What's the next step in managing a patient with erythrocytosis, low Erythropoietin (EPO), functional iron deficiency, and low iron saturation, who tested negative for JAK2 (Janus Kinase 2) mutation?

Next Step: Test for CALR and MPL Mutations

In a patient with erythrocytosis, low EPO, functional iron deficiency, and negative JAK2 testing, the immediate next step is to test for JAK2 exon 12 mutations, followed by CALR and MPL mutations if exon 12 is negative, and perform bone marrow biopsy to establish the diagnosis. 1, 2

Sequential Molecular Testing Algorithm

JAK2 Exon 12 Testing (First Priority)

• Test for JAK2 exon 12 mutations immediately, as these occur in 2-4% of polycythemia vera cases that are JAK2V617F-negative 1, 3
• JAK2 exon 12 mutations are specifically associated with isolated erythrocytosis and low serum EPO levels 4
• These mutations occur in approximately 27% of patients with erythrocytosis and low EPO who are JAK2V617F-negative 4
• Use purified granulocytes rather than whole blood for testing, as exon 12 mutations often have low mutation burden 1

CALR and MPL Testing (If Exon 12 Negative)

• Test for CALR mutations next, though these are primarily associated with essential thrombocythemia and myelofibrosis rather than isolated erythrocytosis 1, 2
• CALR mutations are found in 20-25% of JAK2-negative ET and PMF cases but are rare in isolated erythrocytosis 2
• Test for MPL mutations (particularly MPL W515L/K) if CALR is negative, accounting for 3-5% of JAK2-negative cases 2

Bone Marrow Examination

Mandatory Diagnostic Procedure

• Perform bone marrow biopsy with reticulin staining to evaluate for characteristic myeloproliferative neoplasm histopathology 2
• Look for hypercellularity with panmyelosis, pleomorphic mature megakaryocytes, and trilineage myeloproliferation characteristic of polycythemia vera 1
• Bone marrow examination becomes the definitive diagnostic test in triple-negative cases (JAK2/CALR/MPL negative) 2
• The biopsy can establish MPN diagnosis even without driver mutations when morphology is characteristic 2

Expanded Molecular Panel for Triple-Negative Cases

Additional Clonal Markers

• If all three driver mutations are negative (triple-negative), test for additional clonal markers including ASXL1, EZH2, IDH1/IDH2, SRSF2, TET2, and DNMT3A 1, 2
• These additional mutations support the clonal nature of disease in the 10-15% of cases that are triple-negative 1, 2
• The presence of these mutations helps distinguish true clonal MPN from reactive erythrocytosis 1

Critical Diagnostic Pitfall

Low EPO Does Not Exclude Non-Neoplastic Erythrocytosis

• Approximately 40% of non-neoplastic erythrocytosis (NNE) patients have EPO levels below the normal range, fulfilling a minor diagnostic criterion for PV and raising the possibility of misdiagnosis 5
• Low EPO in JAK2-negative erythrocytosis is not a reliable standalone criterion for distinguishing PV from NNE 5
• This makes molecular testing and bone marrow examination essential rather than optional 5

Functional Testing

Erythroid Progenitor Assay

• Consider testing for EPO-independent (hypersensitive) erythroid progenitors, as these occur exclusively in patients with JAK2 exon 12 mutations (p=0.0002) 4
• This functional assay can help identify patients who should undergo JAK2 exon 12 mutation testing 4

Clinical Context Considerations

Iron Deficiency Pattern

• The functional iron deficiency with low iron saturation in your patient is consistent with polycythemia vera, where iron is consumed by increased erythropoiesis 6
• This pattern supports a myeloproliferative process rather than secondary erythrocytosis 6

Alternative Rare Causes

• If all molecular testing and bone marrow examination are unrevealing, consider rare causes such as EPO-producing tumors or germline mutations in the erythropoietin receptor gene 6, 7
• Germline SOCS3 mutations have been reported in rare cases of erythrocytosis with deregulated JAK2 signaling 7