A 26-year-old male presented with worsening headaches and vomited for two months. MRI of the brain revealed a mass at the right temporal-parietal lobe. T2WI showed a polycystic compartment-like change, and the area is approximately 5.4*4.3cm. A sheet-like low signal and liquid level were found on the inside. The surrounding brain is edema and compressed ( Figure 1 ; left). Brain-enhanced CT (T1W1) showed a solid cystic enhancement of the mass ( Figure 1 ; middle). The entire lesion was surgically removed. Ten years ago, the patient underwent right temporal-parietal tumor resection in a local hospital and was pathologically diagnosed with “atypical central neurocytoma”. CT of the primary lesion showed a round-like 6.3*6.3 cm mixed density shadow in the brain parenchyma of the right parietal lobe with a compressed and deformed ventricle ( Figure 1 ; right). No radiotherapy or chemotherapy was received after the initial surgery. A routine brain scan was performed annually until three years before the recurrence. The patient received a radiotherapy (54Gy) after recurred tumor resection. The whole-body imaging scan found no other lesions, and the patient is in good condition currently.

5μm formalin-fixed paraffin-embedded sections were used for hematoxylin & eosin (H&E) and IHC stained. IHC was performed by an automatic immunohistochemical staining instrument (Ventana BenchMark ULTRA). The primary antibodies of GFAP (MX047), Vimentin (MX034), broad-spectrum Cytokeratin (AE1/AE3), Desmin (MAB-0766), and EMA (E29) were from MXB Biotechnologies. Commercially available prediluted antibodies of ZSGB-BIO, including Syn (EP158), S100 (ZA-0225), CgA (LK2H10), NSE (5E2), NeuN (A60), HMB45 (ZM-0187), Nestin (EP287), Oligo-2 (EP112), INI-1(ZA-0696), Neurofilament (ZM-0198) and Ki67 (UMAB107) were used. Photographs were captured using an Olympus BX51 microscope and an Olympus SC180 camera.

FISH for EWSR1 (22q12) rearrangement was performed on 4μm paraffin sections using the Vysis LSI EWSR1 dual-color break-apart probe (Abbott Molecular, Des Plaines, IL) according to standard protocols. The probe to the 5’ EWSR1 sequence is labeled in green and the probe to the 3’ EWSR1 sequence is labeled in red. A 4’,6-diamidino-2-phenylindole (DAPI) counterstain was used to identify the tumor cell nuclei. Analysis was performed on an epifluorescence microscope using single interference filter sets for red and green. For each interference filter, monochromatic images were acquired and merged using CytoVision (Leica Microsystems). A specimen was considered positive for EWSR1 rearrangement when a minimum of 15% of analyzed cells displayed split red and green signals or a single signal.

Tumor genomic DNA was extracted from FFPE samples using QIAamp DNA FFPE Tissue Kit. Libraries were constructed using the KAPA Hyper DNA Library Prep Kit (KAPA Biosystem, KK8504). The probes (Nanjing Geneseeq Biotechnology) for targeted sequencing cover exons, selected introns and alternatively spliced regions of 82 glioma-related genes (see Supplementary Table ). After hybridization, capture and purification, the library was sequenced as paired 150-bp reads on Illumina HiSeq 4000 according to the manufacturer’s instrument. Base-calling was performed on bcl2fastq v2.16.0.10 (Illumina, Inc.) to generate sequence reads in FASTQ format (Illumina 1.8+ encoding). High-quality reads were mapped to the human genome (hg19, GRCh37 Genome Reference Consortium Human Reference 37) using the BWA aligner 0.7.12 with the BWA-MEM algorithm and default parameters to create SAM files. The Genome Analysis Toolkit (GATK, version 3.4-0) was used to locally realign the BAMs files at intervals with indel mismatches and recalibrate base quality scores of reads in BAM files. The combined fusion results from all tools were manually reviewed on IGV for confirmation.

Both primary and recurrent tumors showed distinct boundaries between the tumor areas and the surrounding brain parenchyma ( Figure 2A ). Fibrous segmentations were seen inside the tumor ( Figure 2B ). There were two distinct tumor cell types: most were oval with unclear cell boundaries and scattered with few vacuolated cytoplasm cells ( Figure 2C ). In focal areas, enlarged epithelioid cell islands were seen with monomorphic, round nuclei, speckled nuclear chromatin, vacuolated cytoplasm, and distinct membrane ( Figure 2D ; upper left corner; arrow means magnification). No myxoid, vascular endothelial cell proliferation, calcifications, or necrosis was found in the primary and recurrent tumors. These tumors were well-circumscribed, and the tumor cells were mild and surrounded thin-walled blood vessels. However, increased and enlarged epithelioid tumor cell islands were found in the recurrent tumor samples.

All the tumor cells were negative for GFAP ( Figure 3A ), Oligo-2 ( Figure 3B ), and EMA ( Figure 3C ). Negative staining was also observed for NSE, NF, CgA, NeuN, Nestin, Desmin, HMB45, and INI-1(not shown). Epithelioid cell islands and scattered vacuolated cytoplasm cells showed positive expression for Cytokeratin ( Figure 3D ). S100 was positive in a patchy pattern ( Figure 3E ), and strong diffuse staining for Vimentin ( Figure 3F ) and Syn ( Figure 3G ) were found for all the tumor areas. Both the primary and the recurrent tumors showed the same immunophenotype including positive expression of Vimentin and Syn, negative for GFAP, Oligo-2, and EMA. However, more Cytokeratin positive epithelioid tumor cells and an increased Ki67 index were found in recurrent tumors. The average percentage of the Ki67 positive tumor cell is 5% for the primary tumor ( Figure 3H ), while 15% for the recurrent tumor ( Figure 3I ).

NGS analysis revealed EWSR1-PLAGL1 fusion ( Figure 4A ) as well as TERT (c.-124C>T, see Supplementary Figure ) promotor mutation. No other gene mutation (Indels or CNVs) was found. The fusion site is exon 8 of the EWSR1 gene and exon 8 of the PLAGL1 gene ( Figure 4B ). FISH with EWSR1 break-apart probe showed red-green separate (blue arrow) or individual red signal (green arrow) in more than 70% of tumor cells, which suggested positive EWSR1 gene rearrangements ( Figure 4C ).