Improved Detection of KIT Exon 11 Duplications in Formalin-Fixed, Paraffin-Embedded Gastrointestinal Stromal Tumors

Abstract

Gastrointestinal stromal tumors (GISTs) are the most common gastrointestinal mesenchymal tumors driven by KIT or PDGFRA mutations. A majority of these mutations affect KIT exon 11 and represent deletions or point mutations, but insertions and duplications have also been reported. The latter have been found exclusively in the 3 part of KIT exon 11. Reported frequency of duplications varies, and a higher frequency has been reported in studies based on frozen tissue. Recently, we have hypothesized that in some cases, the duplications might remain undetected in formalin-fixed, paraffin-embedded GISTs because of the preferential polymerase chain reaction (PCR) amplification of wild-type KIT over the mutant allele. In this study, 16 GISTs initially diagnosed as a wild-type KIT were evaluated using PCR assay amplifying only the 3 part of KIT exon 11, the region commonly affected by duplications. Denaturing highpressure liquid chromatography and direct sequencing analyses revealed duplications in 4 (25%) of 16 analyzed cases. Use of the PCR assay amplifying the specific region affected by duplications and yielding 129 bp in wild-type KIT can substantially improve the detection of these mutations in formalin-fixed, paraffin-embedded GISTs. (J Mol Diagn 2007, 9:89 -94;

Low frequency of MAP kinase pathway alterations in KIT and PDGFRA wild‐type GISTs

“…Twenty‐nine of 78 previously described 27 wild‐type KIT and PDGFRA GISTs were screened for KIT mutations using a PCR assay designed to amplify a region in the 3′ part of KIT exon 11 commonly affected by duplications 18 . The PCR‐SSCP followed by direct sequencing analysis allowed identification of three novel cases harbouring KIT exon 11 duplications: case G6 (579_583dup), case G36 (578_591dup) and case G58 (573_580dup), which ranged from 14 to 42 nucleotides (Figure 1A).…”

“…The reaction consisted of an initial denaturation at 96°C for 10 min, followed by 40 cycles of denaturation at 96°C for 45 s, annealing at 55–60°C for 45 s and extension at 72°C for 45 s, followed by a final extension for 10 min at 72°C, in a Thermocycler (Bio‐Rad, Hercules, CA, USA). Primer sequences for all genes were as previously described 18,29,30 …”

“…Recently, Lasota et al. have suggested that KIT exon 11 duplications may be undetected in formalin‐fixed paraffin‐embedded samples, due to the preferential polymerase chain reaction (PCR) amplification of the wild‐type KIT allele over the large fragment mutant allele 18 . In fact, using a PCR assay specific for the 3′ region affected by duplications at exon 11, the authors showed the presence of these types of mutation in 4/16 previously considered wild‐type KIT formalin‐fixed paraffin‐embedded GISTs 18 …”

A quality control program for mutation detection in KIT and PDGFRA in gastrointestinal stromal tumours

“…The PCR reaction was performed again by this group changing the primer pairs and the KIT exon 9 duplication was detected. As reported by Lasota et al [5] for KIT exon 11 duplication located at the 3 0 end of the exon, a modification of the primers' sequence could increase the rate of detection of mutation in exon 9 of KIT in fixed paraffin-embedded GIST. Centre 11 screened the mutation for a primary imatinib-naïve GIST (sample 36).…”

“…The remaining GIST, referred to as wildtype (WT-GIST), do not carry KIT or PDGFRA mutations. Mutations are found in exons 9, 11, 13 and 17 of KIT and in exons 12, 14 and 18 of PDGFRA [5][6][7][8][9][10].…”