A Novel Denaturing-High Performance Liquid Chromatography (D-HPLC)-Based Method for Kit Mutation Screening of Patients (pts) with Systemic Mastocytosis (SM) Allows the Identification of Unreported Kit Variants.

SM is characterized by activating mutations of the Kit tyrosine kinase receptor. While the so-called ‘enzimatic site’ (ES) type mutation D816V renders Kit resistant to imatinib, ‘regulatory’ type mutations are sensitive to inhibition. Kit mutation screening with sensitive methods is important for appropriate therapeutic management of SM. Our aims were:

• to set up and optimize a D-HPLC-based screening method for mutations in different critical regions of the kit receptor;

• to assess the sensitivity and reliability of our D-HPLC assay as compared to RFLP analysis;

• to characterize additional mutations/polymorphisms.

The analysis was performed on 37 SM pts. For each sample, a RT-PCR product spanning the catalytic and activation loops in the ES was screened in parallel by D-HPLC, followed by sequencing of D-HPLC-positive cases, and by RFLP according to an already reported method for the detection of the D816V. By RFLP analysis, 24/37 (65%) pts were positive for the D816V. By D-HPLC analysis, an abnormal eluition profile was seen in 26/37 (70%) pts - all the 24 pts scored as mutated by RFLP as well as two additional pts. Direct sequencing confirmed the presence of the D816V in all the 24 RFLP-positive cases, and showed that in two of these cases a I798I polymorphism was also present. The two pts scored positive by D-HPLC but negative by RFLP were found to have the same I798I polymorphism. The 11 pts who did not harbour ES type mutations were further investigated by D-HPLC analysis of a RT-PCR product corresponding to the transmembrane (TM) and juxtamembrane (JM) domains. D-HPLC showed an abnormal elution profile in 4 pts. Direct sequencing confirmed the presence of a point mutation in all cases. One patient showed a silent mutation at codon 546. Three pts showed a novel point mutation at codon 541 in the TM domain, resulting in a Met to Leu amino acid substitution. This is the second Kit TM domain mutation reported in a human disease and further supports the hypothesis of a role of the TM domain in regulating the enzymatic activity of an otherwise normal catalytic site. Further characterization of this novel mutant is ongoing. Morphologic and cytofluorimetric analyses of bone marrow biopsies and aspirates will be compared in order to assess whether the pts share any peculiar pathologic feature. Cos-7 cells are currently being transfected with M541L, D816V and wild-type kit in order to evaluate the effects of the M541L on kit enzymatic activity by western blot analysis of total and phosphorylated kit. Patient mast cells will be cultured in the presence or absence of kit ligand or imatinib, dasatinib and nilotinib in order to assess the sensitivity of the M541L to different kit inhibitors. Our novel D-HPLC-based assay proved a straightforward, reliable and sensitive method for kit mutation analysis. It also highlighted the importance of screening for mutations other than the D816V. D-HPLC analysis allowed us to find a novel M541L mutation which is now under characterization.