Analysis of Mutant cMPL in Philadelphia Chromosome-Negative Myeloproliferative Disorders (Ph−MPDs) Using a Novel High-Sensitivity Assay.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2544-2544
Author(s):  
Scott J. Samuelson ◽  
Sabina Swierczek ◽  
Charles J. Parker ◽  
Kenneth Boucher ◽  
Jaroslav Jelinek ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Mutant Allele ◽  
Philadelphia Chromosome ◽  
High Sensitivity ◽  
Primary Myelofibrosis ◽  
Locked Nucleic Acid ◽  
Molecular Heterogeneity ◽  
Gain Of Function ◽  
Allelic Discrimination ◽  
Hematopoietic Stem

Abstract cMPL is a gene encoding for the thrombopoietin receptor that is essential for thrombopoiesis and contributes to pluripotent hematopoietic stem cell expansion. A gain of function cMPL mutation, MPLW515L, was identified in myeloid cells from patients with primary myelofibrosis (PMF). Subsequent studies identified a second gain of function mutation, MPLW515K, in PMF and essential thrombocytosis (ET). The prevalence of MPLW515L and MPLW515K mutations was 5% in PMF and 1% in ET. No mutant cMPL was detected in Polycythemia Vera (PV). The methods utilized in these assays were sensitive to mutant frequencies of >3–5%. We developed a rapid, sensitive, quantitative real time PCR assay based on a unique primer design wherein allelic discrimination was enhanced by the synergistic effect of a mismatch in the −1 position, and a locked nucleic acid nucleoside at the −2 position of the allele-specific primers. An assay of similar design can detect G1849T mutation of JAK2 in <0.1% mutant allele in peripheral blood granulocyte (Nussenzveig Exp Hematol20073:32). We hypothesized that a similar high sensitivity assay would increase detection of mutant cMPL in Ph−MPDs. We analyzed genomic DNA from peripheral blood granulocytes of 197 MPD patients and found that 10/197 (5.1%) carried one of the two cMPL mutations. Further, 5 of these patients were also JAK2V617F positive. cMPL mutations were detected in 1/78 (1.3%) PV patients, 3/56 (5.4%) ET patients, 4/49 (8.2%) PMF patients, and 2/11 (18%) MPD-Unspecified patients. W515L accounted for 9/10 cases, with W515K accounting for only 1. Of the ten positive samples, five (including the patient with PV) had ≤1% mutant alleles. To confirm the validity of our assay, we tested DNA from 96 normal controls. Neither W515L nor W515K was detected (p=0.03 compared to samples from the Ph−MPD patients). Additionally, when DNA from megakaryocytic colonies from a patient with 0.70% mutant alleles was analyzed, 12.5% of colonies were found to be heterozygous for cMPLW515L. These studies demonstrate the sensitivity and accuracy of our assay and show that cMPL activating mutations are more common in ET than previously reported. Mutant allele frequency appears greater in megakaryocytic cultures perhaps indicating a proliferative advantage for the cMPL-mutant clone. That mutant cMPL and JAK2V617F can be found in the same patient demonstrates the molecular heterogeneity of Ph−MPDs and emphasizes the need for prospective studies designed to determine the relationship between genotype and clinical phenotype. Scott J. Samuelson and Sabina Swierczek contributed equally to this project.

scholarly journals Plasma EDA Fibronectin in Primary Myelofibrosis Correlates with Bone Marrow Fibrosis and Predicts Splenomegaly

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1688-1688
Author(s):  
Alessandro Malara ◽  
Cristian Gruppi ◽  
Margherita Massa ◽  
Vittorio Rosti ◽  
Giovanni Barosi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Myeloproliferative Neoplasm ◽  
Primary Myelofibrosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Costal Margin ◽  
Cell Transplant ◽  
Healthy Controls ◽  
Hematopoietic Stem

Introduction: Primary myelofibrosis (PMF) is a Philadelphia chromosome negative myeloproliferative neoplasm with adverse prognosis characterized by bone marrow (BM) fibrosis and extramedullary hematopoiesis. Fibronectin (FN) is an extracellular matrix glycoprotein that plays vital roles during tissue repair and regeneration. It exists in different forms. Plasma FN is synthesized by hepatocytes and secreted into the blood plasma, where circulates at a concentration of 300-600 μg/ml in a soluble, compact form. Differently, cellular FN is synthesized by several cell types, such as fibroblasts, endothelial cells, chondrocytes and myocytes. The alternative splicing of EDA and EDB and more complex splicing of the V domain, during transcription of FN1 gene, allows different isoforms of FN to be expressed in a tissue-dependent and temporally regulated manner. Very low levels (1.3-3 μg/ml) of FN containing EDA and/or EDB are present in plasma. Although its function is not well understood, EDA containing FN (EDA-FN) is known to agonize Toll like receptor 4 (TLR4), resulting in NF-κβ-dependent cytokine release; to induce myofibroblast differentiation during wound healing; and to increase agonist-induced platelet aggregation and thrombus formation in vivo. We previously showed that EDA-FN levels are increased in plasma and BM biopsies of PMF patients. Mechanistically, BM EDA-FN sustains megakaryocyte proliferation through TLR4 binding and confer a pro-inflammatory phenotype to cell niches promoting fibrosis progression in Romiplostim-treated mice. In this work we measured the plasma levels of EDA-FN in 104 well characterized patients with PMF to determine whether elevated levels of EDA-FN predict the occurrence of disease-related events. Methods: Plasma circulating EDA FN was measured with an enzyme linked immunosorbent assay developed at the University of Pavia, by our group. We obtained plasma EDA-FN concentration values and health care data of persons with PMF from the data-base of the Centre for the Study of Myelofibrosis at the IRCCS Policlinico S. Matteo Foundation in Pavia. We sequentially excluded persons treated with disease-modifying drugs at any time before or on the date of base-cohort entry, and those who had been splenectomized or had received a stem cell transplant. We also excluded persons with acute inflammatory diseases, autoimmune diseases, other neoplasms, and severe liver or renal dysfunction. For this study we selected everyone giving written informed consent and the study was approved by the local Ethic Committee. Immunofluorescence was performed on spleen sections from PMF patients who underwent splenectomy either because of anemia or symptomatic splenomegaly, or both; and healthy controls that were splenectomized following traumatic lesion of the spleen. Data were analyzed using STATISTICA software. Results: A homozygous JAK2V617F genotype was the major determinant of elevated plasma EDA-FN. Elevated EDA-FN levels were associated with anemia, increased levels of high-sensitivity C-reactive protein, BM fibrosis and splanchnic vein thrombosis at diagnosis. We interpreted these associations as reflecting the role EDA-FN plays in tissue remodeling, inflammation and vascular injury. Interestingly, EDA-FN levels resulted also associated with spleen size, and elevated levels of EDA-FN at diagnosis predicted large splenomegaly (more than 10 cm from the left costal margin) outcome. The evidence that plasma EDA-FN levels were not associated with the CD34+ hematopoietic stem cells mobilization, drove us to hypothesize that EDA-FN could reflect spleen endothelial cell activation and/or neoangiogenesis. Immunofluorescence analysis of spleen specimens from PMF patients and healthy controls revealed that high levels of EDA-FN were present in pathological spleens in strong association with endothelial neoangiogenesis. Conclusions: Quantification of EDA-FN level in PMF strongly correlates with BM fibrosis and may be the first marker of an altered spleen microvasculature that contributes to splenomegaly. Understanding the role of this FN isoform in PMF would be useful for testing new mechanisms of disease progression and new hypotheses about the treatment of splenomegaly in PMF. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinical Insights into the Origins of Thrombosis in Myeloproliferative Neoplasms

Blood ◽  
2020 ◽  
Author(s):  
Alison R. Moliterno ◽  
Yelena Z Ginzburg ◽  
Ronald Hoffman
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Primary Myelofibrosis ◽  
Vascular Events ◽  
Hematopoietic Stem ◽  
Activating Mutations ◽  
Clinical Burden ◽  
Significant Burden ◽  
Clinical Associations ◽  
Specific Symptoms

The Philadelphia chromosome negative myeloproliferative neoplasms (MPNs), polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are hematopoietic stem cell disorders that are defined by activating mutations in signal transduction pathways and are characterized clinically by the overproduction of platelets, red blood cells and neutrophils, significant burden of disease-specific symptoms, and high rates of vascular events. The focus of this review is to critically re-evaluate the clinical burden of thrombosis in the MPNs, to review the clinical associations between clonal hematopoiesis, JAK2V617F burden, inflammation and thrombosis, and to provide insights into novel primary and secondary thrombosis prevention strategies.

Erythropoietin Signaling Inhibits the Short-Term Marrow Reconstitution.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3028-3028
Author(s):  
Ko-Tung Chang ◽  
Yves D. Pastore ◽  
Roberto H. Nussenzveig ◽  
Josef T. Prchal
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Relative Proportion ◽  
Wild Type Mouse ◽  
Erythropoietin Receptor ◽  
Wild Type ◽  
Short Term ◽  
Gain Of Function ◽  
Hematopoietic Stem

Abstract Murine hematopoietic stem cells (HSCs) transfected with gain-of-function human erythropoietin receptor (EPOR) transgene were reported to have a competitive advantage over wild type mouse HSCs in a bone marrow transplantation (BMT) model (Kirby, Blood; 95(12):3710, 2000). However, EpoRs may not be normally expressed in early progenitor/stem cells, moreover, whether the Epo/EpoR signaling plays a role on homing and repopulating of hematopoietic progenitor/stem cells is also unknown. Our lab has previously created a mouse model harboring either a knocked-in human wild type (wthEPOR) hypomorphic gene or a mutant human gain-of-function EPOR (mthEPOR) gene into the mouse EPOR locus (Divoky, PNAS; 98(3):986, 2001). The wthEPOR mice are anemic, while mthEpoR mice are polycythemic. We tested the possible advantage of mthEPOR HSCs in the competitive bone marrow (BM) transplantation assay using C57/Bl6 congenic mice. BM from wthEPOR (CD45.1/45.2 heterozygous or CD45.1) and mthEPOR (CD45.2) mice were co-transplanted (1:1 ratio) into lethally irradiated (137Cs >11Gy split) normal recipients (CD45.1 or CD45.1/45.2). Unexpectedly, in three independent groups the peripheral blood chimerism derived from wthEPOR-bearing cells significantly out-competed the mthEPOR-bearing cells two weeks after BMT (52% vs. 40%, 52% vs. 45% and 50% vs. 42%, n=11, 8 and 6, p<0.01). Furthermore, administration of exogenous Epo (10U/day/mouse) further impaired peripheral blood engraftment (including Ter119+ erythroid lineages) of mthEPOR cells (45% vs. 39%, n=8, p<0.05) though the relative proportion of erythroid cells within the engraftment was increased. Thus, an enhanced Epo/EpoR signaling interferes with the short-term repopulation of hematopoietic progenitors resulting in a decreased engraftment, possibly by interfering with homing. A possible long-term advantage of mthEPOR HSCs is being evaluated in ongoing studies; however, these short term data suggest that Epo administration to BM transplant patients may impede engraftment. Figure Figure

scholarly journals Targeting Abnormal Hematopoietic Stem Cells in Chronic Myeloid Leukemia and Philadelphia Chromosome-Negative Classical Myeloproliferative Neoplasms

2021 ◽  
Vol 22 (2) ◽  
pp. 659
Author(s):  
Yammy Yung ◽  
Emily Lee ◽  
Hiu-Tung Chu ◽  
Pui-Kwan Yip ◽  
Harinder Gill
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Primary Myelofibrosis ◽  
Hematopoietic Stem

Myeloproliferative neoplasms (MPNs) are unique hematopoietic stem cell disorders sharing mutations that constitutively activate the signal-transduction pathways involved in haematopoiesis. They are characterized by stem cell-derived clonal myeloproliferation. The key MPNs comprise chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). CML is defined by the presence of the Philadelphia (Ph) chromosome and BCR-ABL1 fusion gene. Despite effective cytoreductive agents and targeted therapy, complete CML/MPN stem cell eradication is rarely achieved. In this review article, we discuss the novel agents and combination therapy that can potentially abnormal hematopoietic stem cells in CML and MPNs and the CML/MPN stem cell-sustaining bone marrow microenvironment.

scholarly journals A Practical Guide for Using Myelofibrosis Prognostic Models in the Clinic

2020 ◽  
Vol 18 (9) ◽  
pp. 1271-1278
Author(s):  
Joan How ◽  
Gabriela S. Hobbs
Keyword(s):  
Risk Stratification ◽  
Scoring System ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Primary Myelofibrosis ◽  
Prognostic Models ◽  
International Prognostic Scoring System ◽  
Hematopoietic Stem ◽  
Clinical Presentations ◽  
Version 2.0

Primary myelofibrosis (PMF) has the least favorable prognosis of the Philadelphia chromosome–negative myeloproliferative neoplasms, which also include essential thrombocythemia (ET) and polycythemia vera (PV). However, clinical presentations and outcomes of PMF vary widely, with median overall survival ranging from years to decades. Given the heterogeneity of PMF, there has been considerable effort to develop discriminatory prognostic models to help with management decisions, particularly for the consideration of hematopoietic stem cell transplantation in patients at higher risk. Although earlier models incorporated only clinical features in risk stratification, contemporary models increasingly use molecular and cytogenetic features, leading to more comprehensive prognostication. This article reviews the most widely adopted prognostic models used for PMF, including the International Prognostic Scoring System (IPSS), dynamic IPSS (DIPSS)/DIPSS+, mutation-enhanced IPSS for transplant-age patients (MIPSS70)/MIPSS70+/MIPSS70+ version 2.0, genetically inspired prognostic scoring system, and Myelofibrosis Secondary to PV and ET-Prognostic Model in patients with post-ET/PV myelofibrosis. We also discuss newly emerging prognostic models and provide a practical approach to risk stratification in patients with PMF and post-ET/PV myelofibrosis.

High-activity samarium-153-EDTMP therapy followed by autologous peripheral blood stem cell support in unresectable osteosarcoma

2001 ◽  
Vol 40 (06) ◽  
pp. 215-220 ◽  
Author(s):  
S. Bielack ◽  
S. Flege ◽  
J. Eckardt ◽  
J. Sciuk ◽  
H. Jürgens ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
High Activity ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Blood Stem Cell ◽  
High Dose ◽  
External Radiotherapy ◽  
Primary Tumor Site ◽  
Hematopoietic Stem

Summary Purpose: Despite highly efficacious chemotherapy, patients with osteosarcomas still have a poor prognosis if adequate surgical control cannot be obtained. These patients may benefit from therapy with radiolabeled phosphonates. Patients and Methods: Six patients (three male, three female; seven to 41 years) with unresectable primary osteosarcoma (n = 3) or unresectable recurrent sites of osteosarcomas (n = 3) were treated with high-activity of Sm-153-EDTMP (150 MBq/kg BW). In all patients autologous peripheral blood stem cells had been collected before Sm-153-EDTMP therapy. Results: No immediate adverse reactions were observed in the patients. In one patient bone pain increased during the first 48 hrs after therapy. Three patients received pain relief. Autologous peripheral blood stem cell reinfusion was performed on day +12 to +27 in all patients to overcome potentially irreversible damage to the hematopoietic stem cells. In three patient external radiotherapy of the primary tumor site was performed after Sm-153-EDTMP therapy and in two of them polychemotherapy was continued. Thirty-six months later one of these patients is still free of progression. Two further patients are still alive. However, they have developed new metastases. The three patients who had no accompanying external radiotherapy, all died of disease progression five to 20 months after therapy. Conclusion: These preliminary results show that high-dose Sm-153-EDTMP therapy is feasible and warrants further evaluation of efficacy. The combination with external radiation and polychemotherapy seems to be most promising. Although osteosarcoma is believed to be relatively radioresistant, the total focal dose achieved may delay local progression or even achieve permanent local tumor control in patients with surgically inaccessible primary or relapsing tumors.

Endothelial and Circulating Progenitor Cells in Hematological Diseases and Allogeneic Hematopoietic Stem Cell Transplantation

2018 ◽  
Vol 25 (35) ◽  
pp. 4535-4544 ◽  
Author(s):  
Annalisa Ruggeri ◽  
Annalisa Paviglianiti ◽  
Fernanda Volt ◽  
Chantal Kenzey ◽  
Hanadi Rafii ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Circulating Endothelial Cells ◽  
Hematopoietic Stem ◽  
Hematological Diseases

Background: Circulating endothelial cells (CECs), originated form endothelial progenitors (EPCs) are mature cells not associated with vessel walls and detached from the endothelium. Normally, they are present in insignificant amounts in the peripheral blood of healthy individuals. On the other hand, elevated CECs and EPCs levels have been reported in the peripheral blood of patients with different types of cancers and other diseases. Objective: This review aims to provide an overview on the characterization of CECs and EPCs, to describe isolation methods and to identify the potential role of these cells in hematological diseases and hematopoietic stem cell transplantation. Methods: We performed a detailed search of peer-reviewed literature using keywords related to CECs, EPCs, allogeneic hematopoietic stem cell transplantation, and hematological diseases (hemoglobinopathies, hodgkin and non-hodgkin lymphoma, acute leukemia, myeloproliferative syndromes, chronic lymphocytic leukemia). Results: CECs and EPCs are potential biomarkers for several clinical conditions involving endothelial turnover and remodeling, such as in hematological diseases. These cells may be involved in disease progression and in the neoplastic process. Moreover, CECs and EPCs are probably involved in endothelial damage which is a marker of several complications following allogeneic hematopoietic stem cell transplantation. Conclusion: This review provides information about the role of CECs and EPCs in hematological malignancies and shows their implication in predicting disease activity as well as improving HSCT outcomes.

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