CALR Mutation Detection
Polymerase Chain Reaction with fragment length analysis by capillary electrophoresis
Formalin-fixed paraffin-embedded tissue/bone marrow clot
Lavender BD Hemogard™ K2EDTA Tube
Formalin-fixed paraffin-embedded tissue/bone marrow clot:
The BCR/ABL1-negative myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).1 The JAK2 V617F point mutation occurs in >95% of cases of PV and approximately 50-60% of cases of ET and PMF. In ET and PMF lacking the JAK2 V617F mutation, approximately 10-20% contain a mutation in MPL exon 10 while 60-80% of cases have a mutation in CALR.2,3
The identification of a JAK2, MPL, or CALR mutation is diagnostically useful to separate MPN from a reactive leukocytosis that may mimic a myeloid neoplasm. Cases of ET and PMF with mutations of JAK2, MPL, or CALR may also show prognostic differences.4–7
Approximately 80% of CALR mutations can be classified as either type 1 (a 52-bp deletion) or type 2 (a 5-bp insertion). The remaining mutations represent other, variably sized insertions and deletions. All CALR mutations (type 1, type 2 or other) create a frameshift with the production of an altered C-terminus of the calreticulin protein.
Cleveland Clinic Laboratories has developed, validated and implemented a sensitive PCR assay for the detection of CALR mutations in peripheral blood, bone marrow or formalin-fixed, paraffin-embedded tissues.
CALR mutation testing is useful in the workup of suspected myeloproliferative neoplasms, especially those that are negative for JAK2 V617F.
Normal results are reported as “CALR mutation not detected.”
Positive results are reported as “CALR mutation detected,” and an interpretation is provided that includes a description of the mutation (type 1, type 2, or other).
Genomic DNA is extracted from the sample and CALR exon 9 is amplified by PCR. Fragment length analysis is performed to assess for insertion/deletion mutations.
This assay has a sensitivity of 5% mutant alleles. This assay detects only insertion/deletion mutations in CALR exon 9, and a negative result does not exclude the possibility of a myeloproliferative neoplasm.
1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008.
2. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013;369:2391-405.
3. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369:2379-90.
4. Tefferi A, Lasho TL, Finke CM, et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia. 2014;28:1472-7.
5. Tefferi A, Wassie EA, Lasho TL, et al. Calreticulin mutations and long-term survival in essential thrombocythemia. Leukemia. 2014;28:2300-3.
6. Rumi E, Pietra D, Pascutto C, et al. Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis. Blood. 2014;124:1062-9.
7. Rumi E, Pietra D, Ferretti V, et al. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood. 2013;123:1544-1551.