Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2

Abstract

Background: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge.

Methods: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry.

Results: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients.

Conclusions: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Figure 1. Mutational Profile of 151 Myeloproliferative Neoplasms

Panel A shows the number and type of mutations identified on exome sequencing in each sample obtained from 151 patients with myeloproliferative neoplasms. These patients included 48 with polycythemia vera (PV), 62 with essential thrombocythemia (ET), 39 with myelofibrosis (MF), and 2 with unclassifiable myeloproliferative neoplasms (MPN-U). The type of mutation is indicated in the key in each panel; the circles below the graphs indicate the patients’ mutational status: JAK2, MPL, or no JAK2 or MPL mutation. Also shown are the numbers of somatic mutations in recurrently mutated genes in this study as well as in genes previously reported to be mutated in myeloproliferative neoplasms, according to the type of mutation (Panel B) and subtype of myeloproliferative neoplasm (Panel C). In Panel B, the asterisks denote the significance of recurrently mutated genes (q<0.05). Indel denotes insertion or deletion mutation.

Figure 2. CALR Mutations in Myeloproliferative Neoplasms

Panel A shows the number of CALR mutations found in myeloproliferative neoplasms (polycythemia vera, PV; essential thrombocythemia, ET; or myelofibrosis, MF) that have mutated JAK2, mutated MPL, or nonmutated JAK2 or MPL. Panel B shows the results of validation on polymerase-chain-reaction (PCR) assay and Sanger sequencing of the two most common CALR variants — deletion (L367fs*46) and insertion (K385fs*47) — in patients with myeloproliferative neoplasms. The patients are indicated by their patient-identification numbers above the results of gel electrophoresis of PCR products of patients’ granulocytes (Gran) and T cells. The sequencing traces show the heterozygous mutation of CALR. The shaded region on the left highlights a homologous DNA sequence flanking the common CALR deletion, and the green arrow on the right highlights the inverse tandem duplication of five bases in the common CALR insertion. Panel C shows the genomic location of CALR deletions. The numbers indicate the number of patients with each disease. Panel D shows the genomic location of CALR insertions (shown in red). aCML denotes atypical chronic myeloid leukemia, CMML chronic myelomonocytic leukemia, and MDS myelodysplastic syndromes.

Figure 3. Altered Protein Reading Frame with Novel C-Terminal Associated with CALR Mutations

Panel A shows the functional domains of CALR protein, with (from left to right) signal sequence, N domain (N-terminal), P domain (proline-rich), C domain (C-terminal), and KDEL (endoplasmic reticulum retention signal). The conservation of the affected portion of the C domain across species is depicted by shading, with black indicating conserved regions and gray indicating partially conserved regions. The range of frameshift insertion and deletion mutations in CALR exon 9 are shown, all of which result in a common +1 base-pair–altered reading frame and predict a novel C-terminal peptide sequence lacking the KDEL motif. Panel B shows the mutational spectra of CALR in samples of myeloproliferative neoplasms (MPN) and of common loss-of-function genes such as ASXL1 and TET2 in myeloproliferative neoplasms and acute myeloid leukemia (AML). The numbers of each type of mutation are indicated for each gene. Data regarding myeloproliferative neoplasms are from the exome subgroup in this study, and AML data are from the Cancer Genome Atlas.

Figure 4. Localization of Calreticulin and Clonal Heterogeneity in Patients with CALR Mutations

Panel A shows immunoblotting of transiently transfected human embryonic kidney (HEK) 293T cells analyzed for FLAG (a polypeptide protein tag), CALR (detecting endogenous as well as transfected calreticulin), and beta-actin. The CALR deletion is the L367fs*46 variant, and the CALR insertion is the K385fs*47 variant. The CALR expression construct is shown above the immunoblots. CMV-Pr denotes CMV promoter. Panel B shows confocal photomicrographs of COS-7 cells transiently expressing FLAG-tagged CALR variants and a Golgi reporter (galactosyltransferase fused to yellow fluorescent protein [GalT-YFP]). Red indicates FLAG; green, Golgi; and blue, nucleus (4′,6-diamidino-2-phenylidole dihydrochloride [DAPI]). The images show that nonmutant and mutant CALR have an endoplasmic reticulum localization pattern with no increased accumulation in the Golgi. Panel C shows confocal photomicrographs of COS-7 cells transiently coexpressing nonmutant (NM) CALR tagged with green fluorescent protein (GFP) and FLAG-tagged CALR variants. Merge images show that FLAG-tagged CALR variants colocalize with nonmutant CALR to the endoplasmic reticulum. Panel D shows confocal photomicrographs of myeloid cells from CALR mutated and nonmutated granulocyte–macrophage colony-forming unit colonies derived from a patient with essential thrombocythemia with the CALR K385fs*47 mutation; the cells have been stained for protein disulfide isomerase (PDI, a resident protein of the endoplasmic reticulum) and endogenous CALR. Panel E shows flow cytometric analysis indicating the degree of CALR cell-surface expression in granulocytes (G) and lymphocytes (L) from a healthy control and from a patient with mutated CALR. The graph shows the percentage of viable cells expressing cell-surface CALR in peripheral blood from healthy controls (triangles) and patients with mutated CALR (circles). FSC denotes forward scatter. Panel F shows clonal structures in five patients (indicated by their patient-identification numbers) with myeloproliferative neoplasms with mutated CALR, as determined on genotyping of hematopoietic (erythroid) colonies. Each circle represents a clone, with nonmutant clones shown in white and mutant clones in brown. The earliest detectable clone is represented at the top of each diagram, with subsequent subclones shown below. Somatic mutations that were acquired in each subclone are indicated beside the respective nodes and represent those that were acquired in addition to mutations present in earlier subclones. Numbers of colonies that were identified for each node are shown inside the circles. ET denotes essential thrombo cythemia, and PET-MF post-ET myelofibrosis.