Enrichment of oligodendrocyte precursor phenotypes in subsets of low-grade glioneuronal tumours

Immunohistochemical staining

Representative formalin-fixed paraffin-embedded (FFPE) sections were deparaffinized and stained with haematoxylin and eosin (H&E) and immunohistochemistry (IHC) according to the manufacturer’s instructions of the respective reagents. The following antibodies were used: (i) Astro makers: GFAP (Dako, EP13, 1:150) and S-100 (Dako, 15E2E2 + 4C4.9, 1:4000); (ii) neuronal makers: neuronal nuclei antigen (NeuN) (Zeta, A60, 1:100), neurofilament (NF) (Covance Research Products, 2F11, 1:200), microtubule-associated protein-2 (MAP-2) (Abcam, AP18, 1:100), synaptophysin (SYN) (Neomarkers, EP158, 1:50) and postsynaptic density protein-95 (PSD95, Abcam, EPR23124-118, 1:100); (iii) pan-lineage or stage-specific markers of oligodendrocyte lineage:¹⁹ OLIG2 (Dako, EP112, 1:100), SOX10 (Abcam, EP268, 1:200), NG2 (Cell Signaling Technology, E3B3G, 1:300), PDGFRA (Abcam, polyclonal, 1:100), Nogo-A (Sigma, 1:1000), ENPP6 (Atlas antibodies, polyclonal, 1:50) and MBP (Epitomics, EP207, 1:200); (iv) diagnostic markers: (1) for diffuse glioma: IDH1-R132H (Dianova, H09, 1:60), ATRX (Sigma, HPA001906, 1:800), TP53 (Santa Cruz Biotechnology, BP53.12, 1:200), H3K27M (EMD Millipore, ABE419, 1:3000) and H3K27me3 (Cell Signaling Technology, C36B11, 1:300), (2) CD34 (Dako, QBEnd/10, 1:100) mainly for differential diagnosis of ganglioglioma, (3) Ki-67 (Origene, UMAB107,1:100) for evaluating proliferative activity of tumour cells and (4) anti-B-Raf proto-oncogene, serine/threonine kinase (BRAF) V600E antibody for detecting the BRAF V600E protein expression (VE1, IHC detection kit, Ventana Medical Systems);²² (v) reporter of RAS/MAPK pathway activation: p-Erk1/2 (Cell Signaling Technology, D13.14.4E, 1:400); and (vi) anti-cleaved caspase-3 (Cell Signaling, Asp175, 1:400) for detection of apoptotic cells. The staining was performed using Leica Bond automated staining processors. The anti-BRAF V600E (VE1) immunostaining was automated and conducted on a Benchmark Ultra stainer from Ventana/Roche, utilizing the proprietary antigen retrieval solution provided by the manufacturer.²² Ten diffuse hemispheric gliomas (DHGs), H3 G34-mutant (as known as G34R/V mutant high-grade gliomas), in adolescents and young adults were used as a positive control for PDGFRA staining and negative controls for the other oligodendrocyte lineage markers because these tumours resemble interneuron progenitors and lack oligodendroglial programmes.²³ The white matter adjacent to these tumour, normal brain tissues from traumatic brain injuries and oligodendroglioma (Grades 2 and 3) were used as positive controls for oligodendrocyte lineage markers, including OLIG2, Nogo-A, EPNN6 and MBP. The Luxol fast blue (LFB) staining was performed for detecting myelin sheath structure. The stainings of immunohistochemical diagnostic markers (except Ki-67) were graded according to the percentages of positively stained tumour cells and whether the positive staining was in the nucleus, cytoplasm or cell membrane (0, no staining; 1+, <5%; 2+, 5–25%; 3+, 26–50%; 4+, 51–100%). These ranges are frequently used in reporting the staining results of diagnostic markers.^24,25

Morphometry and statistical analyses of cell numbers

Using the ImageJ programme, the numbers of PDGFRA-, NG2-, OLIG2-, SOX10-, Nogo-A-, NeuN- and Caspase3-positive cells in IHC staining were determined in at least four microscopic fields (0.119 mm²/field) in each sample. The non-tumour regions from the same slide were selected as the control. In the text and table, the mean numbers of cells/mm² ± standard deviation (SD) are presented. The R version 4.0.5 was used for diagram presentation (R package ggplot2 v.3.4.2) and statistical analysis (predefined functions in R). P-values < 0.05, P < 0.01 and 0.001 were indicated with *, ** and ***, respectively.

Targeted exome sequencing

Targeted exome sequencing was performed in 10 DNETs, 3 MGNTs (MGNT 1–3) and 5 RGNTs as previously described.²⁶ Areas of the FFPE specimens enriched in tumour cells were marked by HE staining and isolated for DNA preparation using QIAamp DNA Mini Kit (Qiagen). The quality and concentration of DNA preparations were examined using spectrophotometer (Biophotometer Eppendorf, Germany). Hybridization-based target enrichment was carried out with GeneseeqOne™ pan-cancer gene panel (425-cancer-relevant genes, Geneseeq Technology Inc.) (Supplementary Table 1) and xGen Lockdown Hybridization and Wash Reagents Kit (Integrated DNA Technologies). The libraries were sequenced on the HiSeq 4000 platform (Illumina, San Diego, CA) with 2 × 150 bp pair-end reads. Sequencing data were demultiplexed by bcl2fastq (v2.19) and analysed using Trimmomatic.²⁷ The genome analysis toolkit (GATK)²⁸ was used to perform local realignments around indels and base quality reassurance. SNPs and indels were called using VarScan2²⁹ and HaplotypeCaller UnifiedGenotyper in GATK, with mutant allele frequency (MAF) cut-off of 0.5% for tissue samples and a minimum of three unique mutant reads. Common variants were removed using the reference data from dbSNP and the 1000 Genome project.

For four MGNTs and five RGNTs, PDGFRA mutations were analysed by direct sequencing of PCR products from tumour DNA using the primers reported by Chiang et al.¹¹:

Forward primer: 5′-E8-F GTATGATCCAGACCTTCAGTG-3′

Reverse primer: 5′-E8-R CATCTACAGAGCTAGCATTATCT-3′

Imaging features

Cortical DNETs: preoperative MRI showed irregular or round flaky abnormal signal within thickening cortex, mainly involving temporal lobe and frontal lobe. The lesions were hypointense on T₁-weighted images (T1WI) and hyperintense on T₂-weighted images (T2WI), without obvious contrast enhancement (Fig. 1A). The surrounding tumoural oedema was not obvious.

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Figure 1

Preoperative images of representative cases in this study. (A) Preoperative MRI image of DNET-5 showing a hyperintensity on T₂-weighted images of thickening cortex within temporal lobe without surrounding tumoural oedema. (B) Preoperative MRI image of MGNT-2 showing a well-circumscribed, FLAIR isointensity or hyperintense mass lesion in the interventricular foramen. (C) Preoperative MRI image of RGNT-3 showing a lump mass located in the sellar region with abnormal enhancement. (D) Multiple circular signals in the bilateral basal ganglia and medial temporal lobes were observed in RGNT-4. Arrows indicate the lesion.

MGNTs: preoperative MRI showed that lesions were in the interventricular foramen with clear circular boundary. All lesions showed hypointensity on T1WI, hyperintensity on T2WI and isointensity/hyperintensity on fluid-attenuated inversion recovery (FLAIR) without contrast enhancement (Fig. 1B). Obstructive hydrocephalus was present in all four cases. Based on the imaging data, preoperative diagnoses included subependymoma, neuroepithelial cyst and endodermal cyst.

RGNTs: preoperative MRI showed that lesions were located outside the fourth ventricle, including lateral ventricle, sellar region, basal ganglia and midbrain tectal region. The lesions were hypointense on T1WI and hyperintense on T2WI, Cases 1 and 3 showed enhancement (Fig. 1C and D). Ventricular dissemination was not detected at the time of diagnosis for all MGNT and RGNT cases in this study.

Morphological features and expression of glial and diagnostic markers in LGNTs

DNETs and MGNTs shared highly similar histological characteristics (Fig. 2A and B and Supplementary Fig. 1). These tumours contained OLCs with small monotonous round to oval nuclei, scant to moderate eosinophilic or transparent cytoplasm. All samples contained a prominent mucoid matrix. Mitotic figures were rare or not observed. Neither necrosis nor glomeruloid microvascular proliferation was detected. However, scattered proliferative Astro were identified. Calcifications, rosenthal fibres and eosinophilic granular bodies were not observed. Except for DNET-2, the multinodular intracortical growth pattern was observed in other nine DNET samples examined. However, multinodular architecture was not observed in MGNTs. In two MGNT cases, we observed tumour infiltration into the adjacent white matter in the form of pushing invasion (Fig. 2C). All five RGNT cases showed common morphological features with two distinct regions characterized by a rosette-forming neurocytic component (Fig. 2D and Supplementary Fig. 1) and an astrocytic component (Fig. 2E). The rosette-forming neurocytic component contained neurocytic rosettes and perivascular pseudorosettes, which were embedded in a mucoid matrix in RGNT-1 to RGNT-4; no obvious mucoid matrix was observed in RGNT-5. The astrocytic component was similar to that in pilocytic astrocytoma. Some rosenthal fibres and eosinophilic granular bodies were observed in the astrocytic regions in RGNT-1 to RGNT-3.

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Figure 2

Morphological features of DNET, MGNT and RGNT samples examined. HE stainings of representative DNET case (A) and MGNT case (B). Tumours contained OLCs with small monotonous round to oval nuclei. Proliferative cells were rare. Scattered neurons were found in the mucinous matrix (black arrows) (×400). (C) Tumour tissue was infiltrating into the white matter in the form of push invasion in MGNT-2 (black arrow) (×200). Typical RGNT (RGNT-2) contained a rosette-forming neurocytic component (black arrow indicating neurocytic rosettes and perivascular pseudorosettes) (D) and an astrocytic component (E) (×200). (F) MGNT-1 contained regions with scattered and clustered neurons, which is similar to normal white matter (×200).

The DNET, MGNT and RGNT samples examined presented similar immunohistochemical features (Supplementary Table 2). Staining of Astro marker S-100 was observed in all cases, while GFAP expression was positive only in the astrocytic component of RGNTs and focally positive in three DNETs (DNET-4, -5 and -7). MAP-2 staining was diffusely positive in OLCs in all samples, but its expression was weaker in DNET samples than in MGNT and RGNT samples. Granular SYN staining was observed throughout the background neuropil in all three types of LGNT and especially at the centre of neurocytic rosettes and perivascular pseudorosettes in the RGNT samples (Supplementary Fig. 2). Stainings for IDH1-R132H, BRAF V600E, P53 and H3K27M were negative in all cases. None of cases showed loss of H3K27me3 expression. Except for diffuse staining in RGNT-5 and focal positive staining in DNET-5 and DNET-8, CD34 immunoreactivity was not observed in all other cases. The Ki-67 labelling index was low, ranging from 1% to 4%, and predominantly in the astrocytic region in RGNT samples, indicating most of the LGNTs were not proliferating.

These observations show that our cohort is consistent with previous findings on the overlapping morphological and immunohistochemical features between the LGNT subtypes.^1,2

Mutually exclusive genomic alterations and elevated RAS/MAPK activity in LGNTs examined

To characterize genomic alterations in our cohort, we performed targeted exome sequencing for 425 cancer-related genes and Sanger sequencing for validation of PDGFRA mutation. Alterations in the drivers commonly affected in the other brain tumours, including IDH1/2, TP53, ATRX, TERT, EGFR and QKI, were not detected.¹ Across the LGNT samples, mutual exclusivity was observed between the recurrent genomic alterations in BRAF, FGFR1, FGFR3 and PDGFRA (Fig. 3A). In the 10 cortical DNET examined, 7 harboured different mutations in FGFR1 (6 in exon 18 and 1 in exon 14, with MAF ranging 7.3–31.0%, mean 19.6%), 1 harboured FGFR3 structure variation (SV) (exon17∼FAM184B:exon2, MAF at 34.5%), and another 1 harboured PDGFRA p.K385L mutation with MAF at 21.7%. The remaining one DNET sample harboured BRCA2 mutation (c.10255dup, p.*3419Lfs*19, MAF at 43.17%), with no alterations detected along the RAS/MAPK pathway. All four MGNT cases harboured PDGFRA mutation. Among them, MGNT-2, -3 and -4 carried the typical c.1153-1154AA>TT, p.K385L PDGFRA mutation^10,11,12 with MAF at 38.2% and 44.4% in MGNT-2 and MGNT-3, respectively. The MAF result of MGNT-4 was not available because this sample was only analysed using Sanger sequencing. MGNT-1 harboured an in-frame insertional mutation in PDGFRA (p.E362-I363insW) with MAF at 25.8%, a new variant not reported before in MGNTs. Three of the five RGNTs harboured FGFR1 kinase domain hotspot missense mutation, p.N546K in exon 12 with MAF at 27.3% in RGNT-1, p.N546D and p.K656Q with the respective MAF at 25.7% and 24.7% in exon 12 and exon 14 in RGNT-2 and p.K656E in exon 14 with MAF at 66.7% in RGNT-3.¹⁷ Among them, two harboured NF1 mutation and one harboured CDKN2B mutation. No genetic alteration along the RAS/MAPK pathway was detected in the RGNT-4. BRAF^V600E mutation was found in RGNT-5 with MAF at 23.4%. No copy number variations were detected in these cases.

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Figure 3

Mutually exclusive genomic alterations leading to elevated RAS/MAPK and low apoptotic activities in DNET, MGNT and RGNT tumours. (A) Recurrent genomic alterations in the RAS/MAPK pathway. In 19 LGNTs analysed, 17 LGNTs harboured 1 or 2 alterations along the RAS/MAPK pathway. Except for mutations in NF1, alterations in BRAF and receptor tyrosine kinases occurred in a mutually exclusive manner across the samples. AKT1, v-akt murine thymoma viral oncogene homologue 1. (B) Representative immunohistochemical images of p-Erk1/2 staining in DNET, MGNT and RGNT samples. Strong p-Erk1/2 staining was observed in all DNET and MGNT samples examined and in the neurocytic components of RGNTs (×400, scale bar: 50 μm). (C) Anti-cleaved caspase-3 staining in DNET, MGNT and RGNT samples (×400, scale bar: 50 μm).

To characterize the signalling effects of these genomic alterations, we performed staining for p-Erk1/2, a marker of RAS/MAPK pathway activity. Strong and diffuse nuclear staining of p-Erk1/2 was observed in all cases of DNET and MGNT tumours analysed. In the four RGNT-like tumours analysed (except RGNT-4 due to lack of material), neurocytic regions showed strong and diffuse p-Erk1/2 staining, though p-Erk1/2 staining was relatively weaker and less in astrocytic regions compared with neurocytic regions (Fig. 3B and Supplementary Fig. 3). Interestingly, strong p-Erk1/2 staining was also detected in two samples (DNET-6 and RGNT-4) with no detectable alterations in our panel.

ERK1/2 activation is broadly associated with anti-apoptotic function and thereby promotes cell survival.^34,35 To evaluate the apoptotic level of these tumours, we performed anti-cleaved caspase-3 staining. The proportion of tumour cells with cleaved caspase-3 was 3.30 ± 1.25% in DNET samples (n = 10), 3.00 ± 1.41% in MGNT samples (n = 4) and 3.20 ± 3.90% and 6.00 ± 4.18% in the neurocytic and astrocytic regions of RGNT samples (n = 4), respectively (Fig. 3C and Supplementary Fig. 4). These results indicate a low level of apoptotic death of tumour cells in these LGNT samples.

Together, the mutual exclusivity between the mutations along the RAS/MAPK pathway and their relatively high MAF reinforce the conclusion that the majority of the LGNTs are driven by a single genetic alteration, which stands a good chance to result in elevated RAS/MAPK pathway activity and enhanced cell survival.^2,36

Pervasive axon components in the DNET and MGNT samples examined

Scattered neurons were found in the mucinous matrix in all DNET and MGNT samples analysed by H&E staining (black arrows in Fig. 2A and B, Supplementary Table 2). Interestingly, the pattern of neuron scattering in several regions in MGNT-1 and MGNT-2 was similar to that in normal white matter (Fig. 2F). However, no distinct neurons were observed in the RGNT samples (Fig. 2D and E).

We performed IHC staining to confirm the distribution of neurons. NeuN staining can be used to indicate perikarya and proximal neuronal processes in nearly all neuronal populations.³⁷ MAP-2 staining indicates neuron cell body and dendritic branching,³⁸ NF staining indicates the axon structure,³⁹ and PSD95 is a key postsynaptic protein located preferentially in dendritic spines, as its staining concentrates in the cytoplasm and neurites of neurons.^40,41 In all DNET and MGNT samples analysed, sporadically dispersed neurons, positively stained with NeuN and MAP-2, were observed between the OLC-like cells (Fig. 4, Supplementary Figs. 5 and 6 and Supplementary Table 3). Compared with the DNET samples, less neuronal component was observed in the MGNT samples. However, except for RGNT-1 containing rarely dispersed NeuN-positive neurons, clear and distinct neuronal component was not observed in the other four RGNT samples analysed (Fig. 4). Axon structures were nearly homogeneously spread within the tumour tissues of DNET, but to a less extent or absent in the tumour tissues of MGNT and RGNT (Fig. 4 and Supplementary Fig. 5). Focal positive NF staining was observed in the neurocytic component of RGNT tumours (Fig. 4). In the traumatic brain samples and normal brain regions from tumour samples, PSD95 staining was confined to the cortex. PSD95 staining was weakly positive in DNET samples, further weaker in MGNT and almost absent in RGNT samples (Fig. 4 and Supplementary Fig. 5). SYN, an integral membrane glycoprotein and previously considered specific marker for neuronal lineage,^42,43 showed diffuse positive staining in the background neuropil in all samples examined (Supplementary Fig. 2). Thus, SYN staining is unlikely associated with neuronal component in LGNTs examined. Together, these findings led us to speculate that homogeneously spreading axons may constitute a microenvironment niche towards the OLC-like cells in the DNET and MGNT samples.

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Figure 4

Pervasive axon components in DNET and MGNT samples examined. Representative IHC images of NeuN, MAP-2, NF and PSD95 stainings in DNET, MGNT and RGNT samples are shown (×400). In DNET samples, NeuN- and MAP-2-stained neuronal cell bodies were scattered in the tumour mass, while NF- and PSD95-stained axons were widely dispersed. In MGNT samples, NeuN and MAP-2 staining showed the rare neurons, while NF- and PSD95-stained axons were less widely dispersed. However, NeuN, MAP-2, NF and PSD95 stainings were negative in almost all RGNT samples examined. Scale bar: 50 μm.

We thank our co-workers at Department of Pathology, Beijing Sanbo Brain Hospital, for their valuable support in the collection of this cohort and material preparation.