Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value

Abstract

Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

Figure 1

Generation and characterization of the anti-mutated CALR antibody. In panel a, the sequence of the 17-mer peptide used for generation of the anti-mutated CALR antibody is shown in relation with the amino-acid sequence of wild-type CALR and the predicted sequence of mutated CALR originated from the del52 and ins5 abnormalities. In panel b, gel electrophoresis of the mutated calreticulin prepared in BL21DE3RIPL (lanes 2 and 3) and JM109DE3 (lanes 4 and 5) E. Coli strains; only in the latter strain successful production of calreticulin was obtained. Calreticulin has a molecular weight of 47 kDa, but migrates in these conditions at an apparent higher molecular weight likely owing to a specific conformational pattern. In panel c, western blot analysis shows anti-mutated CALR antibody selectivity for mutated protein vs the wild-type form. CALR recombinant protein expressed in E. Coli was used as a positive control. GAPDH was used as a loading control.

Figure 2

Immunostaining of bone marrow biopsies with the anti-mutated CALR antibody. Representative sections from CALR-unmutated ET/PMF patients (JAK2V617F mutated, MPLW515L mutated and triple-negative mutation) and from CALR-mutated patients (CALRdel52, CALRins5 and CALRindel) are shown in panel a and panel b, respectively. Sections were stained according to the procedure described in the Materials and Methods section using a 1:1000 dilution of the anti-mutated CALR antibody. Pictures were taken with a LEICA DM LS2 microscope using a N-Plan × 40/0.65 objective. The number of immunostained megakaryocytes over total number of morphologically recognizable megakaryocytes was calculated by counting at least 100 megakaryocytes/slide from 10 patients with ET and 10 patients with PMF (n=2 for the CALR indel), and expressed as percent of total megakaryocytes (c); as there was no significant difference between the two groups, all individual data were pooled.

Figure 3

Immunostaining of bone marrow biopsies with the anti-mutated CALR antibody. Panel a shows two megakaryocytes labeled by the anti-mutated calreticulin antibody together with a negative one. In panel b, abnormal, small megakaryocytes in the bone marrow of a CALRins5 PMF patient are shown. Panel c shows a low-resolution picture of an advanced fibrosis in a CALRdel52 patient and d is a high-power field from panel c (square) to show the abnormally shaped large megakaryocytes within buddles of fibers. In panels e and f, the faint label of erythroid and myeloid cells is presented at higher magnification.

Figure 4

Immunostaining of bone marrow biopsies with the anti-wild-type calreticulin antibody. Representative sections from CALR-unmutated ET/PMF patients (JAK2V617F mutated, MPLW515L mutated and triple-negative mutation) and from CALR-mutated patients (CALRdel52, CALRins5 and CALRindel) are shown in panel a and panel b, respectively.

Figure 5

CALR expression in hematopoietic progenitors and differentiated cells. CALR expression profile were analyzed in purified CD34⁺ cells, erythroblasts, megakaryocytes and neutrophils. (a) Heatmap showing the mRNA expression detected by the three different CALR probesets present on the HG-U133A array. Gene expression coloring was based on normalized signals, as shown at the bottom. The expression values were normalized on the median of all samples. (b) Bar graph displaying the average normalized expression detected by the three CALR probesets in the populations examined.

Figure 6

Diagnostic algorithms for CALR-mutated myeloproliferative neoplasms. In scenario 1, a standard ‘genotype-exclusive' diagnostic algorithm is presented, whereas in the flow chart shown in scenario 2 immunostaining with anti-mutated calreticulin is used at the time of bone marrow biopsy to rule out a CALR-mutated MPN before genotyping for JAK2V617F and MPL mutations. See Discussion for details.