Frequent CALR exon 9 alterations in JAK2 V617F-mutated essential thrombocythemia detected by high-resolution melting analysis

Essential thrombocythemia (ET) is a clonal hematopoietic stem cell neoplasm and one of the classic BCL-ABL1-negative chronic myeloproliferative neoplasm (MPN), which also includes polycythemia vera and primary myelofibrosis (PMF). Recently, two seminal studies discovered a high frequency of somatic calreticulin (CALR) mutations in patients with JAK2/MPL-unmutated ET and PMF. The pattern of most CALR mutations in MPN is heterozygous indels in exon 9 causing one-base pair (bp) reading frameshift. CALR mutations have been shown to have important diagnostic and prognostic significance in ET and PMF patients, and will likely be incorporated into the World Health Organization (WHO) diagnostic criteria for MPN. In vitro studies on the molecular pathogenesis of CALR mutations in MPN have shown controversial results in regard to the involvement and/or activation of the JAK/STAT signaling pathway, and the exact pathogenesis of CALR mutations is not yet completely understood at the present time. Several techniques such as Sanger sequencing and polymerase chain reaction (PCR) followed by fragment analysis have been used to detect CALR mutations. High-resolution melting analysis (HRMA) is a well-established method for the screening of mutations, and we have developed a rapid and sensitive HRMA for the detection of CALR exon 9 mutations. In this study, we sought to screen a cohort of 92 Taiwanese ET patients for CALR exon 9 mutations with HRMA and Sanger sequencing independently, and to determine the clinical and molecular correlates. The institutional review board of Mackay Memorial Hospital has approved the screening for mutations. All patients provided written informed consent. Diagnosis of ET was established on the basis of the 2008 WHO criteria. The clinical and laboratory characteristics at the time of diagnosis or referral were collected. Genomic DNAs derived from the bone marrow, peripheral blood and peripheral blood granulocytes and/or mononuclear cells were used for mutation screening. CALR mutations were screened by Sanger sequencing on an ABI 3730 sequencer as preciously described. CALR exon 9 mutations were independently screened by HRMA using a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) as previously described with a maximal sensitivity of 2.5% for both CALR type 1 and type 2 mutants. Briefly, a pair of oligonucleotide primers were used to amplify a 134-bp amplicon (GenBank: NM_004343), which flanked all CALR exon 9 variants reported in MPN. All samples with distinguished melting curves from wild type were confirmed by duplicate studies. Peripheral blood samples from 78 healthy adults were also used to validate the specificity of our HRMA. JAK2 V617F mutation was screened using allele-specific PCR with an analytic sensitivity of 5% and MPL exon 10 mutation using Sanger sequencing as previously described. TA-cloning was performed using the pGEM-T easy vector system (Promega, Madison, CA, USA) as previously described. At least 10 clones in each individual were randomly selected for the screening of CALR exon 9 alterations by Sanger sequencing. All novel single-nucleotide variant that was only detected once was treated as artifact and were excluded. The SPSS Statistics software (IBM, New York, NY, USA) was used for all calculations. P-valueso0.05 were considered significant. Among the 92 ET patients (median age 53 years; 58% females), 59 (64%) patients harbored JAK2 V617F mutation and one (1%)