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

Abstract

The identification of somatic calreticulin (CALR) mutations can be used to confirm the diagnosis of a myeloproliferative disorder in Philadelphia chromosome-negative, JAK2 and MPL wild type patients with thrombocytosis. All pathogenic CALR mutations result in an identical C-terminal protein and therefore may be identifiable by immunohistochemistry. We sought to test the sensitivity and specificity of mutation specific immunohistochemistry for pathogenic CALR mutations using a commercially available mouse monoclonal antibody (clone CAL2). Immunohistochemistry for mutant calreticulin was performed on the most recent bone marrow trephine from a cohort of patients enriched for CALR mutations and compared to mutation testing performed by polymerase chain reaction (PCR) amplification followed by fragment length analysis. Twenty-nine patients underwent both immunohistochemistry and molecular testing. Eleven patients had CALR mutation, and immunohistochemistry was positive in nine (82%). One discrepant case appeared to represent genuine false negative immunohistochemistry. The other may be attributable to a 12 year delay between the bone marrow trephine and the specimen which underwent molecular testing, particularly because a liver biopsy performed at the same time as molecular testing demonstrated positive staining in megakaryocytes in extramedullary haematopoiesis. All 18 cases which lacked CALR mutation demonstrated negative staining. In this population enriched for CALR mutations, the specificity was 100%; sensitivity 82-91%, positive predictive value 100% and negative predictive value 90-95%. We conclude that mutation specific immunohistochemistry is highly specific for the presence of CALR mutations. Whilst it may not identify all mutations, it may be very valuable in routine clinical care.