Dear Editor,
The discovery of mutations in CALR, the gene that encodes calreticulin, in a significant proportion of patients with the myeloproliferative neoplasms (MPN) of essential thrombocythemia (ET) and primary myelofibrosis (PMF) drastically altered the molecular landscape of these diseases (Klampfl et al., 2013[4]; Nangalia et al., 2013[6]). The C-terminal of the calreticulin protein includes an endoplasmic reticulum (ER) KDEL retention signal that has a strong calcium-binding capacity thus acting as a calcium storage protein. Additionally, CALR acts as a molecular chaperone enabling glycoprotein folding in the ER. Nearly all mutations of CALR are either insertions, deletions or insertion and deletions (indels) that result in a loss of the terminal KDEL signal. Mutant CALR interacts with the thrombopoietin receptor MPL by a unique mechanism resulting in constitutive activation of the downstream JAK-STAT pathway and cellular transformation (Edahiro et al., 2020[2]).
Early studies suggested that CALR indel mutations were found inclusively in ET and PMF however expanded screening has uncovered sporadic cases exhibiting a polycythemic phenotype (Broséus et al., 2014[1]; Langabeer et al., 2017[5]). As CALR mutations are absent in polycythemia vera (PV; which is defined by the presence of JAK2 mutations), this most likely represents one end of the phenotypic spectrum of CALR-mutated MPN. More recently, mutations in the 3' untranslated region of CALR, an area not covered by conventional diagnostic approaches, have been identified in patients phenotypically resembling PV (Quattrocchi et al., 2020[7]).
A variety of molecular diagnostic platforms exist to detect CALR mutations such as capillary electrophoresis, real-time PCR, and increasingly, next-generation sequencing. Both deep sequencing and capillary electrophoresis screening have revealed a complexity of CALR mutations as evidenced by co-existing, multiple indels which would not be revealed by use of real-time screening for the most common CALR exon 9 indels (Jeromin et al., 2016[3]; Verger et al., 2020[9]).
A further confounding observation is the low frequency of in-frame CALR indels that would be predicted to retain the terminal KDEL region in some patients with features hematologically suggestive of an MPN (Szuber et al., 2016[8]; Verger et al., 2020[9]). These in-frame mutations have an allelic frequency of approximately 50 %, hence suggestive of a polymorphism, and although not diagnostic of an MPN, require further functional characterization.
Taken together, these more recent findings further underscore the clonal complexity of CALR-mutated MPN and provide a continuing challenge for improvement of molecular diagnostics algorithms.