CALR Mutation

Test Description:
Molecular test for detection of mutations in the calreticulin (CALR) gene

Indication:
Recently, mutations in the calcium-binding endoplasmic reticulin chaperone protein Calreticulin
(CALR) have been detected in the majority of patients with a myeloproliferative neoplasm
(MPN) that lack a mutation in the JAK2 gene (1, 2). CALR has been shown to be somatically
mutated in about 70% in patients with JAK2-negative essential thrombocythemia (ET), ~60-85%
of patients with JAK2-negative primary myelofibrosis, and in 8% of patients with
myelodysplasia. The detection of a CALR mutation may aid in the diagnosis of a
myeloproliferative neoplasm helping to distinguish the clonal disease from a benign reactive
process. In addition, MPN patients with CALR mutation have a more indolent disease course
with a lower thrombotic risk and longer overall survival as compared to those with a JAK2
mutation. Specific targeted therapy for calreticulin-mutated MPN patients has not yet been
described, but the in vitro study suggests that mutant CALR can activate STAT5 signaling and
may convey sensitivity to JAK2 inhibitors (1-3).

CALR mutations are small deletions and insertions in exon 9 of the gene. The most common are
Type 1 (52 bp deletion, c.1092_1143del, p.L367fs*46) and Type 2 (5 bp insertion,
c.1154_1155insTTGCC, p. K385fs*47) mutations that are found in 53% and 32% of all cases
with mutant CALR. All mutations result in a frame shift, generating a novel C-terminus of the
mutated protein.

References:
1. K Thorsten et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J
Med. 2013;369:2379-2390.
2. Nangalia J, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated
JAK2. N Engl J Med. 2013;369:2391-2405.
3. Rotunno G et al. Impact of calreticulin mutations on clinical and hematological phenotype and
outcome in essential thrombocythemia.Blood.2013;11:538983.Fecher LA et al. Toward a
molecular classification of melanoma. J Clin Oncol. 2007 20;25(12):1606-20.

Methodology:
DNA is isolated from submitted specimen using standard laboratory procedure. For surgical
specimens, manual microdissection is performed after selection of appropriate area by a
pathologist. DNA is amplified with primers flanking CALR exon 9 sequence. Bidirectional
Sanger sequencing is performed using the BigDye Terminator Kit on ABI3730 (Applied
Biosystems). The sequencing electropherograms are analyzed for the presence of mutations with
Mutation Surveyor V3.01 (SoftGenetics). The limit of detection is approximately 10% of mutant
alleles present in the background of normal DNA.
Performed:
Wednesday

Reported:
Within 7‐10 business days of receipt.

CPT:
81479

Specimen Requirements:
Peripheral Blood: 3-5 ml, collected in EDTA (purple top) tube, store at room temperature 24
hours
Bone Marrow: 0.5-1ml, collected in EDTA (purple top)tube, store at room temperature, 24
hours
Unacceptable Specimens: Frozen blood or bone marrow specimens are unacceptable as are
tissue samples that have undergone a freeze/thaw cycle(s). Bone marrow/ Peripheral blood
specimens post treatment with very low blast count.

Shipment Must Include:

 Specimen
 Requisition form
 Patient’s pathology report