What are the clinical indications and uses of the JAK2 (Janus kinase 2) assay?

JAK2 Assay Clinical Indications and Uses

The JAK2 assay is primarily used to diagnose Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), specifically polycythemia vera, essential thrombocythemia, and primary myelofibrosis, where the JAK2 V617F mutation is present in >95% of polycythemia vera cases and approximately 50-60% of essential thrombocythemia and primary myelofibrosis cases. 1, 2

Primary Diagnostic Indications

Myeloproliferative Neoplasms (MPNs)

• Test for JAK2 V617F mutation in all patients with sustained erythrocytosis, unexplained thrombocytosis, or suspected MPN. 2 The mutation is found in virtually all polycythemia vera cases (>95%) and constitutes a major diagnostic criterion in the 2016 WHO classification. 3
• Order JAK2 testing when patients present with elevated hemoglobin/hematocrit, persistent platelet elevation (>450 × 10⁹/L), or splenomegaly without an obvious cause. 3, 1
• In JAK2 V617F-negative patients with erythrocytosis, test for JAK2 exon 12 mutations, which account for 2-4% of polycythemia vera cases. 3 These mutations are detected exclusively in polycythemia vera patients who lack the V617F mutation. 3

Splanchnic Vein Thrombosis (SVT)

• Test for JAK2 V617F mutation in all patients with splanchnic vein thrombosis (portal, mesenteric, hepatic, or splenic vein thrombosis), even when peripheral blood counts are normal. 3 The mutation is detected in approximately 20-40% of patients with splanchnic vein thrombosis without overt myeloproliferative disorders. 3
• JAK2 testing is mandatory in patients with Budd-Chiari syndrome or portal vein thrombosis to identify underlying occult myeloproliferative neoplasms. 3 This finding has critical implications for anticoagulation duration (indefinite therapy required). 3

Differential Diagnosis Scenarios

• Order JAK2 testing when distinguishing myelodysplastic syndromes with thrombocytosis from essential thrombocythemia. 3 A positive JAK2 result indicates a myeloproliferative component. 3
• Test for JAK2 in patients with aquagenic pruritus (intense itching after water contact), as this symptom is characteristic of polycythemia vera. 3 JAK2 V617F positivity in this context strongly supports the diagnosis. 3

Sequential Testing Algorithm

First-Line Testing

• Begin with JAK2 V617F mutation testing as the initial molecular diagnostic test. 3, 1 This should be performed using quantitative real-time PCR with sensitivity to detect mutant allele burden as low as 1-3%. 4

Second-Line Testing (JAK2 V617F-Negative Cases)

• If JAK2 V617F is negative in suspected essential thrombocythemia or primary myelofibrosis, immediately test for CALR mutations (found in 20-25% of JAK2-negative cases). 1 The European LeukemiaNet recommends sequential testing: JAK2 first, then CALR, then MPL. 1
• If both JAK2 and CALR are negative, test for MPL mutations (W515L/K), which account for 3-5% of JAK2-negative MPN cases. 3, 1
• In JAK2 V617F-negative polycythemia vera specifically, test for JAK2 exon 12 mutations using high-resolution melt-curve analysis or sequencing. 3, 1

Triple-Negative Cases

• When JAK2, CALR, and MPL are all negative (10-15% of cases), bone marrow biopsy becomes the definitive diagnostic test rather than optional. 1 Proceed with expanded molecular panel including ASXL1, EZH2, IDH1/IDH2, SRSF2, TET2, and DNMT3A to identify additional clonal markers. 1

Risk Stratification and Prognostic Uses

Thrombotic Risk Assessment

• JAK2 V617F-positive essential thrombocythemia patients have higher thrombotic risk compared to CALR-mutated patients. 3 This influences decisions about initiating cytoreductive therapy in younger patients. 3
• Leukocytosis at diagnosis in JAK2-positive patients may indicate higher thrombotic risk, particularly in those aged <60 years. 3

Treatment Monitoring

• Measure JAK2 V617F allele burden when interferon-α therapy is employed, as molecular response guides therapy continuation. 2 Routine allele burden monitoring is not required for other treatments. 2
• Do not use allele burden from flow cytometry for prognostic assessment; morphologic blast percentage by hematopathologist is required. 3

Critical Clinical Pitfalls to Avoid

Diagnostic Errors

• Never rule out polycythemia vera based on normal erythropoietin level alone—EPO sensitivity is only 64-70%. 2 Proceed with JAK2 testing when clinical suspicion exists despite normal EPO. 2
• Do not dismiss polycythemia vera when hemoglobin is normal or borderline-high with microcytosis; iron deficiency can mask expected hemoglobin rise. 2 Test JAK2 if additional clues (thrombocytosis, splenomegaly, pruritus) are present. 2
• Exclude BCR-ABL1 by FISH or RT-PCR before diagnosing chronic myeloproliferative neoplasm; if positive, the diagnosis is chronic myelogenous leukemia, not MPN. 3

Testing Limitations

• Recognize that approximately 10-15% of essential thrombocythemia and primary myelofibrosis cases are triple-negative but still represent true clonal MPN. 1 Bone marrow histology establishes diagnosis in these cases. 1
• Understand that JAK2 mutations occur at lower frequency in myelodysplastic syndromes and de novo acute myeloid leukemia compared to myeloproliferative disorders. 3

Specific Laboratory Considerations

Test Sensitivity Requirements

• Use assays with sensitivity to detect mutant allele burden as low as 0.8-3% for optimal diagnostic yield. 4, 5 Sensitive assays increase detection rates within myeloproliferative neoplasms. 4
• Quantitative real-time PCR is preferred over direct sequencing, RFLP, or ARMS assays for superior sensitivity. 5

Sample Requirements

• Peripheral blood is adequate for JAK2 testing in most cases; mutant allele burden in peripheral blood resembles bone marrow except in patients with very low allele burden. 6
• Bone marrow aspirate and biopsy with cytogenetics should accompany JAK2 testing for complete diagnostic workup. 3