Background
Gastrointestinal stromal tumors (GISTs) are the most common sarcoma affecting approximately 3500 new patients per year in the United States [
1]. Approximately 70–80% of sporadic GISTs are caused by gain-of-function mutations in KIT (c-KIT, CD117); another 5–10% are caused by activating genomic alterations in platelet-derived growth factor receptor alpha (PDGFRA) [
2‐
4]. Even less common are tumors driven by RAS pathway gene mutations (e.g., BRAF, KRAS, NF1; 1–3% combined) or mutations/deficiencies in the four succinate dehydrogenase (SDH subunits (A, B, C, or D; 3% combined) [
2,
3,
5‐
8].
Given the initial understanding that the majority of GISTs were driven by KIT, the application of targeted drugs such as imatinib mesylate (Novartis, Basel, Switzerland) allowed GIST to serve as the paradigm for cancer genotyping and the development of “matched” therapies for solid malignancies. Imatinib and other small molecule tyrosine kinase inhibitors have demonstrated clear anti-GIST activity by targeting oncogenic KIT and PDGFRA mutations, and as a result, these drugs are now firmly established in treatment of GIST patients [
9].
However, approximately 5–15% of adult GIST patients, and most pediatric patients, who tend to be imatinib-resistant, were initially designated as “wild-type (WT)” until other oncogenic mutations were identified. Some have termed this subset of patients “quadruple-WT (qWT)”, or “quadruple-negative GISTs” because they lack oncogenic mutations in any of the aforementioned genes [
10]. Since the designation of qWT GIST, only one study has attempted to define and compare its molecular profile to other GISTs, reporting the overexpression of polycomb target genes (e.g., CDK6, ERG and NTRK2) as potential drivers and identifying potential diagnostic markers (e.g., CALCRL and COL22A1) [
11]. This study provided insight into alternative pathways that may be involved in GIST oncogenesis; however, this report was limited by analyzing only two so-called qWT tumors. Beyond this study, there is a lack of reports addressing the clinical demographics and molecular characteristics of qWT GISTs [
10]. Thus, the patient demographics, pathological data (i.e., tumor location), and genomic profile of qWT GISTs are largely unknown. These deficiencies in our knowledge leave affected patients at a disadvantage by not identifying the genetic abnormalities that could be used to diagnose GIST and delay the development of novel and potentially beneficial precision therapies for qWT GIST.
Given the increasing application of comprehensive genetic profiling (CGP) towards personalized medicine treatments in oncology, we hypothesized that analyzing demographic data, pathological data, and genomic profiles of quadruple-WT GIST would begin to define the pathobiology of this largely undefined GIST subtype, as well as reveal clinically relevant, potentially targetable, genomic alterations (GAs).
Discussion
We report the identification of clinically relevant and previously unreported genomic alterations in gastrointestinal stromal tumors (GISTs) lacking genomic alterations in KIT, PDGFRA, SDH, and the RAS pathway. We profiled patient demographics and tumor characteristics of our cohort and performed CGP of tumors on this subset of GIST patients, including reporting of clinical responses to molecularly matched therapies. Our findings suggest new and potentially targetable alterations in genes such as NTRK3 and FGFR1 in a subset of GIST patients.
The current study focuses on a patient population that we are just beginning to understand more thoroughly. Even without performing whole exome or whole genome sequencing, our study findings expand upon the few reports of the molecular characteristics of WT GISTs [
7,
10,
11,
39,
43]. We compare the incidence of genomic alterations in WT and non-WT GIST, yielding seven genes that appear to be more commonly altered in WT GIST. This includes LTK (lymphocyte receptor tyrosine kinase) and FGFR1, two non-KIT/PDGFRA receptor tyrosine kinases that mainly signal through the RAS-MAPK pathway. In addition, LTK also signals through the PI3K-AKT-mTOR pathway in order to maintain survival signals in tumor cells. These genomic alterations detected in WT GIST affect key pathways such as the PI3K-AKT-mTOR and RAS-MAPK pathways, which overlap with downstream signaling of several known drivers, including KIT and PDGFRA. In addition to cell-cycle regulation, other genomic alterations were seen to affect histone acetyltransferases, transcriptional regulators, and the NFkB pathway, as well as embryonic development and cancer stem cell pathways (e.g., Wnt/β-catenin pathway and Notch pathway). Taken together, the genomic profiles highlight novel genes in WT GIST with similar or intersecting functions as known drivers of non-WT GIST development. Moreover, several of these genomic alterations have potential therapeutic importance. Of note, five mutations in ARID1B, which is part of the SWI/SF chromatin remodeling complex, may be targetable with FDA-approved HDAC inhibitors, including vorinostat and panobinostat. Furthermore, this study uncovered mutations in SUFU, a negative regulator of the Hedgehog signaling pathway. Because SUFU is downstream of the SMO oncogene, FDA-approved agents, which target SMO (e.g., vismodegib and sonidegib) are unlikely to be effective in tumors with downstream SUFU alterations. However, we recently reported that the GLI-family of transcription factors, which are downstream of SUFU, may be targetable in GIST with the FDA-approved agent, arsenic trioxide [
44]. Thus, our new findings have potential therapeutic implications for several subsets of WT GIST patients they may be treated with currently available drugs or those under development.
We detected an FGF6 mutation and four potentially actionable FGFR1 alterations, including one FGFR1 missense mutation, as well as three FGFR1 fusions including a novel FGFR1–HOOK3 fusion and two cases with an FGFR1–TACC1 fusion previously reported in glioblastoma multiforme. These events are predicted to result in constitutive activity of FGFR1 and the downstream tyrosine kinase cascades that promote WT oncogenesis perhaps in the presence or absence of growth factors (e.g., FGF6) or FGFR1 overexpression [
45,
46]. Thus, a subset of GIST, that is similar in size to RAS mutant GISTs in the cohort, may be sensitive to targeted FGFR tyrosine kinase inhibitors, such as lenvatinib (Esai), ponatinib (Ariad), pazopanib (Novartis) or other similar investigational drugs.
The only prior study defining and comparing genomic profiles of 2 qWT GIST to non-qWT GISTs did not identify any genomic alterations in the two tumors but reported mRNA overexpression of polycomb target genes including CDK6, ERG and NTRK2 [
11]. Interestingly, we identified genomic alterations in a related gene, NTRK3 (fusion with ETV6). ETV6–NTRK3 fusions have been reported in infantile fibrosarcoma, secretory breast carcinoma, salivary gland tumors (acinic cell carcinomas, cystadenocarcinomas, and adenocarcinomas), mixed epithelial and stromal tumor of the kidney, leukemias, and thyroid cancer. During the preparation of this manuscript, another group reported an ETV6–NTRK3 fusion in GIST [
39]. These findings have clinical relevance as recent data (including that shown here) suggest that NTRK fusions are sensitive to LOXO-101. Loxo Oncology has reported two other NTRK fusion patients with clinical responses on its Phase I trial (i.e., sarcoma with LMNA–NTRK1 and mammary analogue secretory carcinoma of the salivary gland with ETV6–NTRK3) [
42]. Another report suggests that crizotinib (Pfizer, New York, NY, USA) may be a treatment option for patients with NTRK fusions [
47,
48]. The potential clinical applicability underscores the significant contributions that genomic profiling can add towards clinical decision making and precision therapies.
The strength of the current study includes the large sample cohort and a careful filtering of the mutation identified. The genomic data was evaluated through two different processes, namely bioinformatic analyses associated with the Foundation One™ assays and comparison to dbNSFP, in order to identify all potentially relevant findings. Foundation Medicine assays include multiple gene fusions, which have not been throughly investigated by previous studies. This allowed us to identify deleterious, actionable alteration in FGFR1 and NTRK3 in three patients. Foundation Medicine assays however cannot distinguish rare germline variants from somatic mutations, leaving some ambiguity in the findings specifically in regards to rare germline SDH and NF1 mutations. Finally the CGP approach only screened against genes with known associations with any solid or hematologic cancer, potentially failing to capture truly novel genes that have not yet been linked to cancer development.
Given that 12 patients did not have SDHx mutation testing, we are unable to definitively determine the proportion of these patients that harbor SDHx mutations or are truly WT. Finally, due to the nature of this study, we are unable to match germline and somatic sequencing data in patients, repeat testing with the lattest gene panel that includes SDHx subunits, perform SDHB immunostaining to assess SDHB-competence/-deficiency, or assess for SDHC-epimutant tumors with hypermethylation of the SDHC promoter, which can lead to silencing of expression [
7]. Despite the limitations of this study, we begin to suggest that 17 qWT GIST are biologically distinct from than their non-WT counterparts, providing novel insight into the clinico-pathological features of WT GIST.
Conclusions
In summary, this study builds upon previous work in the GIST field and provides the new insights into the genomic landscape of quadruple-WT GIST. While these tumors historically are considered “wild-type” mainly due to a lack of KIT or PDGFRA mutations, this study showed that the majority of these tumors harbor deleterious genomic alterations in genes participating in crucial cellular activities, such as cell cycle progression, DNA repair, and regulation of gene expression. Alternatively, it is possible that the activity of the canonical genes (i.e., KIT, PDGFRA, KRAS, NF1, BRAF, SDHx, KRAS) may also be altered in WT tumors via epigenetic changes as seen in SDH-deficient tumors. Furthermore, this study identified several actionable mutations, including two ETV6–NTRK3 fusions, and FGF6 or FGFR1 alterations, including three FGFR1 fusions and one known intragenic activating FGFR1 mutation, that may significantly impact tumor responses by assisting in the choice of targeted therapies. Such findings have the potential to change present therapeutic options in GIST, give insight into disease biology, and redefine one of the earliest paradigms in tumor genomics and precision medicine. By providing novel insight into potential genetic drivers for GIST, future studies may further build on this genetic profile of so-called qWT GIST, which are not truly WT, as well as link genomic drivers to therapeutic regimens. In turn, this may lead to individualized treatments that can significantly improve patient outcomes.
Authors’ contributions
Study concept and design: ES, OH, JKS. Acquisition of data: ES, JC, CMT, KW, MCH, GK, CLC, DH, KEF, JDM, MDS, AMB, SM, DM, JSR, OH, JKS. Analysis and interpretation of data: ES, JC, MCH, GK, CLC, DH, KEF, JDM, PTF, SMA, SM, LM, GMH, JCT, RK, DM, JSR, OH, JKS. Drafting of the manuscript: ES, JC, CMT, MCH, DH, KEF, JDM, SM, RK, OH, JKS. Critical revision of the manuscript for important intellectual content: ES, JC, CMT, MCH, DH, SM, JCT, RK, JSR, OH, JKS. Statistical analysis: ES, JC, KEF, JDM, OH. Obtained funding: MCH, CLC, OH, and JKS. Technical, or material support: CMT, KW, GK. Study supervision: OH and JKS. All authors read and approved the final manuscript.
Competing interests
Juliann Chmielecki, Kai Wang, Siraj Ail, Deborah Morosini, and Jeffrey Ross are employees of, and equity holders in, Foundation Medicine, Inc., the provider of the FoundationOne™ and FoundationOne Heme™ assays utilized in this study. Michael Heinrich has an ownership interest in MolecularMD, receives honoraria from Novartis and Pfizer, receives consultant fees from MolecularMD, ARIAD, Novartis, Blueprint Medicines, and Pfizer, as well as receives research funds from Novartis, AROG, Inhibikase, ARIAD, and Deciphera. Christopher Corless receives honoraria from Roche and Asuragen, receives consultant fees from Roche and Asuragen, receives research funds from Roche, as well as receives travel/accommodations/expenses from Roche. David Hong receives research funds from Loxo, Novartis, Genentech, Eisai, AstraZeneca, Pfizer, miRNA Therapeutics, Amgen, Daiichi Sankyo, Merck, Mirati Therapeutics, and Lilly, as well as receives travel/accommodations/expenses from Loxo and miRNA Therapeutics. Lisa Madlensky has a family member that is an employee of Janssen. Gregory Heestand receives consultant fees from Merrimack, as well as receives research funding from EMD Serono, Medimmune, Samumed, and Merck. Jonathan Trent receives consultant fees from GlaxoSmithKline and Bayer/Onyx. Razelle Kurzrock has an ownership interest in RScueRx and receives consultant fees from Sequenom, as well as research funds from Foundation Medicine, Pfizer, Merck Serono, Guardant, Sequenom, and Genentech. Jason Sicklick receives research funds from Foundation Medicine, Novartis, and Blueprint Medicines, as well as consultant fees from Sirtex. The other authors have nothing to disclose.