What are the next steps for a patient in their late 50s with symptoms suggestive of a myeloproliferative neoplasm (MPN) but a negative JAK2 Val617Phe variant analysis result?

Next Steps for JAK2 V617F-Negative MPN Suspicion

In a patient with suspected MPN but negative JAK2 V617F testing, immediately proceed with CALR and MPL mutation testing, followed by bone marrow biopsy if these are also negative. 1, 2

Sequential Molecular Testing Algorithm

First-Line Additional Testing

• Test for CALR mutations immediately as the next diagnostic step, since CALR mutations are found in approximately 20-25% of JAK2-negative ET and PMF cases 1, 3
• Test for MPL mutations (particularly MPL W515L/K) if CALR is negative, as these account for an additional 3-5% of JAK2-negative MPN cases 1, 4
• The European LeukemiaNet guidelines specifically recommend testing JAK2V617F first, then CALR and MPL in that sequential order for suspected ET and myelofibrosis 1

If All Three Driver Mutations Are Negative

• Perform bone marrow biopsy with reticulin staining to evaluate for characteristic MPN histopathology, which remains diagnostic even without driver mutations 1, 2
• Consider expanded molecular panel including ASXL1, EZH2, IDH1/IDH2, SRSF2, TET2, and DNMT3A to identify additional clonal markers that support MPN diagnosis in triple-negative cases 1
• Search for atypical JAK2 mutations in exon 12 (if polycythemia vera is suspected) or exons 19 and 25, as these rare variants occur in approximately 2-3% of V617F-negative cases 1, 4

Critical Diagnostic Considerations

Evaluate for Secondary Causes

• Rule out reactive thrombocytosis from iron deficiency, inflammatory conditions, infectious diseases, malignancy, or hyposplenism, as these account for 72.5% of cases referred with suspected MPN 3
• Measure serum erythropoietin level (typically suppressed in PV), comprehensive metabolic panel including LDH and uric acid, and assess spleen size 2, 5

Bone Marrow Examination Findings

• Look for hypercellularity with panmyelosis and pleomorphic mature megakaryocytes characteristic of PV 2
• Identify megakaryocyte proliferation and atypia with variable fibrosis suggestive of primary myelofibrosis 2
• Bone marrow histology can establish MPN diagnosis even in triple-negative cases when morphology is characteristic 1

Important Caveats

Sensitivity Limitations

• The JAK2 V617F assay used has approximately 1-2% sensitivity, meaning very low allele burdens (<1-2%) may be missed 3
• If clinical suspicion remains high despite negative testing, consider repeat testing with a more sensitive assay or at a reference laboratory 6

CALR Mutation Interpretation

• Distinguish frameshift from non-frameshift CALR mutations, as only frameshift mutations are pathogenic for ET; non-frameshift variants may represent reactive conditions 3
• Newly identified CALR mutations should be confirmed by Sanger sequencing and checked against COSMIC or HGMD databases 3

Triple-Negative MPN

• Approximately 10-15% of ET and PMF cases are triple-negative (JAK2/CALR/MPL negative) but still represent true clonal MPN based on bone marrow histology and clinical features 1
• In these cases, bone marrow biopsy becomes the definitive diagnostic test rather than an optional one 1, 2

Monitoring Strategy

• Perform complete blood counts every 3-6 months while diagnostic workup is ongoing 2, 5
• Reassess clinically for development of MPN-associated symptoms including aquagenic pruritus, erythromelalgia, constitutional symptoms, or progressive splenomegaly 2
• Repeat molecular testing annually or if hematologic parameters change significantly, as clonal evolution may occur 5, 6