Heterogeneity of kinase inhibitor resistance mechanisms in GIST

Abstract

Most GIST patients develop clinical resistance to KIT/PDGFRA tyrosine kinase inhibitors (TKI). However, it is unclear whether clinical resistance results from single or multiple molecular mechanisms in each patient. KIT and PDGFRA mutations were evaluated in 53 GIST metastases obtained from 14 patients who underwent surgical debulking after progression on imatinib or sunitinib. To interrogate possible resistance mechanisms across a broad biological spectrum of GISTs, inter- and intra-lesional heterogeneity of molecular drug-resistance mechanisms were evaluated in the following: conventional KIT (CD117)-positive GISTs with KIT mutations in exon 9, 11 or 13; KIT-negative GISTs; GISTs with unusual morphology; and KIT/PDGFRA wild-type GISTs. Genomic KIT and PDGFRA mutations were characterized systematically, using complementary techniques including D-HPLC for KIT exons 9, 11-18 and PDGFRA exons 12, 14, 18, and mutation-specific PCR (V654A, D820G, N822K, Y823D). Primary KIT oncogenic mutations were found in 11/14 patients (79%). Of these, 9/11 (83%), had secondary drug-resistant KIT mutations, including six (67%) with two to five different secondary mutations in separate metastases, and three (34%) with two secondary KIT mutations in the same metastasis. The secondary mutations clustered in the KIT ATP binding pocket and kinase catalytic regions. FISH analyses revealed KIT amplicons in 2/10 metastases lacking secondary KIT mutations. This study demonstrates extensive intra- and inter-lesional heterogeneity of resistance mutations and gene amplification in patients with clinically progressing GIST. KIT kinase resistance mutations were not found in KIT/PDGFRA wild-type GISTs or in KIT-mutant GISTs showing unusual morphology and/or loss of KIT expression by IHC, indicating that resistance mechanisms are fundamentally different in these tumours. Our observations underscore the heterogeneity of clinical TKI resistance, and highlight the therapeutic challenges involved in salvaging patients after clinical progression on TKI monotherapies.

Figure 1

(A) GIST with spindle cell morphology composed of cells with a pale eosinophilic fibrillary cytoplasm, ovoid nuclei and ill-defined cell borders, with syncytial appearance and palisading (patient 3). (B) GIST with epithelioid cell morphology composed of round cells with eosinophilic cytoplasm arranged in sheets (patient 2). (C) Low-power view of a GIST in the stomach wall, with abrupt transition between two morphologically different tumour components (patient 14; KIT and PDGFR wild-type). Higher-power images of this tumour are demonstrated in (D), showing epithelioid morphology (right) and pleomorphic spindle cell morphology (left). (E) GIST showing unusual morphology, with huge epithelioid cells, vesicular nuclei and prominent nucleoli (patient 7; primary KIT exon 11 mutation). (F) KIT-negative GIST (patient 7). (G) KIT-negative GIST showing strong cytoplasmic staining for caldesmon (patient 7). (H) KIT-negative GIST showing strong cytoplasmic staining for desmin (patient 7)

Figure 2

(A) Summary of secondary KIT resistance mutations detected by D-HPLC in 27/57 tumour samples from 14 patients with progressing GISTs. Associated primary KIT mutations are indicated in red (exon 11 mutations), yellow (exon 9 mutations) and green (exon 13 mutations). (B) Summary of secondary KIT resistance mutations detected by AS–PCR only ( ) or detected by both AS–PCR and D-HPLC (●) in 18 tumour samples. Associated primary KIT mutations are indicated in red (exon 11 mutations), yellow (exon 9 mutations) and green (exon 13 mutations)

Figure 3

(A) FISH analysis of abdominal tumour 1 (spindle cell morphology) from patient 8, with disomic (FISH-negative) pattern. The chromosome 4 centromere probe is shown in orange and the KIT probe in green. (B) FISH analysis of stomach wall (2) tumour (epithelioid morphology) from patient 10, with KIT amplification. Chromosome 4 centromere probe is shown in orange and the KIT probe in green