Expanded molecular detection of MPL codon p.W515 and p.S505N mutations in myeloproliferative neoplasms.

Abstract

Patients negative for the JAK2 p.V617F somatic variant are frequently reflexed to testing for MPL exon 10 variants. Detection of these variants via multiplexed allele-specific PCR followed by fragment analysis has been previously published. The present study builds on this concept by improving the detection of the p.W515A variant, adding a second allele-specific primer to detect the p.W515R variant, and incorporating an improved primer for p.S505N detection. The W515 amplification employs 5'-labeled allele-specific forward primers to detect p.W515K, p.W515L, p.W515R, and p.W515A. The p.S505N amplification includes an allele-specific reverse primer with a tail extension. Fragments were subject to capillary electrophoresis on an ABI 3500 Genetic Analyzer and analyzed using GeneMapper 6.0 (Thermo Fisher Scientific). Thirty MPL-negative and 13 MPL-positive samples previously tested by a reference laboratory were tested with the MPL LDT. Results were 100% concordant. The MPL LDT has a limit of detection of at least 5% VAF for the p.W515 variants and 10% VAF for the p.S505N variant. Current MPL assays are predominantly focused on p.W515L/K and p.S505N mutations. We have engineered an MPL test for detecting p.W515A/L/K/R and p.S505N variants, thereby increasing the diagnostic yield with little additional expense or technician time.