EWSR1-PATZ1 gene fusion may define a new glioneuronal tumor entity

Abstract

We investigated the challenging diagnostic case of a ventricular cystic glioneuronal tumor with papillary features, by RNA sequencing using the Illumina TruSight RNA Fusion panel. We did not retrieve the SLC44A1-PRKCA fusion gene specific for papillary glioneuronal tumor, but an EWSR1-PATZ1 fusion transcript. RT-PCR followed by Sanger sequencing confirmed the EWSR1-PATZ1 fusion. It matched with canonic EWSR1 fusion oncogene, juxtaposing the entire N-terminal transcriptional activation domain of EWSR1 gene and the C-terminal DNA binding domain of a transcription factor gene, PATZ1. PATZ1 protein belongs to the BTB-ZF (broad-complex, tramtrack and bric-à-brac -zinc finger) family. It directly regulates Pou5f1 and Nanog and is essential to maintaining stemness by inhibiting neural differentiation. EWSR1-PATZ1 fusion is a rare event in tumors: it was only reported in six round cell sarcomas and in three gliomas of three exclusively molecular studies. The first reported glioma was a BRAF^V600E negative ganglioglioma, the second a BRAF^V600E negative glioneuronal tumor, not otherwise specified and the third, very recently reported, a high grade glioma, not otherwise specified. In our study, forty BRAF^V600E negative gangliogliomas were screened by FISH using EWSR1 break-apart probes. We performed methylation profiling for the index case and for seven out of the ten FISH positive cases. The index case clustered apart from other pediatric low grade glioneuronal entities, and specifically from the well-defined ganglioglioma methylation group. An additional pediatric intraventricular ganglioglioma clustered slightly more closely with ganglioglioma, but showed differences from the main ganglioglioma group and similarities with the index case. Both cases harbored copy number variations at the PATZ1 locus. EWSR1-PATZ1 gene fusion might define a new type of glioneuronal tumors, distinct from gangliogliomas.

Keywords: DNA methylation profiling; EWSR1; PATZ1; ganglioglioma; glioneuronal tumors.

Figure 1

Pre‐operative MR images for Cases 1 and 2. Case 1 MRI showed a tumor in the left lateral ventricule with a moderate ventricular dilatation suggesting central neurocytoma. Low heterogenous signal in T2 (A). T1‐weighted images with marked gadolinium enhancement of the tumor (B, C). Case 2 MRI revealed a cystic lesion in the right cerebellar hemisphere extending into the 4th ventricule and the tentorial incisure. T2 heterogeneous signal (D). Spontaneous T1 signal hyperintensity may correspond to hemorrhage (E). T1‐weighted images with marked and heterogeneous gadolinium enhancement of the tumor. Dilatation of the ventricular system was observed (F).

Figure 2

Representative histopathology of the Cases 1 and 2. Case 1: Hematoxylin and eosin–stained sections exhibit tumor cell proliferation with a rich vascular network (A) surrounded by poorly arranged cells without clear astrocyte or neuronal differentiation (B). A marked hyalinization is often observed (C). Tumoral cells were immunopositive for olig2 (D) and synaptophysin (E). Neurofilament proteins immunoreactivity is limited to rare axons and few included ganglionic neurons (F). Tumoral cells with irregular or pleomorphic nuclei and clear cytoplasm form lobules surrounded by fusiform astrocyte‐like cells, (G) in a dense reticulin network (H). The fusiform cells are immunonegative for GFAP whereas pleomorphic clear cells within lobules show immunoreactivity (I). Case 2: Hematoxylin and eosin–stained sections show a circumscribed tumor (J). Round cells sometimes with eosinophilic cytoplasm are arranged on a fibrillary matrix. Most of the vessels have a thick hyalinized wall (K). In some areas, large eosinophilic cells form structured sheets interrupted by fibrillary matrix (L). The Gordon Sweet reticulin stain underlines the rich vascular network (M). Strong olig2 expression is observed (N). Diffuse, strong and often dot‐like immunoreactivity for synaptophysin is observed (O).

Figure 3

The EWSR1‐PATZ1 fusion gene. Schematic representation of fusion event between EWSR1 (blue) and PATZ1 (red). The two genes EWSR1 and PATZ1 were on the chromosome 22q. Lines and full boxes represented the intron sequence and the sequence coding exons, respectively. An orange strip depicted the fusion point. The RNA sequencing and the Sanger chromatogram were in line with a schematic view of the fusion mRNA. The RNA sequencing corresponded to an immature form of fusion mRNA and juxtaposed intron 8–9 of EWSR1 gene, and the first exon of PATZ1 from the 966th nucleotide. The Sanger chromatogram corresponded to a mature form of fusion mRNA and juxtaposed the exon 8 of EWSR1, a two bases addition and the first exon of PATZ1 from the 966th nucleotide. The protein fusion combined the whole N‐terminal transcriptional activation domain from EWSR1 and C‐terminal DNA binding domain of the PATZ1 transcription factor. BP: break point, R7BS: RNA polymerase II subunit hsRPB7 binding site, ZF: zinc finger domain, TAD: transcriptional activation domain, BTB: Broad‐Complex, Tramtrack and bric‐à‐brac domain, C2H2: C2H2‐type zinc‐finger protein family, DNA‐BD: DNA binding domain, Chr 22: chromosome 22

Figure 4

Unsupervised hierarchical clustering using the 10 000 top most variably methylated probes across the cohort for the two test cases and selected reference cases from the Heidelberg Molecular Neuropathology platform (https://www.molecularneuropathology.org/mnp). Samples are colored according to the histological type: ganglioglioma green, cortical pilocytic astrocytoma orange, dysembryoplastic neuroepithelial tumor pink, Case 1 (PATZ1‐fused) turquoise blue and Case 2 light green. Clustering was according to Euclidean distance measures and complete linkage. Each row represents a probe; each column represents a sample. Color scale indicates methylation status (blue: unmethylated, red: methylated).