• Diagnose polycythemia vera (PV), essential thrombocythemia (ET), and idiopathic myelofibrosis (MF)

• Monitor patients for therapeutic response and relapse (quantitative JAK2 V617F mutation analysis only)

Chronic myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell malignancies characterized by excessive production of blood cells. ET, MF, and PV are the 3 most common BCR/ABL-negative MPNs and are associated with thrombosis and hemorrhage, splenomegaly, and the risk of transformation to acute myeloid leukemia.

Diagnostic criteria for ET, MF, and PV adopted by the World Health Organization (WHO) include identification of a clonal marker, with a specific recommendation to test for the JAK2 V617F mutation in exon 14.¹ JAK2 V617F is a gain-of-function mutation that leads to clonal proliferation; it is present in about 95% of PV cases and about half of ET and MF cases. The JAK2 allele burden decreases with successful therapy, disappears in some patients, and reappears during relapse.^2,3 Thus, quantitative JAK2 analysis may be useful for diagnosis and patient management. In JAK2 V617F-negative patients, the presence of a JAK2 exon 12 mutation also meets the WHO criterion for establishing clonality. Exon 12 mutations have been found in patients with PV who present with erythrocytosis but are typically not associated with ET or MF (Table 1).^4,5

Because a significant proportion of patients with ET or MF are negative for JAK2 mutations, other somatic mutations were sought; hence mutations in the myeloproliferative leukemia gene (MPL) were identified.⁸ MPL, found at chromosome 1p34, encodes the thrombopoietin receptor that works in concert with thrombopoietin for platelet production. Acquired MPL mutations (eg, W515L and W515K) are associated with severe anemia and have been detected in patients with ET or MF but not in patients with PV (Table 1).^6,7 An inherited MPL mutation (S505N; exon 10) has also been found in a Japanese pedigree with familial ET.⁸

Although mutation analysis testing frequently uses bone marrow or peripheral blood cells, the Leumeta^® tests employed by Quest Diagnostics are performed using plasma samples. For JAK2 mutation testing, plasma was shown to provide a higher mutation detection rate,⁹ to distinguish hemizygous/homozygous from heterozygous mutations,¹⁰ and to detect mutations in RNA that might be missed with DNA cell-based testing.¹¹

For diagnostic purposes, test selection is best based on the hematologic characteristics and relative prevalence of JAK2 and MPL mutations in patients with BCR/ABL-negative MPNs. Typically, mutations in MPL and JAK2 exon 12 are investigated after the JAK2 V617F mutation has been ruled out (Figure).

• Individuals suspected of having PV, ET, or MF

• JAK2 V617F mutation-positive individuals undergoing treatment for PV, ET, or MF

• Reverse transcription-PCR amplification targeting relevant regions of JAK2 or MPL, followed by amplicon sequencing and computer analysis for the presence of mutations

• Analytical sensitivity (qualitative tests): 10% mutant allele in the background of wild-type allele

• Analytical sensitivity (quantitative test): 5 pg JAK2 V617F/μL plasma

• Results reported vary with the test:

• JAK2 V617F qualitative tests: positive (homozygous, homozygous/hemizygous, heterozygous) or negative

• JAK2 V617F quantitative test: pg JAK2 V617F/μL plasma

• JAK2 exon 12 and 13 mutations: positive or negative

• MPL W515 and S505 mutations: positive or negative for each mutation; any additional mutations detected

• CPT codes*:

• 16175X: 83891, 83896, 83898, 83904 x2, 83912

• 16536X: 83891, 83898, 83902, 83904 x2, 83912

• 16539X: 83891, 83898, 83902, 83904, 83912

• 16538X: 83891, 83898, 83902, 83904, 83912

• 16184X: 83891, 83898, 83902, 83904 x2, 83912

Negative for mutation

The presence of a JAK2 V617F or exon 12 mutation is consistent with the diagnosis of PV. JAK2 V617F-positive results may also be associated with ET, MF, or, rarely, with other myeloid neoplasms.¹ In patients with suspected ET or MF, a JAK2 V617F-positive result rules out reactive thrombocytosis and myelofibrosis.¹ A negative result does not rule out the diagnosis of PV, ET, or MF. When monitoring disease, a decrease in JAK2 V617F concentration suggests therapeutic response; an increase suggests relapse.

The presence of a MPL W515 mutation is consistent with ET or MF and meets the WHO diagnostic criterion for establishing clonality; MPL S505N is consistent with familial ET. A negative finding does not rule out ET or MF.

These assays detect mutations only in the exons tested; polymorphisms or mutations at other locations will not be detected. Test results should be interpreted in conjunction with other laboratory and clinical findings. False-negative results may occur when there is a low mutant allele burden in the peripheral blood.