Ultra-Deep Sequencing (UDS) Allows More Sensitive Detection of the D816V and Other Kit Gene Mutations in Systemic Mastocytosis

• Published in: Blood 2014-12, Vol.124 (21), p.1856-1856
• Main Authors: De Benedittis, Caterina, Soverini, Simona, Papayannidis, Cristina, Rondoni, Michela, Colarossi, Sabrina, Dal Pero, Francesca, Zazzeroni, Luca, Zanotti, Roberta, De Matteis, Giovanna, Merante, Serena, Elena, Chiara, Grifoni, Federica Irene, Bonifacio, Massimiliano, Perbellini, Omar, Specchia, Giorgina, Pagano, Livio, Gangemi, Domenica, Bonadonna, Patrizia, Pieri, Lisa, Cavo, Michele, Martinelli, Giovanni
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V124.21.1856.1856

Objectives and background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) examination and is based on a major and four minor criteria. The somatic 'autoactivating’ point mutation D816V in the KIT receptor gene is one of the minor criteria, founded in the great majority of patients (90%) and it plays a central role in the pathogenesis of the disease. Indolent Systemic Mastocytosis (ISM) is the most common variant of SM, characterized by a very low MC burden and associated with very different clinical pictures. A highly sensitive diagnostic methods for D816V detection are required to assure an appropriate diagnosis and to reduce false-negative results. The recent development of “ultra-deep amplicon sequencing” (UDS) technologies has opened the way to a more accurate characterization of molecular aberrations with higher sensitivity of screening for known and unknown mutations. Our aims were: i) to set-up and optimize a UDS-based mutation screening strategy of the KIT gene on the Roche GS Junior Instrument; ii) to test the sensitivity of our UDS assay to detect the D816V mutation; iii) to investigate the presence of additional KIT mutations in SM. Methods: We decided to take advantage of a next generation sequencing approach to perform an UDS KIT gene mutation analysis on 20 bone marrow (BM) samples from patients whit ISM that were negative for the D816V mutation by Sanger Sequencing which has a sensitivity of 20%. Fusion primers were designed to generate ten partially overlapping amplicon covering the whole KIT transcript (exons 1-21) by RT-PCR. To determine the lower detection limit of our UDS-assay, serial dilutions of the HMC-1 cell line (harboring the D816V mutation) into an unmutated K562 cell line in ratios such as to simulate the following mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%. Results and significance: UDS of cell line dilutions showed a high accuracy of D816V mutation detection and linearity of mutation calling over the entire range down to 0.25%. The UDS technology allowed to detected the D816V mutation, below the lower detection limit of Sanger Sequencing, with an abundance from 0.5% to 11%, in 12/20 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame deletion in exon 15 (GenBank X06182.1: c.2164_2166delAGC; p.S715del) found i