Detection of c-kit exons 11- and 17-activating mutations in testicular seminomas by high-resolution melting amplicon analysis

Abstract

A subgroup of testicular seminomas has been reported to contain activating mutations in KIT, the transmembrane tyrosine kinase receptor encoded by the c-kit gene. Most mutations are in exon 17, although exon 11-activating mutations have recently been described. For patients refractory to standard therapeutic protocols for seminoma, the presence of c-kit-activating mutations in some of these neoplasms might suggest an alternative therapy with KIT targeting drugs. We used the novel mutation scanning technique, highresolution melting amplicon analysis, to screen a series of 22 testicular seminomas for c-kit-activating mutations. Four cases (18%) had exon 17-activating mutations and these included D816Y, D816V, Y823N and one case that contained both D816E and D820H. A single case (5%) had an exon 11-activating mutation. Interestingly, the exon 11-activating mutation was L576P, the same mutation that characterizes the rare c-kit mutation-positive cases of malignant melanoma. Fluorescence in situ hybridization (FISH) for c-kit suggested that most seminomas are probably polysomic for c-kit and there was not a significant difference in c-kit FISH characteristics between the mutation-positive and mutation-negative cases. The use of high-resolution melting amplicon analysis as a screening technique will allow for the rapid identification of patients with testicular seminomas whose tumors contain c-kit-activating mutations. This could benefit patients whose tumors are refractory to standard therapeutic protocols.

Somatic KIT mutations occur predominantly in seminoma germ cell tumors and are not predictive of bilateral disease: Report of 220 tumors and review of literature

“…Where constitutional DNA was able to be examined, the c.2394 c>t was present in constitutional DNA with one tumor and matching constitutional DNA showing a c.2394 c>t homozygous variant. The c.2394 c>t; p.Ile798Ile variant was not documented in dbSNP (build 36) but has been identified in other studies of GCTs (Przygodzki et al, 2002;Willmore-Payne et al, 2006).…”

“…Only a few mutations in exon 11 have been reported for germ cell tumors (Sakuma et al, 2003;Rapley et al, 2004;Nakai et al, 2005;Willmore-Payne et al, 2006) (Table 2).…”

Assessing high-resolution melt curve analysis for accurate detection of gene variants in complex DNA fragments

“…Although HRM curve analysis has been tested and shown to be successful in a number of research laboratories for clinical use [Chou et al, 2005;Dobrowolski et al, 2005Dobrowolski et al, , 2007aDobrowolski et al, , 2007bGrievink and Stowell, 2008;Kennerson et al, 2007;Krypuy et al, 2006;Lonie et al, 2006;Margraf et al, 2006;Poláková et al, 2008;Seipp et al, 2008;Smith et al, 2008;Willmore-Payne et al, 2006], we wanted to assess this method for detecting multiple sequence variants, as well as single and multiple variants (including both constitutional and somatic), within GC-rich fragments, as it would be applied in a diagnostic setting, with minimal expertise and time to manipulate assay design. This was achieved by screening five fragments for 13 variants in a total of 35 different combinations using the LightScanner s System and LC Green s PLUS DNA binding dye (Idaho Technology) as well as screening five GC-rich (Z65%) fragments for 12 variants in 22 combinations using the LightCycler s 480 and HRM Master dye (Roche).…”

Validation of High-Resolution DNA Melting Analysis for Mutation Scanning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

“…10, No. 5 already been successfully applied to the analysis of TP53, 22 phenylalanine hydroxylase gene, 23 factor VIII, 24 factor II, factor V, HFE, 11,25,26 C-kit, 27 EGFR HER2, 28 RET, 29 and CFTR. 30 In this last study, the authors report the CFTR scanning by HRM after PCR amplification of 37 exon/intron fragments in 2 panels of 96 random white UK blood donors and 30 blinded DNA samples enriched for CF-causing variants.…”