Four seminal studies published in 2005 showed that the classic Philadelphia-chromosome negative chronic myeloproliferative neoplasms (MPNs) are characterized by a recurrent V617F point mutation in exon 14 of JAK2 . This somatic mutation is found in the vast majority of patients with polycythemia vera (PV) and 60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF). Virtually all patients with post-polycythemia vera (PPV-) and greater than 60% of patients with post-essential thrombocythemia (PET-) myelofibrosis are JAK2V617F mutated. Mitotic recombination of chromosome 9p is frequent in MPNs and leads to the copy-neutral loss of heterozygosity (CN-LOH) for JAK2 that results in homozygosity for the mutated allele. In the minority of PV patients that do not carry JAK2V617F, other JAK2 mutations have been found in roughly half of cases, such as insertions or deletions in exon 12, and overall 99% of PV patients carry a mutation in JAK2. Amongst the 40% of patients with ET and MF that are JAK2V617F negative 3–5% carry mutations at codon 515 of the gene encoding the thrombopoietin receptor (MPL) and CN-LOH of chromosome 1p underlies the transition from heterozygous to homozygous MPLW515 mutations. Both JAK2 and MPL mutations are gain-of-function and promote the over-activation of STAT signaling largely mediated by abnormal and sustained phosphorylation of JAK2. Targeting activated JAK2 with JAK inhibitors is changing the therapeutic landscape of myelofibrosis, and the JAK2 and JAK1 inhibitor ruxolitinib has been approved as the first-in-class drug for the treatment of myelofibrosis; in addition, a number of phase II–III trials with different JAK2 inhibitors are ongoing in PV.
The close association of JAK2V617F mutation with MPNs led to a revision of the WHO diagnostic criteria in 2008, where JAK2V617F and others closely related mutations, such as JAK2 exon 12 and MPLW515 mutations, were elected to represent new major diagnostic criteria for the three classic MPNs . Subsequent studies highlighted an unexpected complexity of the mutational landscape of MPNs with co-mutation of several other genes identified in up to 25–30% of the patients . However, these mutations are not specific to MPNs as they are present in patients with other myeloid disorders such as chronic myelomonocytic leukemia, myelodysplastic syndrome (MDS), and acute leukemia. The spectrum of genes mutated involves epigenetic regulation (EZH2, ASXL1, TET2, DNMT3A, IDH1, and IDH2), the spliceosome (SF3B1, SRSF2, U2AF1) and rarer targets that disrupt JAK-STAT signalling (SH2B3/LNK). As a result of their distribution across myeloid malignancies they do not represent suitable diagnostic markers for classic MPNs but may find application for prognostic assessment, at least in PMF. However, the molecular complexity of MPNs is still far from being fully appreciated and other nonrecurrent mutations are expected to be discovered by application of deep sequencing approaches.
Calreticulin Mutations in MPN: Filling the Gap
The “black box” that comprised 40% of ET and PMF patients lacking a recurrent genetic marker of disease has now largely been resolved by the discovery of mutations in the gene Calreticulin (CALR) reported independently by the research groups of Tony Green/Peter Campbell  and Robert Kralovics . Both studies employed exome sequencing of matched tumor and germline DNA, followed by targeted resequencing in larger cohorts, to identify recurrent mutations in CALR in 60–88% of patients with ET and PMF who were negative for JAK2 and MPL mutations. Therefore, CALR mutations represent an exclusion criterion of PV, that is JAK2 mutated by definition. Large screening of other myeloid disorders revealed infrequent CALR mutations in 8% of MDS, including three of 24 patients with refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T), as well as infrequent cases of rarer myeloid disorders such as atypical chronic myeloid leukemia and chronic myelomonocytic leukemia [2, 3]. CALR mutations were not found in healthy controls, lymphoid neoplasia, acute leukemias or solid tumors, indicating a specificity for ET and PMF. Mutations were all insertions or deletions in CALR exon 9 and the commonest mutations accounting for 80–90% of mutated cases were either a 52-bp deletion (CALRdel52/Type I; c.1092_1143del; L367fs*46; 45-53% of all cases) or a 5-bp insertion (CALRins5/Type II; c.1154_1155insTTGTC; K385fs*47; 32–41% of all cases) (Fig. 1A). CALRdel52 was more frequent in PMF than ET. Various other infrequent insertions and deletions in exon 9 account for up to 15% of CALR mutations (Fig. 1A). Remarkably, all mutations cause a frameshift to a unique alternative reading frame resulting in a novel calreticulin protein C-terminal peptide sequence characterized by the acquisition of a minimal 36 amino acid stretch in place of 27 amino acids that are lost from the normal sequence (Fig. 1B). The C-terminus of the mutant protein contains several positively charged amino acids whilst the equivalent region of the wildtype protein is largely negatively charged. Effective transcription of the mutant protein was confirmed by transient expression in human embryonic kidney cells. Through clonal analysis, CALR mutations were shown to occur in a multipotent progenitor capable of both erythroid and myeloid differentiation and well as in highly enriched CD34+/CD90+/CD38-/CD45RA-/lin- hematopoietic stem cells . Finally, by using hierarchical analysis of individual hematopoietic clones the early acquisition of CALR mutations was demonstrated, consistent with it being an initiating event in the cases characterized [2, 3]. Single nucleotide variants have been reported in rare patients (eg. CALRE379D and CALRE398D) [3, 4], however, whether they are true somatically acquired mutations and what significance they carry remain unclear at present.
Calreticulin: A Multifunctional Protein
Calreticulin (CALR) is a developmentally highly conserved, multicompartmental and multifunctional protein best known for its role as a Ca2+ binding chaperone in the endoplasmic reticulum (ER) lumen . The CALR-1 gene (the product of calreticulin-2 has only been detected in the testis) is located on human chromosome 19 and mouse chromosome 8, spanning 4.6 and 3.6 kb, respectively, and contains nine exons with a highly conserved sequence (96% amino acid identity) between human and mouse. Mature CALR is a 46-kDa protein that consists of three structurally and functionally distinct domains (Fig. 1B). The globular N-terminal domain (residues 1-180) is lectin binding and consists of a signal sequence for targeting to the ER. The middle proline-rich or P-domain (residues 181–290) contains high affinity, low capacity, binding sites for Ca2+, and the highly acidic C-terminal domain (residues 291–400) contains a number of high capacity, low-affinity Ca2+ binding sites. The acidic C-terminal domain is involved in cellular calcium homeostasis—cells that are deficient in CALR have reduced calcium storage capacity in the ER and overexpression of CALR augments ER calcium retention. The C-domain terminates in a KDEL sequence; KDEL receptors function in the retrieval of KDEL-containing proteins from the cis-Golgi back to the ER. The C-terminus of mutant CALR lacks KDEL which raises the possibility of cellular mislocalization, however, preliminary evidence from localization studies of exogenous overexpression of mutant CALR in target cells, as well as localization studies of myeloid cells from CALR mutated patients has shown that mutant CALR largely retains its localization within the ER [2, 3]. This supports the presence of KDEL independent mechanisms for ER retention of calreticulin. Evidence that ER retention may also be determined by the acidic region of CALR (residues 351–359) comes from studies of a calreticulin construct lacking this region where the protein is secreted despite the presence of KDEL. This region may also be involved in the translocation of CALR to the cytosol in a nondegrading process .
Within the lumen of ER, and in concert with other chaperone proteins, CALR plays an essential role in ensuring proper protein and glycoprotein folding. CALR has structural homology with calnexin and both function in concert in the so-called “calnexin/calreticulin cycle”, a N-glycan-dependent quality control process that ensures correct glycoprotein folding and/or degradation and prevents protein aggregation . Amongst other roles, CALR is implicated in the assembly and cell surface expression of major histocompatibility complex (MHC) class I molecules. Disruption of CALR is embryonically lethal with the embryos showing markedly decreased ventricular wall thickness and intertrabecular recesses in the ventricular walls. This appears to be due to impaired myofibrillogenesis as a result of abnormal ER calcium availability. CALR-mediated regulation of calcium homeostasis may influence multiple additional cellular functions including integrin-mediated signaling.
In addition to canonical ER related functions, CALR is reported to be found in the cytosol, nucleus, at the cell surface as well as extracellularly, where it accomplishes multiple functions as a critical mediator of physiological and pathological processes such as the immune response, proliferation and apoptosis, cell phagocytosis, wound healing, and fibrosis . Of interest, upregulation of CALR on the cell surface of leukemia blasts and dysplastic progenitors of MDS has been reported to be involved in an “eat me” signal thereby affecting the phagocytosis of neoplastic cells. However, in CALR mutated MPNs, no significant increase in CALR was found at the cell surface of tumor cells . Interestingly, overexpression of CALRdel52 in interleukin-3-dependent murine Ba/F3 cells led to cytokine independent growth which could be inhibited by the JAK2 inhibitor SAR302503, suggesting the involvement of JAK/STAT signalling that was confirmed by finding increased STAT5 phosphorylation in mutant compared with wild-type cells .
Calreticulin Mutations in MPN: Relevance for Diagnosis and Prognosis
The selective association of CALR mutations with JAK2 and MPL unmutated ET and PMF qualifies CALR genotyping as a first line laboratory test in patients with a suspicion of a MPN who lack the JAK2V617F mutation. Only one patient with concurrent JAK2 and CALR mutations has been reported to date . Polymerase-chain-reaction (PCR) of DNA followed by Sanger sequencing or fragment size analysis have been used in the studies reported thus far and have a sensitivity of 5–10%. It is conceivable that more sensitive tests could be devised in the near future . Whilst the two commonest CALR mutations (CALRdel52 and CALRins5) could theoretically be identified by a sensitive mutant allele-specific PCR method, such an assay would not identify about 15% of CALR mutated patients that carry infrequent variants, thus limiting their diagnostic utility. Moreover, provided such assays allow for more accurate estimation of allele burden than current methods, it is still unclear whether such quantification has diagnostic, phenotypic or prognostic relevance. Nevertheless, it has been reported that the median allele burden for the two commonest CALR mutations was 32% in ET patients at diagnosis compared with 50% in those with PET-MF suggesting that disease progression may be associated with accumulation of mutated CALR alleles .
In the study by Klampfl et al. three of 289 CALR mutated patients were homozygous for the mutation (all had CALRins5) because of acquired chromosome 19p UPD  and similar frequencies of acquired 19p UPD for both CALR insertions and deletions were found by Nangalia et al. (unpublished data). Mutated CALR homozygosity seems to be a rare event in MPNs, in contrast with mutated JAK2 UPD that is associated with distinct phenotypic traits and more frequent evolution to PPV/PET-MF.
The clinical phenotype and prognosis of JAK2 unmutated ET and MF has been extensively characterized previously—JAK2 unmutated patients are younger, with higher platelet counts, reduced rates of thrombosis and possibly better survival. Much of what has been known about this cohort can now be specifically attributed to the patients with CALR mutations. The prognostic impact of CALR mutations was first suggested by Klampfl et al. who found, in a retrospective analysis, that patients with PMF harboring CALR mutations had significantly better overall survival compared with either JAK2V617F and MPLW515 mutated subjects, with a similar trend reported in ET . CALR mutated ET patients were also found to be at lower risk of thrombosis. Nangalia et al. reported that CALR mutated ET patients presented with significantly higher platelet counts and lower hemoglobin than JAK2 mutated counterparts . Findings in ET patients have subsequently been validated in two independent series [9, 10]. In one study that included 576 patients with 2008 WHO-defined ET, 64.1% of whom were JAK2V617F mutated, 4.3% MPLW515 mutated, 15.5% CALR mutated (corresponding to 48.9% of JAK2 and MPL unmutated) and 16.1% triple negative (JAK2, MPL and CALR unmutated) patients , CALR mutated patients were predominantly male with lower leukocyte and hemoglobin levels and higher platelet counts than JAK2V617F mutated counterparts. Of importance, these subjects had longer thrombosis-free survival, and a thrombotic rate that was about one-third lower than JAK2V617F and MPLW515 mutated and similar to triple negative patients. Preliminary evidence, although not in a multivariate analysis, also indicated that CALR mutated ET patients had a lower incidence of transformation to either PV or acute leukemia compared with JAK2V617F mutated , whereas the incidence of PET-MF was similar to JAK2 and MPL mutated patients [9, 10]. In summary, current evidence suggests that ET patients with CALR mutations are at lower risk of thrombosis and hematologic progression than those harboring the JAK2V617F mutations and this may have implications for risk stratification and management. With regards to myelofibrosis, Tefferi et al. studied 254 PMF patients, and found that CALR mutations were associated with younger age, higher platelet count and lower DIPSS-plus score . In a multivariate analysis, CALR mutations, independent of both DIPSS-plus risk and ASXL1 mutation status, predicted for better overall survival when compared to both JAK2V617F mutated or triple negative patients; the latter category had a worse outcome and a shorter leukemia-free survival. All clinical correlates thus far, including those of overall survival, have been retrospective analyses and data from the prospective setting are awaited.
In summary, the high incidence and selective association of newly described CALR mutations with ET and PMF qualifies CALR genotyping as a first line laboratory test in patients with a suspicion of MPN. Its importance for prognosis and, above all, the mechanisms by which it induces a myeloproliferative phenotype remain to be addressed in future studies. Exciting times for research in the field of MPNs.
This study was supported by a special grant from Associazione Italiana per la Ricerca sul Cancro-“AIRC 5 per Mille”- to AGIMM, “AIRC-Gruppo Italiano Malattie Mieloproliferative” (#1005); for a description of the AGIMM project and list of investigators, see at www.progettoagimm.it). Partially supported by Ministero della Università e Ricerca (MIUR; FIRB project #RBAP11CZLK and PRIN 2010NYKNS7). The Green lab is supported by Leukemia and Lymphoma Research, Cancer Research UK, the Kay Kendall Leukaemia Fund, the NIHR Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre, and the Leukemia & Lymphoma Society of America. JN is supported by a clinical research fellowship from the Kay Kendal Leukaemia Fund.