INTRODUCTION

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms that typically originate from interstitial cells of Cajal and most frequently develop in the stomach and small intestine, although they may arise at any site along the gastrointestinal tract.[1] The pathogenesis of GISTs is closely associated with activating mutations in the kinase insert domain receptor (c-KIT) or platelet-derived growth factor receptor alpha (PDGFRA) genes, with the majority harboring mutations in c-KIT, most commonly affecting exon 11.[1,2] The advent of targeted therapies, particularly imatinib, has markedly transformed the clinical management of GISTs. Imatinib functions as a selective tyrosine kinase inhibitor by competitively binding to the ATP-binding site of receptors such as c-KIT, thereby preventing receptor autophosphorylation. This inhibition suppresses the phosphorylation of key downstream signaling mediators, including mechanistic target of rapamycin, Ak strain transforming and mitogen-activated protein kinase, leading to decreased expression of proliferation-associated markers and anti-apoptotic proteins. These molecular alterations collectively impair cellular proliferation, migration, and survival, while promoting tumor cell apoptosis and attenuating tumor progression.[3-5]

Despite initial therapeutic efficacy, resistance to imatinib commonly develops during treatment, often as a result of the emergence of secondary mutations in c-KIT or PDGFRA. These secondary alterations, particularly within exon 17 of KIT, are frequently observed as part of double mutations involving multiple exons and are considered a major mechanism underlying therapeutic resistance.[5,6] Such complex mutational profiles may influence both clinical outcomes and the selection of subsequent therapeutic strategies. Sunitinib, a multi-targeted tyrosine kinase inhibitor, is commonly employed in cases of imatinib-resistant GIST, particularly when secondary mutations are present. Clinical evidence indicates that sunitinib can significantly prolong progression-free survival (PFS) in patients with imatinib-refractory disease.[7,8]

This study describes a clinical case of GIST harboring double KIT mutations with acquired resistance to imatinib. We characterize the molecular alterations underlying resistance and assess the therapeutic response to subsequent treatments. Furthermore, we discuss current and emerging treatment strategies for resistant GISTs, with particular emphasis on optimizing therapeutic approaches in the context of complex mutational profiles. Our findings aim to contribute to the understanding of resistance mechanisms in double-mutant GISTs and to inform the development of more effective, individualized treatment approaches.

CASE REPORT

A 51-year-old man presented to our institution on January 16, 2016, with a 2-week history of left upper abdominal pain and melena. Contrast-enhanced abdominal computed tomography (CT) revealed a pelvic mass involving the small intestine, characterized by irregular margins and heterogeneous enhancement [Figure 1]. Colonoscopic evaluation showed no abnormalities in the ileal or rectal mucosa. Following completion of the necessary diagnostic workup, the patient underwent laparoscopic resection of the small intestinal mass. Histopathological examination demonstrated a moderately cellular spindle cell neoplasm arranged in fascicles, with perinuclear vacuolation noted in some regions [Figure 2a]. Immunohistochemical analysis revealed diffuse positivity for CD34, CD117, and Dog-1 [Figure 2b-d] and a Ki-67 labeling index of 10%. The tumor was negative for smooth muscle actin (SMA) and Desmin [Figure 2e and f]. Final pathology confirmed a high-risk small intestinal GIST measuring between 5.1 and 10.0 cm, with evidence of necrosis, >10 mitoses/50 high-power fields, and negative resection margins. Genetic testing was not performed at that time. The patient was initiated on adjuvant targeted therapy with imatinib (H20133200, Hansoh Pharma, Jiangsu, China) at a dose of 400 mg/day.

Figure 1: Radiological imaging of the primary tumor at initial diagnosis. A contrast-enhanced abdominal computed tomography scan revealed a mass in the pelvic small intestine with irregular borders and heterogeneous enhancement (arrow).

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Figure 2: Histopathological and immunohistochemical staining of gastrointestinal stromal tumor in this case. (a) Histopathological analysis showed moderately dense spindle-shaped tumor cells arranged in bundles, with paranuclear vacuoles present in some areas H&E ×20, scale = 50 μm. (b) CD34 staining was positive on the membranes of most malignant cells (×20, scale = 50 μm). (c) CD117 demonstrated diffuse and strong cytoplasmic positivity (×20, scale = 50 μm). (d) Dog-1 showed cytoplasmic positivity in most malignant cells (×20, scale = 50 μm). (e and f) smooth muscle actin (SMA) and Desmin were negative in the cytoplasm (×20, scale = 50 μm). HE: Hematoxylin and eosin; SI: Staining index.

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During the course of treatment with imatinib, follow-up was conducted by telephone. The patient reported undergoing regular surveillance imaging at a local hospital, with no evidence of recurrence noted. However, 5 years later, on August 16, 2021, a CT scan at the referring facility identified a recurrent pelvic mass. The patient was referred back to our center for further evaluation. Contrast-enhanced abdominal CT revealed a 4.9 × 2.3-cm well-circumscribed, mildly enhancing soft tissue mass located anterior to the rectum [Figure 3]. Histopathological examination of a biopsy specimen showed diffuse proliferation of spindle-shaped tumor cells. Immunohistochemical staining was positive for vimentin, CD117, Dog-1, and CD34 and negative for STAT-6, Desmin, SMA, S-100, and cytokeratin. The Ki-67 labeling index was approximately 20%.

Figure 3: Radiological imaging of the recurrent mass. A contrast-enhanced abdominal computed tomography scan revealed a recurrent mass anterior to the rectum, characterized by well-defined borders and mild contrast enhancement (red arrow).

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Based on histological and radiologic findings, a diagnosis of recurrent GIST was established. First-generation sequencing identified pathogenic mutations in c-KIT exons 11 (V560D) and 17 (N822K) [Figure 4], with no mutations detected in PDGFRA exons. These findings were consistent with acquired resistance to imatinib. Consequently, the patient was transitioned to second-line therapy with sunitinib (H20213753, Qilu Pharmaceutical, Jinan, China) at a dose of 50 mg/day, administered on a 4-week-on, 2-week-off schedule.

Figure 4: Sanger sequencing chromatogram of mutational analysis. Forward and reverse Sanger sequencing identified (a and b) a c.1679T>A (p.V560D) mutation in exon 11 (red arrow) and (c and d) a c.2466T>A (p.N822K) mutation in exon 17 of the KIT gene (red arrow). A: Adenine, C: Cytosine, T: Thymine, G: Guanine, K: G or T (keto bases), Y: C or T (pyrimidine bases).

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At follow-up on December 1, 2021, repeat contrast-enhanced abdominal CT demonstrated an increase in the size of the rectal anterior mass compared with previous imaging, indicating disease progression despite sunitinib therapy [Figure 5]. Considering the progressive disease and the need for advanced oncologic management, the patient was referred to a tertiary care center for continued treatment and further therapeutic planning. No subsequent clinical updates have been reported.

Figure 5: Radiological imaging of the recurrent tumor after sunitinib treatment. A contrast-enhanced abdominal computed tomography scan showed an increase in the size of the mass located anterior to the rectum compared with the previous scan, suggesting that the recurrent tumor had developed resistance to the drug (red arrow).

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This study received approval from the Institutional Review Board of Yantai Yuhuangding Hospital Affiliated with Qingdao University (protocol code: 2021-040). Written informed consent was obtained from the patient or his legal representatives. This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki.[9]

DISCUSSION

Primary mutant GISTs refer to tumors arising in patients who have not received treatment with imatinib or other receptor tyrosine kinase inhibitors. These tumors are typically characterized by a single-driver mutation, most frequently involving c-KIT, with exon 11 mutations being the most prevalent.[1,2] Double mutations, however, are relatively rare among patients with primary mutations. In a molecular analysis of 68 patients with GIST, Haefliger et al. reported one case harboring primary double c-KIT mutations and another case that acquired double mutations following imatinib therapy.[10] Similarly, among 135 cases of micro-GIST, four tumors were identified as double mutant – three with double c-KIT mutations and one with a PDGFRA mutation.[11] In a larger cohort of 1,177 GIST cases, nine double-mutant tumors were reported.[12] By contrast, Yan et al. identified only eight double-mutant cases in a series of 1,530 GISTs, a significantly lower frequency that may be attributed to the use of Sanger sequencing.[13] Furthermore, diagnostic strategies at some institutions involve sequencing only the most commonly mutated exons, with additional testing conducted only if the initial results are wild type. Consequently, if a mutation is detected in the routinely tested exons, a second concurrent mutation may remain undetected.[12]

The majority of GISTs harbor activating mutations in c-KIT, with imatinib demonstrating high efficacy, particularly in tumors with mutations in c-KIT exon 11. In contrast, mutations in c-KIT exon 9 can induce conformational changes that result in constitutive activation of the receptor, thereby limiting imatinib’s inhibitory potential and conferring intrinsic resistance. In addition, c-KIT exon 17 encodes the activation loop of the tyrosine kinase domain, while exons 13 and 14 encode the ATP-binding pocket. Mutations in these regions directly disrupt imatinib binding and may stabilize the receptor in its active conformation, further diminishing drug efficacy. Therefore, the presence of mutations in c-KIT exons 13, 14, or 17 is typically associated with poor therapeutic response to imatinib.[5,7,14,15] Despite initial responsiveness, approximately 40–50% of patients with GIST develop resistance to imatinib within 12–24 months of treatment, primarily as a result of the emergence of secondary mutations in c-KIT or PDGFRA. The presence of double mutations may exacerbate this resistance by enhancing receptor kinase activity or altering its structural conformation, thereby compromising the efficacy of targeted therapies.[6,7] A notable case reported by Conca et al. described a GIST harboring double mutations in KIT exon 11 (W557G and Y578C). Molecular modeling revealed that the double-mutant protein exhibited ATP and imatinib binding affinities comparable to the known activating D559 mutation, suggesting a retained sensitivity to imatinib.[16] Although the Y578C variant is located near the resistance-associated L576P site, it did not appear to impede imatinib binding. This case challenges the prevailing assumption that double-mutant GISTs are inherently resistant to imatinib and underscores the utility of integrating functional assays with structural modeling to inform precision oncology strategies.[16]

In the present case, we described a patient with a double-mutant GIST who demonstrated an initial favorable response to imatinib. However, 5-year post-treatment initiation, the patient experienced tumor recurrence indicative of acquired resistance. Subsequent genetic analysis identified double mutations in c-KIT exons 11 (V560D) and 17 (N822K). As genomic testing was not performed at the time of initial diagnosis, it remains unclear whether the double mutations were present at baseline or acquired during therapy. Notably, exon 11 is the most commonly mutated region in c-KIT, and clinical trials have shown that tumors with exon 11 mutations generally exhibit a favorable response to imatinib and prolonged PFS.[5,7,15] Furthermore, exon 11 mutations are associated with a higher likelihood of acquiring secondary mutations during treatment, particularly within exon 17.[17] Heinrich et al. evaluated the imatinib sensitivity of various c-KIT mutation profiles using V560D as a reference.[5] Their findings demonstrated that the V560D mutation alone was highly sensitive to imatinib, with an IC50 of approximately 100 nmol/L. However, the presence of a double mutation comprising V560D in exon 11 and N822K in exon 17 conferred marked resistance to imatinib. This resistance is thought to stem from structural alterations in the activation loop induced by the exon 17 mutation, which may disrupt the autoinhibitory conformation of the kinase.[5,18] Based on these findings, we hypothesize that our patient initially harbored a single mutation in exon 11 and subsequently acquired the exon 17 mutation during the course of imatinib therapy, ultimately leading to therapeutic resistance.

Following disease progression, the patient was transitioned to sunitinib; however, no clinical benefit was observed, and the tumor continued to enlarge [Figure 5]. This outcome aligns with findings from a phase I/II trial of sunitinib, in which patients with c-KIT exon 11 mutations exhibited a modest clinical benefit rate of 34% and a median PFS of only 5.1 months. Notably, patients with secondary mutations affecting the activation loop (exons 17 and 18) demonstrated even poorer outcomes, with a median PFS of 2.3 months. In vitro analyses further corroborate these findings, showing that secondary mutations in exons 17 and 18 confer resistance to both sunitinib and imatinib, with IC50 values exceeding 1,000 nmol/L.[7]

For patients with GIST who exhibit resistance or intolerance to imatinib, sunitinib represents an effective therapeutic alternative. Clinical studies have demonstrated that sunitinib is particularly efficacious in individuals harboring c-KIT exon 9 mutations and mutations within the ATP-binding pocket. Its therapeutic effect is further potentiated by its anti-angiogenic properties, including the inhibition of vascular endothelial growth factor receptor activity.[7,8] However, its clinical utility is markedly reduced in cases involving kinase activation loop mutations, underscoring the need for alternative treatment strategies. As a fourth-line or later therapy, ripretinib has shown substantial efficacy in refractory GIST, leading to a significant prolongation of PFS.[8] In addition, BPR1J373 has demonstrated the ability to overcome resistance by concurrently targeting c-KIT double mutations (e.g., exons 11 and 17) and inhibiting downstream signaling cascades such as PI3K/AKT and MAPK pathways. This agent also suppresses Aurora kinase A activity, resulting in G2/M phase cell cycle arrest and the induction of apoptosis.[19] Similarly, compound 15a exerts its antitumor effects by binding to the ATP-binding pocket of c-KIT, thereby stabilizing its inactive conformation and preventing autophosphorylation. This compound effectively inhibits both primary mutations (e.g., exon 11 V560G) and secondary mutations (e.g., exon 17 N822K and D816V).[20] Collectively, these findings highlight the therapeutic promise of ripretinib, sunitinib, and investigational agents such as BPR1J373 and compound 15a in the management of treatment-resistant GIST, offering a diverse and expanding array of targeted treatment strategies.

SUMMARY

This study presents a single case report, inherently limiting the generalizability of the findings. The absence of genetic testing at the time of initial diagnosis precludes definitive determination of whether the identified c-KIT double mutations (V560D and N822K) were primary or acquired, introducing the potential for retrospective bias. Furthermore, no functional assays were performed to assess the molecular impact of these mutations on drug sensitivity. The lack of long-term follow-up following second-line treatment with sunitinib further limits the ability to comprehensively evaluate therapeutic efficacy and clinical outcomes. To advance understanding in this area, future research should involve larger patient cohorts to more clearly delineate the clinical and molecular features of double-mutant GISTs. Prospective studies integrating early genetic profiling, longitudinal surveillance, and functional validation are warranted to elucidate the contribution of double mutations to resistance mechanisms.

AVAILABILITY OF DATA AND MATERIALS

The data and materials supporting this work can be obtained upon reasonable request from the corresponding author.

ABBREVIATIONS

c-KIT: The kinase insert domain receptor

CT: Computed tomography

GISTs: Gastrointestinal stromal tumors

PDGFRA: Platelet-derived growth factor receptor alpha

PFS: Progression-free survival

SMA: Smooth muscle actin

AUTHOR CONTRIBUTIONS

GY, PY: Conception and design; GY, WW: Administrative support; NZ: Provision of study materials or patients; NZ, NL, YP, JL: Collection and assembly of data; NL, LS, HZ: Data analysis and interpretation; All authors contributed to manuscript writing and final approval of the manuscript. All authors meet ICMJE authorship requirements.