JAK2 and Myeloproliferative Mutation Panel

The myeloproliferative mutation panel looks for acquired mutations that drive chronic myeloproliferative neoplasms: polycythaemia vera, essential thrombocythaemia and primary myelofibrosis. The key genes are JAK2 (exon 14 V617F and exon 12 variants), CALR (exon 9) and MPL (exon 10).

These mutations switch on cytokine signalling inside blood cells, leading to over production of red cells, platelets or white cells. They act as clonal markers that separate a true neoplasm from reactive causes of high counts, and they enter the diagnostic criteria.

DNA is extracted from peripheral blood or bone marrow in EDTA, and tested by allele specific PCR, quantitative PCR, sequencing or next generation sequencing. A first line test for JAK2 V617F takes 2 to 5 days, and a full panel takes 7 to 14 days.

JAK2 V617F is found in about 95% of polycythaemia vera, and in about 50 to 60% of essential thrombocythaemia and primary myelofibrosis. JAK2 exon 12 variants account for most of the rest of polycythaemia vera. CALR mutations occur in about 20 to 25% of essential thrombocythaemia and primary myelofibrosis, and MPL in 3 to 5% and 5 to 10% of them. About 10% of patients with essential thrombocythaemia or myelofibrosis are "triple negative".

Indications.

• Persistent erythrocytosis (haemoglobin over 165 g/L or haematocrit over 49% in men, and over 160 g/L or 48% in women) with no secondary cause, and low erythropoietin.
• Persistent thrombocytosis (platelets over 450 x 10⁹/L) after iron deficiency, inflammation and other reactive causes are excluded.
• Unexplained thrombosis at unusual sites (splanchnic, cerebral), where JAK2 V617F is found in a large proportion of patients with Budd-Chiari syndrome and portal vein thrombosis.
• Suspected myelofibrosis (leucoerythroblastic film, splenomegaly, marrow fibrosis).
• Unexplained splenomegaly, or erythromelalgia with high platelets.
• Monitoring of allele burden on treatment, in selected cases.

Not indicated for mild, explained reactive changes (iron deficiency, smoking, sleep apnoea, chronic infection) or for screening of healthy people.

Specimen requirements.

• 3 to 5 mL peripheral blood in EDTA (granulocyte DNA gives good representation), or bone marrow aspirate.
• Room temperature transport to the laboratory within 24 to 72 hours.
• A request giving blood count results, erythropoietin, spleen size and thrombosis history.
• Bone marrow biopsy with reticulin staining accompanies the molecular test when a diagnosis is being made.

Normal appearances.

No mutation is detected in a healthy person. Very low level JAK2 V617F clones (under 1 to 2%) are found in some older people without disease (clonal haematopoiesis), and their meaning is uncertain.

Interpretation.

• Polycythaemia vera: JAK2 V617F or exon 12 mutation, with raised haemoglobin or haematocrit, a low erythropoietin and a hypercellular marrow with panmyelosis. A JAK2 mutation is a major diagnostic criterion.
• Essential thrombocythaemia: a JAK2, CALR or MPL mutation with a persistent platelet count over 450 x 10⁹/L and a typical marrow, after exclusion of other myeloid neoplasms (including chronic myeloid leukaemia, by a negative BCR-ABL1).
• Primary myelofibrosis: a driver mutation with marrow fibrosis, anaemia, splenomegaly and a leucoerythroblastic film. CALR type 1 mutations carry a better outcome, and triple negative disease and high risk additional mutations carry a worse outlook.
• Allele burden: a high JAK2 V617F burden is associated with higher thrombosis risk, pruritus, splenomegaly and fibrosis. Serial measurement may show response to treatment.
• Additional mutations (ASXL1, SRSF2, EZH2, IDH1, IDH2, U2AF1, TP53) found by sequencing panels worsen the prognosis in myelofibrosis, and they feed into prognostic scores.
• Negative panel with persistent high counts: a clonal disease is not excluded in a triple negative case, and a marrow examination and sequencing are needed. Reactive causes are checked again.

Critical values and findings needing immediate action.

• Haematocrit over 55% or symptoms of hyperviscosity, or a new arterial or venous thrombosis, with a positive mutation: urgent treatment (venesection, antiplatelet and cytoreductive drugs) and specialist review.
• Thrombosis at unusual sites, with a mutation: anticoagulation and haematology care.
• Rising white cell count, circulating blasts or anaemia with a known neoplasm: suspect transformation to acute leukaemia, and arrange a marrow examination.
• Pregnancy in a patient with a mutation: referral to a specialist unit, since thrombosis and obstetric risk is raised.

Limitations and artefacts.

• Sensitivity: allele specific PCR detects about 1% mutant allele, and sequencing methods detect about 5 to 10%, so a low burden can be missed by a less sensitive assay.
• JAK2 exon 12 and CALR mutations are not found by a V617F-only test, so a full panel is needed when V617F is negative.
• JAK2 V617F is not specific for a neoplasm when the allele burden is very low and the counts are normal.
• Secondary erythrocytosis and reactive thrombocytosis are far more common than neoplasms, so mutation testing is requested after the common reasons are checked, to avoid over diagnosis.
• Recent transfusion, iron deficiency (which masks polycythaemia vera) and dehydration affect haemoglobin and haematocrit.
• A mutation alone does not diagnose a neoplasm, and the full criteria need marrow findings and counts.
• Clonal haematopoiesis can be found in blood after chemotherapy or with age.
• Cost and access: next generation sequencing panels for prognosis are available in few centres.

Special groups.

• Young patients and women: more often essential thrombocythaemia, with CALR mutations, and fertility and pregnancy issues.
• Children: rare, and hereditary or reactive causes are checked first.
• Patients with splanchnic vein thrombosis: JAK2 testing even when blood counts are normal, since masked polycythaemia vera is common.
• Older adults with an incidental low level clone: follow up of counts, with attention to cardiovascular risk.
• Patients on JAK inhibitor treatment: monitoring of allele burden and counts.
• Regions: access to molecular tests is limited, so diagnosis often rests on blood counts, haematocrit, erythropoietin and marrow biopsy. Secondary erythrocytosis from altitude, smoking, lung disease and sickle cell disease is common, and the molecular result helps to separate it from polycythaemia vera.

Follow up tests.

• Serum erythropoietin, ferritin, and iron studies.
• Bone marrow aspirate and trephine with reticulin and cytogenetics.
• BCR-ABL1 testing to exclude chronic myeloid leukaemia.
• Next generation sequencing panel for additional mutations.
• Abdominal ultrasound for spleen size and splanchnic veins.
• Full blood count, lactate dehydrogenase, uric acid and liver function.
• Cardiovascular risk assessment, and thrombosis screening where relevant.
• Specialist haematology review.