The neurotrophic tyrosine receptor kinase (NTRK) family includes NTRK1, NTRK2, and NTRK3 which encode neurotrophin receptors TrkA, TrkB, and TrkC, respectively. Fusions involving the NTRK family are one of the most common mechanisms of oncogenic Trk activation (1) and occur sporadically in low-grade gliomas and glioblastoma (2). A variety of N-terminal gene fusion partners are described (3), which replace the ligand-binding site of the Trk, resulting in ligand-independent dimerization and phosphorylation of the Trk (4).

We describe here a novel NTRK2::LHFPL3 fusion in a Dysembryoplastic neuroepithelial tumor (DNT), a low-grade glioneuronal tumor. While the signaling pathways of TrkB are well-studied, not much is known about the LHFPL3. Lipoma HMGIC fusion partner-like 3 (LHFPL3) is a member of the superfamily of tetra-span transmembrane proteins. RNA-Seq expression data from GTEx showed a dominant LHFPL3 expression in the brain, including amygdala, anterior cingulate cortex, caudate, front cortex, hippocampus, hypothalamus, nucleus accumbens, putamen, and substantia nigra, while its expression is minimal in other non-brain tissues. Two human diseases have been linked to this group of genes. One LHFP-like gene is fused to the high mobility group AT-Hook 2 (HMGA2) in lipomas (5). Mutations in an LHFP-like gene result in deafness (6).

DNTs represent a type of glioneuronal and neuronal tumor (WHO, grade I) in the central nervous system (CNS) (7) and is the second most prevalent CNS neoplasm associated with epilepsy onset (8). DNT was first described by Daumas-Duport in 1988 (9), characterized by a multinodular growth of specific glioneuronal elements, with the columnar architecture of oligodendrocyte-like cells oriented perpendicular to the cortical surface and floating neurons in an abundant mucinous matrix (10, 11). DNT has shown genetic diversity and lacks molecular characteristics (12). Although studies have indicated FGFR1 mutations are more common in DNTs (10), other genetic alterations were also reported (13). While certain pediatric gliomas have been shown to harbor NTRK fusions with frequencies of 5–25% (14), only one case of mixed neuronal-glial tumors (MNGT) with SPECC1L::NTRK2 fusion was reported (12). Due to the exceeding rare NTRK rearrangement in this group of glioneuronal and neuronal tumors, we conducted a comprehensive integrating analysis of imaging, conventional histopathology, and molecular profiling.

A 24-year-old female presented with a history of more than a decade of recurrent and unprovoked seizures. T2 weighted magnetic resonance imaging (MRI) of the brain showed an inhomogeneous hyperintense lesion located in the left frontal lobe ( Figure 1A ), measuring 2.0 × 1.5 × 1.7 cm in dimension, while T1 weighted MR image revealed hypo-intensity without peri-tumoral edema ( Figure 1B ). Contrast-enhanced MRI exhibited no significant enhancement ( Figures 1C, D ).

The patient received extensive tumor resection after careful preoperative planning. The tumor was soft and confined in the lobe ( Figure 1E ). A postoperative MRI confirmed complete resection of the tumor. The fresh tumor tissues were sent for histology and molecular profiling analysis. H&E staining revealed a multinodular glial element, and a honeycomb appearance of oligodendrocyte-like cells was observed, with neurons floating in the matrix ( Figure 1F ). Immunohistochemistry analysis showed positive ATRX nuclear stain and negative P53 expression ( Figures 1G, I ), consistent with the genomic analysis showed wild-type ATRX and TP53. Scattered CD34 expression was seen in tumor cells in addition to vascular endothelial cells ( Figure 1H ), which was consistent with a previous report showing frequent CD34 expression in mixed or diffuse DNT (18). Tumor cells also expressed synaptophysin (Syn) ( Figure 1K ), oligodendrocyte transcription factor 2 (Olig2) ( Figure 1J ), and Ki-67 positivity less than 5%. Neuron-specific enolase (NSE) expression was observed in the neurons as expected ( Figure 1M ). Taken together, the histopathological and molecular pathological profiles were consistent with a complex subtype DNT. Moreover, Anti-Pan TRK antibody staining revealed a strong signal in tumor cells, consistent with the TrkB rearrangement ( Figure 1L ).

An RNA NGS assay with 390 ‘bait’ probes targeting 63 genes commonly rearranged in solid tumors revealed an LHFPL3::NTRK2 transcript ( Figure 2A ). The LHFPL3::NTRK2 fusion gene contained the first two exons of LHFPL3 and the last 6 exons of NTRK2, starting at exon 16 ( Figure 2B ). FISH analysis with a break-apart NTRK2 probe confirmed the NTRK2 rearrangement in tumor cell touch preparation ( Figures 2C, D ).

RT-PCR analysis of tumor RNA with primers specific for LHFPL3 and NTRK2 showed 3 bands. The dominant band ( Figure 2E ) was Sanger sequenced and was identical to the transcript obtained from the RNA NGS. The other 2 minor bands were not sequenced, likely from differential splicing. A targeted DNA NGS ( Figure 2F ) analysis revealed an isocitrate dehydrogenase 1 (IDH1) mutation (p.R132H) ( Figure 3A ) and copy number gains of the chromosomes 7p, 9q, and X ( Figure 3B ). Immunohistochemistry analysis also confirmed the expression of mutant IDH1 (R132H) ( Figure 1N ). IDH1 mutations are often seen in diffuse gliomas including astrocytoma or oligodendroglioma but are rarely reported in DNTs (19). Other mutations, including ATRX and TP53 mutations in astrocytoma and chromosome 1p/19q co-deletion in oligodendroglioma, were not observed in this tumor.

A variety of genomic alterations have been reported in DNTs, although diagnostically specific changes are not established. Nevertheless, fibroblast growth factor 1 receptor (FGFR1) has been frequently activated in DNTs (10, 20). Among FGFR1 alterations, internal tandem duplication (ITD) of the tyrosine kinase domain (TKD) is the most common mutation, which was reported in 40~60% of DNTs. In addition, hotspot missense mutations of FGFR1 were also reported. Other genomic alterations include BRAF p.V600E and copy number chromosome gains (21). The frequency of BRAF alterations in DNTs remains inconclusive among different studies (22, 23), with several studies failing to identify BRAF alterations in their DNTs cohorts (20, 24).

NTRK rearrangements in glioneuronal tumors were extremely rare. STRN1::NTRK2 and ARHGEF2::NTRK1 were reported in a single case of malignant glioneuronal tumor, respectively (25, 26). Alvarez-Breckenridge (27) described a case of low-grade glioneuronal tumor with BCAN::NTRK1 fusion. Surrey (12) reported a case of mixed neuronal-glial tumors (MNGT) with SPECC1L::NTRK2 fusion. Torre et al. reported a cohort of gliomas harboring NTRK fusions, most infantile or adult cases were histologically high-grade (89.7%, 26/29), while pediatric cases demonstrated high-grade histology (15.4%, 2/13) were rare. The median follow-up period after diagnosis was 23 months. During the follow-up period, 57.0% of the cases suffered tumor recurrence or progression (28).

IDH1/2 mutations are rare in DNTs, different from low-grade diffuse gliomas. In a cohort of 100 DNTs, 3 IDH1 mutations were observed (19). Jayapalan (29) reported a rosette-forming glioneuronal tumor with IDH1 mutation, which recurred in situ and progressed to glioblastoma 6 years after partial resection. Authors speculated that IDH1 mutation may lead to the malignant transformation of this type of benign glioneuronal tumor. Nevertheless, the biological and prognostic implications of IDH1/2 mutations in DNTs remain to be clarified.

In conclusion, we report a rare DNT with a novel LHFPL3::NTRK2 fusion and an IDH1 mutation. These findings provide additional evidence for a heterogeneous genomic profile of DNTs. Further functional evaluation of the LHFPL3::NTRK2 fusion oncoprotein will not only detail the oncogenic signaling mechanism but also shed light on the cellular function of LHFPL3, a CNS-specifically expressed gene with no studies.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

This study was reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University. Written informed consent to participate in this study was provided by the participant herself.

YC designed this study. XD, PC, SuZ, and ShZ performed histopathological and molecular pathological examinations. XD and SX were responsible for the pathological diagnosis. QZ, YW, AC, and CD supported clinical data. QL and SX guided this work. All authors contributed to the article and approved the submitted version.

This study was funded by the National Natural Science Foundation of China (81602183).

Authors PC, SuZ, and ShZ were employed by Suzhou Sano Precision Medicine Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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