JAK2 exon 14 c.1849G>T, p.V617F (NM_004972.3) variant is commonly found in Myeloproliferative neoplasms (MPN); approximately 100% of polycythemia vera and 50% of essential thrombocytosis and primary myelofibrosis cases have this pathogenic variant. The pathogenic nature of JAK2 V617F in these disorders have been well-established in the past. The National Comprehensive Cancer Network (NCCN) guidelines for cancer care has indicated molecular testing using blood samples for JAK2 V617F as well as other mutant alleles is needed for MPN diagnosis. Many molecular genetic testing methods are available to detect this single nucleotide sequence change, including Sanger sequencing, quantitative PCR (qPCR), Next-generation sequencing (NGS), and droplet digital PCR (ddPCR). Variants with high allelic fraction (eg, >10%) can be easily determined, however, the low variant allelic fraction (VAF) may not be consistently identified. In this study, we describe a Blocker Displacement Amplification (BDA)-based qPCR approach that has the flexibility to use many existing qPCR platforms to deliver test results at the ddPCR level sensitivity. We are interested at evaluating and applying the BDA, to detect low level and rare gene sequence alterations. The clinical specimens were also tested using other molecular diagnostic methods such as NGS and ddPCR, to compare each method’s detecting limitation of the V617F variant.
We tested 122 clinical specimens (extracted genomic DNA) where the JAK2 gene mutational status has previously been deep sequenced with NGS methods, including 11 samples of low level variants (eg, VAF =<2%), 14 samples of various mutant allelic proportions (eg, VAF>2%), and over 50 samples of no variant identified by NGS testing. A molecular blocker method has been introduced recently to detect gene variants. This novel technology, Blocker Displacement Amplification (BDA), uses competing molecular blockers to enrich low level mutant alleles. Customized BDA assays are designed targeting a specific JAK2 variant (ie, c.1849G>T, p.V617F). The NGS was performed on Illumina NextSeq 550. The sequencing reads of >Q30 score ranged from 81.9– 87.9%, and the average read-depth was at least 1500x at this locus. The ddPCR assay was performed according to BioRad (Hercules, CA) applications guide. The JAK2 probe assay mix was purchased from BioRad.
Our study shows that although NGS is able to identify the c.1849G>T, p.V617F variant at 1% VAF level, it is challenging to detect variants less than 0.5% VAF without ultra-deep sequencing. On the other hand, the BDA approach delivers comparable data as ddPCR, which identifies the variant down to 0.1% VAF. BDA provides a cost-effective alternative mean to identify low level variants without the purchase of capital equipment.
The BDA method gives the flexibility of simultaneously targeting multiple gene alteration events at one time using equipment that is usually present in most laboratories. The successful development of the molecular blocker method will give our pathologists and oncologists a more cost-effective alternative diagnostic tool to identify low level JAK2 c.1849G>T, p.V617F variant or other gene variants.
© 2022 Published by Elsevier Inc.