Background:

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.

Methodology:

Polymerase chain reaction (PCR) of exon 9 of the CALR gene followed by size-based fragment analysis and/or direct DNA sequencing.

Sensitivity:
1% mutant allele burden for calreticulin mutation detection.

Specimen Requirements:

  • 5-10 mL of blood or bone marrow — yellow (ACD) or purple (EDTA) tube.
  • Heparin anticoagulated blood or bone marrow sampels are acceptable.
  • If sending DNA, please send 200ng at a minimum of 25ng/µL
  • 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).
  • Fresh or snap-frozen tissue samples are acceptable. Fresh tissue should be placed into liquid tissue culture media and shipped cold (not frozen). The preferred procedure for tissue samples is to snap-freeze in liquid nitrogen and transport (frozen) on dry ice.
  • Formalin-fixed paraffin embedded (FFPE) tissue will also be accepted (fresh or frozen is always better, if available), but depending on the heterogeneous fixation procedure in individual histology labs, this sample type will be much more likely to yield degraded DNA that will not be diagnostically informative.

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

Test Performed (Days):

Two times per week

Turn Around Time:

7-10 days

Shipment Sensitivity Requirements:

  • 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.

References:

  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.

Additional Info:

The Knight Cancer Institute at Oregon Health & Science University is a pioneer in the field of precision cancer medicine. The institute's director, Brian Druker, M.D., helped prove it was possible to shut down just the cells that enable cancer to grow. This breakthrough has made once-fatal forms of the disease manageable and transformed how cancer is treated. The OHSU Knight Cancer Institute is the only National Cancer Institute-designated Cancer Center between Sacramento and Seattle – an honor earned only by the nation's top cancer centers. It is headquarters for one of the National Cancer Institute's largest research collaboratives, SWOG, in addition to offering the latest treatments and technologies as well as hundreds of research studies and clinical trials.

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