Posterior fossa ependymoma H3 K27-mutant: an integrated radiological and histomolecular tumor analysis

Abstract

Posterior fossa group A ependymomas (EPN_PFA) are characterized by a loss of H3 K27 trimethylation due to either EZHIP overexpression or H3 p.K27M mutation, similar to H3 K27-altered diffuse midline gliomas (DMG), but in reverse proportions. Very little data is available in the literature concerning H3 K27M-mutant EPN_PFA. Here, we retrospectively studied a series of nine pediatric tumors initially diagnosed as H3 K27M-mutant EPN_PFA to compare them to EZHIP-overexpressing EPN_PFA in terms of radiology, follow-up, histopathology, and molecular biology (including DNA-methylation profiling). Seven tumors clustered within EPN_PFA by DNA-methylation analysis and t-distributed stochastic neighbor embedding. Among the two remaining cases, one was reclassified as a DMG and the last was unclassified. H3 K27M-mutant EPN_PFA cases were significantly older than their counterparts with an EZHIP overexpression. Radiological and histopathological central review of our seven H3 K27M-mutant EPN_PFA cases found them to be similar to their counterparts with an EZHIP overexpression. Sequencing analyses revealed HIST1H3B (n = 2), HIST1H3C (n = 2), H3F3A (n = 1), and HIST1H3D (n = 1) K27M mutations (no sequencing analysis available for the last case which was immunopositive for H3K27M). Consequently, HIST1H3C/D mutations are more frequently observed in EPN_PFA than in classic pontine DMG, H3K27-mutant. Overall survival and event-free survival of EZHIP-overexpressing and H3 K27M-mutant EPN_PFA were similar. After surgery and radiation therapy, 5/7 patients were alive at the end of the follow-up. In summary, the diagnosis of EPN_PFA must include tumor location, growth pattern, Olig2 expression, and DNA-methylation profiling before it can be differentiated from DMG, H3 K27-altered.

Keywords: DNA-methylation; Ependymoma; Histones; Posterior fossa.

Fig. 1

Cohort characteristics. Based on H3K27me3 immunohistochemistry, 116 cases were classified as EPN_PFA showing a loss of immunoexpression, and 31 were EPN_PFB with a conserved H3K27me3 expression. From the 116 EPN_PFA, 108 were immunopositive for EZHIP and eight cases were immunopositive for the H3K27M mutation. From the national French Neuropathological Reference network (RENOCLIP-LOC) database, we found one additional pediatric case of EPN_PFA with H3K27M immunopositivity. Scale bars represent 50 µm

Fig. 2

t-Distributed Stochastic Neighbor Embedding plot (t-SNE) analysis of our cases compared with reference samples from the Heidelberg cohort. DMG_EGFR: Diffuse midline glioma, EGFR-altered; DMG_EZHIP: Diffuse midline glioma, EZHIP-overexpressing; DMG_K27: Diffuse midline glioma, H3K27M-mutant; EPN_PFA: Ependymoma of the posterior fossa, subtype A; EPN_PFB: Ependymoma of the posterior fossa, subtype B; EPN_MPE: Myxopapillary ependymoma; EPN_SPINE: Ependymoma of the spine; EPN_YAP: Ependymoma, YAP1-fusion positive; EPN_ZFTA: Ependymoma, ZFTA-fusion positive; GG: Ganglioglioma; SUBEPN_PF: Subependymoma of the posterior fossa; SUBEPN_SPINE: Subependymoma of the spine; SUBEPN_ST: Supratentorial subependymoma

Fig. 3

Radiological and histopathological features of case #8. Axial T2-weighted image showing a circumscribed hemorrhagic lesion of the right cerebellopontine angle (a), with a low signal on T1-weighted images (b), and a peripheral enhancement on T1-weighted image with gadolinium injection (c). Ependymal features with pseudorosettes (d HPS, magnification 400$\times$), associated with no immunoreactivity for Olig2 (e magnification 400$\times$). Neurofilament immunostaining confirming the solid pattern (f magnification 400$\times$). Immunoexpression of H3K27M in all tumor cells (g magnification 400$\times$). Scale bars represent 50 µm. HPS: Hematoxylin Phloxin Saffron

Fig. 4

Radiological and histopathological features of case #6. Sagittal T2-weighted image (a) and FLAIR image (b) showing an infiltrative lesion of the pons with a central liquid part consistent with necrosis, with a peripheral enhancement of the necrosis on T1-weighted image with gadolinium injection (c), and intermediate signal on diffusion-weighted image (d) with partially restricted diffusion on apparent diffusion coefficient map (e). The biopsy highlighted two different histopathological components (f HPS, magnification × 50). The glial diffuse component (g HPS, magnification 400$\times$), associated with an ependymal component with rosettes (h HPS, magnification 400$\times$). Neurofilament immunostaining confirming the infiltrative pattern in the glial component (i magnification 400$\times$) and a more circumscribed pattern in the ependymal component (j magnification 400$\times$). Diffuse immunopositivity for Olig2 in the glial component (k magnification × 400), and partial expression of Olig2 in the ependymal component (l magnification × 400). Diffuse immunoexpression of H3K27M in both tumoral components (m–n magnification 400$\times$). Scale bars represent 1 mm (f) and 50 µm (g–n). HPS: Hematoxylin Phloxin Saffron

Fig. 5

Radiological and histopathological features of case #9. Axial T2-weighted and FLAIR image showing a circumscribed lesion of the medulla oblongata (a,b), without enhancement on sagittal T1-weighted image with gadolinium injection (c before injection, d after injection), and low signal on diffusion weighted image (e) with increased diffusion on apparent diffusion coefficient map (f). A paucicellular tumor with subependymal features (g HPS, magnification 400$\times$). No immunoreactivity for Olig2 except in residual glial cells (h HPS, magnification 400$\times$). Neurofilament immunostaining showing an infiltrative pattern (i magnification 400$\times$). Diffuse immunoexpression of H3K27M in tumor cells (j magnification 400$\times$). Scale bars represent 50 µm. HPS: Hematoxylin Phloxin Saffron

Fig. 6

Age distribution and outcome of PFA_EPN, EZHIP-overexpressing and H3K27M-mutant of our cohort (n = 7) and cases from the literature with available methylation data (n = 2, cases #10 and #11). Median age for EPN_PFA, H3K27M-mutant of 6 years (1.4–12) versus 2.4 years for EPN_PFA, EZHIP-overexpressing (0.1–22.5). Statistical significance was calculated with Mann–Whitney test (median, *p value 0.0168, n = 98) (a). Kaplan–Meier survival estimates of EFS and OS: no significant difference between EPN_PFA, EZHIP-overexpressing (red, n = 89, median EFS 28 months, median OS 123 months) and H3K27M-mutant (orange, n = 9, median EFS 79 months, median OS not reached), log rank test, p value 0.67 and 0.41 respectively (b, c). DMG: diffuse midline gliomas; EPN: ependymomas; EFS: event-free survival; OS: overall survival; PF: posterior fossa