title: Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value.
abstract: Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: A M
authors.initials: AM
authors.lastname: Vannucchi
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Rotunno
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Bartalucci
authors.affiliation: Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Raugei
authors.affiliation: Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Carrai
authors.affiliation: Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Balliu
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Mannarelli
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Pacilli
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Calabresi
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Fjerza
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Pieri
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Bosi
authors.affiliation: Centre for Regenerative Medicine 'Stefano Ferrari', Department of Life Science, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Manfredini
authors.affiliation: 1] Department of Experimental and Clinical Medicine, Laboratorio Congiunto MMPC, University of Florence, Azienda Ospedaliera Universitaria Careggi, Florence, Italy [2] Hematology Unit, Azienda Ospedaliera Universitaria Careggi, Florence, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Guglielmelli
mesh_terms: D037282:Calreticulin / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D019070:Cell Lineage; D006801:Humans; D007150:Immunohistochemistry; D053614:Janus Kinase 2 / Q000235:genetics; D009154:Mutation; D009196:Myeloproliferative Disorders / Q000175:diagnosis / Q000209:etiology / Q000235:genetics*; D053628:Receptors, Thrombopoietin / Q000235:genetics
chemical_list: D037282:Calreticulin; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; C507924:JAK2 protein, human; D053614:Janus Kinase 2