Pediatric high-grade gliomas with concomitant RB1 and SETD2 alterations and Li-Fraumeni syndrome

The diffuse pediatric-type high-grade glioma (pHGG), IDH- and H3-wildtype MYCN subtype (pHGG-MYCN) has been isolated from the DNA-methylation profiling analyses of Primitive Neuroectodermal Tumors (PNET) of the Central Nervous System (CNS) [6] and is characterized by an embryonal-like morphology associated with a poor prognosis [7, 8]. A recent study has suggested the existence of two molecular subtypes: 1/ pHGG-MYCN with a co-amplification of MYCN/ID2 genes (without TP53 germline mutation) and 2/ pHGG-MYCN occurring in a context of Li-Fraumeni syndrome (LFS) (without ID2 amplification) [1]. Herein, we describe, for the first time, two tumors classified as pHGG-MYCN by DNA-methylation profiling that presented several unusual particularities: concomitant TP53 germline mutation, RB1 and SETD2 alterations.

Both cases were supratentorial tumors found in children: an 11-year-old girl who presented with a left fronto-temporal mass (case #1) and a 7-year-old boy with a left frontal lobe lesion (case #2). Both tumors were hemispheric, solid and cystic. One extended to the hypothalamus (#1), with peritumoral edema (Fig. 1). Each showed intense contrast enhancement and intermediate diffusion restriction. Cerebral blood flow by arterial spin labeling was high in one case (#1), and low in the other (#2). This imaging pattern may correspond to a supratentorial ependymoma or a high-grade glioma. Histopathologically, both presented similar histopathological features (Fig. 2a-d) and each had a diffuse growth pattern and were densely cellular with a glial differentiation composed of astrocytic and pleomorphic cells. Tumor #1 also presented a poorly differentiated component (Fig. 2d) without large cells and with distinct nucleoli. There were no eosinophilic granular bodies, perivascular lymphocytic infiltrates or xanthomatous cells. Signs of malignancy were obvious (necrosis, microvascular proliferation, and high mitotic indexes). Using immunohistochemistry, tumor cells expressed OLIG2, but did not stain for neuronal markers. There was no immunoreactivity for IDH1R132H, H3K27M, H3 G34R or BRAFV600E. ATRX, H3K27me3, Mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6) and INI1 expressions were maintained. Both tumors presented RB1 loss (Fig. 2e-f) and p53 overexpression was observed in one tumor (#2) (Fig. 2g). FISH analyses of PDGFRA, MYCN, ID2, MYC and EGFR genes evidenced a MYCN amplification for tumor #1 (Fig. 2h). DNA-sequencing analyses revealed SETD2, RB1 and TP53 mutations for both cases. DNA-methylation profiling classified both samples as pHGG-MYCN (with calibrated scores > 0.9, using the Heidelberg Brain Tumor Classifier v12.8) with several copy number variations (Fig. 2i-j). The familial history of patient #2 identified several cancers (nephroblastoma in his first cousin, leiomyosarcoma and a lung cancer before the age of 44 in his grandmother, pancreatic and lung cancers before the age of 40 in his great uncle). No history of cancer was described for patient #1. A germline pathogenic variant (PV) in the TP53 gene was identified in both patients: patient #1 c.874_875insT, p.(Lys292Ilefs*14) and patient #2 c.114 G > A, p.(Val272Met). Family genetic counselling is currently ongoing in both families.

Fig. 1

Radiological features of cases from this cohort. Axial T2-weighted (a), FLAIR (b), post-contrast T1-weighted (c), coronal T2-weighted (d), ADC map (e) and cerebral blood flow map (f) images of patient #1, showing a large left temporal and hypothalamic mass having a solid and cystic content and avid enhancement. The solid component displayed intermediate diffusion restriction and high cerebral blood flow. Axial T2-weighted (g), FLAIR (h), post-contrast T1-weighted (i), unenhanced CT (j), ADC map (k) and cerebral blood flow map (l) images of patient #2, showing a large left frontal mass having a solid and cystic content and avid enhancement. The solid component displayed intermediate density on CT and intermediate diffusion restriction. Cerebral blood flow was low

Fig. 2

Histopathological and molecular features of cases from this cohort. a-d Both cases presented an astrocytic differentiation with pleomorphic giant cells, necrosis, microvascular proliferation and numerous mitotic figures (HPS, magnification x400). e-f Both showed a loss of RB1 protein expression (magnification x400) and case #2 presented an overexpression of p53 (g, magnification x400). h The FISH analysis evidenced an amplification of both MYCN (orange signals) without ID2 (blue signals) loci amplification (magnification x800) for case #1. Case #1 did not have Li-Fraumeni syndrome. Copy number variations for cases #1 (i) and #2 (j). FISH: Fluorescence in situ hybridization; HPS: Hematoxylin Phloxin Saffron; mut.: mutation. Black scale bars represent 50 μm

After resection, both patients had adjuvant radiation therapy. Patient #1 experienced localized progressive disease one month after the end of radiotherapy, treated with lomustine and bevacizumab for five months, followed by metastatic tumor progression (nodular lesion of the bulb) undergoing treatment with lomustine, vincristine and procarbazine with further progression at the end of follow-up (twelve months after initial diagnosis). Patient #2 was alive at the end of follow-up without recurrence (five months after initial diagnosis).

The two current cases presented atypical histopathological and biological features when compared to previous reports of pHGG-MYCN. Firstly, they did not present an “embryonal” morphology [6,7,8], but the histopathological examination revealed similar features to glioblastoma or pleomorphic xanthoastrocytoma. This morphology may be similar to other pHGG such as diffuse hemispheric gliomas, H3 G34-mutant, and HGG encountered in a context of constitutional mismatch repair deficiency syndrome (CMMRD), but these diagnoses were carried out using immunohistochemistry and molecular analyses.

Genetically, only one of the tumors harbored a MYCN amplification and neither presented an ID2 amplification. The absence of a MYCN amplification in tumors classified as “pHGG-MYCN”, previously reported as being present in only 50% of this subgroup of tumors, did not exclude this diagnosis [2]. However, the two current cases harbored additional alterations of RB1 (associated with a loss of RB1 protein expression) and SETD2 genes. Both our experience and the literature data [2] have shown these alterations to be absent in classical cases of pHGG-MYCN. Moreover, unlike in adults, our experience has found the loss of RB1 expression to be absent in other subgroups of pHGG (n = 60), particularly for differential diagnoses such as pleomorphic xanthoastrocytomas, diffuse hemispheric gliomas, H3 G34-mutant, and HGG encountered in a context of CMMRD) (data not shown). While the two current tumors classified as pHGG-MYCN with high calibrated scores, they did not cluster with pHGG-MYCN associated or not to LFS by t-SNE analysis (t-distributed stochastic neighbor embedding) (Fig. 3). They clustered together and were distinct from all other methylation classes of pediatric gliomas. For both patients, the glioma allowed to identify the presence of TP53 germline PV, which was de novo for one of them. The proportion of de novo LFS has not yet been established for pHGG-MYCN but de novo forms exist in TP53 VP-related cancer predisposition syndrome [3]. Gliomas encountered in a context of LFS typically include mainly pHGG-MYCN, astrocytomas, IDH-mutant and diffuse hemispheric gliomas, H3 G34-mutant [1, 4, 5]. However, a study has evidenced that a subset do not harbor IDH1/2, histones’ genes or MYCN alterations [5], suggesting that other subtypes of pHGG may be encountered in this genetic tumor syndrome. Nevertheless, no DNA-methylation profiling has been performed for these “IDH-/H3-/MYCN-wildtype” gliomas [5].

Fig. 3

T-distributed stochastic neighbor embedding (t-SNE) analysis of the DNA methylation profiles of the investigated tumors alongside selected reference samples. Reference DNA methylation classes: diffuse midline glioma H3 K27M mutant (DMG, H3 K27); diffuse high-grade glioma, H3.3 G34 mutant (DHG, H3 G34); pediatric glioblastoma, IDH wildtype, subclass MYCN (GB, pedMYCN); pediatric glioblastoma, IDH wildtype, subclass RTK1a (GB, pedRTK1a); pediatric glioblastoma, IDH wildtype, subclass RTK1b (GB, pedRTK1b); pediatric glioblastoma, IDH wildtype, subclass RTK1c (GB, pedRTK1c); pediatric glioblastoma, IDH wildtype, subclass RTK2a (GB, pedRTK2a); pediatric glioblastoma, IDH wildtype, subclass RTK2b (GB, pedRTK2b), high-grade astrocytoma with piloid features (HGAP); diffuse paediatric-type high grade glioma, H3 wildtype and IDH wild type, Subtype A (HGG, pedA); diffuse paediatric-type high grade glioma, H3 wildtype and IDH wild type, Subtype B (HGG, pedB); infant-type hemispheric glioma (IHG); pilocytic astrocytoma, hemsipheric (PA, CORT);.pilocytic astrocytoma, infratentorial (PA, INF); pilocytic astrocytoma, midline (PA, MID); pleomorphic xanthoastrocytoma (PXA)

To conclude, we report for the first time two pHGG classified as “pHGG-MYCN” using DNA-methylation profiling, which as of today constitutes the sole method of confirmation for this diagnosis, that present recurrent atypical features. Their histopathological and molecular differences distinguish them from “classic” forms of pHGG-MYCN and their distinction by t-SNE analysis argue to separate them from pHGG-MYCN. This study most likely illustrates that the boundaries of the pHGG-MYCN methylation class are still being determined and that the landscape of pHGG occurring in the context of LFS include potential novel groups of gliomas.