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Mayo Test ID JAK2V JAK2 V617F Mutation Detection, Varies


Specimen Required


Specimen Type: Extracted DNA from blood or bone marrow

Container/Tube: 1.5- to 2-mL tube with indication of volume and concentration of the DNA

Specimen Volume: Entire specimen

Collection Instructions: Label specimen as extracted DNA from blood or bone marrow and indicate volume and concentration of the DNA.

Specimen Stability Information: Refrigerated/Ambient


Useful For

Aiding in the distinction between a reactive blood cytosis and a chronic myeloproliferative disorder using extracted DNA specimens

Method Name

Quantitative Polymerase Chain Reaction (PCR)

Reporting Name

JAK2 V617F Mutation Detection, V

Specimen Type

Varies

Specimen Minimum Volume

50 microliter at a concentration of 20 ng/microliter

Specimen Stability Information

Specimen Type Temperature Time Special Container
Varies Varies

Reject Due To

Bone marrow biopsies
Slides
Paraffin shavings
Frozen tissues and paraffin-embedded tissues
Paraffin-embedded bone marrow aspirates
Moderately to severely clotted		 Reject

Clinical Information

The Janus kinase 2 gene (JAK2) codes for a tyrosine kinase (JAK2) that is associated with the cytoplasmic portion of a variety of transmembrane cytokine and growth factor receptors important for signal transduction in hematopoietic cells. Signaling via JAK2 activation causes phosphorylation of downstream signal transducers and activators of transcription (STAT) proteins (eg, STAT5) ultimately leading to cell growth and differentiation. BCR::ABL1-negative myeloproliferative neoplasms (MPN) frequently harbor an acquired single nucleotide mutation in JAK2 characterized as c.G1849T; p. Val617Phe (V617F). This mutation is identified overall in approximately two-thirds of all MPN,(1-3) but the prevalence varies by MPN subtype. The JAK2 V617F is present in 95% to 98% of polycythemia vera, 50% to 60% of primary myelofibrosis (PMF), and 50% to 60% of essential thrombocythemia (ET). It has also been described infrequently in other myeloid neoplasms, including chronic myelomonocytic leukemia and myelodysplastic syndrome.(4) This mutation is not seen in chronic myelogenous leukemia or in reactive conditions with elevated blood counts. Detection of the JAK2 V617F is useful to help establish the diagnosis of MPN. However, a negative JAK2 V617F result does not indicate absence of an MPN. Other important molecular markers in BCR::ABL1-negative MPN include CALR exon 9 mutation (20%-30% of PMF and ET) and MPL exon 10 mutation (5%-10% of PMF and 3%-5% of ET). Mutations in JAK2, CALR, and MPL are essentially mutually exclusive.

Reference Values

An interpretive report will be provided.

Interpretation

The results will be reported as 1 of the 2 states:

-Negative for JAK2 V617F mutation

-Positive for JAK2 V617F mutation

 

Positive mutation status is highly suggestive of a myeloid neoplasm but must be correlated with clinical and other laboratory features for a definitive diagnosis.

 

Negative mutation status does not exclude the presence of a myeloproliferative neoplasm or other neoplasm.

 

Results below the laboratory cutoff for positivity are of unclear clinical significance at this time.

Cautions

A positive result is not specific for a particular subtype of myeloproliferative neoplasm and clinicopathologic correlation is necessary in all cases. If this test is ordered in the setting of erythrocytosis and suspicion of polycythemia vera, interpretation requires correlation with a concurrent or recent prior bone marrow evaluation.

 

A negative result does not exclude the presence of a myeloproliferative neoplasm or other neoplastic process.

 

In rare cases, a mutation other than JAK2 V617F may be present in an area that interferes with primer or probe binding and cause a false-negative result.

Clinical Reference

1. Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365(9464):1054-1061

2. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature. 2005;434(7037):1144-1148

3. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352:1779-1790

4. Steensma DP, Dewald GW, Lasho TL, et al. The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both "atypical" myeloproliferative disorders and the myelodysplastic syndrome. Blood. 2005;106:1207-1209

5. Stuckey R, Gomez-Casares MT. Recent advances in the use of molecular analyses to inform the diagnosis and prognosis of patients with polycythaemia vera. Int J Mol Sci. 2021;22(9):5042. doi:10.3390/ijms22095042

Method Description

Genomic DNA is extracted and 2 polymerase chain reaction (PCR) amplifications are used for each sample. In each reaction, a short fragment of genomic DNA, including the mutation site, is amplified using quantitative PCR in a real-time PCR instrument (LightCycler 480, Roche). In the first reaction, the 5' terminal base of the reverse primer matches the mutated sequence, and the PCR conditions are such that it will only bind mutated DNA. In the second reaction, the 5' terminal base of the reverse primer matches the wild-type sequence, and the PCR conditions are such that it will only bind the wild-type sequence. In both reactions, the PCR is monitored using TaqMan probe chemistry. The amount of mutated DNA and the amount of wild-type DNA is measured for each sample. In each run, the amount of mutated and wild-type DNA in a calibrator DNA sample is also measured. The calibrator is a mixture of DNA from a positive cell line (HEL) and a negative cell line (HL60) that is frozen in aliquots and expected to give an identical result in each run. Deviations in the calibrator result are assumed to be due to deviations in the run conditions and the sample results are corrected accordingly. Following each reaction, LightCycler 480 Relative Quantification Software is used to calculate the normalized mutated:wild-type ratio, which is expressed as a unitless ratio following correction with the calibrator data.

 

The formula for the normalized ratio is as follows:

 

Normalized ratio =

mutated/wild type (sample)

mutated/wild type (calibrator)

 

 

The final result is reported as percent JAK2 V617F of total JAK2, ie [mutated/mutated + wild type] x 100%, calculated from the normalized mutated:wild-type ratio.(Instruction manual: Roche Applied Science Technical Note No. LC 13/2001. Relative Quantification; LightCycler 480, 2006)

Day(s) Performed

Monday through Saturday

Report Available

2 to 5 days

Specimen Retention Time

3 months

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information

81270-JAK2 (Janus kinase 2) (eg, myeloproliferative disorder) gene analysis, p.Val617Phe (V617F) variant

LOINC Code Information

Test ID Test Order Name Order LOINC Value
JAK2V JAK2 V617F Mutation Detection, V 43399-5

 

Result ID Test Result Name Result LOINC Value
39724 JAK2 Result 53761-3
31160 JAK2 V617F Mutation Detection, V 43399-5