Sensitive Detection of c-kit Point Mutations by D-HPLC in Peripheral Blood and Bone Marrow Samples from Patients with Systemic Mastocytosis.
Authors: Lauber Stephanie , Mueller Martin C. , Erben Philipp , Schenk Thomas , Kruth Jens , Metzgeroth Georgia , Ernst Thomas , Paschka Peter , Hehlmann Rudiger , Reiter Andreas , Hochhaus Andreas 
Published: October 2019

Abstract
The majority of patients (pts) with systemic mastocytosis (SM) is characterized by the presence of the transforming mutation D816V of the c-kit gene, resulting in a factor-independent activation of KIT, a receptor tyrosine kinase on the surface of mast cells. The mutation is regarded the causative event for the pathogenesis of the disease and a potential target for therapeutic intervention. Novel tyrosine kinase inhibitors, like dasatinib, nilotinib (AMN107) and midostaurin (PKC412) have inhibitory effects on c-kit D816V mutant cells according to recent in-vitro findings. However, the sensitivity of screening procedures for mutations by direct sequencing may be compromised by a small proportion of malignant cells in the bone marrow (BM) sample. We therefore sought to establish a sensitive strategy for the detection of c-kit mutations in BM and peripheral blood (PB) samples by D-HPLC (denaturing-high performance liquid chromatography) with fragment collection of the mutated proportion combined with direct sequencing. D-HPLC has been established using serial dilutions of c-kit D816V positive HMC-1 cells in a background of NB4 cells harboring wildtype c-kit and pts mRNA in control mRNA. D-HPLC was optimized for the detection of the D816V mutation down to 0.1–0.5% mutant clone (cell line and RNA dilution). In comparison, the detection limit for D816V mutations by conventional sequencing was 10 to 15%. The method was established for c-kit mutations in exon 17, but is applicable for mutations in exons 9, 11 and 13 as well. In case of a positive D-HPLC signal, the presence of the mutation was confirmed by direct sequencing of c-kit exon 17. Secondly, the D-HPLC eluates containing the mutated products were extracted by fragment collection, enriched by PCR and subsequently sequenced. The technique was applied to BM (n=81) and PB (n=38) samples from 95 pts (53 m, 42 f) meeting the WHO criteria for SM. Median age was 53 yrs (range 24–86). At diagnosis, D-HPLC was positive in 89% of the BM samples (72/81), whereas conventional sequencing revealed the D816V mutation in 69% of the pts (56/81), one patient was positive for the variant D816H mutation. However, the combination of fragment collection and direct sequencing improved the D816V mutation detection to 86% of pts (70/81) compared to sequencing only. The analysis of PB samples revealed D-HPLC positivity in 50% of pts (19/38) with a consecutive detection of the D816V mutation by direct sequencing alone in 34% of pts (13/38). Fragment collection followed by direct sequencing improved the D816V mutation detection to 50% (19/38). In conclusion,D-HPLC is a reliable and sensitive method for the screening of c-kit mutations in SM which is superior to conventional direct sequencing. In case of a positive DHPLC signal, sequencing is recommended to confirm and specify the mutation.The method is suitable for the surveillance of pts during therapy with novel tyrosine kinase inhibitors.Employing sensitive screening techniques, the D816V mutation could be detected in BM as well as in PB samples, but the reliability for a positive result was higher using BM.