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Myeloproliferative neoplasms (MPNs) are a group of clonal hematological disorders that arise from transformation of a multipotent hematopoietic stem cell which includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF).Driver mutation’s confer growth advantage on the cancer cell and most likely is selected in the tissue microenvironment within which the neoplastic cells arise. Three-quarters of these patients carry the unique JAK2V617F mutation, JAK2 exon 12 mutations are found in 5% of patients with PV, MPL exon 10 mutations are present in about 5% ET/PMF and CALR mutations are found in 50-70% patients with ET/PMF.

We analyzed 3000 samples with suspected MPN for JAK2V617F mutation by ARMS-PCR and their allele burdens were reported by RQ-PCR. We have screened a cohort of 500 patients for JAK2/MPL/CALR mutations by a sequential molecular analysis which includes PCR, RT-PCR and fragment analysis.

JAK2V617F mutation is present in 50% of patients with MPN. Among 600 cases submitted for sequential molecular analysis identified 372 cases with JAK2V617F mutation, 70 cases with CALR mutation, and 6 cases with MPL mutations. Allele burden study on JAK2V617F positive patients revealed that patients with ET has the lowest allele burden, those with PV an intermediate one and those with PMF showed the highest burden. Measurement of JAK2V617F allele burden by RQ-PCR for a PMF case after allogeneic transplant (ASCT) reported that allele burden of 2.9% after 20 days of transplant and a negative result after 60 days of transplant vs 13% before ASCT.CALR mutation is found in ET and PMF cases that are mutually exclusive with JAK2V617F and MPL exon 10 mutations in ET whereas 2 cases with PMF found to be positive for JAK2V617F and CALR mutations. We found 40 cases with a 52-bp deletion, 4 cases with a 14bp deletion and 26 cases with a 5bp insertion. CALR variants reported in our cohort were 54% type 1 and 46% type 2 mutations. We found a tendency towards older age among type 2 carriers compared to type 1 carriers (median age at diagnosis: 57 years versus 52 years) or compared to non-type 2 carriers (median age at diagnosis: 57 years versus 53 years). Similarly, platelet count at diagnosis tended to be higher in the subgroup of type 2 mutation carriers than in patients with the type 1 mutation while hemoglobin levels and white blood cell count were lower compared to those with non-type 2 mutation. The mutual allele burden of JAK2V617F /CALR exon indel mutations of two PMF patients found as 10%/65% and 15%/55% respectively. In our cohort, 10% of the patents with CALR mutation had anemia, 21% had splenomegaly, and 43% had megakaryocytes at time of diagnosis. Compared with JAK2 V617F-positive ET and PMF, CALR-mutant ET and PMF are clinically correlated with lower WBC, leukocyte and hemoglobin counts, higher platelet counts, and a reduced risk of thrombosis.

Analysis of JAK2/MPL/CALR genes as molecular marker’s for MPN’s, allows the diagnosis of 95% of patients with MPN.As a novel mutation, CALR testing also has a prognostic significance and it was not mutually exclusive with JAK2V617F mutation. Measurement of JAK2 V617F allele burden early after transplantation is an important predictive parameter in monitoring patients following this treatment.The knowledge of driver mutations can provide valuable information for diagnosis and prognosis, which ultimately can be highly useful for clinical decision making for the management of patients with MPN.