The tropomyosin receptor kinase (TRK) family of receptors is composed of TRKA, TRKB, and TRKC, which are neurotrophic tyrosine receptor kinase (NTRK) proteins encoded by the NTRK1, NTRK2 and NTRK3 genes, respectively (1, 2). These receptors are highly expressed in neural tissue and participate in the development and proper functioning of the central nervous system (CNS). These receptors activate the RAS/MAPK pathway and can also signal via the PI3K/AKT/mTOR and the PLCγ/PLK pathways, depending on which docking protein binds to the kinase domain. Via these pathways, the signal transduction leads to neuronal survival, development, proliferation, synaptic plasticity, neuronal differentiation as well as memory and cognition (3–5). These receptors and their signaling cascade are also implicated in neoplastic cells (6).

Although mutations and alternative splicing occur, fusions are the most common aberrations of NTRK in tumors. Gene fusion events that involve NTRK genes (NTRK1, NTRK2 and NTRK3) occur when the 3’ region of a NTRK gene encoding the tyrosine kinase domain is joined in-frame with 5’-end of a fusion partner gene, either by intra- or inter-chromosomal rearrangement (7, 8). The resulting fusion oncogene leads to the production of chimeric protein, with a constitutive activation of the kinase, due to loss of the extracellular domain, continuous downstream signaling and thus proliferation and cell survival (7).

Among adult and pediatric patients, the incidence of NTRK gene fusions varies between <5% in solid cancers (e.g., lung cancer, colorectal cancer, glioma) and >75% in rare cancers (e.g., infantile fibrosarcoma, secretory breast, secretory carcinoma of the salivary gland) (9). The presence of NTRK gene fusions in both adult and pediatric populations suggests it may be one of the first oncogenic drivers that are both tissue- and age-agnostic (10–12).

Regarding the central nervous system (CNS) tumors, NTRK fusions occur in up to 2% of gliomas in adults (13, 14). The incidence in pediatric high-grade gliomas (HGG) and diffuse infiltrating pontine glioma is around 5% and even 40% in infants with non-brainstem HGG (15–18). NTRK2 is the most common fusion partner of the NTRK family in pediatric brain tumors (7, 8). Nevertheless, the occurrence of NTRK gene fusions in different types of brain tumors in adults remain quite unexplored.

The FGFR family exists of four transmembrane tyrosine kinase receptors (FGFR 1-4). The FGFR plays an important role in embryonal CNS development and in tumorigenesis, regulating angiogenesis, proliferation, differentiation, migration, and survival. FGFR genomic alterations (amplification, mutations, and fusions) occur in ~ 8% of gliomas, with most aberrations occurring in FGFR1 and FGFR3 (19). Chromosomal translocations that fuse the tyrosine kinase domains of FGFR1 or FGFR3 and TACC1 or TACC3 have been identified in 2% to 4% of gliomas (20–22). The IDH1/2 wild-type (3.5%) but none of IDH1/2-mutant grade II and III gliomas harbored FGFR3-TACC3 fusions (21). FGFR-TACC rearrangements are reported in 2.9% of glioblastoma (GBM) (21). These FGFR fusion genes, such as FGFR3-TACC3, are capable of ligand-independent dimerization by virtue of the newly fused coiled-coil domain and have demonstrated oncogenic potential in vitro and in vivo (20). Further, FGFR3-TACC3 fusion has been reported as predictive of response to FGFR tyrosine kinase inhibitors both preclinically (20, 21) and clinically in various solid tumors including gliomas (21). Clinical trials with FGFR inhibitors in brain tumors are being conducted (23, 24). FGFR fusions also have been identified as oncogenic drivers in breast tumors, lung cancer, and bladder carcinomas. FGFR3-TACC3 fusions were identified in a subset of bladder carcinomas (25), raising an interest in FGFR pathway inhibitors (23).

The detection of NTRK and FGFR gene fusions in brain tumors and brain metastases is relevant in clinical practice because these alterations can be predictive biomarkers and therapeutic targets for specific kinase inhibitors. NTRK and FGFR gene fusions result in overexpression of the fusion kinase and its constitutive activation, promoting tumor growth as a driver mutation. Both NTRK and FGFR inhibitors are currently being or already tested in clinical trials with patients who have fusions and available for treatment in tumors, respectively. Therefore, assessing NTRK and FGFR gene fusions in brain tumors and brain metastases could help identify those patients who could receive a clinically relevant response. Clinical outcomes from administering the above-targeted kinase inhibitors for patients could benefit from therapy if the NTRK and FGFR gene fusions were detected.

The incidence of cancer-associated fusions in patients with brain tumors remains poorly documented in France. Although several diagnostic approaches can be used to detect gene fusions, RNA-based next generation sequencing remains one of the most sensitive methods, as it can directly detect the transcribed product of gene fusion at the mRNA level (26). In this retrospective study, we herein report the results of detection of incidence of cancer-associated fusions in different types of brain tumors (gliomas and brain metastases).

Tumors often utilize a recurring mechanism to achieve over-expression of oncogenic kinases, which involves fusing kinases to genes that are highly expressed in tumors or the tissue of tumor-origin (28, 33–35). Since then, targeting the fusion genes has become the primary treatment option for fusion-positive patients, given that tumors with those fusions usually exhibit strong oncogenic addiction (26, 36). In our study, we observed an ETV6 (5)::NTRK3 (15) fusion in 1.69% of diffuse IDH-mutated 1p19q co-deleted oligodendroglioma (grade II), accounting for a significant portion of glioma by this mechanism. Although a prior study observed NTRK1 fusions at an incidence of 1% in glioblastoma (37), we didn’t find any NTRK1 fusion in our series of 59 tumor samples from patients operated for low and high-grade gliomas. NTRK fusions are present in a small percentage of gliomas/neuroepithelial tumors, with an estimated incidence ranging from 0.55% to 2% (13, 14, 38, 39). However, the incidence may be higher in certain groups, such as up to 5.3% in pediatric high-grade gliomas (HGG) (15), 4% of diffuse intrinsic pontine gliomas (DIPG), and up to 40% of non-brainstem HGG in patients younger than 3 years old (33). Gliomas with NTRK fusions have been previously reported to possess co-occurring genetic alterations such as IDH (13, 14, 31), H3.3 K27M (11), H3F3A (40), EGFR amplification (13), EGFRvIII (13), PTEN (13), CDKN2A/2B deletion (11, 37), CDKN2C deletion (37), TP53 mutations/inactivation (13), and ATRX (41), among others (13). A variety of NTRK fusion types (NTRK1, NTRK2, and NTRK3) have also been described in pediatric high-grade gliomas (11). Several downstream signaling pathways, including SHC-RAS-MAPK, PI3K-AKT, PLCγ-PKC, or STAT3, are activated by the three wildtype TRK family members (42), suggesting that most NTRK fusions would use many of these downstream signaling cascades as full-length receptors. It has been demonstrated that the TRK oncogenes induce a transformation of NIH-3T3 fibroblasts and thyroid epithelial cells (43). Similarly, mammary epithelial cells was shown to be transformed using ETV6 (5)::NTRK3 (15) fusion (44). Experiments with ETV6::NTRK3 fusion showed that the fusion protein signals mainly through RAS/MAPK but also activates PI3K/AKT/mTOR. Activation of both pathways might result in a potency oncogene to stimulate proliferation and inhibits apoptosis (45). Taken together, these data support that the ETV6::NTRK3 fusion present in the oligodendroglioma (grade II) could be a driver to promote tumor growth in vivo, suggesting that NTRK inhibitors may be a valuable therapeutic option to delay or avoid the need for radiotherapy in this population.

The NTRK fusion has been previously targeted by different drugs among which the most used are the first-in class TRK-targeting inhibitors are Larotrectinib (selective TRK inhibitor) and entrectinib (pan-TRK, ROS1 and ALK inhibitor). In 2018, the United States Food and Drug Administration (FDA) approved larotrectinib, a highly specific inhibitor of all three TRK proteins, for adult and pediatric patients with solid tumors (46). Larotrectinib’s approval was based on results from three multi-center clinical studies (a phase 1 trial (NCT02122913), SCOUT (NCT02637687), and NAVIGATE (NCT02576431)) (10, 47). According to a clinical trial involving solid tumors positive for NTRK fusion, Larotrectinib showed an overall response rate of 79.1%, with a median duration of response lasting 35.2 months, and a progression-free survival of 28.2 months (48). In 2019, entrectinib, a multi-kinase inhibitor targeting TRK proteins, c-ROS oncogene 1 (ROS1), and anaplastic lymphoma kinase (ALK) was approved by the FDA for adult and pediatric patients (>12 years) with the same indication as larotrectinib (49). Entrectinib’s approval was based on results of the multi-center trials; ALKA-372-001, STARTRK-1, and STARTRK-2 (50, 51). These trials revealed a response rate of 57% in patients with TRK fusion-positive solid tumors across 10 different tumor types (50, 51). The use of inhibitors targeting TRK is associated with high response rates regardless of tumor histology and patient age (1, 2, 52).

Several studies demonstrate the activity of Larotrectinib in TRK fusion-positive primary CNS tumors regardless of histology, which confirms its capacity for blood-brain barrier penetrance as reported previously by the response observed in metastases of extra-cranial tumors to CNS (48, 53). Larotrectinib demonstrated rapid and durable responses in TRK fusion-positive primary CNS tumors, and responses were seen in patients with low- and high-grade gliomas as well as non-gliomas (54). The intracranial efficacy of Larotrectinib has been demonstrated in TRK fusion-positive tumors that have metastasized to the brain (48, 53). These results further support expanded testing for actionable therapeutic targets, including NTRK gene fusions, in patients with primary adult and pediatric CNS tumors and BM.

Members of the receptor tyrosine kinase gene family including EGFR, MET, PDGFRA, and FGFR3 have been known to be heavily involved in the initiation and progression of glioblastoma (20, 55, 56). In the present study, we also identified the presence of FGFR3 (17)::TACC3 (11) fusion in breast carcinomas BM. The constitutively active signal of FGFR3 also transduces via the RAS/MAPK pathway (57). Previously, Singh et al. reported three fusions of FGFR::TACC in 97 glioblastoma examined (20). They demonstrated that the fusion protein has oncogenic activity when introduced into astrocytes and treatment with FGFR inhibitor extends the survival of mice harboring intracranial FGFR::TACC-initiated glioma. Previously, Parker et al. reported that 4 out of 48 glioblastoma samples harbored the FGFR3::TACC3 fusion and remarkably showed that the tumorigenic FGFR3::TACC3 gene fusion escapes miR-99a regulation in glioblastoma due to the loss of the 3’-UTR of FGFR3 (57). FGFR3 is very lowly expressed in normal brain but is highly expressed in fusion positive glioblastoma that is likely due to the loss of microRNA regulation (58, 59). The 3’-UTR of the FGFR3 gene is negatively controlled by microRNAs in the normal brain. In the fusion gene, the region is lost and FGFR3 can no longer be controlled by mir-99a (58). The FGFR3::TACC3 fusion was detected in one out of 72 samples of glioblastoma in the Ivy Center cohort (60), and 2 of 161 samples in the TCGA cohort. FGFR genes fusions are also identified in other cancers, including bladder cancer, cholangiocarcinoma, squamous lung cancer, thyroid cancer, oral cancer, head and neck squamous cell carcinoma, and prostate cancer (25, 61). Clinical trials with FGFR inhibitors in brain tumors are being conducted (23, 24). The FGFR inhibitor ponatinib demonstrate an improved therapeutic activity of temozolomide on DIPG cells derived from patient in in vitro study (62).

The EGFR gene is frequently amplified and rearranged in malignant gliomas with an expression of oncogenic deletion mutants (56). In this study, we found four glioblastoma harboring EGFR exons 2-7 skipping, also known as EGFRvIII, which is constitutively auto phosphorylated and inefficiently down regulated (63).

In this study, we also identified a METex14 skipping within the RNA in pancreas carcinomas BM resulting in an in-frame deletion of the juxta-membrane domain, which normally is a negative regulator of the kinase catalytic activities. Aberrant MET/HGF regulation is observed in a wide variety of human cancers with a dysregulated proliferative and invasive signaling program, epithelial-to-mesenchymal transition, cell motility/migration, angiogenesis, invasion, and metastasis. The METex14 mutations were identified in 221 positive cases (0.6%) out of 38,028 profiled tumors in the largest tumor genomic profiling cohort performed for MET alteration (64).

This study showed also the presence of TMPRSS2 (2)::ERG (4) fusions in prostate carcinomas BM, endometrium carcinomas BM, and oligodendroglioma (grade II), IDH-mutated and 1p19q co-deleted. Studies have shown that the androgen signaling pathway plays a role in facilitating the formation of the TMPRSS2::ERG gene fusion, which is present in approximately 50% of prostate carcinomas. This pathway induces proximity of the TMPRSS2::ERG genomic loci, which are then exposed to gamma irradiation, resulting in DNA double-strand breaks (65). Ongoing clinical trials are further evaluating the prognostic and predictive value of ERG fusions in prostate cancer patients at different stages of the disease or during treatment (e.g., trials evaluating the AR signaling inhibitors enzalutamide and apalutamide, PSMA theranostics, brachytherapy; see ClinicalTrials.gov for reference) and include the analysis of ERG fusion status both in primary and secondary outcome measures.

Genomic rearrangements are the primary way fusions arise in gliomas. Although clinically relevant fusions are rare, RNA-Seq of low- and high-grade glioma samples is a crucial molecular biology technique to discover patient-specific fusions that could guide personalized treatment. FGFR1-3 fusions, like NTRK1-3, offer a therapeutic option for current and forthcoming FGFR inhibitors across various patient subgroups.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by COS (Comité d’Orientation Scientifique) de la Direction Recherche et Enseignement Ramsay Santé. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

PM: Conceptualization, Funding acquisition, Validation, Writing – original draft, Writing – review & editing. CC: Writing – review & editing. EB: Formal analysis, Resources, Validation, Writing – original draft, Writing – review & editing. NB: Methodology, Validation, Writing – review & editing. CV: Methodology, Writing – review & editing. EP: Software, Writing – review & editing. PT: Writing – review & editing. MA: Writing – review & editing. AM: Writing – review & editing. IN: Formal analysis, Methodology, Validation, Writing – review & editing. LO: Conceptualization, Funding acquisition, Writing – original draft, Writing – review & editing.