Diagnostic accuracy of a minimal immunohistochemical panel in at/rt molecular subtyping, correlated to dna-methylation profiling

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tauziède-Espariat et al. Acta Neuropathologica Communications (2023) 11:136 https://doi.org/10.1186/s40478-023-01630-w

in neurogenesis and NOTCH signaling, such as ASCL1, with additional sub-entities recently reported), TYR (overexpressing melanosomal marker genes such as TYR, MITF, and OTX2), and MYC (overexpressing MYC and HOXC clusters).These subgroups seem to be associated with distinct genetic and clinical features [1][2][3] but their significance in terms of prognosis is still unclear given the discrepant results reported in cohorts of patients treated with different therapies [1].Thus, the prognosis and theranostic significance of these molecular subgroups still needs to be performed, which may be facilitated in near future prospective trials, and by using routine subgroupspecific biomarkers.
Their overall distribution within subgroups is depicted by the specificity of t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis (Supplementary Material 1: Fig. S1).Altogether, 42 well-defined AT/RT were considered for further comparison with immunohistochemical subtyping.
This case series is the first to demonstrate the interest for the use of an antibody panel when sub-typing AT/RT.Our results confirm previously published outcomes showing that Tyrosinase expression is correlated to the AT/RT-TYR subgroup but may be encountered in a subset of SHH [5,6].OTX2 immunostaining is routinely used by neuropathologists in medulloblastoma subgrouping and represents a good candidate in the IHC panel for AT/RT subtyping [7].We evidenced for the first time that SOX11 may constitute a good surrogate for the SHH subgroup whereas the sensitivity/specificity of ASCL1 was lowly informative in our series as reported elsewhere [3].We also evidenced that a subset of AT/ RT SHH -defined by methylation profiling -do express proteins that would be expected to relate them to another group based on RNA-expression data.The consistency between transcript and protein expressions suggests that the discrepancies between expression and methylation may reveal an actual diversity within the SHH group predicted by the current version of the classifier, with some AT/RT-SHH harboring markers predicted as being expressed in the MYC subgroup.Remarkably, the concordance between epigenetic and IHC subtyping is perfect for infratentorial AT/RT SHH (10/10 with a concordant sub-typing), but much less so for supratentorial AT/RT SHH (5/11 for cases with available data).This difference may have a potential biological significance and needs to be further explored.
To conclude, an immunostaining panel that includes MYC, SOX11 and Tyrosinase should be included in future clinical trials to study their clinicopathologic relevance and potential for use as surrogate markers.

Fig. 1
Fig. 1 Comparison of molecular sub-typing by DNA-methylation analysis and immunohistochemistry. A: Alluvial diagram showing the assignment to subgroups by IHC subtyping using the panel 5 (left) and DNA-methylation profiling using the version v12.5 of the classifier.Cases with a calibrated score ≤ 0.9 were included in the "no match" group.B: Heatmap of DNA methylation beta-value using the top 5000 most variable probes.Samples are grouped according to the AT/RT subgroup predicted by our IHC method (top annotation).Unsupervised hierarchical clustering was applied to probes using Euclidean metric and Ward linkage.Sample subgroups identified using the DKFZ brain tumor classifier are plotted in the first layer of bottom annotation with the color indicating the AT/RT subgroup (red: TYR, blue: SHH and green: MYC).The height of the bar corresponds to the classification score (from 0 to 1).The three other layers of bottom annotation indicate respectively the expression level of SOX11, MYC and TYR genes in log2(TPM + 1) whenever RNA-seq data were available.C: A representative case with immunohistochemical findings for all subgroups (magnification x400).Black scale bars represent 50 μm Comparison of molecular sub-typing by DNA-methylation analysis and immunohistochemistry IHC: immunohistochemistry; NEC: Not Elsewhere Classified (the tumor was not classified in a subgroup)