Hematological Malignancies JAK2 Exon 12 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); and also present infrequently (3-5%) in myelodysplastic syndrome (MDS) & CMML.

In conjunction with a histopathologic evaluation, the World Health Organization diagnostic criteria for polycythemia vera include a test for the JAK2 V617F mutation in exon 14.  Alternatively, the presence of a JAK2 exon 12 mutation also meets the WHO criterion for molecular evidence of clonality in cases negative for JAK2 V617F.  Typically, JAK2 exon 12 mutation for PV is indicated when the V617F mutation has been ruled out.

Clinical Utility of Direct Quantitative Test for the V617F or Exon 12 Allele Burden:

• To correlate with clinical splenomegaly, pruritus, leukocytosi 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.  Hotspot JAK2 exon 12 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 1-2% 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: 200ng at a minimum of 25ng/µL  (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS)
• Contact Client Services for shipping kit and instructions at (855) 535-1522. 
• If sample cannot arrive at laboratory within 24 hours of draw, refrigerate until sample can be transported.

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 label provided.

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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.
5. Scott, LM. et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. New Engl J Med 2007; 356:459-468.
6. Pardanani A. et al. Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2 V617F-negative polycythemia vera.  Leukemia 2007; 21:1960-1963.