Clinical, pathologic, and genomic characteristics of two pediatric glioneuronal tumors with a CLIP2::MET fusion

A 1-day-old full-term male with meconium aspiration syndrome presented with Escherichia coli sepsis, and initial cranial ultrasound demonstrated 4.8 × 5.3 cm intraparenchymal and intraventricular hemorrhage within the left occipital lobe (Fig. 1A). Once medically stabilized, an MRI/MRA was obtained and demonstrated increased ventriculomegaly with cerebrospinal fluid (CSF) septations and 4th ventricle outflow obstruction. At 3 weeks of age, the patient underwent a ventriculoperitoneal shunt (VPS) placement for hydrocephalus. At 16 months of age, his MRI was stable, and all hemorrhage was resolved without any sign of lesion or mass. (Fig. 1B). At the age of 29 months, he presented to the emergency department with emesis, and underwent revision of the shunt. Post-operative MRI confirmed stable ventricles, loss of parenchymal volume, and a rounded left occipital lesion at the site of the original hemorrhage. The patient was discharged and followed with imaging. At 4-years-old, surveillance imaging demonstrated slow to minimal growth of the lesion measuring 3.2 cm x 2.0 cm (AP x TV). Surgical treatment was pursued at that time at four years post-hemorrhage (Fig. 1C). He underwent gross total resection and did not receive adjuvant therapy. His last follow-up was at 6-months post-operative, where he remained disease-free and at neurological baseline (Fig. 1D).

Chromosomal microarray analysis (CMA) of this tumor sample, using the OncoScan platform (Thermo Fisher Scientific), demonstrated an abnormal copy number profile with copy number losses encompassing most of the short arm of chromosome 1 (1p), 9p, a significant portion of 19q, and a substantial segment of 22q (Fig. 3A). There was also an interstitial deletion in chromosome 7q (Fig. 3B). Additionally, several nonconsecutive segmental deletions along the short and long arms of chromosome 6 were observed, along with the loss of most of 6p (Fig. 3A). Of note, the breakpoints in 1p and 19q were more distal than the typical 1p/19q co-deletions observed in oligodendrogliomas. OncoKids, a comprehensive DNA- and RNA-based next-generation sequencing panel [10], was negative for clinically significant DNA sequence variants, RNA fusions, and gene amplification events. Subsequent whole transcriptome RNA sequencing (RNAseq) analysis [5] of the tumor sample revealed a CLIP2::MET fusion (Fig. 4A). The fusion occurred in-frame, resulting in the expression of a fusion protein encoded by the 5’ portion of the CLIP2 gene (exons 1–11 out of a total of 17 exons) and the 3’ portion of the MET gene (exons 15–21 out of a total of 21 exons), which contained the protein kinase domain of MET. This fusion is predicted to result in the upregulation of the MAPK signaling pathway [8]. As the deletion breakpoints in 7q do not involve the CLIP2 or MET genes by CMA (Fig. 2A), the CLIP2::MET fusion likely results from rearrangements in a primarily balanced form. Further RNAseq analysis of the expression of the MET gene demonstrated higher expression of MET exons 15–21, which contained the tyrosine kinase domain, than that of exons 1–14 (Supplemental Fig. 1). By DNA methylation profiling, no definitive classification can be provided for this tumor, and the calibrated family and/or class scores were below the established in-house methylation class threshold of 0.88. However, the tumor received a suggestive class score of 0.83 for the methylation class “diffuse leptomeningeal glioneuronal tumor (DLGNT)”, wherein loss of 1p, with or without 19q loss, is prevalent [2, 7, 22]. Evaluation of the tumor using version 12.5 of the DKFZ classifier again yielded no match, with equivocal scores for “low grade glial/glioneuronal/neuroepithelial tumor” (0.46) and “diffuse glioneuronal tumor” (0.45) at the family level. The NCI’s Bethesda v2 classifier suggested a superfamily of “low grade glial/glioneuronal tumors” (mean score 0.645), and suggested class of “pilocytic astrocytoma, hemispheric” (mean score 0.991). In this case, the CMA findings of loss of significant portions of chromosomes 1p and 19q could support the diagnosis of DLGNT. Notably, no evidence of IDH1/2 mutation was detected. By UMAP analysis, the tumor clustered with the LGG, PA/GG ST (low grade glioma, subclass hemispheric pilocytic astrocytoma and ganglioglioma) reference samples (Fig. 4B). Overall, in the context of a CLIP2::MET fusion detected by RNAseq, the molecular findings are most consistent with a CLIP2::MET fusion-positive glioneuronal tumor.

An 8-year-old male patient was followed for new onset seizures. Preceding ictal symptoms, he had blurry vision, nausea, and vertigo. Presenting to our clinic, his ictal with episodes involve left head tilting, upward gaze, and bilateral eye twitching. MRI revealed a right parieto-occipital mass (3.7 cm x 5.1 cm x 2.9 cm) (Fig. 1E). Patient was prescribed anticonvulsant due to focal seizures and discussed surgical options.

Two weeks after being seen in clinic he underwent near-total surgical resection. The post-operative MRI revealed a small residual nodule (3–4 mm) (Fig. 1F). Preliminary diagnosis of tissue demonstrated a glioneuronal tumor with no post-operative complications. The patient did not receive any adjuvant therapy. At two-week follow-up, patient’s vision improved, improved from neurological baseline, and discontinued the anticonvulsants. Now, he undergoes annual surveillance imaging. At last follow-up three years post-surgery, patient remains clinically stable, with stable residual disease (Fig. 1G).

CMA of the tumor sample exhibited an abnormal copy number profile with gain of 1q, loss of chromosome 22 (Fig. 3C), as well as several copy number alterations in 7q, including two interstitial deletions, an interstitial gain, and a terminal loss. Notably, the proximal breakpoint of the deletion in 7q11.23q21.11 was within the CLIP2 gene, indicating a potential rearrangement. The MET gene was not included in copy number alterations observed in 7q (Fig. 3D). There was no evidence for monosomy 14 or 1p/19q co-deletion. OncoKids showed no established clinically significant sequence variants, gene fusions, or amplification events. Subsequent RNAseq analysis revealed the same CLIP2::MET fusion as seen in Case 1, with exon 11 of CLIP2 fused to exon 15 of MET. The fusion protein contained the protein kinase domain of MET. The same MET gene expression pattern was noted in this tumor, with higher expression of MET exons 15–21 than that of exons 1–14, using the RNAseq data (Fig. 4). DNA methylation studies did not match this tumor to a known or specific methylation class using the random forest algorithm with the local version of DKFZ 11b4 classifier, but it clustered with the LGG, DNT (low grade glioma, dysembryoplastic neuroepithelial tumor) reference samples by UMAP analysis (Fig. 4B). Additional evaluation with the DKFZ v12.5 also showed no match, with equivocal superfamily scores very similar to Case 1 (0.55 vs. 0.43). The NCI’s Bethesda v2 classifier suggested a superfamily of “low grade glial/glioneuronal tumors” (mean score 0.813) and a suggested class of “glioneuronal tumor, KinF A” (mean score 0.869). The clinical, pathological, and molecular findings of both cases, along with those from published cases with the CLIP2::MET fusion, are summarized in Table 1.