Hematological Malignancies CALRETICULIN (CALR) Gene Mutations in Myeloproliferative Neoplasms

The calcium-binding endoplasmic reticulin chaperone protein Calreticulin (CALR) has recently been shown to be somatically mutated in the majority of patients with a myeloproliferative neoplasm that lack a mutation in the JAK2 gene. In particular, two independent groups (1, 2) have each recently found a variety of insertion or deletion mutations in exon 9 of the calreticulin gene in:

• ~70% of patients with JAK2-negative essential thrombocythemia (ET)
• ~60-80% of patients with JAK2-negative primary myelofibrosis
•  A minority of patients with myelodysplasia (8%)
•  Zero patients with de novo acute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia, or solid tumors

At the protein level, to date, all of the heterogeneous mutations in CALR each creates the same +1 basepair frameshift mutation that truncates the C-terminal calcium-binding acidic domain of the CALR protein (including the KDEL endoplasmic reticulin retrieval signal) and replaces it with a novel C-terminus (1,2).

The availability of a new clinical diagnostic assay for these common exon 9 calreticulin gene mutations, together with the analysis of the JAK2 gene for the common V617F mutation (and to a lesser degree MPL mutations), now allows a specific molecular marker to be identified (and potentially monitored) in the overwhelming majority (>90%) of patients with a myeloproliferative neoplasm.

The detection of a CALR gene mutation may aid in the specific diagnosis of a myeloproliferative neoplasm, and help distinguish this clonal disease from a benign reactive process. After the MPN diagnosis is made, the presence of a CALR gene mutation also predicts a more indolent disease course with a lower thrombotic risk and longer overall survival (relative to those with a JAK2 mutation) (1). Although specific targeted therapy for calreticulin-mutated MPN patients has not yet been described, the in vitro expression of this mutation conveys cytokine-independent growth, activated STAT5 signaling, and sensitivity to JAK2 inhibitors (1).  The presence of a clonal CALR gene mutation may also allow minimal residual disease to be molecularly monitored during the course of therapy.

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 exon 9 in CALR 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.

• 5-10 mL of blood or bone marrow — yellow (ACD) or purple (EDTA) tube.
• If sending DNA, please send 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).
• If sample cannot arrive at laboratory within 24 hours of draw, refrigerate until sample can be transported. Samples refrigerated for up to 7 days will be accepted (but <1 day is ideal).

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

Two times per week

7-10 days

• For fresh blood and/or bone marrow, 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. T Klampfel et al. Somatic mutations of calreticulin in myeloproliferative neoplasms.New Engl J Med (2013) 369:2379-2390.
2. J Nangalia et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. New Engl J Med (2013) 369:2391-2405.