Molecular analysis of pediatric CNS-PNET revealed nosologic heterogeneity and potent diagnostic markers for CNS neuroblastoma with FOXR2-activation

Abstract

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly malignant neoplasms posing diagnostic challenge due to a lack of defining molecular markers. CNS neuroblastoma with forkhead box R2 (FOXR2) activation (CNS_NBL) emerged as a distinct pediatric brain tumor entity from a pool previously diagnosed as primitive neuroectodermal tumors of the central nervous system (CNS-PNETs). Current standard of identifying CNS_NBL relies on molecular analysis. We set out to establish immunohistochemical markers allowing safely distinguishing CNS_NBL from morphological mimics. To this aim we analyzed a series of 84 brain tumors institutionally diagnosed as CNS-PNET. As expected, epigenetic analysis revealed different methylation groups corresponding to the (1) CNS-NBL (24%), (2) glioblastoma IDH wild-type subclass H3.3 G34 (26%), (3) glioblastoma IDH wild-type subclass MYCN (21%) and (4) ependymoma with RELA_C11orf95 fusion (29%) entities. Transcriptome analysis of this series revealed a set of differentially expressed genes distinguishing CNS_NBL from its mimics. Based on RNA-sequencing data we established SOX10 and ANKRD55 expression as genes discriminating CNS_NBL from other tumors exhibiting CNS-PNET. Immunohistochemical detection of combined expression of SOX10 and ANKRD55 clearly identifies CNS_NBL discriminating them to other hemispheric CNS neoplasms harboring "PNET-like" microscopic appearance. Owing the rarity of CNS_NBL, a confirmation of the elaborated diagnostic IHC algorithm will be necessary in prospective patient series.

Keywords: CNS-PNET; FOXR2-activation; Neuroblastoma; SOX10.

Fig. 1

a. Pathological similarity of CNS-PNET samples allocated to various molecular groups (The scale bars: 50 μm). b. Two-dimensional t-distributed stochastic neighbor embedding (tSNE) analysis of reference set composed of various tumor entities including CNS_NBL, GBM_G34, GBM_MYCN and EPN_RELA cohorts. c. tSNE analysis of 84 institutionally diagnosed cPNET (all black spots) revealed distribution their epigenetic profiles among the four reference tumor sets including CNS_NBL (n = 20), GBM_G34 (n = 22), GBM_MYCN (n = 18) and EPN_RELA (n = 24). d. Survival analysis for CNS-PNET cohort shows that both overall survival (OS) and progression-free survival (PFS) were significantly better for patients harboring tumors with CNS_NBL molecular signature (black lines), in comparison to GBM_G34 (red lines), GBM_MYCN (orange lines) and EPN_RELA (green lines)

Fig. 2

Gene expression profiling data obtained after RNA sequencing of CNS-PNET cohort (n = 54). a. Heat-map of unsupervised hierarchical cluster analysis based on 500 differentially expressed genes disclosed that CNS_NBL (blue) were clustered separately from EPN_RELA (red); GBM_G34 (brown), and GBM_MYCN (orange) (Row Z-Scores: green: from 0 to − 6; red: from 0 to 6). Scales of principal component analysis (PCA); b. and two-dimensional t-distributed stochastic neighbor embedding (tSNE); c. analyses also revealed a separate distribution of CNS_NBL from other molecular groups

Fig. 3

a. A set of 30 top most-confident genes differentially overexpressed in CNS_NBL (blue top line); GBM_G34 (brown top line), GBM_MYCN (orange top line), and EPN_RELA (red top line) respectively allows one to discriminate clearly between these tumor cohorts with SOX10 among the top genes for CNS_NBL (Row Z-Scores: green: from 0 to − 6; red: from 0 to 6). b. FOXR2 and SOX10 expression levels were significantly higher in CNS_NBL (blue boxplots) whereas MYCN was overexpressed in GBM_MYCN (orange boxplots) and RELA—EPN_RELA (red boxplots) respectively. c. Gene Ontology analysis disclosed that CNS_NBL transcriptome signatures (blue circles) were associated with neuronal metabolism, synaptic transmission, and neuroendocrine secretion

Fig. 4

Patterns of SOX10 nuclear protein immunoexpression in various CNS tumors (The scale bars: 50 μm). Intense SOX10 expression was found in all CNS NBL, but also in some subtypes of high-grade gliomas. All embryonal CNS tumors were negative for SOX10, whereas glial neoplasms disclosed various degrees of nuclear immunostaining intensity

Fig. 5

a. A set of 25 top most-confident genes differentially overexpressed in CNS_NBL (blue top line) and GBM_MID (pink top line) respectively allows one to discriminate clearly between these tumor cohorts with ANKRD55 on the top genes for CNS_NBL (Row Z-Scores: green: from 0 to − 6; red: from 0 to 6). b. ANKRD55 expression levels were significantly higher in CNS_NBL (blue boxplots) in comparison to other molecular groups. c. Intense ANKRD55 immunostaining was found in CNS_NBL but GBM_MID were negative (The scale bars: 50 μm)