CNS tumors with PLAGL1-fusion: beyond ZFTA and YAP1 in the genetic spectrum of supratentorial ependymomas

Abstract

A novel methylation class, "neuroepithelial tumor, with PLAGL1 fusion" (NET-PLAGL1), has recently been described, based on epigenetic features, as a supratentorial pediatric brain tumor with recurrent histopathological features suggesting an ependymal differentiation. Because of the recent identification of this neoplastic entity, few histopathological, radiological and clinical data are available. Herein, we present a detailed series of nine cases of PLAGL1-fused supratentorial tumors, reclassified from a series of supratentorial ependymomas, non-ZFTA/non-YAP1 fusion-positive and subependymomas of the young. This study included extensive clinical, radiological, histopathological, ultrastructural, immunohistochemical, genetic and epigenetic (DNA methylation profiling) data for characterization. An important aim of this work was to evaluate the sensitivity and specificity of a novel fluorescent in situ hybridization (FISH) targeting the PLAGL1 gene. Using histopathology, immunohistochemistry and electron microscopy, we confirmed the ependymal differentiation of this new neoplastic entity. Indeed, the cases histopathologically presented as "mixed subependymomas-ependymomas" with well-circumscribed tumors exhibiting a diffuse immunoreactivity for GFAP, without expression of Olig2 or SOX10. Ultrastructurally, they also harbored features reminiscent of ependymal differentiation, such as cilia. Different gene partners were fused with PLAGL1: FOXO1, EWSR1 and for the first time MAML2. The PLAGL1 FISH presented a 100% sensitivity and specificity according to RNA sequencing and DNA methylation profiling results. This cohort of supratentorial PLAGL1-fused tumors highlights: 1/ the ependymal cell origin of this new neoplastic entity; 2/ benefit of looking for a PLAGL1 fusion in supratentorial cases of non-ZFTA/non-YAP1 ependymomas; and 3/ the usefulness of PLAGL1 FISH.

Keywords: DNA-methylation; Ependymoma; PLAGL1; Subependymoma.

Fig. 1

DNA methylation-based t-distributed stochastic neighbor embedding distribution. Reference DNA methylation classes (v12.5 of the DKFZ classifier): DMG_K27: diffuse midline glioma H3 K27M mutant/EZHIP overexpressing; DNET: dysembryoplastic neuroepithelial tumor; EPN_MPE: myxopapillary ependymoma; EPN_PFA: ependymoma, posterior fossa group A; EPN_PFB: ependymoma, posterior fossa group B; EPN_PF_SE: subependymoma, posterior fossa; EPN_SP_SE: subependymoma, spinal; EPN_SP: spinal ependymoma; EPN_SP_MYCN: spinal ependymoma, MYCN-amplified; EPN_ST_SE: subependymoma, supratentorial; EPN_ZFTA: ependymoma, ZFTA fusion; EPN_YAP1: ependymoma, YAP1 fusion; GB_RTK1: glioblastoma, IDH wildtype, subclass RTK1; GB_RTK2: glioblastoma, IDH wildtype, subclass RTK2; GB_MES: glioblastoma, IDH wildtype, subclass mesenchymal; HGNET_PLAG: embryonal tumor with PLAG-family amplification; NET_PLAGL1: neuroepithelial tumor with PLAGL1-fusion; PA_CORT: pilocytic astrocytoma, hemispheric; PA_INF: pilocytic astrocytoma, infratentorial; PA_MID: pilocytic astrocytoma, midline; PXA: pleomorphic xanthoastrocytoma

Fig. 2

Histopathological and ultrastructural features. a-c An ependymal component admixed with subependymal features and microcalcifications (case #6, HPS, magnification × 60 for a, magnification × 400 for b-c). d Well-demarcation of the tumor from adjacent brain parenchyma (case #4, HPS, magnification × 200), confirmed using neurofilament staining (e, magnification × 100). f Monomorphous cells with small to medium-sized round nuclei and eosinophilic granular bodies (case #4, HPS, magnification × 400). g Frequent microcystic changes (case #6, HPS, magnification × 400). h Microcystic changes with myxoid substance, and eosinophilic granular bodies (case #8, HPS, magnification × 400) positive with PAS staining (case #8, insert, magnification × 400). i Hemorrhagic modifications with siderophages (case #4, HPS, magnification × 400). j Diffuse GFAP immunoexpression (case #8, magnification × 400). k Diffuse GFAP immunoexpression including in microcystic component (case #4, magnification × 400). l No immunopositivity for Olig2 (case #4, HPS, magnification × 400). m No immunopositivity for SOX10 (case #4, HPS, magnification × 400). n EMA immunoexpression with dot-like or micro-lumens (case #4, HPS, magnification × 400). o MIB-1 labeling index was low, ranged from 1% (case #8, HPS, magnification × 400). Black scale bars represent 500 μm (a), 50 µm (b-c, and f-o), 100 µm (d) and 250 µm (e) HPS: Haematoxylin Phloxin Saffron.

Fig. 3

Ultrastructural findings, ultrathin sections, electron microscopy. a Tumor cells harboring zonula adherens (arrow). b Glial intermediate filaments (*) are present in the cytoplasm of the tumoral cell and in intercellular spaces. We can observe dilated cisternae of the Golgi apparatus and the smooth endoplasmic reticulum (black arrow) and numerous microtubules (white arrow). c Tumoral cell with an intracytoplasmic cilium in transversal section (black arrow). The white arrow shows microtubules. Insert: high magnification of the cilium. d Microvilli-like structures (*) are present in lumen-like spaces between the adjacent cells. The white arrow shows microtubules. e Extracellular eosinophilic bodies. f They are moderately dense, and have a shape of convolutions whose denser lines delimit guts

Fig. 4

Detection of PLAGL1 rearrangements by FISH. a-c FISH images showing positive cases (#2–5-7) and d a negative case (#10) (magnifications × 1000). Representative image of a slide hybridized with a PLAGL1 Break-Apart FISH probe. In positive cases, the images show nuclei harboring a split (red and green signals, yellow arrowheads) and a fused signal or an isolated 3’PLAGL1 signal and a fused signal (grey arrowheads). For the negative case, the images show nuclei harboring two intact fused signals FISH, fluorescence in situ hybridization

Fig. 5

Radiological features on MRI. Illustrative image of case #2 with FLAIR-w (a), T2-w (b), T1-w (c) and post-contrast T1-w (d). MR images showing a large left frontal intra-axial brain lesion with ependymal contact. The lesion shows minimal perilesional edema, T2 hyperintensity, and a hypointense center on T1-w imaging, with subtle enhancements on post contrast imaging. Illustrative image of case #5 with FLAIR-w (e, f), post-contrast T1-w (g) and T2-w (h) axial views of MR images showing a left fronto-parietal intra-axial brain lesion with ependymal contact and extension within the ventricules. The lesion shows minimal perilesional edema, T2 hyperintensity, and barely no enhancement

Fig. 6

Prognosis for our cases. The mean/median PFS were 70.4/27.6 months for ependymomas, ZFTA::RELA fusion-positive, 36.3/not reached months for ependymomas, YAP1 fusion-positive, 24.4/9.2 months for ependymomas, ZFTA non-RELA fused, and 43.9/34.0 months for astroblastomas, MN1-altered, 16.2/12.0 for CNS tumors with BCOR internal tandem duplication and 182.2/277 months for NET PLAGL1 with a significant difference in univariate analysis (p < 0.001). The median OS was not reached for all subgroups except CNS tumors with BCOR internal tandem duplication (76.0 months) and the mean OS was not reached for the ependymomas, YAP1 fusion-positive. The mean OS were 113.5 months for ependymomas, ZFTA::RELA fusion-positive, 39.3 months for ependymomas, ZFTA non-RELA fused, 81.6 months for astroblastomas, MN1-altered, 53.2 months for CNS tumors with BCOR internal tandem duplication and 111.0 months for NET PLAGL1 with a significant difference in univariate analysis (p = 0.002)