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Test 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

Reporting Name

MPN, CALR Gene Mutation, Exon 9

Specimen Type

Varies


Ordering 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.

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 days

Performing 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

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