bluuebunny/crossref_metadata_2025 huggingface dataset:
JAK2 V617F Mutational Status in Myelofibrosis at and before Disease Progression Including Leukemic Transformation: A Longitudinal Study in 68 Patients.
Authors: Mesa Ruben A. , Powell Heather , Lasho Terra , Tefferi Ayalew
Published: April 2020

Background: An acquired point mutations of JAK2 (V617F) was recently described in a substantial proportion of patients with myeloproliferative disorders (MPD), including myelofibrosis with myeloid metaplasia (MMM). The current study examines i) JAK2 V617F mutational status at time of leukemic transformation (LT) of MMM and its clinical correlates, ii) the possibility of changes over time in mutational status in MMM patients that have either undergone leukemic transformation (LT) or experienced disease progression.

Methods: Mutation analysis for JAK2 V617F was performed in DNA derived from either peripheral blood mononuclear cells (PBMC) (i.e. fresh specimens) using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Fluorescent dye chemistry sequencing was performed using the same primers used for amplification.

Results: The study population consisted of 68 patients including 33 patients with disease transformation into AML.

i. MMM patient cohort with LT (n=33). The JAK2 V617F mutant allele was detected in 21 of the 33 patients (64%; homozygous in 6%) at the time of LT. As expected, PPMM was over-represented in the group with the mutation but the presence of the mutation affected neither the clinical presentation at time of LT or overall outcome. Furthermore, in 9 patients (7 heterozygotes and 2 with wild-type allele), archived bone marrow that was collected at a median of 23 months (range, 3–105) before LT was available and disclosed no change in mutational status.

ii. MMM patient cohort with disease progression without LT (n=35; 19 AMM, 9 PPMM, 7 PTMM): Each patient in this group had at least two bone marrow or peripheral blood samples that were collected at two different time points separated by a median of 18.2 months (range (3–82). Of the total 35 patients in this group, 32 (91%) showed no change in JAK2 V617F mutational status over time including 3 patients in whom the first sample was collected before they transformed into MMM from antecedent PV or ET. In the remaining 3 patients, a switch from wild-type to heterozygous state (1 patient), and heterozygous to homozygous (2 patients), was documented after 78, 43, and 83 months, respectively.

Conclusions: In most instances, JAK2 V617F mutational status in MMM remains unchanged over time including during leukemic transformation. Furthermore, the presence of the mutation in MMM-related LT does not appear to affect clinical outcome. These observations undermine the role of JAK2 V617F in either disease progression or LT in MMM.
Thrombocytopenia Is a Strong Adverse Feature in Transplant-Eligible Patients with Myelofibrosis: Utility for an Improved CBC-Based Scoring System.
Authors: Dingli David , Schwager Susan M. , Mesa Ruben A. , Li Chin-Yang , Tefferi Ayalew
Published: April 2020

Background: Allogeneic hematopoietic stem cell transplantation is potentially curative in agnogenic myeloid metaplasia (AMM) but is associated with substantial mortality and morbidity that necessitates accurate identification of patients in whom benefit outweighs risk. The current single institutional study investigates prognostic variables in transplant-eligible patients with AMM with the main objective of improved discrimination between intermediate- and high-risk patient categories.

Methods: Patients diagnosed with AMM before the age of 60 years and seen at Mayo Clinic were identified and the diagnosis confirmed. Relevant demographic, clinical and laboratory characteristics were abstracted and the impact of various parameters on overall survival was evaluated with univariate and multivariate analysis.

Results: A cohort of 159 patients (median age 52 years, range 18–60; 89 males) with AMM is described. Median follow-up from initial diagnosis was 63 months (range 0–300). During this period, 102 patients have died; overall median survival 79 months. Multivariate analysis of parameters measured in all study patients at diagnosis identified thrombocytopenia (platelet count < 100 x 109/L) as the strongest predictor of inferior survival (p=0.002). In addition, a hemoglobin level of <10 g/dL (p=0.003), white blood cell count of either <4 or >30 x 109/L (p=0.03), and older age (p=0.02) were also found to be independent indicators of poor prognosis. However, when the analysis included parameters that were measured in variable proportion of the study population, the independent prognostic factors for poor survival were thrombocytopenia (p=0.0001), anemia (p=0.01), and the presence of unfavorable cytogenetic abnormalities (0.001).

Based on the above findings, we constructed a new complete blood count (CBC)-based prognostic scoring system (Figure 1) that performed better than the Dupriez scoring system in discriminating intermediate- from high-risk patient categories(Figure 2).

Figure Figure

Conclusions: Thrombocytopenia is a strong predictor of poor survival in transplant-eligible patients with AMM. The incorporation of platelet count into the Dupriez prognostic scoring system might allow the construction of an improved, CBC-based scoring system that can accurately identify high-risk as well as intermediate-risk patients with AMM.
Presence of Unfavorable Cytogenetic Abnormalities Is the Strongest Predictor of Poor Survival in Secondary Myelofibrosis.
Authors: Dingli David , Schwager Schwager M. , Ruben Mesa A. , Li Chin Yang , Tefferi Ayalew
Published: April 2020

Post-polycythemic (PPMM) and post-thrombocythemic (PTMM) myeloid metaplasia are consensually referred to as secondary myelofibrosis (sMF). Prognostic variables in sMF are not as well defined as they are for agnogenic myeloid metaplasia (AMM). Such information is particularly crucial for management decisions in transplant-eligible patients. Accordingly, we examined the prognostic impact of several clinical and laboratory variables in 66 young patients (age < 60 years) with sMF including 37 with PPMM and 29 with PTMM. Multivariate analysis of parameters other than cytogenetics identified older age (p=0.02), anemia (hemoglobin level < 10 g/dL; p=0.007), and PPMM (p=0.009) as independent risk factors for shortened survival. However, when such analysis was restricted to patients in whom cytogenetic studies were performed (n=31), the presence of unfavorable cytogenetic abnormalities (i.e. clones other than 20q- and 13q-) became the one and only adverse prognostic factor for survival (p=0.001).

Figure Figure

The prognostic value of cytogenetics was independent of the Dupriez prognostic score (p=0.003).

Figure Figure

A similar analysis in a temporal cohort of 50 age-matched patients with AMM also identified unfavorable cytogenetics as an independent predictor of poor survival along with thrombocytopenia and anemia. The current study suggests an important prognostic role for cytogenetics in both de novo and secondary myelofibrosis.
Estrogen-Based Hormone Therapy and Thrombosis Risk in Women with Essential Thrombocythemia.
Authors: Gangat Naseema , Wolanskyj Alexandra P. , Schwager Susan M. , Mesa Ruben A. , Tefferi Ayalew
Published: April 2020

Background

Estrogen-based hormone treatment (EBHT), in the form of either oral contraceptives or hormone replacement therapy, is generally felt to be associated with an increased risk of thrombosis in women with essential thrombocythemia (ET). However, there is insufficient evidence to support or refute such a concern.

Methods

Data was abstracted from medical records of a consecutive cohort of women with ET seen at the Mayo Clinic. Thrombotic risk was determined by the occurrence of a major thrombotic event namely myocardial infarction, angina, cerebrovascular accident, transient ischemic attack, peripheral arterial thrombosis, pulmonary embolism, or deep venous thrombosis at or after diagnosis of ET.

Results

i. Information regarding EBHT at or after the diagnosis of ET

The study cohort included 307 women with ET (median age, 55 years; range 16–88) with a median follow-up of 133 months from diagnosis (range, 0.2–397). EBHT at or after diagnosis was documented in 93 women (30%); 59 patients were on EBHT at time of diagnosis and treatment was initiated after diagnosis in 34 patients. Among the 59 patients who were receiving EBHT at the time of diagnosis, treatment was discontinued within one month of their ET diagnosis in 17 patients but continued in the other 42 patients for a median of 48 months (range, 2–168). In the 34 patients with EBHT that was initiated after the diagnosis of ET, median treatment period was 44 months (range, 1–144).

ii. Correlation between EBHT and thrombosis at diagnosis

At diagnosis, major thrombosis was documented in a total of 74 patients (24%) that included 11 of the 59 patients (19%) on EBHT and 63 of 248 patients (25%) that were not receiving such therapy at the time of diagnosis (p=0.28).

iii. Correlation between EBHT and thrombosis after diagnosis

A total of 95 patients (31%) experienced a subsequent (i.e. post-diagnosis) thrombotic episode during the study period. These occurred in 65 of 214 patients (30%) that were not exposed to EBHT at or after diagnosis, 5 of 17 patients (29%) in whom EBHT was discontinued with the diagnosis of ET, 13 of 42 patients (31%) in whom EBHT was continued despite the diagnosis of ET, and 12 of 34 patients (35%) in whom EBHT was started after the diagnosis of ET (p=0.95). Furthermore, duration of EBHT, using a cutoff value of 1 year, did not significantly affect the incidence of thrombotic events (p=0.67).

The different comparative groups were similar in age distribution as well as frequency of other cardiovascular risk factors including smoking, diabetes mellitus, hypertension, and hyperlipidemia.

Conclusion

The current study addresses the important issue of safety pertaining to the use of estrogen-based hormones in women with ET and did not reveal a significant alteration of thrombosis risk as a result of hormone therapy.
Protective Effect of Activated Protein C in Murine Endotoxemia: Mechanism of Action.
Authors: Kerschen Edward J. , Cooley Brian C. , Castellino Francis J. , Griffin John H. , Weiler Hartmut
Published: April 2020

Recombinant activated protein C (APC) reduces mortality of patients with severe inflammatory disease associated with multi-organ failure. APC exerts anticoagulant, anti-inflammatory, and cytoprotective effects. The contribution of these distinct APC activities to the overall therapeutic efficacy in septic patients is unknown. The aim of the study is to delineate the mechanism underlying the protective effect of APC in mouse endotoxemia. We first establish an experimental mouse model to demonstrate that recombinant murine APC reduces 6 day mortality of mice subjected to LPS-induced endotoxemia. APC treatment did not alter the extent of inflammatory cytokine release. Recombinant human APC did not exhibit therapeutic efficacy in this model. In contrast, recombinant human and mouse APC reduced to a similar extent experimentally induced arterial thrombus formation. The therapeutic efficacy of wild type recombinant murine APC was abolished in genetically engineered mice with reduced expression of the endothelial protein C receptor (EPCR). Recombinant mutant murine APC with greatly reduced anticoagulant potency was as effective as wild type murine APC in reducing mortality of mice subjected to LPS-induced septicemia. Mice homozygous for the Leiden polymorphism in the factor V (fV) gene, which renders coagulation factor V partially resistant to the anticoagulant effect of APC secondary to blocked fV proteolysis at R504 (R506 in humans), were refractory to the therapeutic benefit conveyed by administration of recombinant wild type mouse APC. In summary, these findings provide evidence that the therapeutic efficacy of recombinant APC is predominantly based on the ability of APC to interact with the endothelial protein C receptor, and that the anticoagulant activity of APC is not sufficient for achieving protection against mortality in a mouse model of endotoxemia. On the other hand, cleavage of fV at R506 appears necessary for retaining therapeutic efficacy in carriers of the fV Leiden allele.
Two Oncogenic Hits Are Required To Initiate Lymphomagenesis in Adult, but Not Neonatal Hosts.
Authors: Fan Alice C. , Giuriato Sylvie , Karlsson Asa , Padua Rose Ann , Felsher Dean W.
Published: April 2020

The hallmark (8:14) translocation of Burkitt’s lymphoma causes MYC overexpression, a primary event in the development of Burkitt’s lymphoma. While Burkitt’s lymphoma comprises nearly one third of childhood malignancies, it is rare in adults. Among many explanations is the possibility that the capacity for a lymphocyte to undergo malignant transformation by MYC activation is influenced by factors related to the developmental state of the host. To address this possibility, we used transgenic mice in which the MYC proto-oncogene is conditionally regulated via the Tetracycline Regulatory System (Tet system). When MYC was activated in cohorts of mice less than 3 weeks of age, mice universally succumbed to lymphoma within 23 weeks. However, when MYC was activated in adult mice (over 7 weeks of age) the mice failed to develop lymphoma within 50 weeks. We were not able to detect differences in the effects of MYC on the proliferation or apoptosis in lymphocytes from adult versus neonatal hosts. We next explored whether the concomitant activation of MYC with a second oncogene could induce lymphomagenesis in adult mice. When we conditionally induced both MYC and BCL2 or MYC and RAS in adult hosts, mice succumbed to lymphoma with a mean latency of 24–27 weeks. Our results suggest that in adult hosts, concurrent activation of at least two oncogenes is required for MYC to initiate tumorigenesis.
Surface-Enhanced Laser Desorption/Ionization Mass Spectral Plasma Profiles Distinguish Individuals of a Protein C-Deficient Family with a Thrombotic Episode before the Age of 40 from Age-Matched Family Members without a History of Thrombosis.
Authors: Svensson Annika , Whiteley Gordon , Callas Peter , Bovill Edwin
Published: April 2020

We tested the hypothesis that differences in the low molecular weight (500–20.000 Da) proteomic profile of plasma may be detectable between members of a protein C-deficient thrombophilic family who have suffered thrombotic events before age <40 compared to family members without a history of venous thrombosis.

Unfractionated plasma samples from members of a previously described large thrombophilic kindred with type I protein C deficiency were applied to ProteinChip weak cation exchange interaction chips (WCX2; Ciphergen Biosystems, Fremont, CA) and subjected to SELDI-TOF (surface-enhanced laser desorption/ionization time-of-flight) mass spectrometry using the Ciphergen PBSII ProteinChip System (Ciphergen Biosystems, Freemont, CA, USA). Profiles were analyzed by a boosted decision-tree algorithm. When individuals who had presented with deep venous thrombosis before the age of 40 (n=21) were compared to age-matched, healthy family members (n=50), the proteomic patterns defined by the decision-tree analysis could classify the entity of DVT before age 40 with 66% sensitivity, at a specificity of 86%. When a small group of cases with history of superficial venous thrombosis (n=6) was added to the case group, the sensitivity was 87.5% at a specificity of 80%. These data support the hypothesis that members of the protein C deficient Vermont kindred II who suffer a thrombotic event before age 40 display significant differences in low-molecular weight proteomics profile compared to those who remain disease-free. This is the first study to apply SELDI-TOF technology in conjunction with a bioinformatics tool to analyze low molecular weight proteomic patterns in patients with venous thrombosis. We are presently pursuing higher resolution analysis with the prOTOF, MALDI orthogonal time of flight mass spectrometer system in order to identify specific peptides
The Role of p53 in C-myc Induced Lymphomagenesis Mediated by the IgH 3′ Enhancers.
Authors: Wang Jinghong , Boxer Linda M.
Published: April 2020

Translocation of the c-myc oncogene to the immunoglobulin heavy chain gene (IgH) locus is the most common chromosomal abnormality in Burkitt’s lymphoma. Our mouse model (IgH-3′E-myc) with the IgH 3′ enhancers integrated 5′ of the c-myc gene demonstrated that the IgH 3′ enhancers play an important role in c-myc deregulation and B cell lymphomagenesis. In this study, we examined the role of the tumor suppressor p53 in c-myc-induced B cell lymphomagenesis by developing IgH-3′E-myc mice with only a single p53 allele (p53+/−-mycE mice). We found that lymphoma development and death were significantly accelerated in these mice compared to the IgH-3′E-myc mice. p53+/−-mycE mice developed Burkitt-like B cell lymphomas at about 5 months of age, and the lymphomas had a clonal origin. In premalignant B cells, p53 expression was comparable to that in wildtype B cells; however, significantly decreased p53 levels were observed in malignant lymphoma cells. In addition, p53 expression was not up-regulated in response to etoposide in lymphoma cells. The remaining p53 allele was present, and no mutations were detected in the coding region. Increased expression of Mdm2 was observed in half of the lymphomas. Premalignant B cells from p53+/−-mycE mice showed higher induced and spontaneous apoptosis due to increased c-myc expression. However, malignant lymphoma cells had a greatly increased survival rate in culture and were more resistant to chemotherapeutic agents. The majority of the lymphomas showed increased expression of the anti-apoptotic protein Bcl-xL. We found that p53 could negatively regulate the bcl-xL promoter in lymphoma cells, and a region that was responsive to p53 in the bcl-xL promoter was identified. These data support the conclusion that decreased p53 levels and overexpression of Bcl-xL play an important role in c-myc-induced lymphomagenesis mediated by IgH 3′ enhancers.
An Activating Mutation (Ser525Pro) within the Transactivation Domain of REL in Two Patients with Human B-Cell Lymphomas Enhances REL’s In Vitro Transforming Activity.
Authors: Trautmann Heiko , Starczynowski Daniel T. , Pott Christiane , Harder Lana , Arnold Norbert , Siebert Reiner , Kneba Michael , Gilmore Thomas D.
Published: April 2020

REL/NF-κB transcription factors are implicated in the control of apoptosis and cell growth particular in hematopoetic lineages. The REL locus at chromosomal region 2p13–16 is frequently amplified in B-cell lymphomas including diffuse-large B-cell lymphoma (DLBCL) and may play a role in lymphomagenesis. Overexpression of wild-type REL can transform chicken lymphoid cells in culture, and several experimentally-generated mutations within the REL C-terminal transactivation domain (TAD) have been previously shown to enhance REL’s transforming ability.

We analysed 83 B-cell lymphomas included in the ‘Deutsche Krebshilfe’ funded network „Molecular Mechanisms in Malignant Lymphoma“ for the presence of activating mutations in the coding region of REL. We performed a systematic dHPLC screening for mutation discovery and identified an identical point mutation in two human B-cell lymphomas (a t(14;18)-positive follicular lymphoma and a mediastinal B-cell lymphoma) that changes Ser525 to Pro within the REL TAD. In the mediastinal B-cell lymphoma, the mutation in REL was proven to be of germline origin. FISH showed an amplification of the REL locus in the tumor cells of this case. Quantitative allelic discrimination of S525P indicates that the mutant REL gene was over-represented in both cases. By in vitro experiments we could show that the S525P mutation enhances the in vitro transforming ability of REL in chicken spleen cells. In addition, REL-S525P differs from wild-type REL in its ability to activate certain κB site-containing reporter plasmids in transient transfection assays. In particular, REL-S525P has a reduced ability to activate the human manganese superoxide dismutase (MnSOD) promoter in A293 cells; however, the MnSOD protein is over-expressed in REL-S525P-transformed chicken spleen cells as compared to wild-type REL-transformed cells. Ser525 of REL falls within a sequence that is similar to other known phosphorylation sites of the IκB kinase, and REL-S525P shows a reduced ability to be phosphorylated by IKKα in vitro. The S525P mutation reduces IKKα- and TNFα-stimulated transactivation by REL, as measured in GAL4 reporter assays. Furthermore, REL-S525P-transformed chicken spleen cells are more resistant to TNFα-induced cell death than cells transformed by wild-type REL. These results represent the first identification of a tumor-derived activating mutation in the REL proto-oncogene, and they suggest that the S525P mutation contributes to the development of human B-cell lymphomas by altering REL’s ability to induce target gene expression by affecting an IKKα-regulated transactivation activity.
Lymphoblastic Leukemia/Lymphoma in Mice Overexpressing the Mer Receptor Tyrosine Kinase.
Authors: Salzberg Dana B. , Keating Amy K. , Anwar Adil , Sawczyn Kelly , Hill Kimberley , Sather Susan , Park Jane , DeRyckere Deborah , Everett Doug , Graham Douglas K.
Published: April 2020

Mer is a transmembrane tyrosine kinase receptor that belongs to a subfamily of tyrosine kinases including Axl and Tyro-3. Within the hematopoietic lineages, Mer is expressed in monocytes/macrophages, dendritic cells and platelets. Normal thymocyte subsets and mature lymphocytes do not express Mer. However, Mer RNA transcript and Mer protein is ectopically expressed in a subset of B cell and T cell acute lymphoblastic leukemia (ALL) cell lines and patient samples. Previous in vitro studies have suggested that constitutive expression of Mer has anti-apoptotic properties and transforming potential. The overexpression of Mer in ALL suggests that this tyrosine receptor kinase may play an important role in leukemogenesis. In an attempt to investigate the oncogenic potential of Mer in vivo, we created a transgenic mouse model in which the expression of the full-length murine Mer cDNA was placed under the control of the Vav promoter. Ectopic expression of Mer has been demonstrated in thymocytes and lymphocytes of the transgenic mice. The ability to activate the ectopic Mer protein with the ligand Gas 6 has confirmed that the protein is functional. Gas 6 stimulation of Mer in lymphocytes from transgenic mice lead to activation of downstream anti-apoptotic signaling pathways including AKT and ERK 1/2. At 12–24 months of age, 60% of the Mer transgenic mice developed lymphadenopathy, hepatosplenomegaly and the presence of circulating lymphoblasts. Histopathological analysis was consistent with a diagnosis of lymphoblastic leukemia/lymphoma. The majority of the lymphomas characterized by flow cytometry were identified as T cell lymphomas with a small subset having co-expression of both T cell and B cell markers. Tumor-free survival in Mer transgenic mice were significantly decreased as compared to the wild type mice (p=0. 003). These data suggest that Mer may play a cooperative role in leukemogenesis and is an attractive candidate marker for biologically targeted leukemia/lymphoma therapy.
Bcl2 Flexible Loop Domain Positively and Negatively Regulates Survival Function through Direct Interaction with p53.
Authors: Deng Xingming , Gao Fengqin , Flagg Tammy , Anderson Jessica , May W. Stratford
Published: April 2020

p53 not only functions as a transcription factor but also has a direct extranuclear apoptogenic role at the mitochondria. We have discovered that DNA damage-induced p53/Bcl2 binding is associated with decreased Bcl2/Bax interaction and increased apoptotic cell death in a mechanism regulated by Bcl2’s flexible loop regulatory domain (FLD) in IL3-dependent myeloid NSF/N1.H7 cells. Purified p53 protein can disrupt the Bcl2/Bax complex by directly binding to a negative regulatory region of the FLD (aa32–68). Deletion of the negative regulatory region (aa32–68) abolishes Bcl2/ p53 binding and enhances its antiapoptotic function. Removal of aa69–87 of the FLD, which contains Bcl2’s phosphorylation site(s) (T69, S70 and S87), enhances Bcl2/p53 binding and abrogates Bcl2’s potent survival activity, indicating this is a positive regulatory region. Phosphorylation of Bcl2 in the FLD inhibits p53 binding since the phosphomimetic T69E/S70E/S87E (EEE) but not the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutant displays a reduced capacity to bind p53 and more potently inhibits p53-induced cytochrome c release. A full-length Bcl2 loop-only protein (aa32–87) can directly bind p53 to impede Bcl2/p53 binding in vitro. Either DNA damage or expression of p53 targeted to mitochondria results in Bcl2/p53 binding followed by exposure of Bcl2’s BH3 domain and inactivation of Bcl2’s antiapoptotic function. Therefore, Bcl2’s FLD contains both a positive and negative regulatory regions which functionally regulate Bcl2’s antiapoptotic activity via differential binding of Bax and p53, respectively.
Phosphorylation of the MCT-1 Oncogene Protein by p42/44 MAPK Is Required for Transformation.
Authors: Nandi Suvobroto , Reinert Line S. , Hachem Ali , Gartenhaus Ronald B.
Published: April 2020

The MCT-1 oncogene has been shown to decrease cell doubling time, dramatically shorten the duration of G1 transit time and/or G1-S transition, as well as transform NIH3T3 fibroblasts. We subsequently demonstrated that there are significantly increased levels of MCT-1 protein in a subset of primary diffuse large B-cell lymphomas (DLBCL) as well as some T-cell lymphomas compared with normal lymphoid tissue. We have mapped MCT-1 to chromosome Xq22-24, a region that has been previously shown to contain amplified DNA in a variety of primary B-cell lymphoid neoplasms using comparative genomic hybridization (CGH). Recent data has shown that MCT-1 is involved in protection against serum-starvation induced apoptosis involving enhanced AKT activity. Phosphorylation is one potential mechanism by which the activity of molecules involved in cell cycle regulation/survival is rapidly modulated. The RAS/MEK/ERK signal transduction cascade plays a prominent role in the regulation of cell growth and proliferation. The MCT-1 protein is predicted to have several putative phosphorylation sites including, a p42/p44 MAPK and cyclinB/cdc2 kinase substrate site. These cdc2 and MAPK phosphorylation sites have been mutated through site-directed mutagenesis and expressed in human dermal fibroblasts (HDF) in order to examine the role of these kinase sites on cell growth and transformation. We demonstrated that the phosphorylation of MCT-1 by p42/p44 MAPK is critical for its ability to stimulate cell proliferation as well as cellular transformation. Our data suggest that targeting the RAS/MEK/ERK signal transduction cascade may be a useful therapeutic approach in those lymphomas and related disorders overexpressing MCT-1.
A New and Rapid Immunoassay for the Detection of 11-Dehydro-Thromboxane B2.
Authors: Geske F. Jon , Hurley Beth , Whittier Amy , Tew Daniel , Ens Gordon , Lopez Luis
Published: April 2020

Aspirin inactivates cyclooxygenase, inhibiting synthesis of thromboxane A2 (TxA2) and other molecules relevant in the development of atherothrombotic vascular events. There is recent evidence suggesting that many individuals are resistant to aspirin’s effects, resulting in increased TxA2 levels. Current methods for detecting aspirin resistance include a number of blood-based assays that measure in vitro platelet aggregation. However, these methods are not quantitative and can be affected by factors that are unrelated to aspirin sensitivity. There is a quantitative commercial immunoassay available for 11-dehydro-thromboxane B2 (11-dh-TxB2), a stable metabolite of TxA2. However, this assay uses a polyclonal antibody to 11-dh-TxB2 and requires an overnight incubation. In order to more quickly and accurately detect aspirin resistance in at-risk patients, we are developing a rapid, quantitative competitive immunoassay for 11-dh-TxB2 using a monoclonal antibody. This new assay can be completed in three hours and utilizes urine as the sample matrix. We compared the monoclonal-based assay with the polyclonal assay using twelve urine controls run multiple times over a period of three months. The controls were derived from aspirin users as well as non-users. The %CV range in the monoclonal assay was 0.3 −12.9% with an average of 4.0%. The %CV range in the polyclonal assay was 0–35.8% with an average of 10.1%. In comparing standard curves, the R2 value of the monoclonal assay averaged 0.9967 compared to 0.9894 for the polyclonal assay. Additionally, although the polyclonal assay was slightly more sensitive (limit of detection was 11.9pg/ml vs. 64.5pg/ml in the monoclonal assay), the monoclonal’s sensitivity is sufficient to detect the lower levels of 11-dh-TxB2 in patients that respond well to aspirin. To validate this, an additional urine panel was established consisting of 10 samples from both aspirin users and non-users, and 11-dh-TxB2 values from the two assays were compared. The correlation (r) between the methods was 0.944, confirming the clinical relevance of the monoclonal assay. Thus, this sensitive, urine-based monoclonal ELISA for 11-dh-TxB2 detection eliminates the potential for analytical variables that can occur in the blood-based assays while offering the sensitivity and quantitation of the ELISA.
The Prevalence and Clinical Significance of IgA Antiphospholipid Antibodies (aPL).
Authors: Shen Yu-Min , Dyszkiewicz-Korpanty Anna , Lee Ray , Balani Jyoti , Frenkel Eugene , Sarode Ravindra
Published: April 2020

AIMS: We investigated the prevalence and clinical relevance and significance of the various immunoglobulin (Ig) isotypes of the different aPLs and lupus anticoagulant (LAC).

METHOD: Our study registry for the year 2003 included 472 patients who had a complete antiphospholipid antibody laboratory work up and clinical evaluation. 137 had the presence of LAC, detected by the dilute Russell’s viper venom time (Dade Behring, Marburg, Germany), PTT-LA (Diagnostica Stago, Asnieres, France), or hexagonal phospholipid neutralization test (Diagnostica Stago, Asnieres, France); 211 had elevated titers of antibodies of IgG, IgM or IgA isotypes against cardiolipin (aCL), phosphatidylserine (aPS), or β2-glycoprotein-I (aβ2GPI), detected by ELISA (Corgenix, Inc, Westminister, CO, USA). 204 had no evidence of either LAC or aPL.

RESULTS: Of the patients with aPL, 67 (32%) had elevated titers of IgG, 126 (60%) had IgM, and 118 (56%) had IgA. Thrombotic events occurred in 61% (41 of 67), 52% (65 of 126), and 61% (72 of 118) of the patients with IgG, IgM, and IgA respectively. Patients with IgG and IgA isotypes of aCL, aPS and aβ2GPI had higher rates (58–67%) of thrombotic events (see table 1) than patients with IgM isotypes (46–50%). Stepwise logistic regression analysis identified elevated titer of IgA of any aPL as an independent risk factor for thrombosis (see table 2), even in the absence of LAC. Thrombotic events observed include deep venous thrombosis (25%), pulmonary embolism (12%), cerebrovascular accident (50%), myocardial infarction (8%), and peripheral arterial occlusion (3%); pregnancy complication, retinal vascular thrombosis, and dialysis access thrombosis were also seen.

CONCLUSION: Our study demonstrates that aPL of IgA isotype is prevalent and significantly associated with thrombotic events.

Proportions of Patients with Thrombosis amongst various aPLs and Ig Isotypes aCL aPS aβ2GPI Total * Two-sided P < 0.05 by McNemar’s test for equality of two dependent proportions IgG 28/43 (65%) 23/36 (64%) 13/21 (62%)* 41/67 (61%) IgM 17/37 (46%)* 28/58 (48%) 45/90 (50%)* 65/126 (52%) IgG 15/26 (58%)* 22/33 (67%) 54/91 (59%)* 72/118 (61%) Total 44/79 (56%) 58/105 (55%) 85/159 (53%) 118/211 (56%) Risk Factors for Thrombosis Antibody Odds Ratio 95% Wald Confidence Limit * p < 0.05 by the Wald chi-square test. Hosmer and Lemeshow goodness-of-fit test was used to assess the model fit. N=472 for the 1st four analyses, N=335 for the last three analyses. LAC 1.627 1.073–2.466* IgG regardless of LAC 1.364 0.781–2.384 IgM regardless of LAC 0.874 0.569–1.344 IgA regardless of LAC 1.587 1.012–2.490* IgG LAC negative 1.790 0.863–3.714 IgM LAC negative 0.878 0.515–1.499 IgA LAC negative 1.812 1.035–3.172*
Comparative Genomic Analysis of the 5′-Upstream Sequences of von Willebrand Factor and 27 Other Endothelial-Specific Genes Identifies Conserved Noncoding Sequences That Are DNase Hypersensitive.
Authors: Bernat John A. , Kondrashov Alexey S. , Crawford Gregory E. , Collins Francis S. , Ginsburg David
Published: April 2020

Von Willebrand factor (VWF), a key blood clotting protein, is a standard histologic marker for endothelial origin. Previously published studies characterizing tissue-specific regulatory elements have concentrated on the proximal promoter region (between −2,645 and the end of murine Vwf intron 1). Transgenic mouse models using these sequences have found elements needed for VWF expression in some, but not all, populations of endothelial cells. To identify potential novel VWF distal regulatory elements, as well as sequences contributing to the more general endothelial-specific expression program, we aligned human and mouse orthologous genomic sequences from VWF and 27 other endothelial-specific genes, extending from the start of transcription to the neighboring 5′-upstream gene (up to 100 kb of sequence), excluding the core promoter, and including intron 1. We analyzed the resulting 262 endothelial cell-specific gene conserved noncoding sequences (EC-CNSs) using a specific quantitative PCR assay for DNase I hypersensitive sites, an indicator of an open chromatin state which suggests the presence of a transcription regulatory complex. 33% of the EC-CNSs (including 6 of 9 upstream of VWF) were hypersensitive (HS) in human umbilical vein endothelial cells (HUVECs), while only 16% were HS in control human fibroblast cells. 20% of the EC-CNSs were HS in HUVECs only, suggesting that they may identify novel endothelial-specific transcriptional elements. When the 262 EC-CNSs were compared to CNSs from non-endothelial genes, a single motif, AGGAA(A/G), was identified as overrepresented in EC-CNSs. This association was replicated in an independent set of 28 additional genes with high HUVEC expression. The motif was also overrepresented in the subset of HS EC-CNSs. The AGGAA(A/G) motif includes the core binding sequence [GGA(A/T)] of the Ets family of transcription factors, which contains several members implicated in tissue-specific gene expression. These results suggest that the AGGAA(A/G) motif may represent the target sequence for a specific Ets family member critical for determining the endothelial-specific gene expression program. We are currently testing this hypothesis by analyzing this motif in Vwf BAC transgenic mice containing 177 kb of upstream sequence, including all ten identified EC-CNSs. Additionally, we have expanded our analysis of the VWF genomic region to include over 700 kb of sequence surrounding and including the gene, searching for additional EC-CNSs conserved across eleven mammalian species. This comparative genomic approach, coupled with functional analysis of putative regulatory elements, should provide new insight into the endothelial cell gene expression program and the more general problem of tissue-specific gene regulation.
Downregulation of P-Selectin from Activated Platelets Involves Both Receptor Internalization and Shedding upon Binding to PSGL-1.
Authors: Dole Vandana S. , Bergmeier Wolfgang , Eichenberger Sarah C. , Wagner Denisa D.
Published: April 2020

P-selectin is present in the alpha granules of platelets and is translocated to the cell surface where it mediates adhesion to leukocytes via binding to PSGL-1. We have previously shown that P-selectin is rapidly lost from the platelet surface when the activated platelets are infused into mice or incubated in vitro with whole blood. This downregulation of surface expression correlates with increased levels of soluble P-selectin in the plasma indicating that some of the protein is shed. The data presented here indicate that the interaction of platelet P-selectin with leukocyte PSGL-1 plays a role in P-selectin shedding. Firstly, P-selectin expression on activated platelets is retained significantly longer (1h) when the cells are infused into PSGL-1−/− mice whereas in the WT recipients, as early as 15 min after infusion P-selectin cannot be detected on the cell surface. Secondly, the concentration of soluble P-selectin in plasma of PSGL-1−/− mice is several fold lower compared to WT mice. In addition, we demonstrate that surface expression of platelet P-selectin is also downregulated by receptor internalization and that internalization is the predominant mechanism in the absence of PSGL-1. When analyzed 4h after infusion into WT mice, approximately 50% of the activated platelets which no longer express P-selectin on the surface can be reactivated to express P-selectin on the cell surface. In contrast, when infused into PSGL-1−/− recipients, most of the platelets can be reactivated to express P-selectin on the cell surface. In summary, engagement with leukocyte PSGL-1 is important for platelet P-selectin shedding and in its absence surface P-selectin is downregulated by receptor internalization. The enzymatic mechanism of P-selectin shedding and its significance needs to be elucidated.
Canonical Notch Signaling Is Dispensable for the Maintenance of Adult Hematopoietic Stem Cells.
Authors: Maillard Ivan , Pross Seth E. , Shestova Olga , Sai Hong , Aster Jon C. , Bhandoola Avinash , Pear Warren S.
Published: April 2020

Canonical Notch signaling operates through a highly conserved pathway that regulates the differentiation and homeostasis of hematopoietic cells. Ligand-receptor binding initiates proteolytic release of the Notch intracellular domain (ICN) which migrates to the nucleus, binds the transcription factor CSL/RBPJk and activates target genes through the recruitment of transcriptional coactivators of the Mastermind-like family (MAML). Notch signaling is essential for the emergence of hematopoietic stem cells (HSCs) during fetal life, but its effects on adult HSCs are controversial. In gain-of-function experiments, activation of Notch signaling in adult HSCs increased their self-renewal potential in vitro and in vivo. However, loss-of-function studies have provided conflicting results as to the role of physiological Notch signaling in HSC maintenance and homeostasis. To address this question, we expressed DNMAML1, a GFP-tagged pan-inhibitor of Notch signaling, in mouse HSCs. We have shown previously that DNMAML1 interferes with the formation of the ICN/CSL/MAML transcriptional activation complex and blocks signaling from all four Notch receptors (Notch1-4) (Maillard, Blood 2004). Transfer of DNMAML1-transduced bone marrow (BM) as compared to control GFP-transduced BM into lethally irradiated recipients gave rise to similar long-term stable expression of GFP for at least 6 months after transplant. DNMAML1 and GFP-transduced cells contributed equally to all hematopoietic lineages, except to the T cell and marginal zone B cell lineages, which are Notch-dependent. Expression of DNMAML1 did not affect the size of the BM progenitor compartment (Lin negative, Sca-1 positive, c-Kit high, or LSK cells), or the proportion of LSK cells that were negative for Flt3 and L-Selectin expression (containing long-term HSCs). The stem cell function of DNMAML1-transduced LSK cells was further assessed with in vivo competitive repopulation assays in lethally irradiated recipients. DNMAML1 and GFP-transduced LSK cells competed equally well with wild-type BM, as judged by their contribution to the myeloid lineage up to 4 months post-transplant, through two successive rounds of transplantation. Our data indicate that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs.
Results of a Phase II Study of 506U78 (Nelarabine) in Refractory Indolent B-Cell or Peripheral T-Cell Lymphoma.
Authors: Thompson Michael A. , Pro Barbara , Sarris Andreas , Hagemeister Fredrick B. , Goy Andre , Bleyer Archie , Cabanillas Fernando F. , Samaniego Felipe , Fayad Luis E.
Published: April 2020

BACKGROUND: Compound 506U78 (Nelarabine), a water soluble pro-drug of 9-β-d-arabinofuranosyl-guanine, is demethoxylated by adenosine deaminase to ara-G in lymphoblasts. Intracellular ara-G is then phosphorylated via deoxycytidine kinase and mitochondrial deoxyguanosine kinase to its active 5′-triphosphate, ara-GTP. Ara-GTP, a potent inhibitor of DNA polymerase, also incorporates into DNA resulting in cell death. Studies in children and adult T-cell leukemia and non-Hodgkin’s lymphoma (NHL) have shown activity, but with reversible neurotoxicity as the most prominent adverse event.

METHODS: We report here the results of a phase II study with 506U78 in adults (median age 64, range 33–81) with relapsed or refractory indolent B-cell or peripheral T-cell NHL. Patients had received a median of 2 prior courses of chemotherapy. 506U78 was given at 1.5 g/m2/day IV on days 1, 3, and 5 every 28 days to a maximum of 6 cycles unless toxicity or progressive disease. If after 4 cycles a complete response (CR) or partial response (PR) was not achieved, then treatment was discontinued.

RESULTS: There were 23 patients enrolled including 13 with T-cell and 10 with indolent B-cell NHL. Among 19 assessable for response, the overall response rate (ORR) in T-cell NHL was 4/9 (44%) with 2 CR and 2 PR, while in indolent B-cell NHL the PR was 3/9 (33%) with no CR. In responders, the median time to progression (TTP) was 8 months (range 2–22 months). Sixteen of 22 (73%) patients evaluable for toxicity had grade 3 or 4 adverse events. Neurotoxicity included one grade 3 and one grade 4 event.

CONCLUSION: Compound 506U78 is active as a single agent in T-cell and B-cell NHL. Our response rate was higher than the ORR of 10.5% reported previously in the Czuczman et al. (2004) Blood 104(11) abstract in adults with T-cell NHL and there was less neurotoxicity in our study. This may be related to different dosing regimens (every 28 days vs. every 21 days). Compound 506U78 should be further evaluated in clinical trials.
Phase I/II Study of Oral FodosineTM, a PNP Inhibitor in Refractory Cutaneous T-Cell Lymphoma Patients.
Authors: Duvic Madeleine , Breneman Debra , Cooper Maureen , Fonseca Gustavo , Bennett J. Claude , Kilpatrick J. Michael , Bantia Shanta
Published: April 2020

Suppression of T-cell immunity seen in children with inherited purine nucleoside phosphorylase (PNP) deficiency supports the potential application of PNP inhibitors for the therapy of T-cell malignancies. Forodesine (FodosineTM) is a specific PNP inhibitor that raises plasma 2′-deoxyguanosine (dGuo) and intracellular dGTP levels, leading to T-cell apoptosis. IV administration of forodesine has shown activity in cutaneous T-cell lymphoma (CTCL) patients. To determine the safety, pharmacokinetics and pharmacodynamics of oral forodesine in patients with refractory CTCL, as well as preliminary evidence of efficacy, five cohorts of patients (≥ 3patients per cohort) were evaluated sequentially, receiving forodesine at 40, 80, 160, 320, and 480 mg/m2 once per day for 28 days. At the end of the treatment cycle (Day 28), all patients with stable or improved disease could enter a long-term, follow-up period. To date, 12 patients have been treated, 11 completing the 28-day cycle. Forodesine is orally absorbed (40%–50%), mean terminal half-life of 12 to 20 h. dGuo levels were elevated in all patients (0.8–2.8 μM, predose levels ≤ 0.004 μM). Forodesine was generally safe and well tolerated in this population. Safety data is available for first 7 patients. No serious adverse events possibly related to the drug occurred. The most common adverse event possibly drug related was nausea (2 patients). Of the 11 patients completing the treatment cycle, 9 began long-term treatment. Of these 9 patients, 4 have received >3 months of treatment, while 1 patient continues to receive for >8 months. Efficacy data are available for 8 patients: 2 with partial response, 3 with minor response, 2 with progressive disease, and 1 with stable disease (Table). Forodesine showed preliminary evidence of clinical activity in refractory CTCL patients. Additional clinical and pharmacodynamic data will be presented.

Clinical and Pharmacodynamic Activity of Oral Forodesine In CTCL Patients Patient Diagnosis Dose (mg/m2) Treatment Plasma dGuo (Cmax, M) μ Clinical Response PR = partial response; MR = minor response; SD = stable disease; PD = progressive disease; N/A = not available 2001 Sezary syndrome (IVA) 40 Completed 0.6 PR 3001 Mycosis fungoides (IVB) 40 Completed 1.7 PD 3002 Mycosis fungoides (IIB) 40 Completed 1.2 MR 3003 Mycosis fungoides (IB) 80 Completed 1.2 MR 3004 Sezary syndrome (IVA) 80 Completed 1.0 MR 3005 Sezary syndrome 80 Completed 1.6 PD 2002 IIA 80 Completed 1.1 PR 2003 III 80 Completed 1.7 N/A 3006 Mycosis fungoides/Sezary syndrome (IVB) 160 Completed 2.8 SD 4001 III 320 Completed 1.8 N/A 1001 Mycosis fungoides (IB) 320 Completed 1.3 N/A 3012 Mycosis fungoides/Sezary syndrome (IVA vs B) 320 Not Completed N/A Not evaluable
Lenalidomide and Novel Immunomodulatory Drugs (IMiDs®): A New Approach to the Regulation of Erythropoiesis and Hemoglobin Synthesis in Anemia.
Authors: de Parseval Laure Moutouh , Brady Helen , Verhelle Dominique , Corral Laura G. , Glezer Emilia , Morris Christopher L. , Chen Roger , Chan Kyle
Published: April 2020

Clinical trial results have demonstrated that lenalidomide (Revlimid®) reduces or even eliminates the need for red blood cell transfusions in some anemic myelodysplastic patients. We have examined whether lenalidomide and Actimid™, members of a new class of immunomodulatory drugs (IMiDs®), which are currently under evaluation for the treatment of hematological cancers could regulate erythropoiesis and hemoglobin synthesis. For this purpose, we used an in vitro culture model to differentiate human erythroid progenitors from bone marrow or peripheral blood CD34+ cells. We demonstrate that lenalidomide and AztimidTM modulate erythropoiesis and increase proliferation of immature erythroid cells. In addition to the regulation of erythroid differentiation, lenalidomide and ActimidTM are potent inducers of fetal hemoglobin. Unlike other inducers of fetal hemoglobin such as 5-aza-cytidine that are cytotoxic, IMiDs® promoted survival of erythroblast cultured with known cytotoxic drug. Gene expression profiling of erythroid differentiated cells showed that IMiDs® regulate specific erythroid transcription factors and genes that participate in hemoglobin synthesis, and genes invoved in cell cycle and cellular differentiation. Globin gene expression is controlled by IMiDs® during erythroid differentiation by inducing fetal hemoglobin synthesis. Our results support the hypothesis that IMiDs® restore effective erythropoiesis in myelodysplastic patients and protect erythroid cells from the cytotoxic effect of chemotherapeutic agents. In conclusion, IMiDs® may represent an interesting new therapy for cancer-related anemia and β-hemoglobinopathies.