CALR Mutations in Myeloproliferative Neoplasms

A novel, specific molecular marker in majority of patients negative for JAK2 and MPL mutations

Mutant CALR is a novel, specific molecular marker detected in majority of patients with myeloproliferative neoplasms who are negative for JAK2 and MPL mutations. Use of this marker in the clinic may improve diagnostic and therapeutic decision making in these patients, according to the late-breaking abstract results presented at the 55th Annual Meeting of American Society of Hematology (ASH) in New Orleans, USA (7-10 December, 2013) by Klampfl Thorsten, PhD of the CeMM Research Centre for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria on behalf of the study team comprised also from researchers of the Department of Internal Medicine I, Division of Haematology and Blood Coagulation, Medical University of Vienna, Austria; Department of Haematology Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; and Department of Molecular Medicine, University of Pavia, Italy.

The classical, BCR-ABL1 negative myeloproliferative neoplasms are polycythaemia vera, essential thrombocythaemia and primary myelofibrosis. The most common genetic alteration in myeloproliferative neoplasms is the JAK2-V617F mutation detected in 95% of patients with polycythaemia vera and in 50-60% of patients with essential thrombocythaemia or primary myelofibrosis. Mutations in exon 12 of JAK2 and in the thrombopoietin receptor gene MPL are found in an additional 5-10% of the cases.

In recent years a number of other genes were shown to be affected in myeloproliferative neoplasms. However, these mutations are not mutually exclusive with JAK2 and MPL mutations and are also found in other myeloid malignancies. A specific molecular marker for the remaining 40% of patients with essential thrombocythaemia or primary myelofibrosis with wild type JAK2 and MPL is still unknown.

The study results

The study team used whole-exome sequencing to identify novel mutations in patients with primary myelofibrosis and wild type JAK2 and MPL. The analysis revealed recurrent somatic insertions and deletions in CALR encoding for calreticulin. All detected mutations resulted in a frame shift and clustered in exon 9 of the gene.

Following up on this finding, the study team developed a PCR based assay to screen 1107 patients with myeloproliferative neoplasms for insertion/deletion mutations in exon 9 of CALR. No mutations were detected in patients with polycythaemia vera. In patients with essential thrombocythaemia and primary myelofibrosis CALR mutations were mutually exclusive with mutant JAK2 and mutant MPL. Of the patients with wild type JAK2 and MPL, 67% of those with essential thrombocythaemia and 88% of those with primary myelofibrosis had mutant CALR. The researchers also tested 19 patients with wild type CALR-exon 9 for mutations in the other exons of the gene, but all were negative.

Furthermore they did not find CALR-exon 9 mutations in 254 patients with de novo acute myeloid leukaemia, 45 patients with chronic myeloid leukaemia, 73 patients with myelodysplastic syndrome or 64 with chronic myelomonocytic leukaemia. Out of 24 patients with refractory anaemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), 3 patients carried CALR mutations. These patients were wild type for JAK2 and MPL.

Types of CALR mutations

In total the researchers detected 36 different types of mutations in CALR. A 52 bp deletion and a 5 bp insertion were the most prominent types found in 53% and 32% of all cases with mutant CALR. All 36 types of mutations result in a frame shift to the same alternative reading frame, generating a novel C-terminus of the mutated protein.

Overexpression of the most common CALR mutation (a 52 bp deletion) in interleukin-3 (IL-3) dependent Ba/F3 cells led to IL-3-independent growth and hypersensitivity to IL-3. Cells overexpressing the mutant were sensitive to the JAK-family kinase inhibitor SAR302503 and showed elevated STAT5 phosphorylation in absence of IL-3. This indicates that JAK-STAT signalling is involved in the observed cytokine independent growth of mutant CALR expressing Ba/F3 cells.

Patients with essential thrombocythaemia and primary myelofibrosis and mutant CALR presented with lower white blood cell counts (p < 0.001 for essential thrombocythaemia, p = 0.027 for primary myelofibrosis) and elevated platelet levels (p < 0.001 in both groups) compared to patients with mutant JAK2. In both diseases, the patients with mutant CALR showed significantly better overall survival than patients with mutant JAK2 (p = 0.043 in essential thrombocythaemia, p < 0.001 in primary myelofibrosis). Patients with essential thrombocythaemia and mutant CALR had a lower risk of thrombosis in comparison to those with mutant JAK2 (p = 0.003).

The authors concluded that mutant CALR represents a novel, specific molecular marker detected in the majority of patients with myeloproliferative neoplasms who are negative for JAK2 and MPL mutations. Detection of this marker in the clinic may improve diagnostic and therapeutic decision-making in these patients.

The authors did not have relevant conflicts of interest to declare.