Clinical validation of a multipurpose assay for detection and genotyping of CALR mutations in myeloproliferative neoplasms.

Abstract

To develop a polymerase chain reaction (PCR)-based approach to detect CALR mutations in myeloproliferative neoplasms (MPNs) in a clinical laboratory. DNA was extracted from bone marrow aspirate samples of 67 JAK2 wild-type MPNs (22 with matched peripheral blood), 54 cases of unclassifiable myelodysplastic syndrome/MPN, and 16 cases of atypical chronic myeloid leukemia. We used genomic DNA to detect somatic mutations in exon 9 of CALR and PCR with fluorescently labeled and M13-tagged primers and subjected the products to capillary electrophoresis (CE) followed by Sanger sequencing. Detailed assay performance characteristics were established. We identified CALR mutations in 19 (28.4%) of 67 JAK2-negative MPNs, including 14 type I (52-base pair [bp] deletion), four type II (5-bp insertions), and one type III (18-bp deletion). All mutations were confirmed by Sanger sequencing. Sensitivity studies showed 2.5% and 5% mutation detection levels by CE and Sanger sequencing, respectively, with high reproducibility. This assay allows for rapid, convenient screening for CALR mutations in MPNs, thereby reducing the number of cases that require assessment by Sanger sequencing, reducing labor and improving turnaround time.