Home
HelpTest Code CALR CALR Mutation Analysis, Myeloproliferative Neoplasm (MPN), Varies
Useful For
Rapid and sensitive detection of insertion and deletion-type mutations in exon 9 of CALR
Aiding in distinguishing between reactive thrombocytosis and leukocytosis versus a myeloproliferative neoplasm (MPN), especially essential thrombocythemia (ET) and primary myelofibrosis (PMF), and is highly informative in cases in which JAK2 and MPL testing are negative
Especially helpful to the pathologist in those bone marrow cases with ambiguous etiology of thrombocytosis, equivocal bone marrow morphologic findings of MPN, and unexplained reticulin fibrosis
Aiding in the prognostication of PMF and thrombosis risk assessment in ET
Special Instructions
Reporting Name
MPN, CALR Gene Mutation, Exon 9Specimen Type
VariesOrdering Guidance
Shipping Instructions
Specimen must arrive within 7 days of collection.
Necessary Information
Specimen source is required
Specimen Required
Submit only 1 of the following specimens:
Specimen Type: Whole blood
Container/Tube: Lavender top (EDTA) or yellow top (ACD)
Specimen Volume: 3 mL
Collection Instructions:
1. Invert several times to mix blood.
2. Label specimen as blood.
3. Send whole blood specimen in original tube. Do not aliquot.
Specimen Stability Information: Ambient (preferred)/Refrigerate
Additional Information: To ensure minimum volume and concentration of DNA is met, the preferred volume of blood must be submitted. Testing may be canceled if DNA requirements are inadequate.
Specimen Type: Bone marrow
Container/Tube: Lavender top (EDTA) or yellow top (ACD
Specimen Volume: 2 mL
Collection Instructions:
1. Invert several times to mix bone marrow.
2. Label specimen as bone marrow.
3. Send bone marrow specimen in original tube. Do not aliquot.
Specimen Stability Information: Ambient (preferred)/Refrigerate
Specimen Type: Extracted DNA from blood or bone marrow
Container/Tube: 1.5- to 2-mL tube
Specimen Volume: Entire specimen
Collection Instructions:
1. Label specimen as extracted DNA from blood or bone marrow
2. Include indication of volume and concentration of the DNA.
Specimen Stability Information: Frozen (preferred)/Refrigerate/Ambient
Specimen Minimum Volume
Whole blood/bone marrow: 1 mL; Extracted DNA: 50 mcl at 20 ng/mcL concentration
Specimen Stability Information
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Varies | Varies | 7 days |
Reject Due To
| Gross hemolysis | Reject |
| Paraffin-embedded bone marrow aspirate clot Bone marrow biopsies, slides, or paraffin shavings Moderately to severely clotted |
Reject |
Clinical Information
The most frequent genetic mutation in BCR-ABL1-negative myeloproliferative neoplasm (MPN), essential thrombocythemia (ET), and primary myelofibrosis (PMF) is the JAK2V617F alteration, which is present in approximately 50% to 60% of patients. It serves as a confirmatory molecular marker of these diseases. Mutations in the MPL gene are found in an additional 5% to 10% of ET and PMF cases. It was recently discovered that somatic mutation (insertions and deletions) in exon 9 of the CALR gene is the second most frequent somatic mutation after JAK2 in ET and PMF patients, and it is mutually exclusive of JAK2 and MPL mutations.(1,2) It has a frequency of approximately 49% to 88% in JAK2 and MPL-wild type (WT) ET and PMF and is not found in polycythemia vera (PV) patients.(1-4) Therefore, the CALR mutation serves as an important diagnostic molecular marker in ET and PMF.
The CALR gene encodes for calreticulin, a multifunctional protein with a C-terminus rich in acidic amino acids and a KDEL endoplasmic reticulum (ER)-retention motif. All the disease-causing CALR mutations reported to date are out-of-frame insertion and/or deletions in exon 9, generating a 1 base pair (bp) frame shift and an altered protein with a novel C-terminus rich in basic amino acids and loss of the KDEL ER-retention signal. The most common mutation types are 52 bp-deletion (c.1092_1143del, L367fs*46) and 5-bp insertion (c.1154_1155insTTGCC, K385fs*47), and they comprise approximately 85% of CALR mutations in MPN.(1,2) CALR mutations have been found in hematopoietic stem and progenitor cells in MPN patients(2) and may activate the STAT5 signaling pathway.(1) They are associated with decreased risk of thrombosis in ET (1,3-5), and better survival in PMF compared to JAK2 mutations.(5)
Reference Values
An interpretive report will be provided
Interpretation
An interpretive report will be issued.
The results will be reported as 1 of the 3 states if DNA amplification is successful (see Cautions):
-Positive. A deletion-insertion-type mutation was detected in CALR, exon 9.
-Negative. No deletion or insertion was detected in CALR, exon 9.
-Equivocal. A small amplicon suspicious for a deletion-insertion type mutation was detected in CALR, exon 9.
Positive mutation status is highly suggestive of a myeloid neoplasm but must be correlated with clinical and other laboratory and morphologic features for definitive diagnosis.
Negative mutation status does not exclude the presence of a myeloproliferative neoplasm or other neoplastic disorders.
Clinical Reference
1. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutation of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369(25):2379-2390
2. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mutation in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013;369(25):2391-2405
3. 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. 2014;123(10):1544-1551
4. Rotunno G, Mannarelli C, Guglielmelli P, et al. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood. 2014;123(10):1552-1555
5. 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(7):1472-1477
6. Greenfield G, McMullin MF, Mils K. Molecular pathogenesis of the myeloproliferative neoplasms. J Hematol Oncol. 2021;14(1):103
Method Description
Polymerase chain reaction (PCR) amplification of CALR exon 9 is performed on DNA isolated from the patient sample. The PCR product is then run on an ABI 3130xl Genetic Analyzer for fragment analysis to detect insertions and deletions. An unmutated CALR will show an amplicon at 266 base pairs (bp), a mutated CALR with insertion will show an amplicon greater than 266 bp, and a mutated CALR with deletion will show an amplicon smaller than 266 bp. This assay has an analytical sensitivity of approximately 6% (ie, 6 mutation-containing cells in 100 total cells) in most mutation types, except for the rare type of 1-bp deletion, which has a sensitivity of approximately 20%. This is a laboratory developed test.(Unpublished Mayo method)
Day(s) Performed
Monday through Friday
Report Available
3 to 5 daysSpecimen Retention Time
Whole blood/Bone marrow: 2 weeks; Extracted DNA: 3 monthsPerforming Laboratory
Mayo Clinic Laboratories in Rochester
Test Classification
This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.CPT Code Information
81219-CALR (calreticulin) (eg, myeloproliferative disorders), gene analysis, common variants in exon 9
LOINC Code Information
| Test ID | Test Order Name | Order LOINC Value |
|---|---|---|
| CALR | MPN, CALR Gene Mutation, Exon 9 | 77174-1 |
| Result ID | Test Result Name | Result LOINC Value |
|---|---|---|
| MP020 | Specimen | 31208-2 |
| 36301 | Final Diagnosis | 22637-3 |
Testing Algorithm
The following algorithms are available:
-Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation
-Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation
Cautions
A positive result is not specific for a particular myeloproliferative neoplasm (MPN) diagnosis, and clinicopathologic correlation is necessary in all cases.
A negative result does not exclude the presence of an MPN or other neoplastic process.
This test is a fragment analysis assay and only detects deletions-insertions (delins). It will not detect point mutations. However, all reported disease-causing mutations in MPN described to date are insertions and/or deletions.
This test may not differentiate between out-of-frame and in-frame delins in rare cases. However, in-frame delin mutations are very rare (<0.5%) and have only been reported in a few healthy individuals and myeloproliferative neoplasm patients with JAK2V617F mutation or out-of-frame CALR mutation. Most of the rare in-frame delins are considered germline variants and represent benign alterations (ie, polymorphisms).
Infrequently, amplification failure can be encountered in a given sample due to inadequate DNA, poor DNA quality, or a polymerase chain reaction inhibitor. In these circumstances, the assay will be reattempted, and if persistently unsuccessful, the report will be issued with an "Invalid" result.
Method Name
Polymerase Chain Reaction (PCR) and Fragment Analysis
Forms
1. Hematopathology Patient Information (T676)
2. If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request (T726) with the specimen.