Improved detection of the C-KIT D816V mutation using allele-specific arms real time PCR assay allows a finer recognition of patients with indolent systemic mastocytosis.

The somatic D816V mutation of the KIT gene is present in more than 90% of Systemic Mastocytosis (SM) and represents one of the minor criteria for its diagnosis by the World Health Organization (WHO) classification. Patients with indolent SM, could have a very low mast cell (MC) burden, especially if lacking skin lesions: in these cases a highly sensitive diagnostic test for D816V detection is necessary. In order to improve detection of D816V c-KIT mutation, we tested BM cells from 110 consecutive adult patients referred with a suspicion of SM to our Multidisciplinary Outpatient Clinic for Mastocytosis in Verona from May 2009 to July 2011 using both RT-PCR+RFLP (Reverse Transcriptase Polymerase Chain Reaction with Restriction Fragment Length Polymor- phism) followed by sequencing, and ARMS-RTq-PCR (Amplification Refractory Mutation System-Reverse Transcriptase and Real Time PCR), respectively in Bologna and Verona laboratories. All patients underwent to a BM evaluation with histology/cytology and flow cytometry. For both D816V c-KIT mutation methods RNA was extracted on BM mononuclear cell fraction and reverse transcribed. Then for PCR+RFLP/sequencing assay a RT-PCR product of 287 bp, spanning exons 16-18, was digested with HinfI to detect D816V (variant cell pop- ulation must be at least 4% to be detectable). For patient samples that did not harbour D816V mutation, the RT-PCR product was further investigated by direct Sanger sequencing to detect other site mutations (sen- sitivity of about 25%). These methods revealed D816V mutations in 47 patients. Simultaneously BM samples were analyzed by ARMS-RTq-PCR, setting a cut-off of positivity at 0.001% by MC line HMC-1 RNA dilutions: D816V was identified in 77 patients, corresponding to 100% of cases showing CD25+MC. SM was diagnosed in 76 cases with ARMS-RTq-PCR, Cutaneous Mastocytosis (CM) in 1 and Monoclonal MC activation syndrome (MMAS) in 1. Seventeen out 76 SM patients would not have satisfied sufficient WHO criteria for the diagnosis of SM on the basis of RT-PCR+RFLP and would diagnosed as CM (6), MMAS (10), idiopatic myelofibrosis (1). Patients with discordant D816V results had significantly lower serum tryptase levels, lower amount of CD25 MC and less frequently fulfil major histological criteria. We suggest that a ARMS-RTq-PCR technique, along with a highly sensitive flow cytometry assay, could improve the recognition of patients with indolent SM with a very low MC burden; then negativity assessment must be verified by d-HPLC/sequencing.