Although relatively rare, low-grade gliomas and glioneuronal tumors account for approximately 30% of pediatric CNS tumors (Ostrom et al., 2022). Many tumor types and subtypes are included in the pLGG/GNTs group (pediatric low-grade gliomas/glioneuronal tumors), showing histological diversities that were recognized and described over years of microscopical and immunohistochemical studies. However, these tumors frequently show overlapping morphological features, and in some cases, the salient aspects may also be absent due to limited tumor sampling (Bale and Rosenblum, 2022). Over the past decades, the development of novel molecular techniques has led to revolutionary insights into the genetic drivers of these tumors (Ryall et al., 2020a). Among novel molecular diagnostics, methylome profiling is of particular interest in pLGG/GNTs (Qaddoumi et al., 2016). MAP kinase pathway alterations are almost universally present across pLGG/GNTs, even though they may occur in different forms. In fact, specific genetic alterations may have different meanings in the diagnostic algorithm of pLGG/GNTs. In this regard, fifth edition of the 2021 WHO Classification of Tumors of the Central Nervous System put together the current knowledge regarding the clinical, histopathological, immunohistochemical, and molecular features of these tumors, opening the doors for further precision in classification and treatment of these tumors (WHO Classification of Tumours Editorial Board, 2021). In the 2021 WHO, the pLGG/GNTs group has been subclassified into three different families: pediatric-type diffuse low-grade gliomas, circumscribed astrocytic gliomas, and glioneuronal and neuronal tumors (Table 1).

The term circumscribed refers to the growth pattern, which is opposed to the “diffuse” tumors. They include pilocytic astrocytoma, high-grade astrocytoma with piloid features (HGAP), pleomorphic xanthoastrocytoma (PXA), subependymal giant cell astrocytoma (SEGA), chordoid glioma, astroblastoma, and MN1-altered (Table 1).

Glioneuronal tumors are rare tumors composed of both neural and glial components in different proportions. All neuronal and glioneuronal tumors are immunoreactive for neuronal cell markers, such as synaptophysin or neuron-specific enolase (NSE). However, in addition to neuronal marker positivity, only the glioneuronal subgroup of tumors are immunoreactive for markers of glial differentiation, such as glial fibrillary acidic protein (GFAP) or oligodendrocyte transcription factor 2 (Olig2). The fifth CNS WHO improves the role of molecular diagnostics in CNS tumor classification, combining them with traditional histology and immunohistochemistry (Krauze, 2021). The fifth CNS WHO comprises 14 different subtypes, including 3 new entities: multinodular and vacuolating neuronal tumor (MNVNT) which was only mentioned in the 2016 classification, diffuse glioneuronal tumor with oligodendroglioma-like features and nuclear clusters (DGONC), which is a provisional type, and myxoid glioneuronal tumor (MGT) (WHO Classification of Tumours Editorial Board, 2021). Multinodular and vacuolating neuronal tumor (MVNT) is a CSN WHO grade 1 neoplasm, arising in the temporal/frontal lobe of adult patients, with few pediatric examples. Most patients present with seizures, headache, episodic confusion, and dizziness. Histologically, MVNT shows clear hypomyelinated nodules with a fibrillary matrix, prominent vacuolar alteration, and monomorphic neuronal cells, which is haphazardly distributed or aligned along capillary vessels. Neoplastic cells are positive for OLIG2, doublecortin, and non-phosphorylated NFP and may express synaptophysin, and MAP2. Ki-67 is frequently low (<1%). CD34 expression may be observed in ramified neural elements and GFAP-positive reactive astrocytes of the adjacent cortex (Barresi et al., 2022). Molecular analyses may reveal MAPK pathway-activating abnormalities. Less commonly, they are associated with BRAF mutations or FGFR2 fusions. Generally, they do not recur after gross total resection and remains stable also in the case of subtotal resection (Bale and Rosenblum, 2022). Diffuse glioneuronal tumor with oligodendroglioma-like features and nuclear clusters (DGONC) represents a new entity, which is included as a provisional type to the group of glioneuronal tumors. Its incidence is unknown but is presumed to be exceptionally rare. DGONC mainly occurs in pediatric age, with no sex predilection, and is localized in the cerebral hemispheres, mainly in the cortical or subcortical area of the temporal lobe. Histopathological features represent the hallmark of this tumor type, which are characterized by a diffuse growth of oligo-like and multinucleated cells, with nuclear clusters disposed as “pennies on a plate.” Tumor cells display diffuse positivity for OLIG-2 and synaptophysin, focal positivity for Neu-N and MAP2, and negativity for GFAP. The mitotic index is variable, and the Ki-67 labeling index can be up to 30%. The molecular hallmark of DGONC is monosomy of chromosome 14, which has been found in all the cases reported (Bale and Rosenblum, 2022). These tumors present a specific DNA methylation profile; however, if DNA methylation profiling is unavailable, morphological and immunohistochemical features may suggest the diagnosis. Due to the low number of cases with an available follow-up, DGONC was not assigned to a CNS WHO grade. To date, outcome data are only available for 26 patients, indicating a 5-year progression-free survival rate equal to 81% and 5-year overall survival rate equal to 89% (Barresi et al., 2022). Myxoid glioneuronal tumour (MGT) represents a newly introduced entity, which is located in septum pellucidum and deep periventricular white matter and classified as CNS WHO grade 1 due to its favorable outcome (Bale and Rosenblum, 2022). These are uncommon primary brain tumors with a peak incidence in the second and third decades of life. Histologically, MGT is circumscribed tumor composed of oligo-like cells immersed in a myxoid stroma. Some cases may show floating neurons and perivascular neuropil, which are similar to DNTs. Mitoses are very rare or absent, and the proliferative index is low. The oligo-like cells are immunoreactive for OLIG2, SOX10, GFAP, and MAP2 and negative for synaptophysin. Floating neurons, perivascular neuropil, and neurocytic rosettes are synaptophysin-positive. MGT shows a recurrent PDGFRA p.K385L/I dinucleotide somatic mutation, typically occurring in the absence of accompanying PDGFRA gene amplification. Outcome is good, even in the cases showing local recurrence or dissemination throughout the ventricular system (Barresi et al., 2022).

The tumor family defined as “pediatric-type diffuse high-grade gliomas” represents one of the main changes in the fifth WHO Classification of CNS Tumors (WHO Classification of Tumours Editorial Board, 2021; Gianno et al., 2022a). The term “pediatric-type” has been introduced to distinguish these tumors from the adult-type counterpart. In fact, compared with adult-type diffuse high-grade gliomas, these tumors present different clinico-pathological characteristics, with diverse prognostic and therapeutic implications (Annese et al., 2022; Tamma et al., 2023). The term “diffuse” reflects the growth pattern of these tumors, even if some cases of H3-wildtype and IDH-wildtype glioma and infant-type glioma may show a circumscribed growth pattern (Guerreiro Stucklin et al., 2019; Clarke et al., 2020). The term high-grade reflects both morphology and biologic behavior of these tumors, even though occasionally, especially in some cases of H3 K27-altered diffuse midline glioma, they may show a misleading low-grade morphology, not corresponding to their undoubtful aggressive behavior (Buczkowicz et al., 2014). Finally, even though defined as “gliomas” and surely belonging to this category, these tumors may sometimes show neuronal or embryonal differentiation (Andreiuolo et al., 2019a; Tauziède-Espariat et al., 2019a). Pediatric-type diffuse high-grade gliomas are subdivided into four different clinico-pathological entities: diffuse midline glioma, H3 K27-altered; diffuse hemispheric glioma, H3 G34-mutant; diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype; and infant-type hemispheric glioma. All of these tumors represent novel entities, which is first included in the 2021 CNS WHO. The only exception is diffuse midline glioma, H3 K27-altered, which has been revised and renamed with respect to 2016 CNS WHO (WHO Classification of Tumours Editorial Board, 2021).

Diffuse midline glioma (DMG) is an aggressive tumor occurring in the midline structures of the CNS. It is recognized as CNS WHO grade 4, due to its dismal prognosis, independently from its microscopical appearance. It is characterized by the loss of H3 K27me3 and is subdivided into three molecular subtypes: (1) DMG, H3 K27-mutant (encompassing K27M or K27I mutation in H3.3, H3.1, or H3.2); (2) DMG, with EZHIP overexpression (H3-wildtype); and 3) DMG, EGFR-mutant (including either EGFR mutation or amplification) (2021 CNS WHO). The H3 K27-mutant subtype is characterized by somatic heterozygous mutation in one of the genes encoding histone H3 variants (in order of frequency: H3.3, H3.1, and H3.2) (Castel et al., 2015, 2018). The DMG subtype with EZHIP overexpression represents the rarest subtype. The increased expression of EZHIP protein may be assessed both by immunohistochemistry or molecular analyses (Castel et al., 2020). In both subtypes, the final result is a loss of H3 K27 trimethylation (H3 K27me3) due to the inhibition of the methyltransferase activity of EZH2, which is the catalytic subunit of PRC2. In the first subtype, this inhibitory effect is a consequence of the H3 mutation (Bender et al., 2013; Lewis et al., 2013), whereas in the second subtype, this inhibitory effect is probably mediated by EZHIP overexpression, acting as an endogenous mimic of mutated H3 genes (Jain et al., 2019). EGFR-mutant subtype commonly shows small in-frame insertions/duplications within exon 20, which encodes the intracellular tyrosine kinase domain (TKD). Alternatively, it may also present missense mutations in other exons. In some cases, also EGFR gene amplification has been reported, which may co-occur with EGFR mutation or in absence of it, hence it might be more precisely defined as EGFR-altered subtype. Furthermore, in this specific subtype, EGFR abnormalities seem to always co-occur with H3 mutations or alternatively with EZHIP overexpression and are more frequently observable in DMG with a bithalamic or monothalamic presentation (Mondal et al., 2020; Sievers et al., 2021b). DMG is considered as a rare tumor, preferentially occurring in children. When located in the pons, DMG is also defined as “diffuse intrinsic pontine glioma” (DIPG) (Ostrom et al., 2022). Another common location is represented by the thalamus. In children, it usually presents with a bilateral thalamic involvement, while in adolescents and young adults, DMG tends to prefer a monothalamic or spinal location (Hoffman et al., 2016; Chai et al., 2020, 2023). On MRI, DMG appears as a solid mass, with diffuse infiltration of the surrounding structures. It is hypointense on T1, hyperintense on T2, and may show variable or no contrast enhancement on FLAIR (Poussaint et al., 2011; Giagnacovo et al., 2020). Histologically, DMG usually presents as a hypercellular diffusely infiltrative tumor, composed of neoplastic glial cells, showing a variegate spectrum of morphologies (Solomon et al., 2016). Mitosis, necrosis, and microvascular proliferation are frequently observable but are not essential for diagnosis. Immunohistochemically, they are usually positive for GFAP, OLIG2, and MAP2, with a variable expression. IDH1 is negative, while 50% of the cases show overexpression of p53, and 15% of cases present a loss of ATRX nuclear expression. In most of the cases, immunohistochemistry is sufficient to reach the diagnosis because it may assess the loss of H3 K27me3 expression (the cutoff is at least 80%) and H3 K27M or EZHIP positivity (Huang et al., 2018; Castel et al., 2020). In cases with loss of H3 K27me3, but not showing positivity for neither H3 K27M nor EZHIP, molecular analyses are mandatory in order to obtain a diagnosis. In rare cases, molecular analyses may reveal infrequent co-occurring alterations, such as IDH1/2 mutations, CDKN2A/B homozygous deletions, TERT promoter mutations, and MGMT promoter methylation (Mackay et al., 2017, 2018). It is noteworthy to mention the possible co-occurrence of H3 K27M and BRAF V600E mutations, which has been described not only in DMG (Gestrich et al., 2022) but also in glioneuronal tumors with a midline location (Nguyen et al., 2015; Pagès et al., 2017). Moreover, even though its significance still needs to be fully understood, the eventual presence of BRAF mutations or other MAPK pathway-related genetic alterations, such as FGFR1 mutations, may be related to a better outcome and might also be predictive of response to BRAF or MEK inhibitors (Schüller et al., 2021). Considering the wide morphological spectrum and the possible presence of co-occurring mutations, DMG presents numerous differential diagnoses. The appropriate application of 2021 CNS WHO criteria is a fundamental aid to correctly diagnose this tumor. In difficult cases, molecular analyses may be a useful tool to correctly diagnose DMGs and identify the specific subtype. Moreover, molecular results may also be useful for predicting prognostic differences and, hopefully in the very next future, might suggest possible target therapies. Unfortunately, DMG prognosis currently remains invariably poor, with a 2-year survival rate of <10% (Mackay et al., 2017). Up-to-date, the therapeutic approach is based on surgery, frequently limited by location, radiotherapy, and chemotherapy (Vallero et al., 2023). CAR-T cells have yielded very promising preclinical and clinical results but are not yet part of the standard of care (de Billy et al., 2022; Majzner et al., 2022; Vitanza et al., 2023). Different clinical trials obtained promising results for the treatment of DMG with CAR T cells using different targetable antigens, such as GD2 (Majzner et al., 2022; Mackall, 2023), which are recently found to be highly expressed in DMG/DIPG. For these reasons, pathologists and oncologists should always try to obtain extensive information on each and every DMG case, not only for a precise diagnosis of specific tumor subtype but also for possible prognostic and therapeutic implications, widening the understanding of this rare and still lethal neoplasm.

Diffuse hemispheric glioma, H3 G34-mutant is a diffusely infiltrative tumor, arising in the cerebral hemispheres and assigned to CNS WHO grade 4 (WHO Classification of Tumours Editorial Board, 2021). Although classified as a glioma, it may show morphological and immunohistochemical aspects of neuronal differentiation, which is also confirmed by transcriptomic and epigenomic studies, suggesting a possible neuronal origin (Chen C. C. L. et al., 2020). The characteristic pathogenic alteration is a missense mutation of the H3-3A gene, resulting in a substitution of glycin 34 with an arginine (G34R) or less frequently with a valine (G34V) in the H3.3 protein (Wu et al., 2012; Korshunov et al., 2016), and to a consequent inhibition of SETD2 methyltransferase (Jain et al., 2020) and KDM2A lysine demethylase activity (Cheng et al., 2014). Studies on H3 G34-mutant cells demonstrate that differential binding of H3 K36me3 induces a transcriptional reprogramming, recapitulating that of the developing forebrain, and causes prominent upregulation of the proto-oncogene MYCN (Bjerke et al., 2013). Co-occurring alterations are TP53 and ATRX mutations (present in 90–95% of cases), MGMT promoter methylation (Korshunov et al., 2016), and PDGFRA mutations (present in 50–70% of cases) (Chen C. C. L. et al., 2020). This tumor occurs at a median age of 15 years and is mainly located in the temporal or parietal lobe (Mackay et al., 2017). On MRI, it exhibits features comparable to those of other high-grade gliomas (Kurokawa et al., 2022b). Histologically, H3 G34-mutant diffuse hemispheric glioma typically presents as a highly cellular, infiltrative astrocytic tumor with brisk mitotic activity. Some cases may show an alternative pattern, which is morphologically similar to CNS embryonal tumors (Andreiuolo et al., 2019a). Immunohistochemically, GFAP expression may be variable. The embryonal-like variant usually expresses synaptophysin in a diffuse and strong manner. The negativity for OLIG2, together with ATRX loss of expression and p53 overexpression, is highly suggestive for this entity, though not specific. Hence, the demonstration of H3 G34 mutations is mandatory to diagnose this tumor, as specified in the 2021 CNS WHO diagnostic criteria. In the majority of the case, H3 G34R and H3 G34V mutations may be detected immunohistochemically by the two respective antibodies. However, false negative cases have been described (Gianno et al., 2021). Therefore, in cases with negative immunohistochemistry, but presenting the appropriate clinico-pathological context, molecular analyses are needed to demonstrate the presence of H3 G34 mutation or address possible differential diagnoses. Molecular investigations may be also useful to stratify the prognosis in a more precise manner. In fact, better prognosis is associated with the presence of MGMT promoter methylation and MUC gene mutations, while a worse prognosis is associated with PDGFRA mutations and the amplification of oncogenes, such as PDGFRA, EGFR, CDK4, and MDM2 (Korshunov et al., 2016; Hu et al., 2022; Vuong et al., 2022). Furthermore, the demonstration of alterations in PDGFRA and MUC genes might be potentially useful to open up new therapeutic options for these patients (Lucas et al., 2021; Hu et al., 2022).

Diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (pHGG H3/IDH WT) represents a heterogeneous group of tumors, which is characterized by histological high-grade features, absence of histone H3 and IDH mutations, and aggressive biological behavior (CNS WHO grade 4) (WHO Classification of Tumours Editorial Board, 2021). The pHGG RTK1 subtype is characterized by PDGFRA amplifications. The pHGG RTK2 presents EGFR amplifications and TERT promoter mutations. The pHGG MYCN subtype, as its name suggests, is enriched for MYCN amplifications (Korshunov et al., 2017). Hence, in order to obtain a diagnosis of pHGG H3/IDH WT, the identification of PDGFRA, EGFR, or MYCN alterations is essential. Alternatively, this diagnosis may be obtained by demonstrating the alignment of the tumor methylation profile with the pHGG RTK1, pHGG RTK2, or pHGG MYCN subtypes. Some cases of pHGG H3/IDH WT may develop the following therapeutic radiation, or in the context of Li Fraumeni syndrome or germline mismatch repair deficiency (i.e., CMMRD or Lynch syndrome), and usually belong to the pHGG RTK1 subtype (López et al., 2019). On MRI, pHGG H3/IDH WT is similar to other high-grade gliomas, which usually appears as contrast-enhancing tumors with mass effect. Differently from other subtypes, pHGG MYCN tumors may show more specific characteristics, being better circumscribed, with slight perilesional edema and homogeneous contrast enhancement (Tauziède-Espariat et al., 2019a, 2020). At microscopical examination, pHGG H3/IDH WT may show either a glioblastoma-like or a primitive, undifferentiated morphology that may also co-exist in the same tumor. Giant cells may be variably present and might raise the suspicion of mismatch repair deficiency, which may be also assessed by immunohistochemistry. Therefore, in cases of pediatric high-grade gliomas presenting severe pleomorphism and/or giant cells, pathologists should always ask for immunohistochemical evaluation of mismatch repair (MMR) proteins (MLH1, PMS2, MSH2, and MSH6), in order to exclude a constitutional mismatch repair deficiency (CMMRD) (Guerrini-Rousseau et al., 2019). Immunohistochemically, they show at least focal positivity for glial markers, such as GFAP and/or OLIG2, even though MYCN subtype may also be completely negative for glial markers, expressing neuronal markers. As a defining feature, these tumors are always negative for IDH1 (R132H) and H3K27M antibodies and show a preserved nuclear expression of H3K27me3. The number of possible differential diagnoses is high. The application of 2021 CNS WHO diagnostic criteria is greatly helpful for managing differential diagnoses, although some critical issues may be raised (Gianno et al., 2022a). First, the characteristic genetic alterations of the three pHGG H3/IDH WT subtypes are not exclusive of this entity and may be frequently found in other CNS tumors. Moreover, in a considerable percentage of pHGG H3/IDH WT tumors, the subtype-specific genetic alterations (PDGFRA, EGFR, TERT, and MYC) are absent, and the diagnosis may be reached, only demonstrating an aligned methylation profile (Korshunov et al., 2017; Tauziède-Espariat et al., 2020). Hence, neither the absence nor the presence of these alterations alone should never suggest to certainly exclude or confirm this diagnosis, without an appropriate clinico-pathological and molecular context. At the state of the art, prognosis for these tumors remains unfavorable, with some differences as regard the three subtypes: worst for MYCN (median OS of 14 months), intermediate for RTK1 (21 months), and better for RTK2 (44 months). PDGFRA and EGFR alterations may represent potential therapeutic targets in these tumors but yet to be demonstrated and validated. Another therapeutic chance may be represented by immunotherapy, especially in the context of mismatch repair deficiency-related cases (Korshunov et al., 2017; Mackay et al., 2018).

Infant-type hemispheric glioma is a high-grade diffuse glioma arising in cerebral hemispheres during early childhood. CNS WHO grade has not been assigned for this new entity due to the lack of prospective outcome data. This tumor is characterized by receptor tyrosine kinase (RTK) fusions regarding NTRK family, ROS1, ALK, or MET (WHO Classification of Tumours Editorial Board, 2021). These fusions lead to an aberrant expression of a kinase domain, which drives tumorigenesis via the activation of PI3K and/or MAPK pathways. Generally, co-occurring genetic alterations are missing, even though NSD1 mutations, with still uncertain significance, have been reported in rare cases (d’Amati et al., 2023b). Most of the cases occur very early in childhood, in particular during the first year of life. At histological examination, they appear highly cellular and composed of astrocytic cells, with mild-to-moderate nuclear pleomorphism. Rarely, gemistocytic elements, ganglion cells, or ependymal differentiation may be present (Olsen et al., 2015; Guerreiro Stucklin et al., 2019; Clarke et al., 2020). High mitotic activity, necrosis, and microvascular proliferation are frequently observable, even though biphasic tumors with low-grade and high-grade components have been reported (Ng et al., 2019; Valera et al., 2020). Immunostaining is scarcely useful for the dentification of gene fusions, as ALK positivity can be found only in some ALK-fused tumors, and NTRK shows high expression also in the normal brain tissue. Molecular analyses are fundamental in order to demonstrate the presence of a specific RTK fusion or an aligned DNA methylation profile. However, methylation profiling only recognizes a common subgroup, regardless of RTK fusion type, whereas the identification of these fusions is potentially useful for targeted therapeutic options (Olsen et al., 2015; Guerreiro Stucklin et al., 2019; Clarke et al., 2020). Finally, it is important to note that the identification of an RTK fusion in a morphologically high-grade glioma does not correspond to the diagnosis of infant-type hemispheric glioma. In fact, RTK rearrangements are also occasionally found in adult-type glioblastoma, IDH-wildtype, probably representing additional molecular events as a consequence of clonal evolution (Ferguson et al., 2018; Woo et al., 2020). Regarding prognosis, although data are currently limited for this new entity, the onset in early childhood is historically related to better outcomes as compared with pediatric-type diffuse high-grade gliomas, occurring in older children. Furthermore, each specific fusion seems to be associated with distinct prognosis. On the basis of data reported in a single study, ALK-rearranged tumors show the best prognosis (53.8% 5-year OS) as compared with the intermediate prognosis of NTRK-fused tumors (42.9% 5-year OS) and the poorer prognosis of tumors harboring ROS1 alterations (25% 5-year OS) (Guerreiro Stucklin et al., 2019). However, to support these preliminary findings, prospective studies with bigger cohorts are required. The same consideration may be true regarding the effectiveness of small-molecule inhibitors that are showing promising responses in these tumors harboring RTK-activating fusions, although further studies are required for a complete validation (Drilon et al., 2017; Ziegler et al., 2018).

Ependymal tumors are a heterogeneous group of neuroepithelial neoplasms arising from the progenitors of the ependymal cells, which line the inner cavities of the CNS. These are rare tumors, accounting for only 2–3% of all primary CNS neoplasms (Ostrom et al., 2022). They may arise along the whole neuroaxis, but most of the pediatric cases usually occur intracranially, whereas the spinal cord represents the preferential location among adults (McGuire et al., 2009). During the last years, significant novel molecular data allowed to identify distinct tumor subtypes, which are characterized by specific DNA methylation profile and genetic alterations. These molecular developments led to a substantially revised classification of the ependymal tumors, which are included in the 2021 WHO Classification of the CNS Tumors. The current classification, which is based on a combination of clinical, histological, immunohistochemical, and molecular features, subdivides ependymal tumors into three main groups, according to their location: supratentorial, infratentorial, and spinal.

Supratentorial ependymomas account for approximately 30% of all intracranial ependymomas, arising more frequently in pediatric age (Vera-Bolanos et al., 2015; Elsamadicy et al., 2020). Histologically, they show characteristic morphologic features of ependymomas, which appear similar across different anatomic sites. Perivascular pseudorosettes represent the hallmark feature and are composed of tumor cells organized around a central blood vessel. True ependymal rosettes, composed of tumor cells around an ependymal channel, are rarer to be observed. Immunohistochemically, ependymomas arising in other locations, are characterized by positivity for GFAP, negativity for OLIG2, and dot-like or ring-like cytoplasmic positivity for EMA. Regarding grading, ependymomas are classified as CNS WHO grade 2 or 3, principally on the basis of mitotic activity and independently from their location. However, there is no established cutoff, and the association between histological grading and outcome is not consistent. Supratentorial ependymomas are subclassified into ZFTA- and YAP1-fusion positive; therefore, molecular evaluation is necessary to demonstrate the presence of these entity-defining gene fusions (Andreiuolo et al., 2019b; Pagès et al., 2019). A significant part of supratentorial ependymomas do not show fusions involving ZFTA or YAP1 genes and is currently classified as “Supratentorial Ependymomas, NEC” (Louis et al., 2018). In this context, a recent study reported that some supratentorial tumors preferentially occurred in pediatric age, showed ependymoma-like morphological and immunohistochemical characteristics, and characterized by recurrent fusions in PLAGL1 genes (Sievers et al., 2021a).

Posterior fossa (PF) ependymomas, as suggested by the name, arise intra-axially in structures located in the posterior cranial fossa, mainly in the fourth ventricle or in the cerebellopontine angle (Witt et al., 2011). Numerous subgroups of posterior fossa ependymomas were identified through analysis of DNA methylation profiling and DNA/RNA NGS data, but two main subgroups, PFA and PFB, were reliably confirmed in independent investigations and have been included in the 2021 WHO classification (Mack et al., 2014, 2018). In most cases, immunostaining for H3 K27me3 serves as a reliable surrogate of DNA methylation profiling, being a crucial marker for the diagnostic categorization of PF ependymomas into two PFA and PFB subgroups (Panwalkar et al., 2017). As for other CNS tumors, the inability to perform appropriate immunohistochemical and/or molecular analyses prompts the addition of “NOS” (Louis et al., 2018). Posterior fossa ependymomas can be assigned CNS WHO grade 2 or 3. As for ependymomas arising in other sites, brisk mitotic activity and microvascular proliferation seem to be more reliable in defining histological grading, compared with necrosis and pleomorphism, but inconsistent results have been reported in the literature (Godfraind et al., 2012). Regarding prognosis, PFA show a poorer prognosis compared with PFB (Pajtler et al., 2015). Independently from the molecular group, the identification of a chromosome 1q gain is a reproducible indicator of adverse outcome (Kilday et al., 2012).

According to 2021 WHO Classification of CNS Tumors, the spinal location has been recognized three types of ependymomas: (1) spinal ependymoma (morphologically similar to other ependymomas); (2) MYCN-amplified spinal ependymoma (characterized by MYCN-amplification and poorer prognosis); (3) myxopapillary ependymoma (identifying morphological features and usually localized in conus medullaris/filum terminale) (WHO Classification of Tumours Editorial Board, 2021).

Subependymoma is a rare ependymal tumor, with an excellent prognosis, assigned to CNS WHO grade 1 (WHO Classification of Tumours Editorial Board, 2021). It is more common in adults and sometimes discovered as incidental findings (Nguyen et al., 2017). Their typical location is fourth or lateral ventricles (Bi et al., 2015). At microscopic examination, subependymomas appear composed of small neoplastic glial cells, typically forming nuclear clusters within a fibrillary matrix and associated with microcysts and calcifications. In some cases, these tumors may be admixed with more classic ependymoma-like areas. These so called “mixed ependymoma-subependymoma” cases are considered to have a more aggressive behavior, comparable to conventional ependymomas (Rushing et al., 2007). Their immunophenotype is similar to ependymomas. Molecular analyses are usually not required for diagnosis, though they have shown interesting epigenetic results, presenting distinct site-specific DNA methylation profiles (Neumann et al., 2020). Outcome is usually excellent, with very rare recurrences, even after subtotal surgical resection. A single study reported some tumors with brainstem location, showing subependymoma morphology and H3 K27M mutations. Interestingly, these tumors seem not to be associated with the adverse outcome of DMG, although data are limited to the few cases of this study (Yao et al., 2019).

Embryonal tumors of the CNS are characterized by genetic driving events, which are extremely aggressive, mainly affecting children (Sturm et al., 2016). DNA analysis and gene expression profiling allowed the identification of novel entities, leading to a reclassification of these tumors (Louis et al., 2014). One example is the diagnosis of medulloblastoma, which combines histopathological and molecular features (Taylor et al., 2012). Atypical teratoid/rhabdoid tumor (AT/RT), usually characterized by SMARCB1 (or alternatively SMARCA4) inactivation, includes three genetically, epigenetically, and clinically different molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC (Ho et al., 2020). Embryonal tumor with multilayered rosettes (ETMR) typically harbors the amplification of a microRNA cluster on chromosome 19 (C19MC). In the new WHO classification, ETMR received an updated designation due to the newly discovered DICER1 mutation in this tumor. Two newly introduced entities are FOXR2-activated CNS neuroblastoma and CNS tumor with BCOR internal tandem duplication (ITD) (WHO Classification of Tumours Editorial Board, 2021).

Medulloblastoma is categorized in WHO CNS5 based on a combination of molecular and histological characteristics. Extensive transcriptome and DNA profile studies have led to the present molecular classification, which reflects the clinico-biological variability of this neoplasm (Ellison, 2020). Children are most commonly affected by medulloblastomas, which can occur at any age. This tumor accounts for approximately 20% of intracranial neoplasms in this age group, which is second only to high-grade gliomas (Ostrom et al., 2022). Several inherited cancer syndromes are associated with medulloblastomas (Waszak et al., 2018). A variety of germline mutations can be found in ELP, SUFU, PTCH1 (naevoid basal cell carcinoma syndrome/Gorlin syndrome), TP53, APC, PALB2, and BRCA2. Medulloblastoma may grow in the fourth ventricle or be located in the cerebellar parenchyma (Blaser and Harwood-Nash, 1996), displaying symptoms and signs of elevated intracranial pressure caused by non-communicating hydrocephalus. Medulloblastomas have the ability to spread regionally, the leptomeninges, or occasionally outside the CNS. The majority of metastases are discovered adhering to the pia mater. The cerebrospinal fluid (CSF) or a hematogenous way is two possible mechanisms, mainly for SHH and non-WNT/non-SHH groups. Moreover, non-WNT/non-SHH medulloblastomas virtually always have distant CNS metastases at the time of the recurrence (Hill et al., 2020). Despite the fact that some molecular groupings and subgroups of medulloblastoma, such as WNT-activated tumors, exhibit a very good response to current therapy regimens and almost all of these individuals can be treated, all types of medulloblastomas are classified as embryonal tumors and CNS WHO grade 4. The histology is dominated by small, poorly differentiated cells with a high N:C ratio and high levels of mitotic activity and apoptosis. Architectural and cytological variation, on the other hand, classifies medulloblastomas into four histological subtypes: classic, desmoplastic/nodular, medulloblastoma with extensive nodularity, and large cell/anaplastic (Giangaspero et al., 1992; McManamy et al., 2007; Smolle et al., 2012). Such a wide range of morphological traits can be found in medulloblastoma molecular groupings. The new classification preserves the original four key molecular groups defined by the previous CNS WHO classification; wingless activated (WNT), sonic hedgehog (SHH) activated, and non-WNT/non-SHH. SHH tumors are classified based on TP53 status in TP53-mutant and TP53-wildtype tumors (Taylor et al., 2012; Table 2). DNA methylation profiling, on the other hand, has resulted in the detection of 12 subgroups (four subgroups for SHH medulloblastoma and eight subgroups for groups 3 and 4) (Cavalli et al., 2017). This segmentation of molecular subgroups has important biological and clinical implications for prognosis and treatment options (Massimino et al., 2013; Goschzik et al., 2018). To distinguish between WNT, SHH, and non-WNT/non-SHH medulloblastomas, immunohistochemistry is even useful. The nuclear immunoreactivity for beta-catenin, which is found in the majority of malignant, helps to identify the WNT-activated group. The GAB1 and YAP1 protein immunostaining in the cytoplasm identifies the SHH-activated group. The medulloblastoma WNT and SHH groups both have cytoplasmic positivity for filamin A. Non-WNT/non-SHH tumors are immunonegative for GAB1 and YAP1 (Gianno et al., 2022b). Nevertheless, the gold standard for assessing the status of a medulloblastoma subgroup is DNA methylation profiling (Schwalbe et al., 2017). The best prognostic and predictive data come from combining morphological interpretation with molecular analysis. The level of diagnostic accuracy is further improved by incorporating information into genetic changes. To increase accuracy, additional genetic changes, such as MYC amplification, currently employed in the risk categorization, are incorporated into an integrated diagnosis. When a medulloblastoma develops in the context of a hereditary tumor syndrome, an integrated approach to diagnose with commentary provides a chance to focus on the clinical implications of germline. New potential treatment options derived from recent studies regarding metabolic changes during cancer progression. Indeed, MB subgroups demonstrate different gene expression, leading to dysregulated metabolic pathways (lipid metabolism, nucleotide metabolism, and oxidative phosphorylation) (REF indicate) associated with different prognosis. These contributed to metabolic clustering and further risk stratification groups. In particular, high-risk metabolic clusters comprise G3/G4 methylation subgroup, MYC-amplified. MYC amplification displayed upregulation of genes related to nucleotide metabolism and oxidative phosphorylation, making them a potential therapeutic target (Gwynne et al., 2022; Funke et al., 2023).

AT/RT is a high-grade neoplasm characterized by the ability to differentiate along three germ layer lines, making this tumor unique. AT/RT is distinguished genetically by biallelic inactivation of SMARCB1 (known as INI1 or BAF47), less frequently, SMARCA4 (BRG1) (Hasselblatt et al., 2014). It is assigned to grade 4 CNS WHO classification. The incidence rate of AT/RTs is 1.6% of all pediatric CNS tumors, and they can occur as familial cases in the context of rhabdoid tumor predisposition syndromes 1 (Hasselblatt et al., 2014), although there have been reports of de novo germline mutations (Bourdeaut et al., 2011). The median age of the patients is 20 months (Ostrom et al., 2022). Adult occurrence is uncommon (Chan et al., 2018). These tumors are located mainly in the supratentorial compartment, but the whole neuraxis can be involved. Immunohistochemistry reveals positivity for synaptophysin, EMA, and AML. Loss of INI1 or BRG1 expression represents the surrogate of the underlying gene mutations. DNA methylation analysis combined with gene expression profiling has identified three molecular subgroups: AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC. These subgroups stratify patients based on their age, place of origin, and gene alteration pattern (Frühwald et al., 2020; Table 3). AT/RT-SHH is characterized by the upregulation of proteins in the SHH and Notch signaling pathways. Heterozygous SMARCB1 point mutations are frequently observed (Ho et al., 2020). AT/RT-TYR is distinguished by an increase in the expression of proteins involved in the melanosomal system (tyrosinase), the bone morphogenetic protein (BMP) pathway, and transcription factors related to the development. The deletion of the SMARCB1 gene is caused mostly by a mutation in one allele, and the second hit leads to a total or partial loss in the second allele of chromosome 22 (Ho et al., 2020). AT/RT-MYC expresses the MYC oncogene and the HOX cluster genes. It arises more frequently in the supratentorial compartment, although they rarely happen in the spinal cord. This group also includes the uncommon AT/RTs, affecting adults restricted to the sella (Ho et al., 2020; Broggi et al., 2022). A recent study also found a high correlation between histological patterns and molecular grouping (Zin et al., 2021). The prognosis for patients with AT/RT is often dismal. Clinical studies have revealed, however, that AT/RTs do not always result in a bad outcome. High-dose chemotherapy combined with stem cell rescue and radiation was linked to a 4-year survival rate of 43% (Reddy et al., 2020). Epigenomic landscapes of AT/RT subtypes may be associated with varied treatment response so that it may be possible to stratify patients with AT/RT (Mittal and Roberts, 2020).

ETMR is an embryonal rare malignancy with characteristic morphological features, which is characterized by a C19MC alteration or, less commonly, a DICER1 mutation (CNS WHO grade 4). The median age of children affected by ETMR is less than 4 years. Three main histological arrangements can be observed: embryonal tumor with abundant neuropil and true rosettes (ETANTR), ependymoblastoma, and medulloepithelioma. On DNA methylation profile and gene expression, these three patterns cluster together. Different patterns of epithelial or mesenchymal development can be recognized (WHO Classification of Tumours Editorial Board, 2021). ETMRs exhibit widespread immunopositivity for LIN28A, which represents a very helpful marker for the identification of these tumors (Spence et al., 2014). The molecular detection of C19MC amplification or DICER1 mutations is mandatory. Only ETMRs have the C19MC microRNA cluster mutation at 19q13.42, which is present in 90% of cases (Korshunov et al., 2010). Copy number profiling array FISH analysis is effective ways to identify C19MC changes. Only 5% of ETMRs do not show C19MC amplification, harboring DICER1 mutations. Rare ETMRs that do not have a DICER1 mutation or C19MC change should be categorized as NEC. The survival rates for ETMR are still extremely low, despite rigorous multimodal treatment.

CNS neuroblastoma FOXR2-activated is a rare recently-described embryonal tumor displaying various degrees of neuroblastic/neuronal development and foci of ganglion elements and neuropil-rich stroma. It typically harbors rearrangements activating the transcription factor FOXR2 (CNS WHO grade 4). It arises in children classically within the supratentorial compartment, rarely with intraventricular location (Sturm et al., 2016). On MRI, this tumor typically manifests as a delineated mass with a cystic and a solid component, exhibiting a mild contrast enhancement (Holsten et al., 2021). Histologically, CNS NB-FOXR2 exhibits embryonal architecture-organized sheets. Homer-Wright rosettes and vascular pseudorosettes can also be observed. The immunoprofile shows a significant positivity for OLIG2. Synaptophysin is positive in regions with neurocytic/ganglionic differentiation. The majority of cases also have TTF1 overexpression (Holsten et al., 2021). CNS NB-FOXR2 is a recent addition to CNS 2021 WHO, discovered by using DNA methylation analysis, which revealed that several tumors may have belonged to distinct entities. This novel entity has chromosomal rearrangements with overexpression of FOXR2 gene (Sturm et al., 2016; Louis et al., 2020). Next-generation sequencing is required for the discovery of FOXR2 rearrangements, but copy-number analysis may be able to reveal changes to the FOXR2 locus on chromosome Xp11.21. However, DNA methylation profiling greatly aids in the diagnosis of these cancers. Data on the prognosis of CNS NBFOXR2 are limited, but studies show that they have strong response to the current treatment (von Hoff et al., 2021).

CNS tumor with BCOR internal tandem duplication (ITD) is a malignant CNS neoplasm that has an ITD in exon 15 of the BCOR gene. The reported patients’ median age at presentation is 3.5 years (the range is 0 to 22 years). The cerebral or cerebellar hemispheres are most frequently involved (De Lima et al., 2020). On MRI, they show a central cystic area and inhomogeneous contrast enhancement (Bremer et al., 2020). Some regions may exhibit a glioma-like appearance, and compact fascicular patterns are commonly connected with a branching capillary network. Myxoid or microcystic region is quite distinctive. Mitosis and palisading necrosis can also commonly occur. They diffusely express vimentin and CD56, while absent or sparse expression of OLIG2, GFAP, or S100 supports the diagnosis (Louis et al., 2020). Although nuclear expression of BCOR is a sensitive marker, it is not specific (Kao et al., 2016; Ferris et al., 2020). The molecular detection of the specific ITD is required for a conclusive diagnosis. It is possible to distinguish CNS tumors with BCOR ITD from other CNS tumors by DNA methylation profiling and gene expression patterns. Patients with these malignancies have low survival rates (Wen and Packer, 2021).