Molecular Advances in the Treatment of Advanced Gastrointestinal Stromal Tumor

Abstract Most gastrointestinal stromal tumors (GIST) are driven by activating mutations in Proto-oncogene c-KIT (KIT) or PDGFRA receptor tyrosine kinases (RTK). The emergence of effective therapies targeting these mutations has revolutionized the management of advanced GIST. However, following initiation of first-line imatinib, a tyrosine kinase inhibitor (TKI), nearly all patients will develop resistance within 2 years through the emergence of secondary resistance mutations in KIT, typically in the Adenosine Triphosphate (ATP)-binding site or activation loop of the kinase domain. Moreover, some patients have de novo resistance to imatinib, such as those with mutations in PDGFRA exon 18 or those without KIT or PDGFRA mutation. To target resistance, research efforts are primarily focused on developing next-generation inhibitors of KIT and/or PDGFRA, which can inhibit alternate receptor conformations or unique mutations, and compounds that impact complimentary pathogenic processes or epigenetic events. Here, we review the literature on the medical management of high-risk localized and advanced GIST and provide an update on clinical trial approaches to this disease.


Implications for Practice
The management of advanced GIST has profoundly changed over the past 2 decades. This review highlights improvements in the understanding of molecular pathogenesis of both primary and TKI resistant GIST and the sequential development of effective targeted therapies. It emphasizes the importance of upfront molecular testing to personalize treatment and optimize outcomes. It summarizes clinical trial results to provide an evidence base for current practice and highlights unique drug associated toxicities. It concludes with summary of the pipeline of novel agents being evaluated in this disease.

The Emergence and Advancements of TKI Therapy
Novel TKIs, developed to inhibit aberrant KIT and PDGFRA mutations, have sequentially been approved for treatment of advanced GIST. Differential response to TKIs based on primary and secondary mutations highlights the potential importance of mutation testing with treatment initiation and at times of progression.
Imatinib, a relatively selective oral kinase inhibitor, approved for its BCR-ABL inhibition in chronic myelogenous leukemia, 43 also exhibited inhibitory potential against mutated forms of KIT and PDGFRA. It competitively inhibits ATP from binding the ATP-binding domain and stabilizes the inactive conformation. Imatinib was found effective in 2 international phase III trials 44,45 and Food and Drug Administration (FDA) approved in 2001 as the first treatment for patients with advanced GIST. 46 After the approval of imatinib, subsequent research showed differential responses based on molecular subtype. Imatinib was found to be most active in KIT exon 11 mutations. 24,47 KIT exon 9 mutations, however, confer some degree of primary resistance, which may be overcome with higher doses. 25 PDGFRA D842V mutations, and those without KIT or PDGFRA driver mutations, showed no response. 34,47 Despite initial efficacy, most patients develop resistance to imatinib within 2 years. This is typically due to secondary KIT mutations within the kinase domain by either interfering with drug binding within the ATP-binding domain (exon 13/14 mutations) or b modifying the activation loop to stabilize the active conformation (exon 17/18 mutations). 23,26,48-51 Figure 1 shows the current understanding of primary and secondary resistance to select TKIs in KIT or PDGFRA mutant GIST.
Additional oral multitargeted TKIs were approved in sequential fashion in attempt to overcome secondary resistance.
Sunitinib and regorafenib work similarly to imatinib; however, they have a broader spectrum of activity that includes anti-angiogenic inhibition of vascular endothelial growth factor receptors 1-3 (VEGFR1-3). Sunitinib delayed time to tumor progression (TTP) compared to placebo (27 vs. 6 weeks) in phase III testing after imatinib failure 27,52,53 and was FDA approved in 2006 to treat imatinib-refractory or intolerant patients. 54 Regorafenib improved progression-free survival (PFS) compared to placebo (4.8 vs. 0.9 months) and was FDA approved in 2013 for patients after sunitinib failure. 55,56 Both sunitinib and regorafenib have shown some efficacy against secondary KIT mutations. In the clinical setting, sunitinib was found to have activity against ATP-binding domain mutations (exon 13/14), but not activation loop mutations (exon 17/18). 28 In the preclinical setting, regorafenib has activity against exon 17 mutations with less impact on exon 13 mutations. 29 Ripretinib is a switch-control targeting agent which inhibits KIT and PDGFRA by stabilizing their inactive conformation. Preclinical models demonstrate a wide spectrum of activity against several KIT mutations, including within the kinase domain. 30 Ripretinib significantly improved PFS compared to placebo (7.0 vs. 1.0 months) in the fourth line and beyond setting, 57,58 and was FDA approved in 2020. 59 PDGFRA D842V mutations on exon 18, which occur in roughly two-thirds of PDGFRA mutant GIST, showed no response to any approved TKIs. 34,47 Early-generation TKIs competitively inhibit the ATP pocket, thus stabilizing the inactive conformation; however, the D842V mutation shifts the PDGFRA kinase protein into the activated conformation, which renders the ATP pocket unavailable. 31, 60,61 Avapritinib, an oral, selective KIT and PDGFRA inhibitor, readily fits into the ATP binding pocket, with the activation loop in an open, aspartate (D)-phenylalanine (F)-glycine (G) motif (DFG)-in conformation 23,31,60 In a phase I study, avapritinib was found to be active in patients with PDGFRA D842V mutant GIST with an ORR of 88%. 32,62 This study led to FDA and European Medicines Agency approvals in 2020 use in GIST with PDGFRA exon 18 and with PDGFRA D842V mutations, respectively. 57,58 The 15% of patients with GIST that do not harbor a mutation in either KIT or PDGFRA have fewer treatment options. This GIST subtype does not respond to imatinib, 24,25,47 and has some, but limited, response to alternate TKIs, such as sunitinib. 63,64 Patients with SDH-deficient GIST and NF-related GIST are generally referred for clinical trial participation. Patients whose GIST harbors BRAF or NTRK alterations may benefit from BRAF/MEK 40,41 and NTRK 42 inhibition, respectively.
The optimal length of postoperative imatinib is unclear. For patients tolerating therapy, in some countries, it is not uncommon to continue postoperative imatinib beyond 3 years. This is especially the case with evidence that imatinib is not cytotoxic, but rather suppresses GIST growth, and most recurrences in adjuvant trials occurred within a year of discontinuing postoperative imatinib. 69,[72][73][74] Molecular testing has emerged as a critical tool to optimize decision making for adjuvant treatment in high-risk patients. For example, patients without KIT or PDGFRA mutant GIST, and those with PDGFRA D842V mutations do not benefit from postoperative imatinib. 69,70

Imatinib for the Majority
Imatinib remains the standard-of-care frontline treatment for most patients with advanced GIST.
One phase III study evaluated standard-dose (400 mg daily) compared with highdose (800 mg daily) in 746 patients. After a median follow-up of 4.5 years, the median PFS and OS did not differ between groups (18 vs. 20 months for PFS, and 55 and 51 months for OS, respectively). 45 Imatinib was well tolerated in both groups. Common toxicities included fatigue, diarrhea, rash, mild cytopenias, and fluid retention. Patients had higher rates of grade 3+ toxicities with 800 mg imatinib (63%) compared with 400 mg imatinib (43%). Dose reductions and delays were more common with 800 mg imatinib (58% and 59%, respectively) than 400 mg imatinib (16% and 38%). 45 Patients who crossed over from 400 to 800 mg imatinib at progression saw some effect, with 3% achieving partial response (PR) and 28% stable disease (SD), and a postcrossover median PFS of 5 months. This benefit in dose escalation, however, was limited to primary KIT exon 9 mutant tumors. 45 A separate phase III trial (EORTC-ISG-AGITG) of 946 patients, also randomized to imatinib 400 and 800 mg arms, had similar results upon crossover with 2% achieving PR and 27% achieving SD, and postcrossover median PFS of 3 months. 75 Post hoc analyses revealed patients with KIT exon 11 mutation had better response to imatinib therapy compared to those with KIT exon 9. 24,47 A meta-analysis of both phase III trials showed that patients with KIT exon 9 mutation who received high-dose imatinib had 42% higher median PFS and numerically, but not significantly better OS. 25 Patients with PDGFRA D842V and without KIT or PDGFRA mutant GIST were confirmed imatinib nonresponders. 24,25,34,47 In summary, imatinib remains first-line treatment for most patients with GIST. The standard-dose (400 mg/day) is appropriate for patients with KIT exon 11 mutant tumors; however, patients with KIT exon 9 mutated GIST may benefit from a higher starting dose (800 mg/day). A small portion of patients with KIT exon 9 mutation who progress on 400 mg may respond to escalation. Patients with PDGFRA D842V mutant GIST or without KIT or PDGFRA mutant GIST should not receive imatinib.

Avapritinib for PDGFRA Exon 18 Mutation
As discussed above, patients with PDGFRA exon 18 D842V mutations do not respond to imatinib. 76,77 Avapritinib was initially evaluated in NAVIGATOR, an open-label phase I trial of 250 patients with unresectable GIST, 56 of whom harbored a PDGFRA exon 18 D842V mutation. 32, 62 In the initial report, 49 D842V patients (88%) had an objective response with 5 patients (13%) achieving a CR. At a median follow-up of 15.9 months, the estimated 1-year and 2-year OS were 91% and 81%, respectively. 32 A subsequent analysis, performed at median follow-up of 27.5 months, showed similar ORR of 91% with median duration of response of 28 months, median PFS of 34 months, and median OS not yet reached. 62 Avapritinib had similar toxicities to other TKIs (fatigue, diarrhea, peripheral edema, cytopenias); however, it produced notable safety signals for cognitive effects (eg, impairment, mood changes, hallucinations, disordered sleep) and for intracranial hemorrhage. Among participations, 40% had cognitive effects of any grade, a majority (58%) grade 1, and 2% had intracranial hemorrhage (grade 3, nonfatal). The trial established 300 mg as the recommended dose, though 84% of patients did require at least one dose reduction or hold. 32 A longer-term analysis reaffirmed these findings with 96% requiring dose modification or delay, 46% of participants developing cognitive effects (any grade), and 3% developing intracranial hemorrhage. 62 Avapritinib is the first drug approved for a specific molecular subtype of GIST establishing the standard-of-care for patients with PDGFRA D842V, exon 18 mutant GIST.

Subsequent Lines of Treatment
Several TKIs have been approved for treatment of imatinib-refractory GIST.

Sunitinib
Sunitinib is FDA-approved for patients who are refractory or intolerant to imatinib therapy. 54 Sunitinib was evaluated in a randomized, placebocontrolled phase III trial of 312 patients with imatinibresistant GIST. Due to significant treatment effect at interim analysis, the trial was unblinded early. The median TTP was longer with sunitinib (27.3 weeks) compared with placebo (6.4 weeks), as was median PFS (24 vs. 6 weeks). Sunitinib was generally well-tolerated with infrequent dose reductions (11%) and interruptions (28%). 27 A treatment-use study followed patients with imatinibresistant GIST who received sunitinib as part of an expanded access program. In total, 1124 patients were recruited across 34 countries and had median TTP of 8.3 months and median OS of 16.6 months. 52 Common toxicities (any grade) included fatigue, diarrhea, hand-foot syndrome/skin changes, hypertension, and mild cytopenias. 27,52,53 In a longitudinal follow-up analysis, some patients (13%) developed clinically significant hypothyroidism, mostly grade 1 to 2 (11%). Adverse cardiac events, such as reduced ejection fraction (EF) and left ventricular (LV) dysfunction, occurred (11%); however, grade 3 or higher events were uncommon (<1%). 52 Sunitinib was initially studied at dosing of 50 mg in a 4-weeks on/2-weeks off regimen which remains the FDAapproved dose; however, subsequent trials evaluated continuous 37.5 mg daily which suggested similar outcomes and remains a guideline-recommended option. 78 Molecular analyses demonstrated sunitinib is active against multiple KIT mutations. Interestingly, median PFS and OS were significantly longer in KIT exon 9 than KIT exon 11 (PFS: 19.4 vs. 5.1 months; OS: 26.9 vs. 12.3 months, respectively). Patients with secondary KIT exon 13/14 mutations had longer median PFS than patients with secondary KIT exon 17/18 (7.8 vs. 2.3 months). 28,29 Sunitinib is the only second-line therapy approved for molecularly unselected patients with GIST. It is well-tolerated with a manageable side effect profile.

Regorafenib
Regorafenib is FDA-approved for patients refractory or intolerant to imatinib and sunitinib after demonstrating efficacy in phase II and III studies. 55,56 GRID was a randomized, placebo-controlled phase III trial of patients with GIST after prior imatinib and sunitinib. Median PFS was 4.8 months in the regorafenib group compared to 0.9 months in the placebo group (HR 0.27, CI 0.19-0.39). No difference was noted in OS (HR 0.77, CI 0.42-1.41); however, 85% of participants in the placebo arm crossed over to receive regorafenib. Regorafenib was dosed at a starting dose of 160 mg daily for 3-weeks on/1-week off and generally well-tolerated. The most common treatmentrelated grade 3 or higher events were hypertension, handfoot syndrome, and diarrhea. Dose modifications (72%) were common, but permanent discontinuation due to toxicity was infrequent (6%). 56 Post hoc molecular analysis demonstrated regorafenib is active against certain KIT secondary mutations, notably exon 17/18. Regorafenib has some activity against KIT exon 13/14 mutations, with the exception of exon 13 V654A mutation. 29 Regorafenib is the only TKI approved in the third line for advanced GIST.

Ripretinib
Ripretinib 30 has been studied in 2 phase III trials, 33,79 and is FDA-approved for treatment in the fourth-line setting. 59 INVICTUS was a 129-patient, double-blind, placebocontrolled phase III trial in advanced, multidrug refractory GIST. After a median follow-up of 6.3 months, the median PFS was greater for ripretinib (6.9 months) than for placebo (1 month) (HR 0.15, CI 0.09-0.25). Sixty-six percent of patients assigned to the placebo arm crossed over to ripretinib upon progression. The median OS was significantly higher for ripretinib (15 months) compared to placebo (6.6 months) (HR 0.36, CI 0.21-0.62). 79 The starting dose of ripretinib is 150 mg daily. The phase I study of ripretinib allowed for dose-escalation to 150 mg BID at disease progression. Of the 142 total participants, 67 participants in the second (n = 10), third (n = 17), and fourth (n = 40) line setting underwent dose-escalation and had postescalation median PFS of 5.6, 3.3, and 4.6 months, respectively. 80 Additionally, a preplanned analysis of INVICTUS demonstrated that dose-escalation in 43 participants led to a postescalation median PFS of 3.7 months. 81 Given the benefit seen in INVICTUS, ripretinib was investigated in the second-line setting. The INTRUIGE study was a double-blind, randomized phase III trial of 453 patients comparing ripretinib to sunitinib after imatinib failure. The median PFS did not differ between the ripretinib and sunitinib arms in the total population (8 vs. 8.3 months, respectively) and in the KIT exon 11 cohort (8.3 vs. 7.0 months, respectively). However, patients with KIT exon 9 mutation had greater benefit with sunitinib over ripretinib (13.8 vs. 5.5 months, HR 2.85, CI 1.48-5.48). 33 Though ripretinib did not demonstrate PFS benefit in the total population, an exploratory circulating tumor DNA (ctDNA) analysis of INTRUIGE revealed differential PFS based on mutational profile. Namely, patients with ctDNA detection of KIT exon 11 and 17/18 mutations (n = 52) had significantly improved PFS on ripretinib (n = 27) compared to sunitinib (n = 25) (14.2 vs. 1.5 months, HR = 0.22, P < .001). Alternatively, patients with KIT exon 11 and 13/14 mutations (n = 41) had shorter PFS on ripretinib (n = 21) compared to sunitinib (n = 20) (4.0 vs. 15.0 months). 82 Based on these analyses of INTRIUGE, the National Comprehensive Cancer Network (NCCN) recommends ripretinib as an option for patients intolerant to sunitinib. 83 Moreover, a randomized phase 3 trial is planned to compare sunitinib versus ripretinib in KIT exon 11/17/18-mutant GIST. 84 Ripretinib is generally well-tolerated with common toxicities including fatigue, nausea, alopecia, hand-foot syndrome, and diarrhea. The most common grade 3+ toxicities included lipase elevation, hypertension, fatigue, and hypophosphatemia. 79 Avapritinib Avapritinib is approved for use in PGFRA exon 18 mutated GIST 57 based on results from the NAVIGATOR trial described above. 32, 62 The VOGAGER phase III study evaluated avapritinib compared to regorafenib in a molecularly unselected treatment-refractory GIST population of patients with KIT or PDGRA mutant GIST, and avapritinib was not found to be superior to regorafenib. 85

Future Direction
Nearly all advanced GIST with KIT or PDGFRA mutations will eventually develop resistance to currently available TKIs. Developmental therapeutic efforts are focused on strategies to overcome secondary resistance mutations and to identify active treatments for patients without KIT or PDGFRA mutant GIST.
In addition, the role of circulating tumor DNA (ctDNA) continues to evolve with potential to: monitor for disease relapse, assess treatment response, and identify resistance mutations. Prospective efforts to optimize its role in care and research are ongoing, but with promising initial results. 82,[86][87][88][89][90][91] Overcoming KIT Resistance Mutations Resistance to KIT inhibition is typically due to secondary mutations within the ATP-binding domain (exon 13/14 mutations) or activation loop (exon 17/18 mutations). Most approved TKIs, such as imatinib and sunitinib, are type II inhibitors and block the kinase domain only when in its inactive confirmation; however, certain activation loop mutations, such as KIT exon 17 D816V, stabilize the active confirmation. Type I inhibitors hold promise as they can access and inhibit the active conformation. 23,26,[48][49][50][51] Bezuclastinib Bezuclastinib (CGT9486, previously PLX94986: Cogent Biosciences), is an oral type I KIT inhibitor with high in vitro activity against primary KIT exon 9 and 11 mutations, as well as secondary KIT exon 17/18 mutations, including exon 17 D816V mutation. 92 A 2-part phase Ib/IIa trial of bezuclastinib in imatinibresistant GIST demonstrated single-agent safety and efficacy. Common AEs, mostly grade 1-2, included fatigue, AST elevation, diarrhea, and nausea; grade 3 or higher AEs included anemia and hyperuricemia. Single-agent ORR, CBR, and median PFS at the recommended phase II dose (RP2D) of 1000 mg daily, was 8%, 50%, and 5.75 months, respectively. Combined sunitinib and bezuclastinib at RP2D were confirmed safe, with anticipated adverse events for each agent, and had heightened efficacy with ORR 20%, CBR 80%, and PFS 12.1 months. ctDNA analysis demonstrated single-agent bezuclastinib reduced circulating KIT exon 17/18 mutations and combined therapy further suppressed KIT exon 13/14 mutations 93 A phase III randomized open-label trial of CGT9486 and sunitinib versus sunitinib alone (NCT05208047) is currently underway in patients with imatinib resistance or intolerance. 94

IDRX-42
IDRX-42 (Previously M4205: IDRx, Inc.), is a highly selective, type II KIT inhibitor designed to have broad activity against common primary and secondary KIT mutations, such as exon 13 V654A. Preclinical testing of IDRX-42 in patient-derived, multidrug-resistant GIST xenograft mice models showed tumor shrinkage and reduction in mitoses. 95 IDRX-42 is currently in first-in-human phase I testing (NCT05489237). 96

THE-630
THE-630 (Theseus Pharmaceuticals) was also designed as an oral pan-KIT inhibitor with activity against primary and secondary KIT mutations. Preclinical testing of THE-630 in GIST demonstrated potent inhibition against both ATP binding domain and activation loop mutations. In ATP binding domain mutations, such as V654A, THE-630 produced greater tumor growth inhibition compared with ripretinib (86% vs. 26%). In activation loop mutations, such as N822K and D820A, THE-630 compared favorably with sunitinib (88% vs. 25%) and ripretinib (59% vs. 1%), respectively. 97 THE-630 is currently undergoing first-in-human phase I/II doseescalation and dose-expansion testing (NCT05160168). 98 NB003 NB003 (Previously AZD3229: Ningbo Newbay Technology Development Co., Ltd), was also designed as a broad inhibitor of KIT and PDGFRA mutant GIST. Preclinical in vitro testing demonstrated superior potency against both primary and secondary KIT mutations compared to imatinib and other approved agents. 99 NB003 is currently undergoing phase 1 testing in patients with advanced GIST who progressed on or were intolerant to imatinib and other subsequentline agents. 100

Manipulating Related Pathways
Preclinical models have shown that expression of ETV1, a member of the ETS family of transcription factors, is necessary for GIST tumor growth, and KIT activation promotes expression and stability of ETV1. 101,102 Combined inhibition of KIT and ETV1 with imatinib and binimetinib (MEK-162), respectively, showed synergistic effect in in vitro and in vivo models. 103 Phase I and II (NCT01991379) testing found this combination to be safe and well-tolerated in treatment-naïve patients. 104,105 The ORR was 69%, and median PFS was 30 months. 105 Heat-shock protein 90 (Hsp90), a chaperone molecule necessary for KIT and PDGFRA protein folding, emerged as a compelling target. 23 Preclinical models of pimitespib (TAS-116), a novel Hsp90 inhibitor, showed efficacy in both imatinib-naïve and resistant cell lines. 106 Phase I testing of pimitespib demonstrated safety with lower ocular and hepatotoxicity than prior Hsp90 inhibitors. 107 CHAPTER-GIST-301 was a randomized, placebocontrolled phase III trial of pimitespib in 86 patients with multi-drug refractory GIST. Patients in the pimitespib arm had higher median PFS than placebo (2.8 vs. 1.4 months, HR 0.51, P = .0006) and were 58% less likely to die. 108 A phase I trial of pimitespib in combination with imatinib (NCT05245968) is currently recruiting. 109 mTOR inhibitors, such as everolimus, act downstream of mutant KIT and PDGFRA to reduce the activity of the PI3K/ Akt/mTOR pathway. Preclinical testing demonstrated efficacy of combined mTOR and KIT inhibition in imatinib-resistant GIST. 110 A phase I/II study of everolimus 2.5 mg daily and imatinib 600 mg daily, respectively, demonstrated an acceptable safety profile and potential efficacy with lengthened PFS after imatinib and sunitinib failure. 111 New Treatments for SDH-Deficient GIST SDH-deficient GIST is characterized by epigenetic changes, in particular global DNA methylation, which leads to upregulation of fibroblast growth factor receptor (FGFR). [112][113][114] This overexpression of FGFR has been implicated in the pathogenesis of SDH-deficient GIST, and FGFR inhibition in patient-derived, SDH-deficient xenograft mice models led to significant tumor reduction. 102 Rogaratinib (BAY 1163877: Bayer), an oral pan-FGFR inhibitor (FRFR 1-4) is currently undergoing phase II testing in patients with FGFR1-4 altered sarcomas or SDH-deficient GIST (NCT04595747). 115

Conclusions
The management of advanced GIST has been revolutionized by improved understanding of molecular pathogenesis and emergence of oral targeted therapies. The majority (85%) of GIST are characterized by activating mutations in KIT or PDGFRA. Primary driver mutations can occur in the extracellular (KIT exon 9), juxta-membrane (KIT exon 11, PDGFRA exon 12), and ATP-binding site (KIT exon 13, PDGFRA exon 14) or activation loop (KIT exon 17, PDGFRA exon 18).
Imatinib was found to inhibit KIT and PDGFRA and became the first approved treatment for patients with GIST. Most patients benefited; however, treatment responses varied based on driver mutation with some, such as PDGFRA D842V and without KIT or PGDFRA, showing no response.
Nearly all patients eventually develop imatinib resistance as secondary mutations develop, typically in the ATP-binding domain or activation loop of the kinase domain. Sunitinib, regorafenib, and ripretinib were approved in response to imatinib resistance. Avapritinib was found to have impressive activity and approved as treatment in PDGFRA D842V mutations.
Future advances will hopefully come from the development of next generation KIT inhibitors, such as bezuclastinib (CGT9486), THE-630, and IDRX-42, and from inhibition of pathogenic processes, such as ETV1 (with binimetinib), Hsp90 (with pimitespib), and the PI3K/Akt/mTOR pathway. Finally, targeted treatments for SDH-deficient GIST, such as FGFR inhibition (with rogaratinib), are sorely needed.

Author Contributions
All authors contributed to the conception/design, manuscript writing, and final approval of manuscript.

Data Availability
No new data were generated or analyzed in support of this research.