With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (PH-) myeloproliferative neoplasms (MPNs) in 2005, important advances in the diagnosis of MPNs have been made in their pathogenesis involving the JAK/STAT pathway and have been used as a new targeted therapy. Nevertheless, the presence of mutations in approximately one-third of patients with non-JAK2 or MPL mutations in primary platelets (ET) as well as primary myelofibrosis (PMF) remained unknown at that time. It was not until late 2013 that two studies using whole-exome sequencing methods identified a recurrent mutation in the gene encoding the calcium network protein (CALR). This mutation was present in most ET and PMF patients with non-JAK2 or MPL mutations, but not in patients with true erythroblastosis (PV).Somatic 52-bp deletions (type 1 mutations) and recurrent 5-bp insertions (type 2 mutations) in exon 9 of the CALR gene (the last exon encoding the C-terminal amino acid of the protein calreticulin) were detected and were always found to produce code-shifting mutations. All detected mutants in calreticulin share a novel amino acid sequence at the C-terminus. calr mutations are acquired early in the clonal phase of the disease and lead to JAK/stat activation. calr mutations are the second most common mutation in patients with MPNs after the JAK2V617F mutation, and their detection has significantly improved the diagnostic approach for ET and PMF. This article focuses on the characteristics of CALR mutations and their impact in the diagnosis, clinical and pathogenesis of the disease. Classical Philadelphia chromosome-negative (PH-) myeloproliferative neoplasms (MPNs) include true erythroblastosis (PV), primary thrombocythemia (ET) and primary myelofibrosis (PMF). In 2005, significant progress was made with the discovery of the JAK2V617F mutation, a JAK/STAT pathway activation caused by MPNs The JAK2V617F mutation appears in 95% of PV, 50% of ET and 60% of PMF patients. Two other mutations (JAK2 exon 12 and mutations in the gene for the thrombopoietin receptor, myeloproliferative leukemia, MPL) were subsequently found to also directly affect this pathway. Mutations in JAK2 exon 12 were present in 2% of PV patients; for MPL mutations were present in non-JAK2 mutations in 5% of ET and 10% of PMF patients. Looking at somatic mutations in MPNs, other genes such as TET2, ASXL1, DNMT3A and EZH2 were found in patients with JAK2 and MPL mutations and their presence was non-specific. Until recently, mutations in approximately one-third of non-JAK2 or MPL mutations in ET and PMF that enable motility mutations were not recognized.In 2013, a study by Klampfl and Nangalia identified the occurrence of recurrent mutations in genes encoding calreticulin (CALR) in patients with non-JAK2 or MPL mutations, and their role in the development of MPNs. Further studies have looked at the role of CALR mutations in the pathogenesis of MPNs and have reported and the role of clinical relevance of these mutations. Discovery of CALR mutations Klampfl et al. used exon sequencing to detect DNA from peripheral blood granulocytes (tumor samples) and DNA from matched CD3+ T lymphocytes (control samples) in six JAK2 and MPL-negative PMFs. Somatic mutations of 2-12 were identified in each patient, and only the CALR (located on chromosome 19p13.2, containing 9 exons and encoding calreticulin) gene was reproducible. Two cases had somatic deletions and four patients had a reproducible 5-bp insertion in exon 9 of CALR (encoding the C-terminal amino acid of the protein). PCR detected insertional and deletional mutations in exon 9 of CALR in 896 patients with MPNs. no CALR mutations were detected in any of the PV patients. 25% of ET and 35% of PMF patients had detectable CALR mutations. All patients with CALR mutations were non-JAK2 and MPL mutated (CALR mutations are mutually exclusive with JAK2 and MPL mutations). Less than 10% of patients with ET or PMF were negative for JAK2, MPL, and CALR mutations. Several studies analyzing CALR mutations in MPN have been reported, with 10-23% of ET patients reporting triple negatives (negative for JAK2, MPL and CALR mutations). Nangalia et al. examined 151 MPN samples by exome sequencing. Among 31 ET or PMFs with non-JAK2 or MPL mutations 26 were positive for CALR somatic mutations. Mutations in JAK2V617F and CALR exon 9 are thought to be mutually exclusive. However three cases (two ET and one PMF) with double mutations (JAK2V617F and CALR mutations) have been reported. The true frequency, pathogenicity and clinical significance of the double mutations are not known. Patients with other myeloid diseases (acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome (MDS), chronic granulocytic leukemia, refractory anemia with cyclic iron granulocytosis with marked thrombocytosis (RARS-T)) were tested separately for CALR exon 9, and three RARS-T patients were screened for CALR mutations. 8% of MDS patients had CALR mutations were present in 1 of 524 healthy volunteers. Thirty-six types of somatic CALR mutations (insertions and deletions) were detected that resulted in code shifting to replace the reading frame. type 1 (52 bp deletion) and type 2 (5-bp insertion) CALR mutations accounted for 53.0% and 31.7%, respectively. type 1 mutations were more frequent in PMF than ET. All other mutation types were observed at much lower frequencies. Calreticulin Calreticulin is a multifunctional protein. In the endoplasmic reticulum, the proteins ensure proper folding of newly synthesized glycoproteins and regulate calcium dynamic homeostasis. Calcium reticulin is also found in intracellular, cell surface and extracellular compartments where it has been implicated in many biological processes including proliferation, apoptosis and immunogenic cell death. Calreticulin has three major structural and functional domains: an N-terminal lectin-binding domain, a proline-rich P domain, and an acidic C-terminal domain containing multiple calcium-binding sites. Calreticulin contains an endoplasmic reticulum lagging motif at the C-terminus (KDEL motif). This KDEL motif appears in some endoplasmic reticulum proteins, allowing these proteins to return from the Golgi apparatus to the endoplasmic reticulum. CALR protein mutants with C-terminal alterations All detected CALR protein mutants share a new amino acid sequence at the C-terminus. The non-mutant CALR C-terminus is predominantly negatively charged, while the CALR mutant C-terminus contains many positively charged amino acids. type 1 mutations eliminate almost all negatively charged amino acids, while type 2 mutations retain about half of the negatively charged amino acids. Since the negatively charged C-terminal structural domain of calcium reticuloproteins is a low-affinity, high-volume, calcium-binding structural domain, the function of calcium-binding protein mutants may be diminished. In addition, the KDEL motif at the C-terminus is lost in all mutants. Thus, mutant calreticulin can have altered subcellular localization. Investigating whether the clonal cell history of patients with CALR mutations is early or late, analysis of hematopoietic precursor cell clones obtained from MPN patients revealed that CALR mutations are predominantly found in early clones. Pathogenic role of CALR mutations in MPN Non-mutant and type 1 mutant CALR were transferred into IL-3-dependent murine cell lines, respectively. Cells expressing type 1 CALR mutations showed growth, which was IL-3 independent and sensitive to IL-3. Cells expressing non-CALR mutant or type 1 mutant CALR showed similar sensitivity to JAK2 kinase inhibitors, suggesting that IL-3-dependent growth of CALR mutant cells is dependent on JAK2 or JAK family kinases. To confirm the hypothesis, STAT5 phosphorylation was assayed in control and CALR-transfected cell lines in the presence or absence of IL-3. Increased phosphorylation of STAT5 was detected in type 1 mutant CALR cells in the absence of IL-3 and in low concentrations of IL-3, but not in non-mutant CALR cells. These findings support the hypothesis of activation of JAK/STAT signaling in type 1 CALR mutations. Familial MPN Although most MPNs are disseminated, familial MPNs (at least 2 members with MPN) are well described. Family members with MPNs may have acquired somatic mutations. It is believed that a patient’s familial MPN inheritance “susceptibility” stems from somatic mutations in MPN. A study found that two members of 21 familial MPNs had CALR mutations, suggesting that these mutations can also occur in familial cases. Clinical relevance of CALR mutations in MPN patients CALR mutations are associated with low age, male patients with ET, and low age in patients with PMF. In patients with ET, those with CALR mutations had lower hemoglobin levels, lower white blood cell (WBC) counts, and higher platelet counts than those with JAK2 mutations at diagnosis. patients with JAK2V617F mutations were associated with lower serum erythropoietin than patients with CALR mutations. Among PMF patients, those with the CALR mutation at diagnosis had lower WBC and higher platelet counts than mutant JAK2. In a univariate analysis of 254 PMF, CALR mutations were associated with higher platelet counts (P<0.0001), and anemia and leukocytosis were relatively mild in patients with CALR mutations. The incidence of thrombosis was lower in ET patients with CALR mutations than in those with JAK2 mutations, and in a comparative cohort study of 144 patients at risk for visceral vein thrombosis (SVT), the incidence was 18.8% in those with JAK2V617F mutations; none of the patients with mutations in CALR exon 9. This finding supports the lower risk of thrombosis in patients with CALR mutations compared to those with JAK2 mutations. Erythrocytic transformation A 15-year follow-up found that patients with erythrocytosis without CALR mutations had a 29% cumulative risk of JAK2 mutation ET. Myelofibrosis conversion The incidence of conversion to myelofibrosis (MF) according to somatic mutation status remains controversial. In one study, the incidence of conversion from ET to MF was significantly higher in patients with CALR mutations than in those with JAK2 mutations. In other studies, there was no significant difference in the incidence of MF conversion between the two groups. Overall survival A multivariate analysis study by Klampfl et al. showed that patients with JAK2 and MPL mutations in MPN died more than those with CALR mutations. nangalia et al. reported no significant survival differences between the two different ET mutation groups. In a cohort study looking at 576 patients with ET, the results showed that CALR mutations did not affect survival. Another study on the effect of CALR mutations on long-term survival in ET, looking at 299 patients diagnosed before 2006, showed that the shortest survival was in patients with MPL mutations. median survival was 19 years in JAK2 patients and 20 years in CALR mutations (p = 0.32). The unique feature of this study is the very long follow-up period, which provides an accurate estimate of long-term survival in ET and adds to the updated information on specific mutation phenotypes and prognosis. In PMF, CALR mutations were favorable for survival, independent of the Dynamic International Prognostic Scoring System (DIPSS) and ASXL1 mutation status. Triple-negative patients showed less leukemic transformation. These findings suggest that "CALR(-)/ASXL1(+)" and "triple-negative" are high-risk molecular features for PMF. In a subsequent study, 570 PMF patients were derived (N = 277) and validated (N = 293) for molecular prognostic observation of CALR and ASXL1 mutations. Survival was longest for CALR(+)/ ASXL1(- ) and shortest for CALR(-)/ ASXL1(+). the CALR/ ASXL1 prognostic model was independent of DIPSS and was effective in identifying survival in patients in the low-intermediate risk-1-group and in the high-medium risk-2-group. Current status of ET and PMF diagnosis and treatment Considerable progress has been made in the diagnosis of MPN in just a few years after the discovery of the JAK2V617F mutation. An important consequence of the new findings has led to a revision of the World Health Organization (WHO) classification and diagnosis of these diseases. patients with JAK2-positive MPNs share certain features, but the currently available data do not firmly support the management of the classification with or without JAK2 mutations, so the WHO classification remains the diagnostic tool used in clinical practice. the discovery of the CALR mutation is another milestone in our understanding of the pathogenesis of MPNs. The discovery of CALR mutations is another milestone in our understanding of the pathogenesis of MPNs. The evaluation of CALR mutations has significantly improved the diagnostic approach to ET and PMF. The initial mutation screening for suspected ET or PMF should begin with JAK2V617F, with negative JAK2V617F followed by screening for CALR mutations. negative CALR mutations are screened for MPL mutations. Triple negatives are patients who test negative for all three of these mutations. CALR mutations should be included in the diagnosis of MPN and integrated into subsequent versions of the WHO classification system. Similar to the JAK2V617F mutation, it is still too early to attempt a molecular diagnosis based on the JAK2/ MPL/ CALR mutation, but it may be a harbinger of the future of MPN. Patients with primary thrombocythemia are at lower risk for thrombotic complications with CALR mutations compared to JAK2 mutations, however, no treatment modification recommendations based on mutation status have been implemented. In two patients with CALR-mutated primary thrombocythemia, treatment with alpha interferon resulted in a sustained hematologic complete response, and the load of allelic mutants continued to decrease after discontinuation of maintenance therapy, suggesting that alpha interferon can target therapeutic cells via CALR mutations and that the therapeutic response is sustained. results from two clinical trials of ruxolitinib for PMF suggest that this JAK1 and JAK2 inhibitor is effective in most patients, regardless of the presence of the JAK2V617F mutation. The fact that the vast majority of patients with non-JAK2 mutations have CALR mutations means that ruxolitinib is also effective in patients with CALR mutations. Based on the molecular prognostic model for CALR and ASXL1 mutations, not only DIPSS plus high-risk myelofibrosis should be transplanted with stem cells, but CALR(-)/ASXL1(+) mutation status for any degree of risk disease. I believe that in future risk stratification scores for myelofibrosis, information on mutations will be integrated, which will facilitate This will facilitate the selection of high-risk patients for allogeneic transplantation. Outlook More information is still needed to understand the role of CALR mutations in the development and progression of this disease. Peptide sequences obtained from the alternative reading frame of the C-terminal structural domain of the CALR mutation represent cancer-specific epitopes that provide opportunities for use as immunotherapeutic targets.