KIT Mutations In Gastrointestinal Stromal Tumors Research Articles

Clinical efficacy of avapritinib in gastrointestinal stromal tumors (GIST) with different KIT genotypes: Post hoc analysis of the phase 1 NAVIGATOR and phase 1/2 CS3007-101 trials.

11523 Background: Avapritinib, a novel KIT/platelet-derived growth factor receptor α (PDGFRA) kinase inhibitor, is approved in patients (pts) with GIST harboring PDGFRA activation loop (AL) exon 18 mutations, based on unprecedented clinical antitumor activity. Despite available treatments, there remains a great unmet medical need for pts with KIT-AL-mutant GIST receiving 2nd-line (2L) standard of care (SOC) sunitinib, and pts with primary KIT exon 9 ( KIT 9)-mutant GIST receiving 4th-line (4L) SOC ripretinib. This post hoc analysis assessed the clinical benefit of avapritinib in pts with KIT mutations, especially with AL exons 17 and 18, or primary KIT 9 mutations. Methods: Tumor tissue and liquid biopsy samples were collected and analyzed to determine baseline mutational status. KIT molecular subgroups and outcomes were determined in pts with primary KIT mutations treated with oral avapritinib 300 mg once daily (starting dose) enrolled in the phase 1 NAVIGATOR (NCT02508532) and phase 1/2 (China bridging) trials (NCT04254939, CS3007-101). Pts with PDGFRA mutations and PDGFRA- KIT co-mutations were excluded. Progression-free survival (PFS) and objective response rate (ORR) were compared using Cox and logistic regression, respectively. Results: A total of 160 pts with KIT-mutant GIST were evaluated (NAVIGATOR, n= 131; CS3007-101, n= 29). At data cutoff (NAVIGATOR: March 31, 2021; CS3007-101: June 30, 2021), median follow-up duration was 22.0 months (95% CI 18.3–27.4). KIT-AL mutations were more frequently detected (n = 74, 46.3%) than KIT ATP-binding pocket (ABP) mutations (n = 34, 21.3%). Sixty pts (37.5%) had KIT-AL mutations without KIT-ABP mutations (ALposABPneg group); the remaining 100 pts were designated as KIT OTHERS. Across all lines of therapy, the adjusted (inverse probability weighting of baseline characteristics) median PFS (mPFS) was longer for the ALposABPneg group than for the KIT OTHERS group (9.1 vs 3.4 months; HR 0.47, 95% CI 0.32–0.68; P< 0.0001), and the ORR was higher (31.4% vs 12.1%; odds ratio 3.31, 95% CI 1.44–7.58; P= 0.0047). mPFS and ORR in the KIT ALposABPneg group were 19.3 months and 38.5%, respectively, in the 2L setting (n = 13) and 11.0 months and 36.4%, respectively, in Chinese pts (3–9 lines, n = 11). In pts with KIT 9 mutations in the 4L (n = 14) and > 4L settings (n = 19), mPFS was 5.6 and 3.7 months, respectively. Conclusions: This post hoc analysis demonstrated significantly more robust avapritinib antitumor activity in pts with KIT ALposABPneg GIST versus pts with other KIT mutation profiles. The results suggest that avapritinib could confer meaningful clinical benefit in pts with GIST and specific KIT mutation types, especially KIT-AL or KIT 9 mutations. Avapritinib might be a potential 2L option for pts with KIT ALposABPneg GIST, and in later lines of therapy for pts with ALposABPneg or KIT 9 mutation profiles. Clinical trial information: NCT02508532 , NCT04254939 .

Fluorine-18 fluorodeoxyglucose (18FDG)-uptake assessment of PDGFRA mutant gastrointestinal stromal tumors (GIST): A retrospective multicenter Italian study.

11539 Background: D842V PDGFRA mutation identifies a molecular subtype of GIST, primarly resistant to imatinib and characterized by higher indolent behavior and a prominent immune profile. Although functional imaging with 18FDG-PET plays a proven role in GIST, especially in early assessment tumor response, less is known about 18FDG-uptake according to the GIST molecular subtypes. Taking into account of clinical and molecular features of D842V mutant GIST, we assumed that this subset of GIST could also have a different 18FDG-uptake. Therefore, the aim of the present study has been to investigate the degree of FDG uptake of PDGFRA mutant GIST, focusing on D842V ones, in order to better define the role of functional imaging in this rare and peculiar subset of GIST. Methods: Patients with PDGFRA mutant GIST underwent 18FDG-PET were retrospectively included from seven GIST Italian reference centers. Data on maximum standardized uptake (SUVmax) value of primary tumor or metastatic disease were collected. Results: 71 patients have been included: 37 (55.1%) with D842V PDGFRA mutant GIST (group A) and 34 (47.9%) with PDGFRA non-D842V mutant GIST (group B). Additionally, 30 patients with exon 11 KIT mutant GIST have been included, as control (group C). SUVmax values were obtained from primary tumor and metastatic lesions in 55 (54,4%) and 46 (45,6%) patients, respectively. Considering the whole population of 101 patients, the global median SUVmax was 3,7 (IQR 0-9.1), while the median SUVmax for group A, B, and C was 0, 3.6, and 10.4, respectively. The median SUVmax of PDGFRA mutant GIST was significantly lower than the median value of exon 11 KIT mutant GIST (p < 0.001). Notably, median 18FDG-uptake was significantly lower in D842V PDGFRA mutant GIST compared to PDGFRA non-D842V mutant ones (p = 0.021). Conclusions: PDGFRA D842V-mutant GIST present an overall lower 18FDG uptake compared to other GIST subgroups. This feature could be related to their specific molecular background and is consistent with their higher tendency to an indolent behavior. Therefore, the role of functional imaging with 18FDG-PET in this subset of GIST is limited. Finally, the prognostic value of the 18FDG-uptake degree within all PDGFRA mutant GIST will be investigated in the future.

Precision Oncology in Gastrointestinal Stromal Tumors

GIST (gastrointestinal stromal tumors) represent 20% of sarcomatous tumors and 1-2% of primary gastrointestinal cancers. They have an excellent prognosis when localized and resectable, though their prognosis is poor in the metastatic setting, with limited options after the second line until recently. Four lines are now standard in KIT-mutated GIST and one in PDGFRA-mutated GIST. An exponential growth of new treatments is expected in this era of molecular diagnostic techniques and systematic sequencing. Currently, the main challenge remains the emergence of resistance linked to secondary mutations caused by selective pressure induced by TKIs. Repeating biopsies to tailor treatments might be a step in the right direction, and liquid biopsies at progression may offer a non-invasive alternative. New molecules with wider KIT inhibition are under investigation and could change the catalog and the sequence of existing treatments. Combination therapies may also be an approach to overcome current resistance mechanisms. Here, we review the current epidemiology and biology of GIST and discuss future management options, with an emphasis on genome-oriented therapies.

Abstract 4872: DCC-3116, a first-in-class selective ULK1/2 inhibitor of autophagy, in combination with the KIT inhibitor ripretinib induces complete regressions in GIST preclinical models

Abstract Background: Cancer cells activate autophagy as an adaptive stress response (ASR) mechanism to therapies targeting the RTK/RAS/MAPK/PI3K pathways, limiting antitumor response. Autophagy is initiated through ULK1/2 kinases and is triggered by inhibitors of the MAPK and PI3K pathways. Most gastrointestinal stromal tumors (GIST) are driven by mutations in KIT kinase. KIT signals through MAPK/PI3K pathways, suppressing ULK1/2 kinases and autophagy1,2,3. Inhibition of mutant KIT reverses this suppression, activating autophagy and cancer cell survival. Approved therapies for GIST include imatinib, sunitinib, regorafenib, ripretinib, and avapritinib. Treatment with these inhibitors is initially successful, but drug resistance can develop either through KIT secondary mutations or ASR pathways including autophagy. DCC-3116 is a selective, potent, first-in-class investigational inhibitor of the ULK1/2 in clinical development in combination with targeted therapies that activate the autophagic ASR pathway. Herein we demonstrate that ULK1/2 and autophagy are activated upon treatment with ripretinib in KIT mutant GIST models A combination of ripretinib with DCC-3116 inhibits autophagy in vitro and leads to complete tumor regressions in preclinical models of GIST. Methods: Inhibition of ULK1/2 in cell assays was measured using an ELISA for the ULK substrate phospho-ATG13 (pATG13). Autophagic flux was measured by monitoring mCherry/GFP tagged LC3 protein in GIST cells. Xenograft studies were performed at CROs. Results: Ripretinib treatment led to the activation of ULK1/2 by 2-3-fold in mutant KIT GIST cell lines. DCC-3116 inhibited both ripretinib-induced and basal pATG13 with IC50 values of 12-32 nM. Treatment of GIST-T1 cells with ripretinib increased autophagic flux 3-fold. DCC-3116 potently inhibited flux with an IC50 value of 38 nM. Ripretinib also induced pATG13 and autophagic flux (1.5-2.5-fold) in multiple imatinib-resistant cell lines, which was inhibited by DCC-3116 with IC50 values between 8-189 nM. In a GIST T1 PK/PD model, DCC-3116 inhibited ULK1/2-mediated pATG13. The combination of DCC-3116 with ripretinib resulted in complete tumor regressions in comparison to single agent treatment in GIST preclinical models. Conclusions: These data demonstrate preclinically that, like other receptor tyrosine kinase inhibitors1, ripretinib activates ULK1/2-mediated autophagy as an ASR resistance mechanism which is inhibited by DCC-3116, providing the rationale to study the combination of DCC-3116 with ripretinib in GIST patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors (NCT04892017).

Abstract 3887: ATP-binding pocket substitutions as secondary or tertiary in-cis mutations are major on-target ripretinib resistance mechanisms in gastrointestinal stromal tumor

Abstract Introduction: Ripretinib (Rip) is a kinase inhibitor with broad preclinical activity against mutant KIT. Based on the INVICTUS trial, Rip was approved for patients with Gastrointestinal Stromal Tumors (GIST) after treatment with 3 or more kinase inhibitors. Most patients in this ≥4th-line setting progress on Rip within one year. Here, we characterized Rip-progressing GIST samples to identify resistance mechanisms. Methods: Progressing lesions in 25 patients were analyzed by NGS after Rip failure. KIT mutations (muts) were recapitulated by gene editing. Activities of Rip, sunitinib (SU), and novel TKIs were characterized by cell viability assays and immunoblot. Mutagenesis experiments were performed using ENU. SU- and Rip-resistant cell lines were pooled and treated with various regimens, with clonal composition deconvoluted by cDNA-based amplicon sequencing. Results: 19/25 Rip-progressing GISTs displayed muts in the ATP-binding pocket (ATP-BP; e13/14). Four of these had pre-existing muts in the activation loop (AL; e17/18), which were confirmed to be in-cis with the primary and ATP-BP muts (triple in cis; TIC). Mutagenesis screens using a double KIT-mutant GIST line (e11 + AL) as a starting point confirmed that TIC-muts are a predominant escape mechanism when treated with Rip. A GIST subline (T1-triple) with TIC-muts in e11, e18 (A829P), and e13 (V654A) was highly resistant to Rip (GR50 > 2µM). Structural analyses of ATP-BP muts suggest steric interference and loss of van der Waals interactions that impede Rip binding and thereby confer Rip resistance. Another 3/25 Rip-progressing GISTs harbored pathogenic KIT muts in e9 only. A novel GIST cell line with primary KIT e9 mut (T1-e9) was 14-fold less sensitive to Rip than isogenic e11-driven cells (GR50 = 115 vs 8nM). Notably, adding a typical AL mut to T1-e9 (T1-e9-N822K) sensitized the cells to Rip (GR50 = 20nM). Immunoblots showed >95% inhibition of phospho-KIT in AL-mutant cell lines at Rip 100nM, whereas inhibition was weaker in T1-e9 (77%) and T1-V654A (46%), and absent in T1-triple. A compound screen of FDA-approved kinase inhibitors identified Nintedanib (NIN) as the most active compound against T1-triple. Clonal outgrowth assays of mixed cultures revealed SU (93% inhibition), and a weekly switch between Rip and either SU (96%) or NIN (79%) as the most effective inhibitors of pooled cell growth. Conclusions: KIT e9 primary muts and e13/14 (ATP-BP) secondary muts are enriched in post-progression biopsies following Rip treatment. KIT TIC-muts are novel frequent events driving GIST clinical progression and confer a high degree of resistance. Strategies to overcome resistance may include combinations or sequences of approved drugs, and novel drugs that more efficiently inhibit TIC-mutant KIT. Citation Format: Thomas Mühlenberg, Johanna Falkenhorst, Tom Schulz, Benjamin S. Fletcher, Alina Teuber, Dawid Krzeciesa, Isabella Klooster, Jonas Lategahn, Wen-Bin Ou, Meijun Lundberg, Margaret von Mehren, Susanne Grunewald, Alicia I. Tüns, Mehdi Brahmi, Michael C. Heinrich, Cesar Serrano, Hans-Ulrich Schildhaus, Sonja Sievers, Jürgen Treckmann, Lydia Wilson, Chandrajit P. Raut, Adrian Marino-Enriquez, Suzanne George, Daniel Rauh, Jonathan A. Fletcher, Sebastian Bauer. ATP-binding pocket substitutions as secondary or tertiary in-cis mutations are major on-target ripretinib resistance mechanisms in gastrointestinal stromal tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3887.

TKI Treatment Sequencing in Advanced Gastrointestinal Stromal Tumors.

Prior to the early 2000s, patients with advanced gastrointestinal stromal tumors (GIST) had very poor prognoses owing to a lack of effective therapies. The development of tyrosine kinase inhibitors at the turn of the century significantly improved the overall survival for patients with GIST. The resounding success of imatinib in the first clinical trial of a tyrosine kinase inhibitor to treat GIST led to its approval for first-line therapy for advanced GIST; this study was open to all comers and not restricted to any GIST subtype(s). The trials that led to the approvals of second-, third-, and fourth-line therapy for advanced GIST were also open to all patients with advanced/metastatic GIST. Only in retrospect do we realize the role that the molecular subtypes played in the results observed in these studies. In this review, we discuss the studies that led to the US Food and Drug Administration approval of imatinib (first line), sunitinib (second line), regorafenib (third line), and ripretinib (fourth line) for advanced KIT-mutant GIST. In addition, we review how information about GIST molecular subtypes has been used to accelerate the approval of other targeted therapies for non-KIT mutant GIST, leading to the approval of five additional drugs indicated for the treatment of specific GIST molecular subtypes. We also discuss how our understanding of the molecular subtypes will play a role in the next generation of therapeutic approaches for treating advanced GIST.

A phase 1, multicenter, open-label, first-in-human study of DS-6157a in patients (pts) with advanced gastrointestinal stromal tumor (GIST).

11512 Background: GPR20 is selectively and abundantly expressed in GISTs, the most common sarcoma of digestive tract. DS-6157a is an anti-GPR20 antibody-drug conjugate with a novel tetrapeptide-based linker and DNA topoisomerase I inhibitor exatecan derivative (DXd). The target drug-to-antibody ratio is ̃8. The DXd payload, when released, inhibits topoisomerase I and induces cell apoptosis. Methods: This study conducted in the US and Japan was designed to include a dose-escalation (Part 1) and a dose-expansion (Part 2) in adult pts with advanced GIST (NCT04276415). Part 1 evaluated safety, tolerability, efficacy, and pharmacokinetics (PK) to determine the maximum-tolerated dose (MTD) and/or recommended dose for expansion. Key eligibility criteria included ECOG PS of 0 or 1, measurable disease per RECIST v1.1, and adequate bone marrow, hepatic, and renal function. DS-6157a was administered every 3 weeks as monotherapy. Pts had tumor assessments per RECIST 1.1 every 6 weeks (wks) for 36 wks, then every 9 wks thereafter. Results: At data cutoff, 34 pts were available for full analysis: median treatment duration was 9.9 wks (range, 3-56 wks) and 5 pts (14.7%) were ongoing; median age 60.5 years (y) (range, 29-81 y); 19 pts (56%) were male; 47% of pts were Asian and 47% were white. Baseline GPR20 expression was highly prevalent, and dose-dependent PK exposure was confirmed within the range of dose levels. The MTD was 6.4 mg/kg. One pt with SDH-deficient GIST with both SDH B and NF1 mutations, had a confirmed PR with shrinkage of -75%; 18 pts (53%) experienced stable disease and 10 pts (29%) experienced progressive disease as best response. Median progression-free survival across all dose levels was 3.6 months (95% CI, 1.6, 6.9). Treatment-emergent AEs (TEAEs) occurred in all pts and treatment-related TEAEs (TRAEs) in 32 pts (94%). The most common (≥25%) all-grade (Gr) TEAEs were nausea (82%), decreased appetite (59%), fatigue (47%), anemia (44%), constipation (38%), decreased platelets (35%), and vomiting (32%). Gr ≥3 TRAEs occurred in 14 pts (41%); the most common were decreased platelets (21%) and anemia (18%). Serious TEAEs (SAEs) occurred in 9 pts (27%) and related SAEs in 4 pts (12%). Related Gr 4 SAEs in 2 pts included abnormal hepatic function, neutropenia, thrombocytopenia and leukopenia. There was 1 treatment related death (hepatic failure). Further investigations are on-going to understand the modest clinical efficacy observed. The study did not proceed to Part 2, because the data from Part 1 did not meet efficacy targets. Conclusions: DS-6157a was generally well tolerated with early signs of moderate clinical activity. While there were no objective responses in pts with KIT-mutant GIST, tumor shrinkage was observed in all 4 pts with KIT wild-type GIST treated at different doses, including a confirmed PR at the MTD in a pt with SDH-deficient GIST with both SDH B and NF1 mutations. Clinical trial information: NCT04276415.

Circulating tumor DNA (ctDNA) analyses of the phase III VOYAGER trial: KIT mutational landscape and outcomes in patients with advanced gastrointestinal stromal tumor (GIST).

101 Background: The genotype of primary mutations predicts imatinib response in untreated metastatic GIST. However, the sequence of salvage treatments in metastatic GIST is based solely on the chronological order of registration trials. ctDNA sequencing offers a powerful diagnostic tool to detect resistance mutations in GIST but has not been shown to correlate with outcomes in clinical trials of pretreated patients (pts). We analyzed ctDNA samples collected at baseline in the phase III VOYAGER trial (NCT03465722) to describe the landscape of KIT alterations and its association with outcomes of pts treated with avapritinib or regorafenib. Methods: In VOYAGER, 476 pts with advanced KIT-mutant GIST were randomly assigned to avapritinib (240 pts) or regorafenib (236 pts) in 3rd-4th line. Baseline plasma was collected and ctDNA analyzed with the Guardant 360 (G360), 74-gene panel. KIT molecular subgroups were determined and correlated with outcomes. PDGFRA-mutant GISTs were excluded from outcomes analysis. Results: Baseline ctDNA analysis was performed in 386/476 pts (81%). ctDNA was detected in 333 pts (86%), with 250 and 18 pts showing at least one KIT (75%) or PDGFRA (5%) variant, respectively. KIT primary mutations were detected in 71% pts (exon 11, 56%; exon 9, 14%; exon 13, 1%) and KIT secondary mutations in 55% of pts. Activation loop (AL, exons 17 and 18) was more commonly affected (44%) than the ATP-binding pocket (ABP, exons 13 and 14; 23%). Among KIT-mutant tumors, multiple KIT mutations were commonly detected within individual tumors (mean, 2.56; range, 1-14). Notably, 17% of pts had > 3 mutations (mean, 6.07; range, 4 to 14). Median PFS and OS were shorter for patients whose ctDNA was positive for V654A or T670I (ABP hot spots) when treated with avapritinib vs. regorafenib: mPFS, 1.9 mo vs. 7.4 mo; log-rank p <.001; mOS, 8.3 mo vs. 11.7 mo; log rank p =.0651. mPFS was shorter for patients with ctDNA positive for KIT exon 17 mutation if concurrently KIT V654A/T670I was absent when treated with avapritinib, with no difference in OS: mPFS, 4.7 mo vs. 6.7 mo; log-rank p =.03; mOS, 19.2 mo vs. NR; log-rank p =.628. mPFS on avapritinib was longer when ABP mutations were absent when compared to those with ABP present (5.6 vs. 1.9 mo; log-rank p <.001). There were no differences considering AL mutations vs. no AL mutations (3.8 vs. 3.9 mo; log-rank p =.622) when treated with avapritinib. Regorafenib showed similar activity regardless of KIT mutational status and the location of KIT mutation. Conclusions: Hybrid capture-based plasma sequencing detects ctDNA in the majority of patients with advanced TKI-resistant GIST, including heterogeneity of KIT mutations. This study is the first to show that ctDNA sequencing correlates with outcomes in pretreated GIST. Identification of ABP (exon13/14) KIT mutations negatively correlates with avapritinib activity.

Circulating Tumor DNA Mutations in Progressive Gastrointestinal Stromal Tumors Identify Biomarkers of Treatment Resistance and Uncover Potential Therapeutic Strategies.

Liquid biopsy circulating tumor DNA (ctDNA)-based approaches may represent a non-invasive means for molecular interrogation of gastrointestinal stromal tumors (GISTs). We deployed a customized 29-gene Archer® LiquidPlex™ targeted panel on 64 plasma samples from 46 patients. The majority were known to harbor KIT mutations (n = 41, 89.1%), while 3 were PDGFRA exon 18 D842V mutants and the rest (n = 2) were wild type for KIT and PDGFRA. In terms of disease stage, 14 (30.4%) were localized GISTs that had undergone complete surgical resection while the rest (n = 32) were metastatic. Among ten patients, including 7 on tyrosine kinase inhibitors, with evidence of disease progression at study inclusion, mutations in ctDNA were detected in 7 cases (70%). Known somatic mutations in KIT (n = 5) or PDGFRA (n = 1) in ctDNA were identified only among 6 of the 10 patients. These KIT mutants included duplication, indels, and single-nucleotide variants. The median mutant AF in ctDNA was 11.0% (range, 0.38%–45.0%). In patients with metastatic progressive KIT-mutant GIST, tumor burden was higher with detectable KIT ctDNA mutation than in those without (median, 5.97 cm vs. 2.40 cm, p = 0.0195). None of the known tumor mutations were detected in ctDNA for localized cases (n = 14) or metastatic cases without evidence of disease progression (n = 22). In patients with serial samples along progression of disease, secondary acquired mutations, including a potentially actionable PIK3CA exon 9 c.1633G>A mutation, were detected. ctDNA mutations were not detectable when patients responded to a switch in TKI therapy. In conclusion, detection of GIST-related mutations in ctDNA using a customized targeted NGS panel represents an attractive non-invasive means to obtain clinically tractable information at the time of disease progression.

Identification of New Tumor-Related Gene Mutations in Chinese Gastrointestinal Stromal Tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. As the main GIST drivers, gain-of-function mutations in KIT or PDGFRA are closely associated with not only tumor development and progression but also therapeutic response. In addition to the status of KIT and PDGFRA, little is known about other potential GIST-related genes. In this study, we identified the mutation profiles in 49 KIT-mutated GIST tumors using the whole exome sequencing (WES) method. Furthermore, some representative mutations were further validated in an independent GIST cohort using the SNaPshot SNP assay. We identified extensive and diverse mutations of KIT in GIST, including many undescribed variants. In addition, we revealed some new tumor-related gene mutations with unknown pathogenicity. By enrichment analyses of gene function and protein-protein interaction network construction, we showed that these genes were enriched in several important cancer- or metabolism-related signaling pathways, including PI3K-AKT,RTK-RAS, Notch, Wnt, Hippo, mTOR, AMPK, and insulin signaling. In particular, DNA repair-related genes, including MLH1, MSH6, BRCA1, BRCA2, and POLE, are frequently mutated in GISTs, suggesting that immune checkpoint blockade may have promising clinical applications for these GIST subpopulations. In conclusion, in addition to extensive and diverse mutations of KIT, some genes related to DNA-repair and cell metabolism may play important roles in the development, progression and therapeutic response of GIST.

Abstract 1292: Preclinical characterization of THE-630, a next-generation inhibitor for KIT-mutant gastrointestinal stromal tumors (GIST)

Abstract Introduction: GIST is the most common sarcoma of the GI tract, with most cases driven by activating mutations in KIT Ex11 or Ex9. Prognosis is poor for patients whose tumors progress following first-line (1L) imatinib treatment, despite availability of 3 approved drugs (sunitinib [2L], regorafenib [3L], ripretinib [4L]). In most patients, resistance is driven by secondary mutations in KIT, with nearly all occurring in either the ATP binding pocket (Ex13/14) or activation loop (Ex17/18). No approved drug has potent activity against both classes of resistance mutations and most patients appear to have multiple resistance mutations. There remains a need for a pan-mutant KIT inhibitor, which has potent activity against all classes of mutations. Experimental Procedures: Drug activity was determined by assessing effects on tumor cell lines and BaF3 cells expressing mutant forms of KIT. Mice were dosed orally once (THE-630 and sunitinib) or twice (ripretinib) daily at doses that did not exceed the MTD. Results: THE-630 potently (IC50 ~3 nM) inhibited KIT-driven viability and downstream signaling in GIST-T1 cells, which contain an activating deletion in Ex11 (Ex11Del). Similar results were observed in engineered BaF3 cells containing Ex11Del or an activating insertion in Ex9 (both IC50s ≤3 nM), whereas potency of imatinib, regorafenib, and ripretinib, was reduced by ≥5-fold against the Ex9 variant. Consistent with available clinical data, in engineered BaF3 cells containing a primary Ex11Del mutation and a resistance mutation, none of the 4 approved drugs had potent activity against both classes of resistance mutations. In contrast, THE-630 potently inhibited ATP binding pocket mutants (V654A and T670I) and activation loop mutants (D816G/H, D820A/G, N822K, Y823D, A829P), with IC50s all below 25 nM. THE-630 also had potent activity against V654A and D816H in the context of an Ex9 primary mutation (IC50s 10 and 33 nM). In vivo, THE-630 strongly inhibited tumor growth (by 86%) in a model containing the most common ATP binding pocket mutant (V654A) as a secondary mutation, while ripretinib achieved only modest inhibition (26%). THE-630 was also highly active in models expressing secondary activation loop mutants, regressing tumors containing N822K by 88% (compared to 25% tumor growth inhibition by sunitinib) and regressing tumors containing D820A by 59% (compared to 1% tumor regression by ripretinib). Conclusions: We have identified a next-generation pan-KIT inhibitor that has potent activity against all major classes of activating and resistance mutations observed in KIT-mutant GIST patients. A phase 1 clinical trial of THE-630 is planned to begin in 2021. Citation Format: Victor M. Rivera, Wei-Sheng Huang, Mengrou Lu, Justin R. Pritchard, David Dalgarno, William C. Shakespeare. Preclinical characterization of THE-630, a next-generation inhibitor for KIT-mutant gastrointestinal stromal tumors (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1292.

PDGFRA Immunohistochemistry Predicts PDGFRA Mutations in Gastrointestinal Stromal Tumors.

Platelet-derived growth factor receptor A (PDGFRA) is a receptor tyrosine kinase that is activated by mutations in 10% of gastrointestinal stromal tumors (GISTs) and 55% to 70% of inflammatory fibroid polyps. PDGFRA-mutant GISTs are usually epithelioid and occur predominantly in the stomach. Succinate dehydrogenase-deficient GISTs also arise in the stomach and are usually epithelioid, as are some KIT-mutant GISTs. Recently, avapritinib was approved to treat PDGFRA D842V-mutant GISTs, which do not respond to conventional targeted therapy. Here, we evaluate the utility of PDGFRA immunohistochemistry (IHC) to predict PDGFRA mutations to direct targeted therapy. PDGFRA IHC was performed at 1:3000 and 1:10,000 dilutions on a tissue microarray containing 153 GISTs (126 KIT-mutant, 17 PDGFRA-mutant, and 10 succinate dehydrogenase-deficient). The "positive" staining threshold was defined as 50% of neoplastic cells staining at moderate intensity. PDGFRA IHC was 75.0% and 80.9% specific for PDGFRA mutations at 1:3000 and 1:10,000 dilutions, respectively, and it was 100% sensitive at both. On the basis of its higher specificity, a 1:10,000 dilution was used to stain whole-tissue sections of GISTs and other gastric tumor types. Combining tissue microarray and whole-tissue data, PDGFRA IHC was 94.4% sensitive and 81.0% specific for PDGFRA-mutant GIST among all 210 GISTs, and it was 84.1% specific among 149 GISTs with an epithelioid component. PDGFRA was positive in a subset of inflammatory fibroid polyps (15/30; 50%), monophasic synovial sarcomas (5/10; 50%), inflammatory myofibroblastic tumors (5/10; 50%), and plexiform fibromyxomas (2/8; 25%). It was negative in poorly differentiated adenocarcinoma (0/20), diffuse large B-cell lymphoma (0/10), glomus tumor (0/10), gastrointestinal neuroectodermal tumor (0/10), leiomyoma (0/10), gastric schwannoma (0/8), and gastroblastoma (0/3). Among GISTs, PDGFRA IHC is highly sensitive and moderately specific for PDGFRA-mutant tumors; it also can be positive in inflammatory fibroid polyp and some other mesenchymal tumor types. PDGFRA positivity could be used to triage epithelioid GISTs for PDGFRA sequencing to determine optimal therapy.

Gene Expression Landscape of SDH-Deficient Gastrointestinal Stromal Tumors.

Background: About 20–40% of gastrointestinal stromal tumors (GISTs) lacking KIT/PDGFRA mutations show defects in succinate dehydrogenase (SDH) complex. This study uncovers the gene expression profile (GEP) of SDH-deficient GIST in order to identify new signaling pathways or molecular events actionable for a tailored therapy. Methods: We analyzed 36 GIST tumor samples, either from formalin-fixed, paraffin-embedded by microarray or from fresh frozen tissue by RNA-seq, retrospectively collected among KIT-mutant and SDH-deficient GISTs. Pathway analysis was performed to highlight enriched and depleted transcriptional signatures. Tumor microenvironment and immune profile were also evaluated. Results: SDH-deficient GISTs showed a distinct GEP with respect to KIT-mutant GISTs. In particular, SDH-deficient GISTs were characterized by an increased expression of neural markers and by the activation of fibroblast growth factor receptor signaling and several biological pathways related to invasion and tumor progression. Among them, hypoxia and epithelial-to-mesenchymal transition emerged as features shared with SDH-deficient pheochromocytoma/paraganglioma. In addition, the study of immune landscape revealed the depletion of tumor microenvironment and inflammation gene signatures. Conclusions: This study provides an update of GEP in SDH-deficient GISTs, highlighting differences and similarities compared to KIT-mutant GISTs and to other neoplasm carrying the SDH loss of function. Our findings add a piece of knowledge in SDH-deficient GISTs, shedding light on their putative histology and on the dysregulated biological processes as targets of new therapeutic strategies.

Tumor genotype, location, and malignant potential shape the immunogenicity of primary untreated gastrointestinal stromal tumors.

Intratumoral immune infiltrate was recently reported in gastrointestinal stromal tumors (GISTs). However, the tumor-intrinsic factors that dictate GIST immunogenicity are still largely undefined. To shed light on this issue, a large cohort (82 samples) of primary untreated GISTs, representative of major clinicopathological variables, was investigated by an integrated immunohistochemical, transcriptomic, and computational approach. Our results indicate that tumor genotype, location, and malignant potential concur to shape the immunogenicity of primary naive GISTs. Immune infiltration was greater in overt GISTs compared with that in lesions with limited malignant potential (miniGISTs), in KIT/PDGFRA-mutated tumors compared with that in KIT/PDGFRA WT tumors, and in PDGFRA-mutated compared with KIT-mutated GISTs. Within the KIT-mutated subset, a higher degree of immune colonization was detected in the intestine. Immune hot tumors showed expression patterns compatible with a potentially proficient but curbed antigen-specific immunity, hinting at sensitivity to immunomodulatory treatments. Poorly infiltrated GISTs, primarily KIT/PDGFRA WT intestinal tumors, showed activation of Hedgehog and WNT/β-catenin immune excluding pathways. This finding discloses a potential therapeutic vulnerability, as the targeting of these pathways might prove effective by both inhibiting pro-oncogenic signals and fostering antitumor immune responses. Finally, an intriguing anticorrelation between immune infiltration and ANO1/DOG1 expression was observed, suggesting an immunomodulatory activity for anoctamin-1.

Lower-dosing ponatinib in pre-treated GIST: Results of the POETIG phase II trial.

11536 Background: Despite long-lasting responses to imatinib most metastatic gastrointestinal stromal tumors (GIST) eventually progress and subsequent treatments are associated with limited duration of disease control. Ponatinib is a potent KIT inhibitor with a strong activity against secondary mutations in exon 17, including the highly resistant D816 mutations of KIT. Based on the dose-depending toxicity profile we sought to evaluate the safety and activity of lower dosing (30mg) ponatinib in pretreated patients with KIT-mutant GIST. We here report safety data for the whole cohort and first efficacy data for the last line cohort within a planned interim analysis. Methods: This multicenter phase 2 trial (NCT03171389) recruited patients with advanced, unresectable GIST progressing after imatinib (Cohort A/B: absence/presence of KIT Exon 13/14 mutations by plasma sequencing) or imatinib, sunitinib and regorafenib (Cohort C). Patients were treated with 30mg oral ponatinib daily in 4-week-cycles. The primary endpoint was the clinical benefit rate at 16 weeks as measured by mRECIST1.1 criteria. Results: At the cutoff date of 31st Jan 2020, 39 patients were evaluable for safety analysis (25 male, 14 female, median age 60 (38-86) years). Median duration of treatment was 65 (14-699) days. 66.7% of all patients observed Grade 3/4 adverse events (AEs), most common were pain (10/39), hypertension (6/39), GGT or lipase increase (both 5/39), and fever (3/39). One AE of special interest was observed (myocardial infarction, rated not related to study drug) and 20/39 patients experienced at least 1 severe AE (6/39 possibly related to ponatinib). Within the last line cohort, 20 patients were evaluable for efficacy. Clinical benefit rate was 35% (CI 15.4-59.2%). Median progression-free survival was 86 days with single patients yielding long-lasting responses (75% quartile 210 days, maximum: 420 days). Conclusions: Treatment with ponatinib was tolerable at a dose of 30mg qd with a toxicity profile comparable to other TKIs used in GIST. The majority of grade 3/4 AEs were hypertension or asymptomatic increases of laboratory values and thromboembolic events were rare. In a heavily pretreated patient population that lacks alternative treatment options the clinical activity was notable. An updated analysis including predictive biomarkers will be presented. Clinical trial information: NCT03171389 .

The Emerging Role of the FGF/FGFR Pathway in Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumors (GIST) are rare neoplasms of mesenchymal origin arising in the gastrointestinal tract. The vast majority are characterized by mutually exclusive activating mutations in KIT or Platelet-derived growth factor alpha (PDGFRA) receptors, or less frequently by succinate dehydrogenase complex (SDH) or NF1 inactivation, with very rare cases harboring mutant BRAF or RAS alleles. Approximately 5% of GISTs lack any of such mutations and are called quadruple wild-type (WT) GISTs. Recently, deregulated Fibroblast Growth Factor (FGF)/FGF-receptor (FGFR) signaling emerged as a relevant pathway driving oncogenic activity in different molecular subgroups of GISTs. This review summarizes all the current evidences supporting the key role of the FGF/FGFR pathway activation in GISTs, whereby either activating mutations, oncogenic gene fusions, or autocrine/paracrine signaling have been detected in quadruple WT, SDH-deficient, or KIT-mutant GISTs.

Abstract 263: KITlow cells mediate Imatinib resistance and disease persistence in gastrointestinal stromal tumor

Abstract INTRODUCTION: Gastrointestinal stromal tumor (GIST) is commonly driven by oncogenic KIT mutations that are effectively targeted by Imatinib (IM). However, IM does not cure GIST; adjuvant therapy only delays recurrence in high-risk tumors. Therefore, we hypothesized that GIST contains cells with primary IM resistance, representing a critical target for overcoming disease persistence. METHODS: Gene Set Enrichment Analysis (GSEA) was performed with publicly available data (GSE1596636). Human GIST cell lines (GIST-T1 and 882) and primary KIT-mutant GIST (IRB #090401) were analyzed by fluorescence-activated cell sorting (FACS) to sort KITloCD34+ (KITlo) vs KIThiCD34+ (KIThi) subpopulations using anti-human KIT and CD34 antibodies. RNA was extracted from cell lysates for analysis by quantitative RT-PCR. Cell viability was assessed by CellTiter-Glo or MTT assays following drug treatments. RESULTS: We performed GSEA on 27 matched GISTs comparing pre- and post-neoadjuvant IM treatment (RTOG S0132). Post-IM samples had 50% lower KIT expression (P=0.002) and were enriched in “cancer stem cell” and Axl/Gas6/NF-κB signaling gene signatures. Similarly, in vitro IM treatment of GIST cell lines resulted in 2-fold reduction of KIT expression and 1.9 to 4.4-fold increased expression of stem-associated transcription factors (SATFs: OCT4, SOX2, KLF4, NANOG). Parallel FACS analysis of IM-treated GIST cell lines (125 nM, 72-h) demonstrated the presence of a KITlo subpopulation (GIST-T1: 0.75%; GIST882: 4%) while untreated cells had 3- to 11-fold fewer KITlo cells. Primary human GISTs also had KITlo cells by FACS (4-8%; N=4), suggesting that the population is not an artifact of in vitro culture. KITlo GIST882 cells were IM-resistant (IC50: KITlo 3641 nM vs KIThi 180 nM) and significantly overexpressed all SATFs by qRT-PCR (2.0 to 4.1-fold; P<0.001) consistent with RTOG S0132 analysis. Moreover, RNAseq confirmed that KITlo cells (relative to KIThi) are enriched in factors present in the “cancer stem cell” gene signature identified by GSEA (KLF4, CCL5, ATF3, JUN, IFIT1, PMP22). Lastly, we tested candidate drugs against targets overexpressed in IM-treated tumors. Unsorted cells were pre-treated with IM (185 nM, 48-h) to enrich for KITlo cells. Subsequent treatment with R428 (AXL inhibitor) or bardoxolone (NF-κB inhibitor) resulted in 70% (R428, 5 μM) and 80% (bardoxolone, 5 μM) cell death. Finally, FACS-sorted KITlo cells were sensitive to both drugs (50% and 88% killing, respectively). CONCLUSIONS: KITlo cells are a distinct subpopulation in human GIST with intrinsic IM-resistance and may represent a novel mechanism of GIST persistence. These cells overexpress stem cell transcriptional programs, including the Axl/Gas6/NF-κB pathway, which represent novel therapeutic targets in vitro. Further studies are needed to explore the in vivo efficacy of combination or sequential targeting of KITlo cells in GIST. Citation Format: Sudeep Banerjee, Chih-Min Tang, Mayra Yebra, Kwat Medetgul, Adam M. Burgoyne, Pablo Tamayo, Robert Wechsler-Reya, Jason K. Sicklick. KITlow cells mediate Imatinib resistance and disease persistence in gastrointestinal stromal tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 263.

Differential immune profiles distinguish the mutational subtypes of gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma, frequently characterized by an oncogenic mutation in the KIT or platelet-derived growth factor receptor alpha (PDGFRA) genes. We performed RNA sequencing of 75 human GIST tumors from 75 patients, comprising the largest cohort of GISTs sequenced to date, in order to discover differences in the immune infiltrates of KIT and PDGFRA-mutant GIST. Through bioinformatics, immunohistochemistry, and flow cytometry, we found that PDGFRA-mutant GISTs harbored more immune cells with increased cytolytic activity when compared to KIT-mutant GISTs. PDGFRA-mutant GISTs expressed many chemokines, such as CXCL14, at a significantly higher level when compared to KIT-mutant GISTs and exhibited more diverse driver-derived neoepitope:HLA binding, both of which may contribute to PDGFRA-mutant GIST immunogenicity. Through machine learning, we generated gene expression-based immune profiles capable of differentiating KIT and PDGFRA-mutant GISTs, and also identified additional immune features of high PD-1 and PD-L1 expressing tumors across all GIST mutational subtypes, which may provide insight into immunotherapeutic opportunities and limitations in GIST.

Genomic aberrations in cell cycle genes predict progression of KIT-mutant gastrointestinal stromal tumors (GISTs)

BackgroundActivating mutations of the receptor tyrosine kinase KIT are early events in the development of most gastrointestinal stromal tumors (GISTs). Although GISTs generally remain dependent on oncogenic KIT during tumor progression, KIT mutations alone are insufficient to induce malignant behavior. This is evidenced by KIT-mutant micro-GISTs, which are present in up to one-third of normal individuals, but virtually never progress to malignancy.MethodsWe performed whole exome sequencing on 29 tumors obtained from 21 patients with high grade or metastatic KIT-mutant GIST (discovery set). We further validated the frequency and potential prognostic significance of aberrations in CDKN2A/B, RB1, and TP53 in an independent series of 71 patients with primary GIST (validation set).ResultsUsing whole exome sequencing we found significant enrichment of genomic aberrations in cell cycle-associated genes (Fisher’s Exact p = 0.001), most commonly affecting CDKN2A/B, RB1, and TP53 in our discovery set. We found a low mutational tumor burden in these 29 advanced GIST samples, a finding with significant implications for the development of immunotherapy for GIST. In addition, we found mutation of spliceosome genes in a minority of cases, implicating dysregulation of splicing as a potential cancer promoting mechanism in GIST. We next assessed the prognostic significance of CDKN2A, RB1 or TP53 mutation/copy loss in an independent cohort of 71 patients with primary GIST. Genetic events (mutation, deletion, and/or LOH) involving at least one of the three genes examined were found in 17% of the very low-risk, 36% of the low-risk, 42% of the intermediate risk, 67% of the high-risk/low mitotic-count, and in 86% of the high-risk/high mitotic-count group. The presence of cell cycle-related events was associated with a significantly shorter relapse-free survival (median 67 months versus not reached; p < 0.0001) and overall survival (Log Rank, p = 0.042).ConclusionOur results demonstrate that genomic events targeting cell cycle-related genes are associated with GIST progression to malignant disease. Based on this data, we propose a model for molecular pathogenesis of malignant GIST.

LMTK3 is essential for oncogenic KIT expression in KIT-mutant GIST and melanoma.

Certain cancers, including gastrointestinal stromal tumor (GIST) and subsets of melanoma, are caused by somatic KIT mutations that result in KIT receptor tyrosine kinase constitutive activity, which drives proliferation. The treatment of KIT-mutant GIST has been revolutionized with the advent of KIT-directed cancer therapies. KIT tyrosine kinase inhibitors (TKI) are superior to conventional chemotherapy in their ability to control advanced KIT-mutant disease. However, these therapies have a limited duration of activity due to drug-resistant secondary KIT mutations that arise (or that are selected for) during KIT TKI treatment. To overcome the problem of KIT TKI resistance, we sought to identify novel therapeutic targets in KIT-mutant GIST and melanoma cells using a human tyrosine kinome siRNA screen. From this screen, we identified lemur tyrosine kinase 3 (LMTK3) and herein describe its role as a novel KIT regulator in KIT-mutant GIST and melanoma cells. We find that LMTK3 regulated the translation rate of KIT, such that loss of LMTK3 reduced total KIT, and thus KIT downstream signaling in cancer cells. Silencing of LMTK3 decreased cell viability and increased cell death in KIT-dependent, but not KIT-independent GIST and melanoma cell lines. Notably, LMTK3 silencing reduced viability of all KIT-mutant cell lines tested, even those with drug-resistant KIT secondary mutations. Furthermore, targeting of LMTK3 with siRNA delayed KIT-dependent GIST growth in a xenograft model. Our data suggest the potential of LMTK3 as a target for treatment of patients with KIT-mutant cancer, particularly after failure of KIT TKIs.