Ultra-deep sequencing (UDS) allows more sensitive detection of the D816V and other KIT gene mutations in systemic mastocytosis

Background: According to the World Health Organization (WHO) classification, the diagnosis of Systemic Mastocytosis (SM) relies on bone marrow (BM) exam- ination and is based on a major and four minor criteria. The somatic ‘autoacti- vating’ point mutation D816V in the KIT receptor gene is one of the minor cri- teria, 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 accu- rate characterization of molecular aberrations with higher sensitivity of screen- ing for known and unknown mutations. Aims: Our aims were: i) to set-up and optimize a UDS-based mutation screen- ing 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 24 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 fol- lowing mutation loads were sequenced: 50%, 37.5%, 25%, 12.5%, 5%; 2.5%, 1.25%, 0.5%, 0.25%. 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 15/24 ISM patients. Two additional sequence variations were detected in a large proportion of patients. These two variations included a 3bp in-frame dele- tion in exon 15 (GenBankx06182.1: c.2164_2166delAGC; p.S715del) found in 12/24 patients and a 12bp in frame-deletion in exon 9 in all patients, whit an abundance ranging from 83% to 97% (GenBankx06182.1: c.1550_1561del- GTAACAACAAAG; p.G510_K513del). Previously published studies indicate that the KIT Gly-Asn-Asn-Lys510-513+/- alternatively spliced located immediately downstream to the extracellular KIT domain and KIT Ser715+/-, an interkinase KIT domain, are expressed in normal human hematopoietic cell, leukemic cell lines, acute myeloid leukemia blast and GISTs and represent rather a splice variant of KIT transcript. Interestingly our results showed the presence of the transmembrane domain M541L (GenBankx06182.1: c.1642A>C; p.Met541Leu) KIT-activating mutation in exon 10, with an abundance of 50%, in addition to D816V, in 3/24 ISM. This mutation is known to retain sensitivity to imatinib mesylate.Summary and Conclusions: Our preliminary results suggest that our-UDS based KIT gene mutation screening assay might be a reliable and sensitive alter- native to conventional sequencing methods for the detection of the D816V. We are now planning to investigate whether the greater sensitivity of UDS allows to detect the D816V mutation in peripheral blood mononuclear cells from patients with a suspected clonal mast cell disorder. These results could represent a start- ing point to plan other extensive studies to better understand the exact role of KIT receptor alterations in SM.