# CNS neuroblastoma, FOXR2-activated and its mimics: a relevant panel approach for work-up and accurate diagnosis of this rare neoplasm

Arnault Tauziède-Espariat, Dominique Figarella-Branger, Alice Métais, Emmanuelle Uro-Coste, Claude-Alain Maurage, Benoît Lhermitte, Aude Aline-Fardin, Lauren Hasty, Alexandre Vasiljevic, Dan Chiforeanu, et al.

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Arnault Tauziède-Espariat, Dominique Figarella-Branger, Alice Métais, Emmanuelle Uro-Coste, Claude-Alain Maurage, et al. CNS neuroblastoma, FOXR2-activated and its mimics: a relevant panel approach for work-up and accurate diagnosis of this rare neoplasm. Acta Neuropathologica Communications, 2023, 11 (1), pp.43. 10.1186/s40478-023-01536-7. hal-04086974

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# CNS neuroblastoma, *FOXR2*-activated and its mimics: a relevant panel approach for work-up and accurate diagnosis of this rare neoplasm

Arnault Tauziède-Espariat<fnref n="1" />,<fnref n="2" />*, Dominique Figarella-Branger<fnref n="3" />, Alice Métais<fnref n="1" />,<fnref n="2" />, Emmanuelle Uro-Coste<fnref n="4" />,<fnref n="5" />,<fnref n="6" />, Claude-Alain Maurage<fnref n="7" />, Benoît Lhermitte<fnref n="8" />, Aude Aline-Fardin<fnref n="9" />, Lauren Hasty<fnref n="1" />, Alexandre Vasiljevic<fnref n="10" />, Dan Chiforeanu<fnref n="11" />, Guillaume Chotard<fnref n="12" />, Homa Adle-Biassette<fnref n="13" />, Alexandra Meurgey<fnref n="14" />, Raphaël Saffroy<fnref n="15" />, Delphine Guillemot<fnref n="16" />, Gaëlle Pierron<fnref n="17" />, Philipp Sievers<fnref n="18" />,<fnref n="19" /> and Pascale Varlet<fnref n="1" />,<fnref n="2" /> on behalf of the RENOCLIP-LOC

The “neuroblastoma of the central nervous system” (CNS) has been referenced in the World Health Organization’s (WHO) classification of brain tumors since 1993 [2]. However, in recent years, thanks to molecular analyses, *forkhead box R2 (FOXR2)* alterations have become associated with this tumor type and as a result, the WHO classification renamed the neoplasm “CNS neuroblastoma (NB), *FOXR2*-activated” [4, 5]. These alterations are comprised of complex structural rearrangements

for which routine testing is not easily implemented [4]. Consequently, the diagnosis is confirmed using advanced molecular techniques (Next-generation sequencing or DNA-methylation profiling) [3]. In this context, routine biomarkers for neuropathologists are needed to facilitate a NB-FOXR2 diagnosis. The aims of this report are to present the French national RENOCLIP-LOC network’s experience diagnosing NB-FOXR2 and to propose an alternative diagnostic approach.

Arnault Tauziède-Espariat

a.tauziede-espariat@ghu-paris.fr

<footnote>*Correspondence:</footnote>

<footnote n="1">¹ Department of Neuropathology, GHU Paris-Psychiatrie et Neurosciences, Sainte-Anne Hospital, 1, rue Cabanis, 75014 Paris, France</footnote>

<footnote n="2">² INSERM, UMR 1266, IMA-Brain, Institut de Psychiatrie et Neurosciences de Paris, Paris, France</footnote>

<footnote n="3">³ Department of Anatomopathology and Neuropathology, CNRS, INP, Inst Neurophysiopathol, and APHM, La Timone Hospital, Aix-Marseille Univ, 13385 Marseille, France</footnote>

<footnote n="4">⁴ Department of Pathology, Toulouse University Hospital, Toulouse, France</footnote>

<footnote n="5">⁵ Cancer Research Center of Toulouse (CRCT), INSERM U1037, Toulouse, France</footnote>

<footnote n="6">⁶ Université Paul Sabatier, Toulouse III, Toulouse, France</footnote>

<footnote n="7">⁷ Institute of Pathology, Centre de Biologie Pathologie, Lille University Hospital, 59000 Lille, France</footnote>

<footnote n="8">⁸ Department of Pathology, Strasbourg Hospital, Strasbourg, France</footnote>

<footnote n="9">⁹ Department of Pathology, Fort-de-France Hospital, Fort-de-France, France</footnote>

<footnote n="10">¹⁰ Department of Pathology and Neuropathology, GHE, Hospices Civils de Lyon, Lyon, France</footnote>

<footnote n="11">¹¹ Department of Pathology, Ponchaillou Hospital, Rennes, France</footnote>

<footnote n="12">¹² Department of Pathology, Pellegrin Hospital, Bordeaux, France</footnote>

<footnote n="13">¹³ Department of Pathology, Lariboisière Hospital, APHP, 75475 Paris, France</footnote>

<footnote n="14">¹⁴ Department of Biopathology, Léon Bérard Cancer Center, Lyon, France</footnote>

<footnote n="15">¹⁵ Department of Biochemistry and Oncogenetics, Paul Brousse Hospital, 94804 Villejuif, France</footnote>

<footnote n="16">¹⁶ Curie Institute Research Center, INSERM U830, Paris-Sciences-Lettres, Paris, France</footnote>

<footnote n="17">¹⁷ Laboratory of Somatic Genetics, Curie Institute Hospital, Paris, France</footnote>

<footnote n="18">¹⁸ Department of Neuropathology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany</footnote>

<footnote n="19">¹⁹ Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center DKFZ, Heidelberg, Germany</footnote>
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24 tumors histologically suspect of NB-FOXR2

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DNA-methylation profiling and genetic analyses

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Evaluation of sensitivity/specificity of different technical panels to
improve the hypothesis of NB-FOXR2 before molecular confirmation

<figure><img src="image_8.png" /><figcaption>**Fig. 1** Flowchart of the study. Different technical panels used: panel 1 (Olig2 and synaptophysin immunopositivities, and vimentin negativity); panel 2 (Olig2, synaptophysin and SOX10 immunopositivities); panel 3 (Olig2, synaptophysin and SOX10 immunopositivities, and vimentin negativity); panel 4 (Olig2, synaptophysin and SOX10 immunopositivities, and vimentin negativity, and 1q gain). The sensitivity and specificity for each panel were: 100 and 71% (panel 1); 100 and 50% (panel 2); 100 and 86% (panel 3); and 100 and 100% (panel 4)</figcaption></figure>
Our study included a total of 24 cases initially sus-
pected to be NB-FOXR2, based on histopathology alone,
and reviewed by the French national network. All tumors
were pediatric (except one in a young adult) and located
in the supratentorial region. We performed an immu-
nohistochemical (IHC) panel (including Olig2, syn-
aptophysin, vimentin, and SOX10), fluorescent in situ
(FISH) analysis of chromosome 1, and DNA-methyla-
tion profiling for all tumors. Each maintained BRG1 and
INI1 expression and showed no immunoexpression for
Lin28A.

DNA-methylation profiling using the CNS tumor Clas-
sifier supported the NB-FOXR2 diagnosis in 9/24 cases
(37%), with a calibrated score (> 0.9) for nine of the sam-
ples. The other diagnoses having scores > 0.9 included:
high-grade gliomas, *IDH*- and H3-wildtype (n = 2); gli-
oneuronal tumors not otherwise specified, subtype A
(n = 2); a diffuse glioneuronal tumor with oligodendro-
glioma-like features and nuclear clusters (DGONC);
an anaplastic neuroepithelial tumor with condensed
nuclei (ANTCON) [1]; a CNS embryonal tumor with
*BRD4::LEUITX* fusion; a CNS tumor *BCOR*-altered; and
a neuroepithelial tumor, *PLAGL1*-fused. An orthogonal
validation, using different molecular techniques, con-
firmed these diagnoses (cf. Additional file 1: Table 1). The
six remaining cases presented a low calibrated score for
different methylation classes. All cases were included in
a t-distributed stochastic neighbor embedding analysis to
better characterize tumors with low scores. Finally, 10/24
cases were confirmed as NB-FOXR2.

From these results, we tested the sensitivity/specific-
ity of different technical approaches using IHC, FISH,
or combinations of these different techniques. The best
diagnostic panel included an IHC panel (showing Olig2,
synaptophysin, and SOX10 immunopositivities without
vimentin immunoexpression), and FISH analysis (pres-
ence of 1q gain) with a very high sensitivity/specificity
(Fig. 1 and Additional file 2: Figure 1).

In accordance with the WHO Classification, our work
evidenced that a diagnosis of NB-FOXR2 based on mor-
phology alone is currently not possible. Indeed, many dif-
ferential heterogeneous diagnostic pitfalls exist, including
glial, glioneuronal, embryonal and new emerging tumor
types. Many of these have been very recently described
in the literature and are not yet listed in the latest WHO
classification, highlighting the velocity with which CNS
tumors are being deciphered in recent years thanks to
DNA-methylation profiling, and thus, their increasing
complexity. The essential diagnostic criteria of the WHO
classification defines NB-FOXR2 as an embryonal tumor
having foci of neuroblastic or neuronal differentiation
and a *FOXR2* activation or a DNA-methylation profile
aligned with this diagnosis. These genetic or epigenetic

diagnostic techniques are not routinely used or available
in all countries worldwide. However, IHC and FISH anal-
yses are well established, can be automated, are relatively
easy to standardize, are less expensive, and are widely
available in most pathology laboratories worldwide. In
this context, the national French histopathological net-
work's experience showed that IHC (confirming the
necessity of a co-expression of Olig2 and synaptophysin,
and the interest of SOX10 immunopositivity) and FISH
analyses (confirming the necessity of chromosome 1q
gain) may improve the diagnosis of NB-FOXR2, and help
eliminate potential mimickers [3, 4].

In conclusion, the diagnosis of NB-FOXR2 may be
ameliorated by using an algorithmic approach that
includes several criteria based on histopathology, IHC
and FISH analysis. This diagnostic panel can be tested in
further series in order to be validated as a working for-
mula that facilitates the diagnostic approach and reaches
an accurate diagnosis in a resource-limited environment.

**Supplementary Information**

The online version contains supplementary material available at https://doi.org/10.1186/s40478-023-01536-7.

**Additional file 1.** Histopathological and molecular features of differential diagnoses of CNS neuroblastoma, *FOXR2*-activated.

**Additional file 2: Figure 1.** Immunohistochemical and FISH analyses results of the differential diagnoses.

Author contributions

ATE, and PV conducted the neuropathological examinations; ATE, PS, EUC, RS, GP, DG, and AM conducted the molecular analyses; ATE, and PV drafted the manuscript. All authors read and approved the final manuscript.

Funding

No funding.

Declarations

Ethics approval and consent to participate

This study was approved by the GHU Paris Psychiatry and Neurosciences, Sainte-Anne Hospital's local ethic committee.

Competing interests

The authors declare that they have no conflict of interest directly related to the topic of this article.

Received: 27 January 2023 Accepted: 25 February 2023

Published online: 14 March 2023

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