Aiding in the distinction between a reactive cytosis and a chronic myeloproliferative disorder
Evaluating for variants in JAK2, CALR, and MPL genes in an algorithmic process
| Test Id | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| CALX | CALR, Gene Mutation, Exon 9, Reflex | No, (bill only) | No |
| MPLR | MPL Exon 10 Mutation Detection, R | No, (bill only) | No |
This reflex test sequentially evaluates for the common major gene variants associated with non-BCR-ABL1-positive myeloproliferative neoplasms until a variant is identified. The testing sequence is based on the reported frequency of gene variants in this disease group. Initial testing evaluates for the presence of the JAK2 V617F variant. If this result is negative or very low positive (0.06%-2%), testing proceeds with assessment for CALR gene variants. If the CALR result is also negative, then testing proceeds to evaluate for variants in exon 10 of the MPL gene. If either JAK2 V617F (>2%) or CALR variants are detected in the process, the testing algorithm ends; therefore, the complete reflex is followed only in the event of sequential negative variant. An integrated report is issued with the summary of test results.
For more information the following algorithms are available:
-Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation
-Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation
Quantitative Polymerase Chain Reaction (qPCR)
83872-JAK2B
Janus kinase 2 gene
Tyrosine Kinase Mutation
CALR
Calreticulin
Essential Thrombocythemia
JAK2-negative Myeloproliferative Neoplasm
Myelofibrosis
Myeloproliferative Disorder
Myeloproliferative Neoplasm (MPN)
Primary Myelofibrosis
MPL S505
MPLW515
Myeloproliferative leukemia virus oncogene
This reflex test sequentially evaluates for the common major gene variants associated with non-BCR-ABL1-positive myeloproliferative neoplasms until a variant is identified. The testing sequence is based on the reported frequency of gene variants in this disease group. Initial testing evaluates for the presence of the JAK2 V617F variant. If this result is negative or very low positive (0.06%-2%), testing proceeds with assessment for CALR gene variants. If the CALR result is also negative, then testing proceeds to evaluate for variants in exon 10 of the MPL gene. If either JAK2 V617F (>2%) or CALR variants are detected in the process, the testing algorithm ends; therefore, the complete reflex is followed only in the event of sequential negative variant. An integrated report is issued with the summary of test results.
For more information the following algorithms are available:
-Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation
-Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation
Varies
Specimen must arrive within 7 days of collection.
The following information is required:
1. Pertinent clinical history
2. Clinical or morphologic suspicion
3. Date of collection
4. Specimen source
| Question ID | Description | Answers |
|---|---|---|
| MP023 | Specimen Type |
Submit only 1 of the following specimens:
Specimen Type: Whole Blood
Container/Tube: Lavender top (EDTA) or yellow top (ACD solution B)
Specimen Volume: 3 mL
Collection Instructions:
1. Invert several times to mix blood.
2. Send whole blood specimen in original tube. Do not aliquot.
3. Label specimen as blood.
Specimen Stability Information: Ambient (preferred)/Refrigerate 7 days
Specimen Type: Bone marrow
Container/Tube: Lavender top (EDTA) or yellow top (ACD solution B)
Specimen Volume: 2 mL
Collection Instructions:
1. Invert several times to mix bone marrow.
2. Send bone marrow specimen in original tube. Do not aliquot.
3. Label specimen as bone marrow.
Specimen Stability Information: Ambient (preferred)/Refrigerate 7 days
Specimen Type: Extracted DNA from blood or bone marrow
Container/Tube: 1.5- to 2-mL tube
Specimen Volume: Entire specimen
Collection Instructions: Label specimen as extracted DNA from blood or bone marrow and provide indication of volume and concentration of the DNA.
Specimen Stability Information: Frozen (preferred)/Refrigerate/Ambient
If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request (T726) with the specimen.
Blood, bone marrow: 0.5 mL; Extracted DNA: 50 mcL at 20 ng/mcL concentration
| Gross hemolysis | Reject |
| Paraffin-embedded bone marrow aspirate clot or biopsy blocks Slides Paraffin shavings Moderately to severely clotted | Reject |
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Varies | Varies | 7 days |
Aiding in the distinction between a reactive cytosis and a chronic myeloproliferative disorder
Evaluating for variants in JAK2, CALR, and MPL genes in an algorithmic process
This reflex test sequentially evaluates for the common major gene variants associated with non-BCR-ABL1-positive myeloproliferative neoplasms until a variant is identified. The testing sequence is based on the reported frequency of gene variants in this disease group. Initial testing evaluates for the presence of the JAK2 V617F variant. If this result is negative or very low positive (0.06%-2%), testing proceeds with assessment for CALR gene variants. If the CALR result is also negative, then testing proceeds to evaluate for variants in exon 10 of the MPL gene. If either JAK2 V617F (>2%) or CALR variants are detected in the process, the testing algorithm ends; therefore, the complete reflex is followed only in the event of sequential negative variant. An integrated report is issued with the summary of test results.
For more information the following algorithms are available:
-Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation
-Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation
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 variant in JAK2 characterized as c.G1849T; p. Val617Phe (V617F).
The JAK2 V617F variant is present in 95% to 98% of patients with polycythemia vera, 50% to 60% of patients with primary myelofibrosis (PMF), and 50% to 60% of patients with essential thrombocythemia (ET). It has also been described infrequently in other myeloid neoplasms, including chronic myelomonocytic leukemia and myelodysplastic syndrome. Detection of JAK2 V617F helps establish the diagnosis of MPN. However, a negative JAK2 V617F result does not indicate the absence of MPN. Other important molecular markers in BCR::ABL1-negative MPN include CALR exon 9 variant (20%-30% of PMF and ET) and MPL exon 10 variant (5%-10% of PMF and 3%-5% of ET). Variants in JAK2, CALR, and MPL are essentially mutually exclusive. A CALR variant is associated with decreased risk of thrombosis in both ET and PMF and confers a favorable clinical outcome in patients with PMF. A triple negative (JAK2 V617F, CALR, and MPL-negative) genotype is considered a high-risk molecular signature in PMF.
An interpretive report will be provided.
The results will be reported as 1 of the 4 following states:
-Positive for JAK2 V617F variant
-Positive for CALR variant
-Positive for MPL variant
-Negative for JAK2 V617F, CALR, and MPL variants
Positive variant status is highly suggestive of a myeloid neoplasm but must be correlated with clinical and other laboratory features for definitive diagnosis.
Negative variant status does not exclude the presence of a myeloproliferative neoplasm or other neoplasms.
Results below the laboratory cutoff for positivity are of unclear clinical significance currently.
A positive result is not specific for a particular subtype of myeloproliferative neoplasm and clinicopathologic correlation is necessary in all cases.
A negative result does not exclude the presence of a myeloproliferative neoplasm or other neoplastic process.
In rare cases, a variant other than JAK2 V617F may be present in an area that interferes with primer or probe binding, which may cause a false-negative result.
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.
Analytical sensitivity is determined at 0.06% (by dilution of a JAK2 V617F-positive cell line into a negative cell line DNA).
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(17):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(4):1207-1209
5. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutation of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369(25):2379-2390
6. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mutation in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013;369(25):2391-2405
7. Pikman Y, Lee BH, Mercher T, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Med. 2006;3(7):e270
8. Pardanani AD, Levine RL, Lasho T, et al. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood. 2006;108:3472-3476
9. Kilpivaara O, Levine RL. JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science. Leukemia. 2008;22(10):1813-1817
10. Defour JP, Chachoua I, Pecquet C, Constantinescu SN. Oncogenic activation of MPL/thrombopoietin receptor by 17 mutations at W515: implications for myeloproliferative neoplasms. Leukemia. 2016;30(5):1214-1216. doi:10.1038/leu.2015.271
Genomic DNA is extracted, and 2 polymerase chain reaction (PCR) reactions are used for each sample. In each reaction, a short fragment of genomic DNA, including the variant site, is amplified using quantitative PCR in a real-time PCR instrument. In one reaction, the reverse primer matches the altered sequence, and the PCR conditions are such that it will only bind altered DNA. In the second reaction, 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 altered DNA and the amount of wild-type DNA is measured for each sample. In each run, the amount of altered and wild-type DNA in a calibrator DNA sample is also measured.
The final result is reported as % JAK2 V617F of total JAK2.(Unpublished Mayo method)
Monday through Friday
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.
81270-JAK2 (Janus kinase 2) (eg, myeloproliferative disorder) gene analysis, p.Val617Phe (V617F) variant
81219-CALR (calreticulin) (eg, myeloproliferative disorders), gene analysis, common variants in exon 9 (if appropriate)
81339-MPL (MPL proto-oncogene, thrombopoietin receptor) (eg, myeloproliferative disorder) gene analysis; sequence analysis, exon 10 (if appropriate)
| Test Id | Test Order Name | Order LOINC Value |
|---|---|---|
| MPNR | MPN (JAK2 V617F, CALR, MPL) Reflex | In Process |
| Result Id | Test Result Name |
Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
|
|---|---|---|
| 36687 | Final Diagnosis | 22637-3 |
| 39725 | MPNR Result | No LOINC Needed |