Hematological Malignancies JAK2 V617F Quantitative Mutation Analysis

Chronic myeloproliferative diseases (CMPDs) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages in the bone marrow and increased numbers of mature and immature cells in the peripheral blood.  CMPDs include polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myelofibrosis (IMF), and chronic myeloid leukemia (CML). 

Most patients with myeloproliferative disorders carry a V617F JAK2 mutation:

• 74 - 97% of patients with polycythemia vera (PV)
• 33 - 57% of patients with essential thrombocythemia (ET)
• 35 - 50% of patients with myelofibrosis (IMF)
• Infrequently (3-5%) in myelodysplastic syndrome (MDS) & CMML
• JAK2 V617F mutation (auto-inhibitory domain) activates the kinase

 Clinical Utility of Direct Quantitative Test for the V617F JAK2 Allele Burden:

• To correlate with clinical splenomegaly, pruritus, leukocytosis or myelofibrosis
• To monitor disease burden post-treatment or as a marker for therapeutic intervention
• To identify prognostically relevant clonal proliferation
• JAK2 targeted therapies may soon be available

This test is performed by PCR-based Next Generation Sequencing of DNA extracted from formalin fixed paraffin embedded (FFPE) tissue or fresh tissue including peripheral blood and bone marrow.  The JAK2 V617F exon is sequenced using massively parallel sequencing (next-generation sequencing) with a combination of multiplexed PCR (customized QIAseq Targeted DNA panel) and sequencing on an Illumina platform.  An in-house bioinformatics analysis pipeline has been used that employs multiple established variant calling tools (FreeBayes, MuTect2 and Scalpel) and variant annotation tools.  The genomic variants have been interpreted in accordance with the 2017 guideline recommendations by AMP/ASCO/CAP (PMID: 27993330).  The assay is validated in accordance with the AMP guidelines (PMID: 28341590)

Sensitivity:
The lower detection limit of this assay is 1% VAF depending on read quality and quantity.

Specificity:
A clinical correlation is imperative for the interpretation of a result of this test.

• 5-10 mL of blood or bone marrow — yellow (ACD) or purple (EDTA) tube. 
• DNA: 100ng at minimum of 100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS)
•  If sample cannot arrive at laboratory within 24 hours of draw, refrigerate until sample can be transported.

A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES.  Please include detailed clinical information.

Weekly

7-10 days

• Keep specimen cold during transit, but do not ship on dry ice. 
• Please use the cold pack provided in the KDL shipping kit. 
• Ship the specimen overnight express, using the FedEx priority overnight label provided.
• Contact Client Services for shipping kit and instructions at (855) 535-1522.

1. Baxter, EJ.  Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders.  Lancet 2005; 365: 1054 – 1061.
2. Kralovics, R.  A gain-of-function mutation of JAK2 in Myeloproliferative disorders. N Engl J Med 2005; 352: 1779 – 1790.
3. Jones, A. et al.  Widespread occurrence of the JAK2 V617F Mutation in Chronic Myeloproliferative Disorders.  Blood 2005; 106: 2162 - 2168.
4. Tafferi, A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH, and IKZF1. Leukemia 2010; 24: 1128-1138.