Refinement of diagnostic criteria for pediatric-type diffuse high-grade glioma, IDH- and H3-wildtype, MYCN-subtype including histopathology, TP53, MYCN and ID2 status

Diffuse pediatric-type high-grade glioma (pHGG), IDH- and H3-wildtype are currently subdivided into three subgroups: pHGG-RTK (Receptor Tyrosine Kinase) 1, pHGG-RTK2, and pHGG-MYCN [2]. Each subgroup is known to present recurrent gene amplifications such as PDGFRA, EGFR and MYCN, respectively for RTK1, RTK2, and MYCN [2] but these amplifications are not specific to these subgroups. Although recurrent histopathological (such as nodules composed of large cells with prominent nucleoli and expression of glial and neuronal markers) [4, 5], and genetic (TP53 mutations, particularly in a context of Li-Fraumeni syndrome) features [1] have been identified in pHGG-MYCN, the diagnosis of this subgroup is currently confirmed only by DNA-methylation analysis. Because a subset of pHGG-MYCN have been found to harbor an amplification of the ID2 gene with (12/36, 33%) or without (1/36, 3%) a concomitant MYCN amplification (MYCN+/ID2+) [2], we have formulated a FISH (Fluorescence in situ hybridization) technique that targets both loci. Taking a monocentric series of 29 pHGG, IDH- and H3-wildtype, we studied the status of MYCN and ID2 for each tumor, and correlated the data with histopathological and genetic features (including TP53 status, somatic and germline), and DNA-methylation profiling.

The results are detailed in Supplementary Fig. 1 and Supplementary Table 1. The integrative histopathological, genetic and epigenetic analyses, including t-Distributed Stochastic Neighbor Embedding analysis (t-SNE) (Supplementary Fig. 2) segregated tumors into: pHGG-RTK1 (n = 5), pHGG-RTK2 (n = 11), and pHGG-MYCN (n = 13). All DNA-methylation proven pHGG-MYCN, except one (#11), harbored an amplification of MYCN, and five of them an ID2 amplification (Fig. 1). A MYCN amplification was also observed in 31% (5/16) of pHGG-non MYCN (four RTK2 and one RTK1). However, none of them had an ID2 amplification. Ten/13 pHGG-MYCN presented the histopathological features previously reported for this subgroup, whereas the three remaining cases showed features typically associated with diffuse astrocytic gliomas. Somatic mutations of TP53 were present in 12/13 pHGG-MYCN, and four of them (with available data) harbored a germline mutation (Fig. 1). The outsider case (#11) presented the histopathological features of pHGG-MYCN, a TP53 somatic mutation and a MYC amplification (Supplementary Fig. 3).

Fig. 1

Histopathological and molecular features of pediatric high-grade gliomas, MYCN subgroup. The case #3 presented classical features of HGG-MYCN such as a dense proliferation composed of large cells with prominent nucleoli (HPS, magnification x400) with overexpression of p53 (magnification x400). The FISH analysis evidenced an amplification of both MYCN (orange signals) and ID2 (blue signals) loci (magnification x800). There was no Li-Fraumeni syndrome in this case. The case #7 presented classical features of HGG-MYCN such as a dense proliferation composed of large cells with prominent nucleoli and numerous mitoses (HPS, magnification x400) with overexpression of p53 (magnification x400). The FISH analysis evidenced an amplification of MYCN locus without amplification of ID2 gene (magnification x800). There was a context of Li-Fraumeni syndrome. FISH: Fluorescence in situ hybridization; HGG: high-grade glioma; HPS: Hematoxylin Phloxin Saffron; mut.: mutation. Black scale bars represent 50 μm

Fig. 2

Diagnostic approach for pediatric high-grade glioma, with MYCN-amplification. Co-amp.: co-amplification; HGG: high-grade glioma; LFS: Li-Fraumeni syndrome; ped: pediatric; Wt: wildtype. Black scale bars represent 50 μm

As previously reported [2], a subset of pHGG-MYCN presented a co-amplification of MYCN and ID2 (MYCN+/ID2+), which was specific for this subgroup of pHGG, and another subset of pHGG harbored an amplification of MYCN without ID2 amplification (MYCN+/ID2-). The current series showed that all pHGG-MYCN cases, except one, harbored a somatic TP53 mutation and a subset of them presented a germline mutation for TP53, as previously reported [1]. Interestingly, our results seem to show that pHGG-MYCN MYCN+/ID2 + are not associated with TP53 germline mutations, whereas Li-Fraumeni syndrome is present in the subgroup of MYCN+/ID2-. Moreover, the current series described for the first time a pHGG-MYCN without amplifications of MYCN and ID2 but harboring a MYC amplification. This example may illustrate the phenomenon previously described where a spinal ependymoma classified as “MYCN amplified” by DNA-methylation profiling but harbored a MYC amplification [3]. DNA-methylation analysis is still only limited to a small number of centres worldwide or may not be contributive (8/29 cases from this cohort presented a low calibrated score for a methylation class), so alternative methods for routine practice need to be validated. Therefore, histopathology (including p53 overexpression correlated to TP53 mutation), and FISH MYCN/ID2 may help diagnose pHGG-MYCN, using a simple algorithm approach (Fig. 2).

While a subset of pHGG-MYCN is found in the tumoral spectrum of Li-Fraumeni syndrome, for exclusion purposes, the co-amplication MYCN/ID2 seems not to be associated with this genetic predisposition. The association of histopathological and genetic features may potentially represent alternative diagnostic criteria for pHGG-MYCN, particularly if DNA-methylation profiling is not available or not conclusive. FISH analyses of MYCN/ID2 genes may constitute an interesting diagnostic tool for routine neuropathological practice.