A new monoclonal antibody (CAL2) detects CALRETICULIN mutations in formalin-fixed and paraffin-embedded bone marrow biopsies

Abstract

Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method.

CAL2 Immunohistochemical Staining Accurately IdentifiesCALRMutations in Myeloproliferative Neoplasms

“…However, as reported by others and shown in the present work, mutated CALR is preferentially expressed in megakaryocytes. 12,13 The reason for this has been elucidated. 14 Mutant CALR has been reported to deregulate JAK/ STAT signaling pathways by interacting with the thrombopoietin receptor (MPL), and the extracellular domain of MPL is crucial for activation by CALR mutants.…”

“…We analyzed the performance of a recently published and commercially available mutant CALR specific monoclonal antibody (CAL2) suitable for immunohistochemistry in formalin-fixed, decalcified bone marrow biopsy specimens. 13 Using double immunofluorescence, we also further characterize immunoreactivity of CAL2 in bone marrow cells.…”

“…11 Recently, two groups worked on developing antibodies for immunohistochemical staining of bone marrow biopsy specimens to detect mutant CALR. 12,13 Currently, molecular methods are the standard for detection of these mutations. An immunohistochemical stain that consistently detects the mutation in formalin-fixed bone marrow biopsy specimens would be a rapid and inexpensive way to identify patients with these mutations in routine practice.…”

Mutation specific immunohistochemistry is highly specific for the presence of calreticulin mutations in myeloproliferative neoplasms

“…4,5 Given that the many different pathogenic mutations result in an identical epitope, mutation specific immunohistochemistry directed to the C-terminus of mutated calreticulin would be a logical and cost effective way to screen for these mutations. Indeed, recently both Vannucchi et al, 7 using a rabbit polyclonal antibody, and Stein et al, 8 using a mouse monoclonal antibody, have demonstrated excellent sensitivity and specificity in the research laboratory setting with this approach.…”

“…A mouse monoclonal anti-mutant calreticulin antibody (clone CAL2, the same as that used by Stein et al 8 ) has recently become commercially available. Therefore, we sought to test its sensitivity and specificity in a diagnostic surgical pathology laboratory to determine if mutation specific immunohistochemistry could supplement or replace molecular testing for the identification of pathogenic calreticulin mutations in the routine clinical setting.…”