Molecular detection of Janus Kinase 2 (JAK2) and Calreticulin (CALR) mutations in myeloproliferative neoplasm using high resolution melting analysis

Abstract

Introduction: Myeloproliferative neoplasm (MPN) is a group of myeloid disorder in which there is excess proliferation of myeloid lineage with efficient maturation that leads to erythrocytosis, thrombocytosis and leucocytosis. MPN with breakpoint cluster region-Abelson (BCR-ABL) positive is chronic myeloid leukaemia (CML) while BCR-ABL negative MPN includes polycythaemia vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). One of the major criteria for diagnosis of BCR-ABL negative MPN is the presence of mutations. The most common mutation is JAK2-V617F mutation which is positive in 95% of PV and around 60% of ET and PMF patients. Second most common mutation is calreticulin (CALR) mutation which accounts for 20-30% in ET and 25-35% in PMF. Since Formalin-Fixed Paraffin-Embedded (FFPE) marrow specimen can be kept for a long period of time, it can be used as an alternative to whole blood for detection of these mutations. Unfortunately, FFPE produces low quality DNA that put challenge for successful amplification of DNA. Objective: The aim of this study was to assess the utility of high resolution melting (HRM) analysis for detection of JAK2-V617F mutation from FFPE specimens and to determine the frequency of JAK2-V617F and CALR exon 9 mutations in MPN patients. Methodology: This study is a descriptive cross-sectional study. Forty FFPE marrow specimens were retrieved from the year 2014-2016. Bio-Rad Precision Melt Analysis software was used for analysis of HRM data. Allele-specific PCR (AS-PCR) was done for validation of JAK2-V617F results followed by Sanger sequencing in the case of positive results. Results: JAK2-V617F mutation was positive in 3 out of 4 in PV, 1/2 in ET, 4/4 in PMF, 4/10 in unclassified MPN and 1/20 in CML patients. Level of agreement between HRM and AS-PCR for detection of JAK2-V617F mutation was 95%. However, no individual carry CALR mutation. Conclusion: HRM is a powerful diagnostic assay which is suitable for detection of JAK2-V617F mutation in FFPE marrow specimens. Despite its small sample size, the prevalence of JAK2-V617F mutation in PV and ET is comparable with international data. CALR mutation was not found in MPN cases.