JAK2^V617F Mutation Analysis, Quantitative With Reflex to CALR Mutation Analysis, JAK2 Exon 12-15 Mutation Analysis and MPL Mutation Analysis by NGS

Molecular testing of blood or bone marrow is useful in the evaluation of suspected myeloproliferative neoplasms (MPN). Mutations in the JAK2, MPL and CALR genes are present in virtually all MPNs and their presence help distinguish benign reactive processes from clonal neoplasms. These mutations are generally considered mutually exclusive, although concurrent clones have been reported in rare patients. This test will assess for the JAK2^V617F (exon 14) mutation first and will reflex to CALR mutation analysis, MPL mutation analysis, and JAK2 exon 12 to 15 mutation analysis if the JAK2^V617F mutation is negative.

The JAK2 (Janus kinase 2) gene encodes for a non-receptor protein tyrosine kinase that activates cytokine and growth factor signaling. The V617F (c.1849 G>T) mutation results in constitutive activation of JAK2 and downstream STAT5 and ERK signaling. The V617F mutation is observed in approximately 95% of polycythemia vera (PV), 60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). It is also infrequently present (3-5%) in myelodysplastic syndrome, chronic myelomonocytic leukemia and other atypical chronic myeloid disorders. A small percentage of JAK2 mutation positive patients (3.3%) contain other non-V617F mutations within exons 12 to 15. In particular, mutations in exon 12 of JAK2 have been described in approximately 3% of patients with PV. JAK2 allele burden correlates with clinical phenotype, with low levels of mutant allele characterized by thrombocytosis, intermediate levels with erythrocytosis and high mutant allele burden, correlating with enhanced myelopoiesis of the BM, leukocytosis, increasing spleen size and circulating CD34-positive cells.

The CALR (Calreticulin) gene encodes for a multifunctional calcium-binding protein involved in many cellular activities such as growth, proliferation, adhesion and programmed cell death. Among patients with JAK2-negative MPNs, CALR mutations are found in approximately 70% of patients with JAK2-negative essential thrombocythemia (ET) and 60-88% of patients with JAK2-negative primary myelofibrosis(PMF). Only a minority of patients (approximately 8%) with myelodysplasia have mutations in the CALR gene. CALR mutations are rarely detected in patients with de novo acute myeloid leukemia, chronic myelogenous leukemia, lymphoid leukemia or solid tumors. CALR mutations are not detected in polycythemia and generally appear to be mutually exclusive with JAK2 mutations and MPL mutations. The majority of mutational changes involve a variety of insertion deletion mutations in exon 9 of the calreticulin gene: approximately 53% of all CALR mutations are a 52 bp deletion (type-1) while the second most prevalent mutation (approximately 32%) contains a 5 bp insertion (type-2). Other mutations (non-type 1 or type 2) are seen in a small minority of cases. CALR mutations in PMF tend to be associated with a favorable prognosis compared to JAK2^V617F mutations, whereas primary myelofibrosis negative for CALR, JAK2^V617F and MPL mutations (so-called triple negative) is associated with a poor prognosis and shorter survival.

The MPL (myeloproliferative leukemia virus oncogene) gene encodes the thrombopoietin receptor, which regulates hematopoiesis and megakaryopoiesis. Activating MPL mutations are associated with a subset of myeloproliferative neoplasms and acute megakaryoblastic leukemia. MPL W515 mutations are present in approximately 5-8% of patients with primary myelofibrosis (PMF) and 1-4% of patients with essential thrombocythemia (ET). The S505 mutation is detected in patients with hereditary thrombocythemia.