Intratumoral KIT mutational heterogeneity and recurrent KIT/ PDGFRA mutations in KIT/PDGFRA wild-type gastrointestinal stromal tumors

Abstract

Objective: Gastrointestinal stromal tumors (GISTs) with no mutations in exons 9, 11, 13, and 17 of the KIT gene and exons 12, and 18 of the PDGFRA gene were defined as KIT/PDGFRA wild-type and they accounted for ~15-20% of GISTs. However, some KIT/PDGFRA wild-type GISTs with KIT mutations in other exons were occasionally reported. We therefore assessed GISTs to understand the whole genomic genotypes of KIT or PDGFRA genes in KIT/PDGFRA wild-type GISTs.

Methods: A cohort of 185 KIT/PDGFRA wild-type GISTs from 1,080 cases was retrospectively assessed. Thirty-nine patients were excluded due to insufficiency of genomic DNA data or failure of library preparation, and 146 patients were analyzed by targeted next-generation sequencing (NGS) followed by validation.

Results: For hot spots in KIT and PDGFRA genes, 23 out of 146 KIT/PDGFRA wild-type cases carried mutations according to NGS; there were 19 KIT mutations and 4 PDGFRA mutations, and these were exclusive. Intratumoral KIT mutational heterogeneity was observed in 4 of 19 samples which potentially triggered mechanisms of polyclonal evolution and metastasis and drug sensitivity. Eleven patients treated with imatinib were evaluable for clinical response, and 2 of 3 patients with KIT mutations achieved partial response (PR), while only 1 of 8 patients without KIT mutations reached PR.

Conclusion: NGS had the potential property to identify partial mutant tumors from a subset of GISTs regarded as KIT/PDGFRA wild-type tumors using Sanger sequencing, and provided a better understanding of KIT/PDGFRA genotypes as well as identified patients eligible for imatinib therapy.

Keywords: KIT/PDGFRA mutation; imatinib; intratumoral heterogeneity; next-generation sequencing; wild-type GISTs.

Figure 1. Mutations located in other exons of KIT/PDGFRA genes

The distribution of missense or deletion mutations identified by NGS in other exons of KIT (A) or PDGFRA (B) genes.

Figure 2. Intratumoral KIT mutational heterogeneity of 4 patients

FFPE sections of 19 patients identified to carry hot spots mutations of KIT by NGS were macrodissected into four regions followed by PCR amplification and Sanger sequencing. Four of 19 patients demonstrated intratumoral KIT mutational heterogeneity with concurrent wild-type and mutant tumor cells.

Figure 3. Patient screening flow chart

From 1,080 patients studied for KIT/PDGFRA mutations, 185 were KIT/PDGFRA wild-type and 146 were analyzed using targeted next-generation sequencing (NGS). There was insufficient genomic DNA or the library preparation failed for 39 patients.