1. APMG Pathologist, MD FCAP
Emerging Concepts in the Workup of Polycythemia and Thrombocythemia: JAK2
APMG Pathologist, MD FCAP

2. Clinical Question HGB over 18.5 in ♂ or 16.5 in ♀?
Platelets over 1 million?
Are the hematologic abnormalities reactive or the result of an underlying neoplastic process?

3. JAK-STAT Pathway Cell signaling pathway
Allows extracellular chemicals to effect nuclear DNA expression
Erythropoietin signals through the JAK-STAT pathway.

4. JAK2
In Spring 2005, four separate groups independently published discovery of a point mutation (V617F) in the JAK2 gene of patients with the PV and ET
Subsequent studies have identified JAK2 mutations as key molecular events in development of the myeloproliferative neoplasms (MPNs)
Incidence of JAK2 Mutation in MPNs
Polycythemia vera (PV)
99%
Essential thrombocythemia (ET)
60%
Primary myelofibrosis (PMF)
40%
Chronic myelogenous leukemia (CML)
<1%

5. JAK2: wild-type and mutant
EPO
No signal
Signal
Signal
Campbell P, N Engl J Med 2006, 355:

6. Clinical Dilemma HGB over 18.5 in ♂ or 15.5 in ♀?
JAK2 Testing
HGB over 18.5 in ♂ or 15.5 in ♀?
JAK2 abnormal in 99% of PV
Platelets over 1 million?
85% are not Essential Thrombocytosis (ET)
JAK2 abnormal in 60% of ET
Detection of JAK2 means neoplastic process.
JAK2 Testing

7. Mutations Most common mutation is V617F
Substitutes a phenylalanine for valine at codon 617
Other JAK2 exon 12 mutations (not V617F)
Found in PV that does not have V617F
Thus nearly 100% PV have some JAK2 abnormality
ET and CIMF can also have MPL mutations
Found in up to 5%
Not seen in PV

8. Polycythemia vera: WHO 2008
Diagnosis requires meeting both major criteria and one minor criterion OR the first major criterion and two minor criteria.
Major criteria
Hemoglobin > 18.5 g/dL in men, 16.5 g/dL in women or other evidence of increased red cell volume.*
Presence of JAK2V617F or other functionally similar mutation such as JAK2 exon 12 mutation.
Minor criteria
Bone marrow biopsy showing hypercellularity for age with trilineage growth (panmyelosis) with prominent erythroid, granulocytic, and megakaryocytic proliferation.
Serum erythropoietin level below the reference range for normal.
Endogenous erythroid colony formation in vitro.
Reference:
Thiele J, Kvasnicka HM. The 2008 WHO diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Curr Hematol Malig Rep Jan;4(1):33-40.
*Hemoglobin or hematocrit > 99th percentile of method-specific reference range for age, sex, and altitude of residence; or hemo- globin > 17 g/dL in men, 15 g/dL in women if associated with a documented and sustained increase of at least 2 g/dL from an individual’s baseline value that cannot be attributed to correction of iron deficiency; or elevated red cell mass > 25% above mean normal predicted value.

9. PV Dx Algorithm
*Clinical clues for PV include splenomegaly, thrombosis, aquagenic pruritus, and erythromelalgia. Laboratory clues for PV include thrombocytosis, leukocytosis, and increased leukocyte alkaline phosphatase score. Janus kinase 2 (JAK2) screening is to detect the V617F mutation that occurs in most patients with PV.
Reference:
Tefferi A, Barbui T. bcr/abl-negative, classic myeloproliferative disorders: diagnosis and treatment. Mayo Clin Proc Sep;80(9): Review.

10. Essential Thrombocythemia: WHO 2008
Diagnosis requires meeting all four criteria.
Sustained* platelet count ≥ 450 × 109/L.
Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged, mature megakaryocytes. No signi- ficant increase or left-shift of neutrophil granulopoiesis or erythropoiesis.
Not meeting WHO criteria for polycythemia vera,† primary myelofibrosis,‡ BCR-ABL1–positive chronic myelogenous leukemia,§ or myelodysplastic syndrome¶ or other myeloid neoplasms.
Demonstration of JAK2V617F or other clonal marker, or in the absence of JAK2V617F, no evidence for reactive thrombocytosis.**
Reference:
Thiele J, Kvasnicka HM. The 2008 WHO diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Curr Hematol Malig Rep Jan;4(1):33-40.
*Sustained during the workup process. †Requires the failure of iron replacement therapy to increase hemoglobin level to the polycythemia vera range in the presence of decreased serum ferritin. Exclusion of polycythemia vera is based on hemoglobin and hematocrit levels, and red cell mass measure- ment is not required. ‡Requires the absence of relevant reticulin fibrosis, collagen fibrosis, peripheral blood leukoerythroblastosis, or markedly hypercellular marrow accompanied by megakaryocyte morphology that is typical for primary myelofibrosis (small to large megakaryocytes with an aberrant nuclear/cytoplasmic ratio and hyperchromatic, bulbous, or irregularly folded nuclei and dense clustering). §Requires the absence of BCR-ABL1. ¶Requires the absence of dyserythropoiesis and dysgranulopoiesis. **Causes of reactive thrombocytosis include iron deficiency, splenectomy, surgery, infection, inflammation, connective tissue disease, metastatic cancer, and lymphoproliferative disorders. However, the presence of a condition associated with reactive thrombocytosis does not exclude the possibility of essential thrombocythemia if the first three criteria are met.

11. ET Diagnostic Algorithm
Reference:
Tefferi A, Barbui T. bcr/abl-negative, classic myeloproliferative disorders: diagnosis and treatment. Mayo Clin Proc Sep;80(9): Review.
*In addition to clinical history, laboratory tests that are helpful in distinguishing reactive thrombocytosis from ET include serum ferritin, peripheral blood smear, and C-reactive protein.

12. JAK2 Testing JAK2 V617F Detection JAK2 Exon 12 Mutation Analaysis
Performed on peripheral blood or bone marrow
Highly sensitive and specific assays
Should be performed in CAP Accredited lab
JAK2 Exon 12 Mutation Analaysis
Used when suspect PV but V617F not detected

13. Quantification of JAK2? diagnosis monitoring “clinical/hematologic/
Detection is sufficient.
Quantification not necessary
diagnosis
“clinical/hematologic/
prognostic correlates”
Controversial
Response to therapy
Utility not well defined clinically
monitoring

14. APMG Pathologist, MD FCAP
Questions
Contact pathologist with questions or to sort out appropriate testing on a patient:
APMG Pathologist, MD FCAP
(888)

15. About these slides Provided by the CAP as an aid to pathologists
Intended as a “starting point” from which customization / modifications can be made for personal use
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Content has been reviewed by experts at the CAP, but does not necessarily reflect the official opinion of the College of American Pathologists.
Version 0.3, rev. 8/3/2012
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