Calreticulin mutations Are Present in Polyclonal As Well As Clonal ET

First and second authors contributed equally

During female embryogenesis, most of the genes in either the maternal or paternal X-chromosome are randomly inactivated in each cell, a process that remains remarkably constant in their progeny. X-chromosome inactivation has been used to define clonality in myeloproliferative neoplasms (MPNs) such polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET). One such method to determine clonality uses a quantitative, transcriptional clonality assay based on conservative exonic polymorphisms in five X-chromosome genes (MPP1, FHL1, IDS, BTK, and G6PD). Females who are heterozygous for any of these polymorphisms are considered “informative” and can be studied for clonality by interrogating their platelets’ and granulocytes’ RNA allelic usage ratio.

JAK2 mutations occur in >95% of PV and 50-60% of ET and PMF; cMPL mutations are found in another 5-10% of ET and MF. Somatic calreticulin (CALR) mutations have been identified in a majority of patients with ET and MF who lack JAK2 and cMPL mutations. CALR mutations are reported to be associated with a more favorable prognosis and are believed to be acquired early in the disease course. More than 30 CALR mutations have been described, but type 1 (52-bp deletion; c.1092_1143del) and type 2 (5-bp insertion; c.1154_1155insTTGTC) mutations are the most frequent.

We analyzed 61 females informative for a transcriptional clonality assay and 44 males with unexplained thrombocytosis or marrow fibrosis and no detectable JAK2 or cMPL mutations for CALR mutations in their granulocytes. With the exception of an absence of a clonal marker, these patients met WHO criteria for ET or PMF. A CALR mutation (20 type 1 and 17 type 2) was present in 37 of these 105 patients (22 females and 15 males). One of the CALR mutated females had a paternal grandmother with JAK2^V617F –positive PV, confirming a previous report that, in familial clustering of MPNs, affected individuals may carry different disease-defining somatic mutations. In those CALR positive patients who had available T cells, no detectable CALR mutations were found in their T cells. In one of these subjects, CD34+ cells were available and had a similar mutation level as in the granulocytes.

Of the 22 females with a CALR mutation, 19 had clonal hematopoiesis, but 3 had polyclonal hematopoiesis; all 3 had previously unexplained thrombocytosis. None of these patients had any prior treatment for thrombocytosis. Clonal hematopoiesis was present in 26 of the 39 females without a CALR mutation. All female patients with myelofibrosis had clonal hematopoiesis, regardless of CALR mutation status. In contrast to the polyclonal hematopoiesis seen in some CALR positive ET patients, 166 informative PV and JAK2^V617F-positive ET or PMF females all had clonal hematopoiesis.

We report that CALR mutations are associated with polyclonal hematopoiesis in some ET patients. This finding differs from JAK2^V617F-positive ET and PMF and PV females, where clonal hematopoiesis was always seen. This indicates that CALR mutated clones have a weaker suppressive effect on residual normal hematopoietic stem cells than JAK2 mutated clones and may contribute to the possibly more benign course of CALR mutated ET. The CALR mutation was not detected in T cells, which also differs from JAK2^V617F mutated MPNs, where a small level of the JAK2 mutation is often detected in T cells. Similar to other reports, we found a lower prevalence of the CALR mutation in JAK2 or cMPL non-mutated ET and PMF than initially described.