
Authors: Latella Jennifer , Desmarais Sylvie , Kahn Susan ,   Veto Investigators
Published: April 2020

Background: The pathophysiology of the post-thrombotic syndrome (PTS) is postulated to involve persistent venous thrombosis and valvular reflux. We prospectively studied if d-dimer levels or valvular reflux were associated with subsequent development of the PTS after objectively confirmed deep venous thrombosis (DVT).

Methods: Consecutive patients with objectively diagnosed acute symptomatic DVT were recruited at 8 hospital centres in Quebec and Ontario, Canada. Patients attended study visits at Baseline, 1, 4, 8, 12, and 24 months. Blood was taken to measure d-dimer (VIDAS d-dimer; cut off <500 ug/L) at the 4 month visit. A standardized ultrasound assessment for popliteal venous valvular reflux was performed at the 12 month visit. Standardized assessments for PTS (using Villalta scale) were performed at each follow-up visit. Subjects were classified as having developed PTS if the ipsilateral Villalta score was >5 on at least 2 visits starting at the 4 month visit or later or was >5 at the final follow-up visit. Statistical analyses assessed associations between d-dimer level, ipsilateral reflux and PTS.

Results: 387 patients were recruited and followed. Mean age was 56 years and 51% were male. PTS developed in 45% of patients. Mean d-dimer was significantly higher in patients who developed PTS compared with those who did not (712.0 vs. 444.0 ug/L; p= 0.02). In logistic regression analyses adjusted for age and warfarin use at the time of d-dimer determination, d-dimer levels significantly predicted PTS (p=0.03). Ipsilateral venous valvular reflux was more frequent in patients with moderate/severe PTS than in patients with no PTS or mild PTS (65% vs. 40% vs. 43%, respectively; p=0.013). Finally, mean d-dimer was higher in patients who developed recurrent VTE during follow-up (n=31) than in those who did not (1126.8 vs. 514.9 ug/L; p=0.05), and d-dimer was an independent predictor of recurrent VTE (p=0.04) after adjustment for other known predictors of recurrence.

Conclusion: D-dimer levels measured 4 months after DVT are associated with subsequent development of PTS and are predictive of VTE recurrence. Venous valvular reflux was associated with moderate/severe PTS. Further studies are required to assess whether d-dimer or valvular reflux may be useful in determining which patients are most at risk of developing PTS or severe PTS and who may thus benefit from preventive strategies.
R93W Mutation in Orai1 Causes Impaired Calcium Influx in Platelets.
Authors: Bergmeier Wolfgang , Oh-hora Masatsugu , McCarl Christie-Ann , Roden R. Claire , Bray Paul F. , Feske Stefan
Published: April 2020

The intracellular Ca2+ concentration of non-excitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmatic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1R93W). Platelets expressing Orai1R93W were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca2+]i. Orai1R93W platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1R93W platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1R93W platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets pro-coagulant response rather than for other Ca2+-dependent cellular responses.
Determinants for Iron Overload-Related Disease in Siblings of Probands with Clinically Detected HFE Hereditary Hemochromatosis: The Hemochromatosis Family Study.
Authors: Jacobs Esther M.G. , Hendriks Jan C.M. , Kreeftenberg Herman G. , de Vries Richard A. , Marx Joannes J.M. , van Deursen Cees Th. B. M. , Stalenhoef Anton F.H. , Verbeek Andre L.M. , Swinkels Dorine W.
Published: April 2020

The clinical expression of HFE-associated hereditary hemochromatosis (HH) gen is highly variable and may be influenced by nongenetic factors and coinherited genetic modifiers, complicating early screening options to prevent iron-overload related disease. The aim of this study was to verify the existence of HH-related disease in C282Y homozygous siblings of C282Y homozygous probands with clinically detected HFE-related HH and to identify factors predictive for the iron-related disease within these siblings. To this end, C282Y homozygous (n=110, males n=53) and non-homozygous siblings (n=318, males n=145) of 224 probands were compared for levels of serum iron parameters, and self-reported environmental and lifestyle factors and previously diagnosed HH-related diseases. Compared to non-homozygous C282Y siblings, C282Y homozygous siblings more often mentioned to have been diagnosed with arthropathy (Odds Ratio [OR] 2.76, 95% Confidential Interval [CI] 1.71–4.46) and liver disease (OR 2.90, 95%CI 1.27– 6.62). Using multivariate logistic regression modelling, genotype (OR 2.29, 95%CI 1.04– 5.02), age (OR 1.07, 95% CI 1.04–1.09) and gender (OR 1.71, 95%CI 1.04–2.80) were found predictive for the development of iron-associated organ disease. With genotype in the model, there was neither an additive predictive value of the serum iron parameters, nor of body mass index (BMI) or alcohol intake. However, when the predictive value of the iron parameters was analyzed in siblings above 55 yrs, the input of the serum ferritin levels was also significant, with a less prominent influence of gender. In conclusion, our results show that the prevalence of hemochromatosis-attributed morbid conditions is increased in the C282Y homozygous siblings compared to their non-homozygous counterparts. Results furthermore suggest that age and gender, but not BMI and alcohol intake, add to the identification of C282Y homozygous siblings most at risk to develop hemochromatosis-associated disease. These findings will be instrumental in the definition of a high-risk group for iron overload-related disease among siblings of clinically detected C282Y homozygous probands and may contribute to the cost-effectiveness of family screening.
CD36 Mediated Reactive Oxygen Species Formation Contributes to Thrombosis.
Authors: Li Wei , Febbraio Maria , Silverstein Roy L.
Published: April 2020

We previously showed that mice in which the CD36 gene was deleted had significantly prolonged time to form an occlusive thrombus in response to FeCl3-induced vascular injury than wild type (WT) mice. In other studies we and others have shown that CD36 promotes reactive oxygen species (ROS) formation in murine models of cerebral and cardiac ischemia. In this study, we examined the formation and roles of ROS in FeCl3-induced carotid artery injury and thrombosis using CD36 null and WT mice. Hydroethidine, a fluorescent superoxide indicator, was injected via the jugular vein and then carotid artery injury was induced in the contralateral artery by direct topical application of FeCl3 at concentrations of 7.5% or 12.5%. Fluorescence images were monitored with intravital microscopy. FeCl3 dose dependently induced significant accumulation of ROS in the vessel wall; however, the degree was significantly less in CD36 null mice than in WT mice exposed to the lower dose of FeCl3. Direct injection of Edavarone (1 mg/Kg body weight), a free radical scavenger, significantly elongated the time to thrombosis in WT mice. Thrombosis time was also increased in the CD36 null mice, but not to the same extent as WT. We also found fewer endothelial cell-derived microparticles (CD105 positive) in plasma of CD36 null mice 3 minutes after injury with the low dose of FeCl3. Immunoblots of lysates prepared from carotid arteries with thrombi or from untreated normal carotid arteries showed that levels of peroxiredoxin 2 (Prx 2), an antioxidant enzyme known to detoxify ROS, were lower in the thrombi formed from CD36 null mice, but higher in the normal vessel wall, suggesting that an anti-oxidative system may exist in the vessel wall of CD36 null mice. We conclude that CD36 plays a role in ROS formation during thrombosis and may also sensitize the vessel to injury induced by FeCl3. High Prx 2 expression in the vessel wall in CD36 null mice may contribute to the anti-oxidative effect observed in these mice.
Select γ-Secretase Inhibitors Induce Apoptosis in Pre-B ALL Cells and Disrupt the Balance Between Constitutive Notch Signaling and Repression.
Authors: Wilson Bridget S. , Meng Xiangbing , Mazel Tomas , Willman Cheryl L. , Atlas Susan , Harvey Richard , Chen I-Ming , Hunger Stephen P. , Oliver Janet M , Winter Stuart S.
Published: April 2020

Several γ secretase inhibitors (GSIs) were tested for the ability to induce apoptosis in precursor B acute lymphoblastic leukemia (pre-B ALL) cells. Of five GSI’s tested, treatment with two compounds resulted in effective killing of both pre-B lymphoblasts and cells from multiple pre-B ALL lines. Since Notch receptors represent an important group of γ secretase targets, we evaluated expression and activation status of Notch receptors in CD19+ lymphoblasts from pediatric pre-B ALL patients, as well as cultured pre-B ALL cells. We found that, unlike T-ALL where activating mutations are common, pre-B ALL cells appear to drive constitutive Notch signaling through autocrine signals. Blasts from 11 patients expressed 3 Notch receptors and multiple Notch counter-ligands. Expression of Notch pathway genes was also confirmed by microarray analysis of genes expressed in 207 children with high risk B precursor ALL. GSI treatment of pre-B ALL cells led to dephosphorylation of AKT and Foxo3, Bim expression and caspase activation. GSI treatment also blocked cleavage of Notch 1 and 2 to their active forms and inhibited expression of Notch targets, Hey2 and Myc. In contrast, increased expression of Hes1 and Hey1 was correlated with GSI-induced loss of the co-repressor, SMRT. GSI treatment appears to induce precursor B cell death by disrupting the balance between constitutive Notch signaling and repression.
Toxicities in Adults with Acute Lymphoblastic Leukemia (ALL) Treated with Regimens Using Pegasparaginase.
Authors: Rytting Michael , Earl Marc , Douer Dan , Muriera Brenda , Advani Anjali , Bleyer Archie
Published: April 2020

Background: The current therapeutic strategy of applying pediatric-based regimens for acute lymphoblastic leukemia (ALL) to adults with ALL exposes these patients to multiple doses of asparaginase (ASP). Exposure to long-acting or pegylated ASP is particularly prominent due to dosing convenience, since pegylated ASP can be administered intravenously and requires fewer doses than shorter-acting forms. Previously, adult patients were much less likely to be treated with ASP-containing regimens due to reports from the 1970s of increased toxicity from ASP in adults compared with children. We report on the toxicities encountered in 3 protocols that include multiple doses of pegylated ASP as part of therapy for ALL in adult patients.

Methods: Thus far, the 3 protocols have enrolled 92 patients between the ages of 14 and 71 years. The pegylated ASP dose ranges from 2000–2500 IU/m2. Approximately 330 doses of pegylated ASP have been given.

Results: Grade 3–4 hepatic toxicity is the most prominent; grade 3–4 transaminase elevation occurred in 47 (51%) patients, and grade 3–4 hyperbilirubinemia was seen in 22 (24%) patients (Table). Hyperglycemia was grade 3–4 toxicity in 30 (33%) patients. Grade 3–4 allergic reactions to pegylated ASP occurred in 5 (5%) patients. Twelve (13%) patients developed thromboses. Of note, 3 (3%) patients have had leukoencephalopathy on magnetic resonance imaging scans with reversible stroke-like symptoms. The majority of hepatic toxicities resolve spontaneously, allowing patients to continue chemotherapy. All of the patients with stroke-like symptoms have fully recovered.

Conclusions: Considerable hepatotoxicity and hyperglycemia occur in adult ALL patients treated with polychemotherapy that includes long-acting ASP. Other toxicities occur with a frequency similar to that seen in pediatric patients treated with a long-acting ASP. This toxicity profile warrants close monitoring and continued data collection from clinical trials that use pegylated ASP in adults with ALL.

USC Cleveland Clinic M.D. Anderson Total *No. of patients with grade 3–4 toxicities. Median age (years) 33 46 20 33 Age range (years) 18–57 20–71 14–34 14–71 No. doses/patients 127/39 56/25 147/28 330/92 Toxicity* Elevated liver enzymes 23 7 17 47 Hyperbilirubinemia 7 6 9 22 Hyperglycemia 12 5 13 30 Clinical pancreatitis 5 N/A 3 8 Fatigue 3 1 7 11 Thrombosis 3 (SVC only) 2 7 12 Hypofibrinogenemia N/A 8 N/A 8 Elevated PT / INR N/A 1 N/A 1 Bleeding 0 N/A 0 0 Nausea / vomiting 1 4 2 7 Allergy / hypersensitivity 0 2 3 5 Neuropathy 1 1 N/A 2 CNS stroke-like syndrome 0 0 3 3
Platelets in Storage: Altered S-Nitrosothiol Metabolism and Decreased Responsiveness May Be Related.
Authors: Kanter Julie , McLaughlin Dk , Dimitry J , Rogers S. , Kell Pj , Frazier WA , Doctor A.
Published: April 2020

Under normal physiologic conditions, platelets circulate in an inactivated state and require only minimal stimulation ensuring their immediate availability for hemostasis. Previous research has shown that during platelet pheresis and storage, platelets undergo early activation resulting in expression of the surface activation marker CD62, release of α granule contents, and a decrease in inducible activation. In addition to regulating vascular tone, nitric oxide (NO) groups in plasma may provide tonic inhibition to circulating platelets thereby preventing the inappropriate adhesion and aggregation seen in stored platelets. S-Nitrosothiols (RSNOs) may serve as a reservoir for nitric oxide (NO) in the normal circulation. We hypothesize that platelets are prematurely activated during the process of apheresis in part due to the lack of RSNOs in the microenvironment during collection and storage. Furthermore, RSNO metabolism by stored platelets is changed by the process of apheresis itself. To test this hypothesis, we monitored the activation status of platelets using CD62 flow cytometry and quantified intracellular NO content and uptake using the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA). Both measurements were made on freshly isolated platelets (Fl-Plts) and on apheresis platelets (A-Plts). In addition, we employed these techniques to evaluate the responsiveness of Fl-Plts and A-Plts to agonists [thrombin receptor activating peptide (TRAP), ADP, and collagen] and to NO-donating molecules after varying storage times. In preliminary studies we have found that Fl-Plts bind RSNOs and demonstrate steroselectivity in their degree of uptake. Fl-Plts incubated with L-RSNOs produce over 2-fold the fluorescence compared to Fl-Plts incubated with D-RSNOs. A-Plts lose this stereoselectivity in RSNO uptake following the collection and storage process. We have also found that A-Plts have up to a 10-fold increase in uptake of nitric oxide from RSNOs compared to Fl-Plts. This significant increase and loss of stereoselectivity in RSNO uptake in A-Plts correlates with the loss of inducible activation over storage time. These data suggest RSNO metabolism in platelets is fundamentally altered during apheresis, collection, and storage and provides a novel target with therapeutic potential for further investigation into the loss of platelet function during storage.