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.

Endogenous megakaryocytic colonies underline association between megakaryocytes and calreticulin mutations in essential thrombocythemia

“…In the same way, it is consistent with the almost complete absence of CALR mutations in polycythemia vera (PV) 13 (despite the fact that rare PV cases with CALR mutations have recently been reported 14 ), the higher platelet counts in CALR patients, 11 and the preferential expression of CALR-mutated protein in megakaryocytes. 15 Our results also confirm that EEC is a characteristic feature of JAK2V617F mutation. No EEC was observed in MPL-mutated patients and only one patient carrying CALR mutation was EEC positive.…”

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis

“…28 However, the diverse mutations likely involve additional abnormalities in other metabolic pathways; for instance, mutant calreticulin might have peculiar effects on megakaryocyte biology. 29 As previously observed in ET, 16,22 JAK2 (V617F) appears to be highly thrombophilic also in patients with PMF, indicating that this mutation likely causes thrombosis through multiple mechanisms, including activation of platelets and granulocytes. 30,31 On the contrary, despite the fact that calreticulin mutations involve high platelet counts also in PMF, the risk of thrombosis of these patients is relatively low, at least compared with that of JAK2-mutant patients.…”

Molecular determinants of pathogenesis and clinical phenotype in myeloproliferative neoplasms

“…43 Differences in cytosolic calcium mobilization have been reported with the 52 base pair deletion, 44 suggesting that this may be one mechanism by which mutant CALR exerts its effect, and expression of the mutant protein does appear to be particularly restricted to megakaryocytes on immunohistochemical evaluation of bone marrow specimens. 45 More recently, it has been shown that CALR mutations can impart TPO-independence in both cell lines 46,47 and retroviral mouse models, 48,49 in a MPL-and JAK2-dependent manner, mimicking the effect of activating MPL mutations. This has been shown to be mediated by direct binding of MPL by the N domain of CALR, a phenomenon that uniquely occurs in the presence of the mutated C-terminus, 48,49 leading to autocrine activation of MPL, JAK2 dimerization and downstream STAT5 and ERK phosphorylation.…”