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Incorporating Advances in Molecular Pathology Into Brain Tumor Diagnostics

Velázquez Vega, José, E., MD*; Brat, Daniel, J., MD, PhD

Advances In Anatomic Pathology: May 2018 - Volume 25 - Issue 3 - p 143–171
doi: 10.1097/PAP.0000000000000186
Review Articles

Recent advances in molecular pathology have reshaped the practice of brain tumor diagnostics. The classification of gliomas has been restructured with the discovery of isocitrate dehydrogenase (IDH) 1/2 mutations in the vast majority of lower grade infiltrating gliomas and secondary glioblastomas (GBM), with IDH-mutant astrocytomas further characterized by TP53 and ATRX mutations. Whole-arm 1p/19q codeletion in conjunction with IDH mutations now define oligodendrogliomas, which are also enriched for CIC, FUBP1, PI3K, NOTCH1, and TERT-p mutations. IDH-wild-type (wt) infiltrating astrocytomas are mostly primary GBMs and are characterized by EGFR, PTEN, TP53, NF1, RB1, PDGFRA, and CDKN2A/B alterations, TERT-p mutations, and characteristic copy number alterations including gains of chromosome 7 and losses of 10. Other clinically and genetically distinct infiltrating astrocytomas include the aggressive H3K27M-mutant midline gliomas, and smaller subsets that occur in the setting of NF1 or have BRAF V600E mutations. Low-grade pediatric gliomas are both genetically and biologically distinct from their adult counterparts and often harbor a single driver event often involving BRAF, FGFR1, or MYB/MYBL1 genes. Large scale genomic and epigenomic analyses have identified distinct subgroups of ependymomas tightly linked to tumor location and clinical behavior. The diagnosis of embryonal neoplasms also integrates molecular testing: (I) 4 molecularly defined, biologically distinct subtypes of medulloblastomas are now recognized; (II) 3 histologic entities have now been reclassified under a diagnosis of “embryonal tumor with multilayered rosettes (ETMR), C19MC-altered”; and (III) atypical teratoid/rhabdoid tumors (AT/RT) now require SMARCB1 (INI1) or SMARCA4 (BRG1) alterations for their diagnosis. We discuss the practical use of contemporary biomarkers for an integrative diagnosis of central nervous system neoplasia.

*Department of Pathology and Laboratory Medicine, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA

Department of Pathology, Feinberg School of Medicine and Robert H Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL

The authors have no funding or conflicts of interest to disclose.

Reprints: Daniel J. Brat, MD, PhD, Department of Pathology, Northwestern University Feinberg School of Medicine, Ward 3-140, 303 East Chicago Avenue, Chicago, IL 60611 (e-mail: daniel.brat@northwestern.edu).

Molecular-genetic analysis is now an integral component of contemporary surgical neuropathology.1 A critical conceptual shift that materialized with the publication of the revised fourth edition of the WHO Classification of central nervous system (CNS) tumors was the incorporation of molecular alterations into the diagnosis of specific neoplastic entities, in distinction to the prior practice of recognizing them as mere associations.2,3 In this new era, diagnostic impressions from microscopic examination of hematoxylin and eosin (H&E) stained slides are interpreted in the setting of molecular-genetic testing.4 These changes ushered in substantial reclassification of the infiltrating gliomas and embryonal tumors, and also led to the introduction of new entities and to the revision or removal of others.2 Below we discuss how molecular pathology has changed the practice of diagnostic surgical neuropathology.

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INFILTRATING GLIOMAS IN THE POST-ISOCITRATE DEHYDROGENASE (IDH) ERA

Gliomas are a broad category of CNS tumors that affect patients of all ages and are highly variable with respect to location, histologic features, genomic alterations, clinicobiological behavior, and treatment responses. The subset with an infiltrative growth pattern, namely the diffuse astrocytomas and oligodendrogliomas, frequently arise within the cerebral hemispheres of adults and have a tendency toward clinical progression, albeit at varying rates, and are ultimately fatal, as they are not amenable to complete surgical excision and do not completely respond to adjuvant therapies. The infiltrating gliomas are currently assigned to grades II to IV based on histologic criteria within the WHO Classification.2,5,6

For the past 90 years, CNS tumors have been classified by their appearance under the microscope. Infiltrating astrocytomas have been recognized by their hyperchromatic, elongated nuclei embedded within a highly fibrillary background.5 Depending on the size of the evaluated biopsy/excision material, the presence of 1 or several mitotic figures have been used to discriminate between a WHO grade II diffuse astrocytoma and a WHO grade III anaplastic astrocytoma. The presence of either microvascular proliferation or necrosis is required for a diagnosis of GBM, WHO grade IV, the highest grade form of an infiltrating astrocytoma.3 For oligodendrogliomas, the classic morphologic features include monomorphic rounded tumor nuclei with perinuclear halos and a network of delicate branching capillaries. Differentiating between a low-grade oligodendroglioma (WHO grade II) and an anaplastic oligodendroglioma (WHO grade III) has relied on identifying ≥6 mitoses per 10 high power fields, florid microvascular proliferation, or areas of necrosis.7,8 Ever since the original brain tumor classification by Bailey and Cushing in 1926, it had been clear that assigning tumor lineage based only on histologic assessment was not always straightforward and diagnoses such as “mixed glioma,” “oligoastrocytoma,” and “glioblastoma with oligodendroglioma component (GBM-O)” were rendered when tumors appeared to have a mixture of glial cell types or had ambiguous morphology. Both the subjective nature of histologic classification and the lack of firm criteria for “mixed” gliomas led to suboptimal diagnostic reproducibility that caused confusion in patient care.9

Molecular-genetic testing is currently used to subdivide gliomas into discrete diagnostic categories.10–14 Landmark publications by Parsons et al15 and Yan et al16 established IDH-mutant diffuse gliomas as a form of disease that could not be recognized histologically, yet had clinically distinct behavior compared with those that were IDH-wild-type (wt). Somatic heterozygous IDH 1/2 mutations are now regarded as an initiating event in gliomagenesis that stratifies diseases with highly distinct biological behaviors.17 Mutations within the enzymatic active sites of IDH 1/2 were initially thought to result in an enzymatic loss-of-function pertaining the conversion of isocitrate to α-ketoglutarate, yet further studies demonstrated a gain-of-function leading to the accumulation of the oncometabolite 2-hydroxyglutarate.18–20 2-hydroxyglutarate inhibits DNA and protein demethylation, resulting in DNA hypermethylation and genome-wide epigenetic alterations that promote gliomagenesis.21,22 The subset of gliomas with the highest level of DNA methylation is referred to as CpG island methylator phenotype (G-CIMP), which is directly related to IDH mutations.20–26

More than 80% of WHO grades II and III infiltrating astrocytomas and secondary GBMs are IDH-mutated and, by current definitions, all oligodendrogliomas are IDH-mutated and 1p/19q codeleted.2,10,11,15,16,27–31 IDH1 and IDH2 mutations result in a substitution for a key arginine at codons R132 and R172, respectively.16,30,31 The most frequent IDH mutation, accounting for 92.7%, occurs at codon 132 of the IDH1 gene and is known as the R132H variant (histidine is substituted for arginine).31 A highly sensitive and specific monoclonal antibody (IDH1R132H) that recognizes the mutant protein was quickly developed and is now widely used in diagnostic practice.32 IDH1R132H mutations are followed in frequency by R132C (4.1%), R132S (1.5%), R132G (1.4%), and R132L (0.2%).31 Although IDH2 mutations represent ∼3% of all IDH mutations, they are more frequent in oligodendrogliomas than astrocytomas.31 Residue R172 in exon 4 of the IDH2 gene is homologous to R132 in the IDH1 gene, and R172K (65%), R172M (19%), and R172W (16%) are the most common IDH2 mutations.31 IDH mutations are almost always stable events that persist through the course of disease, which can be exploited to evaluate residual/progressive disease; however, loss of the IDH-mutated allele has been documented in some cases at the time of recurrence and appears to be associated with a transition to high-grade behavior.19,33,34

The finding of an IDH mutation in a glial proliferation strongly supports the diagnosis of an infiltrating glioma since they are only rarely, if ever, found in other CNS neoplasms.30 In addition, identifying an IDH mutation in surgical material, where the main differential diagnosis is gliosis, is of crucial importance as it confirms a neoplastic rather than a benign reactive process.35 Adult patients with IDH-mutant infiltrating gliomas are significantly younger when compared with the histologically similar IDH-wt counterparts; however, IDH mutations are exceedingly rare in tumors of children under the age of 14 years.10,11,15,16,29,36–40

The family of adult diffuse gliomas can now be divided into 3 dominant classes: (1) oligodendrogliomas, with IDH mutations, 1p/19q codeletions, and TERT-p mutations; (2) IDH-mutant astrocytomas, with IDH mutations, TP53 mutations, and α-thalassemia/mental retardation syndrome X-linked (ATRX) alterations; or (3) and IDH-wt astrocytomas.10 These categories have been codified in the 2016 WHO Classification of CNS tumors. For the diagnosis of IDH-mutant astrocytomas, the finding of an IDH mutation by immunohistochemistry (IHC) or sequencing is followed by IHC for p53 and ATRX. In the setting of an IDH mutation, strong p53 immunoreactivity and loss of nuclear expression with ATRX IHC are excellent surrogates of TP53 mutation and ATRX loss at the genetic level (Figs. 1, 2).28,29,41,42 When the morphology of the tumor is in keeping with the prototypic oligodendroglioma or the p53/ATRX IHC results suggest an oligodendroglial genotype, 1p/19q test is performed by fluorescent in situ hybridization (FISH) or by cytogenomic molecular inversion probe (MIP) array to support the integrated diagnosis of oligodendroglioma, IDH-mutant, 1p/19q codeleted (Figs. 3–5).2,4

FIGURE 1

FIGURE 1

FIGURE 2

FIGURE 2

FIGURE 3

FIGURE 3

FIGURE 4

FIGURE 4

FIGURE 5

FIGURE 5

The absence of a molecular signature for oligoastrocytomas has been confirmed by many, with these lesions representing IDH-mutant astrocytomas, oligodendrogliomas or IDH-wt gliomas at the molecular level.10 Similarly, genomic and transcriptomic studies of GBM-O have concluded that these are either anaplastic oligodendrogliomas, IDH-mutant GBMs, or IDH-wt GBMs when analyzed genetically.43 The revised fourth edition of the WHO Classification still recognizes oligoastrocytoma as a histologic diagnosis but its use is discouraged and, if used, it should be followed by a not otherwise specified (NOS) classifier to emphasize that molecular testing was not performed or that its results were inconclusive.2 Dual-genotype gliomas are rare but have been described as case reports.44

New questions have arisen in the post-IDH era that require further attention and are the focus of ongoing investigations, such as the optimization of risk-stratification within genetic subsets of infiltrating gliomas. For example, recent studies have shown that histologic grade (WHO grades II vs. III) and conspicuous mitotic activity are not highly informative of clinical outcome among IDH-mutant infiltrating astrocytomas.45 It remains uncertain whether grading criteria for IDH-mutant astrocytomas will differ from those of IDH-wt counterparts or whether molecular markers will play a role in grading infiltrating gliomas. Another dilemma is that IDH-wt anaplastic astrocytoma (WHO grade III) has a poorer outcome than IDH-mutant GBMs (WHO grade IV), further highlighting the need to optimize grading.46

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OLIGODENDROGLIOMAS ARE DEFINED BY IDH MUTATIONS AND 1P/19Q CODELETIONS

The WHO defines oligodendrogliomas as having both IDH mutations and 1p/19q codeletions. As such, this tumor type has the longest median survival among the diffuse gliomas.10,28,29 Reifenberger et al47 first recognized the tight association of the oligodendroglial phenotype with the loss of 1p/19q and Cairncross et al,48 later showed that this signature was associated with enhanced response to chemotherapy. The codeletion is now known to be mediated through an unbalanced translocation t(1;19)(q10;p10) followed by the loss of the derivative chromosome, resulting in whole-arm losses of 1p and 19q.8,49,50 FISH for 1p/19q became a popular method for assessing codeletions and is still widely used. However, FISH recognizes focal losses specific to the probes and high-grade astrocytomas can also show focal losses in the setting of genomic instability, potentially leading to misclassification.28,51 In a study of 491 GBMs, Clark et al52 showed that 5.7% demonstrated 1p/19q codeletion by either FISH or polymerase chain reaction-based loss of heterozygosity, but that the vast majority of these (over 90%) also had chromosome 10q loss of heterozygosity or EGFR amplifications, which are genetic features of IDH-wt GBM known to inversely correlate with whole-arm losses of 1p/19q. Clinical tests that evaluate the entire arms, such as MIP array and next-generation sequencing (NGS) methods, reduce the risk of false positives on testing for 1p/19q codeletion.28,53

The genomic spectrum of IDH-mutant, 1p/19q codeleted oligodendrogliomas also include inactivating mutations of the tumor suppressor genes far-upstream binding protein 1 (FUBP1) gene and in the human homolog of the Drosophila capicua (CIC), on chromosomes 1p31.1 and 19q13.2, respectively. FUBP1 and CIC mutations occur following the unbalanced translocation at an estimated frequency of 20% to 30% and 46% to 83%, respectively. These mutations are mutually exclusive with TP53 and ATRX mutations and are exceedingly rare in diffuse astrocytomas.8,10,54–58 The prognostic or predictive significance of FUBP1 and CIC mutations in oligodendrogliomas remains to be elucidated although recent studies found that outcomes of 1p/19q codeleted gliomas were not altered by these mutations.57,59

IDH-mutant, 1p/19q codeleted oligodendrogliomas also carry highly specific mutations in the telomerase reverse transcriptase (TERT) gene promoter (C228T or C250T), upstream of the TERT ATG start site.11,60–66 Diffuse gliomas harboring IDH and TERT-p mutations in conjunction with whole-arm losses of chromosomes 1p and 19q are associated with prolonged overall survival (OS).11,67 TERT-p mutations are rare in IDH-mutant astrocytomas, yet there have been scattered reports of cases with both or neither ATRX and TERT-p mutations.11,57,66,68 In contrast to the alternative lengthening of the telomeres (ALT) phenotype characteristic of IDH-mutant astrocytomas, activating mutations in the TERT-p upregulate TERT expression resulting in enhanced telomerase activity and maintenance of telomere length.69 Most studies have shown that 95% to 100% of oligodendrogliomas with IDH mutations and 1p/19q codeletions carry TERT-p mutations and those that are TERT-p-wt have significantly worse outcomes.10,70 Interestingly, 90% of IDH-wt (primary) GBMs also carry TERT-p mutations, and these have an unfavorable prognosis compared with the TERT-p-wt counterparts.11,60–65,70 Loss of chromosome 4 follows 1p/19q codeletion as the second most common copy number alteration seen in ∼25% of cases, but very few other recurring CNAs have been described in oligodendrogliomas.10,71 In our recent analysis of oligodendrogliomas in the low-grade glioma data set of The Cancer Genome Atlas (TCGA), we found that gains of chromosomes 7p and 11p occurred in 8.9% and 11.2%, respectively, whereas losses of 14q and 15q occurred in 11.8% and 16.6%, correspondingly. Interestingly we saw a tendency of these CNAs to occur in oligodendrogliomas with features of clinical progression.72

Other genetic alterations characterizing the molecular landscape of oligodendrogliomas include NOTCH1, PIK3CA, PIK3R1, ZBTB20, and ARID1A mutations.10 Inactivating mutations in NOTCH1 occur in a significant subset of molecularly defined oligodendrogliomas (31% in the TCGA analysis) and are exceedingly rare in IDH-mutant or IDH-wt astrocytomas.10,37,56 Activating mutations in the PI3K pathway genes PIK3CA and PIK3R1 occur in 20% and 9% of the cases, respectively.10 NOTCH1 and PI3K mutations, as well as other pathways including MYC, may play a role in oligodendroglioma progression and have prognostic value.73 Aoki et al59 recently reported that NOTCH1 mutations were associated with shorter survival of patients with oligodendroglioma. In our recent analysis of the TCGA data set, we also found that NOTCH1 mutations and RBPJ alterations, both key components of the canonical Notch pathway, correlated with markers of disease progression including high cellular density, proliferation, and contrast enhancement in magnetic resonance imaging.72 Currently, we separate low-grade oligodendrogliomas from anaplastic oligodendrogliomas on the basis of mitotic figures, microvascular proliferation, and necrosis (Figs. 3, 4) but molecular-genetic abnormalities may also be of utility in grading and risk-stratification.7,74

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IDH-MUTANT ASTROCYTOMAS ARE CHARACTERIZED BY TP53 MUTATIONS AND ATRX ALTERATIONS

One large scale analysis of infiltrating gliomas showed that approximately two thirds of the IDH-mutant WHO grade II and III diffuse gliomas had intact 1p/19q; of these 94% had mutations in TP53 and 86% had a functional loss of ATRX, establishing this combination of alterations as the genetic signature of IDH-mutant astrocytomas.10 Among IDH-mutant tumors, inactivating mutations of ATRX appear to be restricted to those with TP53 mutations and this combination is almost mutually exclusive with whole-arm 1p/19q codeletions.10,28,37,39,42,75,76 It is thought that TP53 mutations predispose toward the acquisition of ATRX alterations.37

Loss of nuclear ATRX expression by IHC in diffuse astrocytomas is strongly associated with both IDH and H3F3A mutations.41 Together with death-domain associated protein (DAXX), ATRX is a core mediator of a chromatin remodeling complex necessary for the incorporation of histone variant H3.3 into the nucleosome. Alterations of the ATRX/DAXX complex have been implicated in telomere stability through activation of the ALT mechanism, a telomerase-independent pathway for telomere maintenance, and telomere length has been positively correlated with ATRX mutations.68 The ATRX/DAXX complex normally represses ALT and its alteration appears to be permissive of telomere maintenance by homologous recombination.69,77,78 DAXX or ATRX mutations impair the heterochromatic state of the telomeres, probably in part by reduced incorporation of chromatin onto H3.3 histones.79 TP53 mutations play a complimentary role with genomic instability and ALT, as tumor cells presumably acquire the capacity to evade apoptosis and become immortalized.37,54,69,75,77,78,80–82 The ALT phenotype in astrocytomas correlates with a younger patient age, loss of ATRX expression by IHC, p53 immunoreactivity, IDH mutations, platelet-derived growth factor receptor alpha (PDGFRA) amplifications, and absence of epidermal growth factor receptor (EGFR) amplifications.54,79,83

ATRX alterations have been investigated as potential prognostic markers in IDH-mutant astrocytomas. Some studies have shown that IDH-mutant astrocytomas with ATRX loss have improved outcomes [longer median progression-free survival (PFS) and/or median OS] compared with the ATRX-wt subgroup.29,41,54,84 More recently, however, multivariate analysis showed no statistically significant survival associations.70

A clinically relevant subset of IDH-mutant astrocytomas has a distinctive pattern of genome-wide DNA methylation changes that is associated with a poor prognosis. This subset has a hypomethylation pattern (G-CIMP-low epigenetic signature) compared with the other IDH-mutant astrocytomas and appears to be enriched for alterations in cell cycle pathway genes such as CDK4 and CDKN2A.68 Other groups have also now reported that alteration of retinoblastoma (Rb) pathway genes (RB1 mutation, CDKN2A deletion, and CDK4 amplification) were associated with poor survival.59,71 It has been suggested that increased cMYC expression may be an adverse prognostic factor among IDH-mutant gliomas, as cMYC protein overexpression by IHC has been associated with shortened time to malignant transformation and OS in both univariate and multivariate analyses.85 Others have suggested that increased CNAs and genomic instability may be associated with more rapid progression and unfavorable outcomes in IDH-mutant astrocytomas.86 Additional study of biomarkers for their prognostic significance among the IDH-mutant diffuse astrocytomas are needed, as grading by pre-IDH era morphologic features remains suboptimal.45

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IDH-WILD-TYPE DIFFUSE GLIOMAS: A HETEROGENOUS GROUP, WITH MOST HAVING AGGRESSIVE FEATURES

The vast majority of primary (or de novo) GBMs (95%) and a smaller percentage of WHO grades II and III infiltrating gliomas (20% to 25%) that occur in adults are IDH-wt.10,15,16,28 With current histologic grading criteria, IDH-wt WHO grades II and III astrocytomas that lack necrosis and microvascular proliferation fall short of the morphologic definition required for a diagnosis of GBM; however, their molecular-genetic profiles and clinical behavior are strikingly similar to IDH-wt GBM, with 1 study showing a median OS of 1.7 years.10 These lower grade IDH-wt infiltrating astrocytomas could represent either undersampled or incipient GBMs. Notably, there is an important subgroup of IDH-wt infiltrating gliomas that genetically and epigenetically resemble pediatric pilocytic astrocytomas (PA) and carry a favorable outcome, and therefore not all IDH-wt gliomas should be lumped together as homogenous and highly aggressive.68 This “PA-like” group of IDH-wt infiltrating glioma lacked the prototypic genetic signatures of primary IDH-wt GBMs, such as EGFR, CDKN2A/B, and PTEN alterations and displayed euploid DNA copy number profiles.68 These findings and others indicate the need to further investigate IDH-wt infiltrating gliomas at the population level, as well as the need to more fully characterize them for clinical care purposes.

Reuss and colleagues recently studied 160 IDH-wt WHO grades II and III infiltrating astrocytomas and found that 78% were molecular equivalents to conventional primary IDH-wt GBM, on the basis of similar frequencies in TERT-p mutations, chromosome 7p gain/10q loss, and EGFR amplifications or combined 10q/13q/14q codeletion. A median survival of 19.4 months was observed for this subset. Moreover, when H3 K27M-mutant diffuse gliomas (Fig. 6) were included in the analysis, then 87% of these IDH-wt astrocytomas were molecularly and clinically indistinguishable from GBM.87 Notably, nearly all IDH-wt infiltrating gliomas with gain of chromosome 7/loss of chromosome 10 harbor TERT-p mutations or exhibit upregulated TERT expression.68 TP53 mutations were noted in only 25% of IDH-wt infiltrating grade II to III gliomas, almost identical to the frequency observed in IDH-wt GBMs.16,36 Similarly, FGFR-TACC fusions were noted in 3.5% of lower grade IDH-wt infiltrating astrocytomas, a frequency similar to that seen in primary GBMs. In cases described thus far, these fusion events are mutually exclusive with IDH mutations and EGFR, PDGFRA, and MET mutations or amplifications, though they often co-occur with CDK4 amplifications.88–90

FIGURE 6

FIGURE 6

When compared with the IDH-mutant gliomas, the IDH-wt counterparts have greater activation of signaling through EGFR, MET, and BRAF; upregulated cell cycle activators; and reduced cell cycle inhibitors.91 Approximately 1% of adult low-grade infiltrating IDH-wt gliomas harbor activating BRAF V600E somatic mutations, which drives the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway.92–94 BRAF V600E-mutant infiltrating gliomas are thought to represent a distinct clinicopathologic entity with better outcomes; however, this mutation is far more frequent in grade I, noninfiltrative gliomas including PAs, pleomorphic xanthoastrocytomas (PXA), gangliogliomas, pediatric diffuse gliomas, and epithelioid GBMs. The presence of a BRAF V600E mutation may have therapeutic implications, although larger scale investigations are needed.38,92,93 Additional studies of IDH-wt infiltrating gliomas are necessary to further elucidate subgroups that may exhibit less aggressive clinical behavior and the biomarkers that characterize them.

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Key Points: Diffuse Astrocytomas and Oligodendrogliomas

  • Diffuse gliomas in adults can be classified on the basis of IDH mutations and whole-arm 1p/19q codeletions
  • IDH mutations and whole-arm codeletions of 1p/19q are definitional of oligodendroglioma
  • IDH-mutant astrocytomas are characterized by TP53 mutations and ATRX alterations
  • IDH-wt diffuse astrocytomas harboring genetic alterations such as TERT-p mutations, chromosome 7p gain/10q loss, and H3 K27M mutations have been associated with worse outcomes
  • IDH-wt diffuse astrocytomas lacking the classic genetic alterations of IDH-wt GBM may represent different biological subtypes and include those that are BRAF V600E mutants
  • NOTCH1 mutations are associated with worse outcomes in oligodendroglioma, IDH-mutant, and 1p/19q codeleted. Alterations within the Rb pathway are associated with worse outcomes in IDH-mutant astrocytomas
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GLIOBLASTOMAS ARE A DIVERSE GROUP OF AGGRESSIVE BRAIN TUMORS

GBMs account for nearly half of all intrinsic brain tumors and are clinically aggressive, with survivals ranging from 6 to 30 months and with very few surviving over 5 years.95,96 GBMs are often referred to as “primary” when they present as grade IV disease as the first clinical manifestation, and as “secondary” when they have evolved over time from a lower grade infiltrating astrocytoma. The distinction between primary and secondary GBMs has been emphasized by the WHO Classification and the correlation with IDH status is well established.2 Primary GBMs typically arise in older patients with a rapid onset of clinical symptoms and are nearly all IDH-wt. In contrast, secondary GBMs commonly arise in patients below 45 years and progress more slowly, with nearly all being IDH-mutant.15,27,95

IDH-wt GBMs are characterized by PTEN, TP53, NF1, RB1, and TERT-p mutations, the combination of gain of chromosome 7/loss of chromosome 10, deletions of CDKN2A/B, and amplifications of protooncogenes, most notably, EGFR, PDGFRA, or c-MET (Fig. 7). Regarding the sequence of oncogenic events in IDH-wt primary GBMs, it has been suggested that TERT-p mutations, present in up to 90% of adult GBMs, may precede the characteristic co-occurrence of chromosome 7 gain/chromosome 10 loss, seen in 60% of primary IDH-wt GBM.68,97 IDH-wt GBMs nearly always harbor alterations of the following 3 core signaling pathways: (I) the receptor tyrosine kinase pathway (RTK/RAS/PI3K), (II) the p53 pathway, and (III) the Rb pathway, which are altered in 88%, 87% and 78% of GBMs, respectively.96,98 The most frequently altered genes in the RTK/RAS/PI3K pathway include PTEN, NF1, EGFR, PIK3R1, PIK3CA, and PDGFRA. Among genes in the Rb pathway CDK4, CDK6, CCND2, CDKN2A/B, and RB1 are the most commonly altered. Disruptions in the p53 pathway include alterations of the MDM2, MDM4, and TP53 genes.13,15,27,95,96,98–100

FIGURE 7

FIGURE 7

Brennan et al99 has shown that 57% of GBMs had evidence of mutation, rearrangement, altered splicing, and/or focal amplification of EGFR, highlighting its role as a key oncogenic event. Among EGFR-amplified GBMs, ∼50% also harbor the variant III (EGFRvIII) deletion leading to constitutive tyrosine kinase activation.2,13,101 PDGFRA amplifications occur in ∼15% to 18% of primary IDH-wt GBM’s. MDM2 and CDK4 gene amplifications occur correspondingly in 5% to 15% and 14% to 18% of primary IDH-wt GBMs, functioning as alternative mechanisms of Rb and p53 pathway disruption.2,13,15,95,99 Although much less frequent, high-level amplifications of the MET protooncogene are noted in 4% of GBMs.2,95

Homozygous CDKN2A deletions at chromosome 9p21 are present in up to 50% of GBMs and lead to the loss of tumor suppressor proteins p16 (INK4a) and p14 (ARF), and inactivation of Rb and p53, respectively.2,12–15 Mutations and deletions of TP53 and RB1 are seen in 28% to 35% and 8% to 12% of primary IDH-wt GBMs, correspondingly, whereas activating mutations of the PI3K subunits (PIK3CA or PIK3R1) are present in 12% to 25%.2,13,15,99 PTEN, the primary negative regulator of the PI3K/AKT signaling pathway, is mutated or deleted in ∼25% to 35% of GBMs, whereas mutations or deletions of NF1, a Ras antagonist, occur in ∼10% to 18% of GBMs.2,13,15,95,96,98,99 Although not completely understood, cogains of chromosomes 19 and 20 have been associated to long-term survival in GBM, warranting further investigation.71,102

The revised fourth edition of the WHO Classification recognizes several variants and patterns of IDH-wt GBM.2 Giant cell GBM (Fig. 8) is characterized by markedly pleomorphic and multinucleated giant cells with a tendency for better circumscription and a slightly better prognosis than conventional IDH-wt GBM.103 A recent study highlighted some key differences in the genetic profile of giant cell GBM when compared with conventional IDH-wt GBM: ∼84% of giant cell GBMs were TP53-mutant, 25% were TERT-p-mutated, and only 6% exhibited EGFR amplifications, which contrasts with the frequency of TP53 and TERT-p mutations and EGFR amplifications in conventional IDH-wt GBM (23%, 72%, and 56%, respectively).103 Gliosarcomas represent another variant of IDH-wt GBM and are characterized by a biphasic pattern of gliomatous and sarcomatous components highlighted by glial fibrillary acidic protein and reticulin stains, respectively. The genetic profile of gliosarcomas is very similar to that of conventional IDH-wt GBM, however, EGFR amplifications are rare.103

FIGURE 8

FIGURE 8

Although the frequency of BRAF and H3F3A mutations is much lower in adult gliomas when compared with their prevalence in childhood tumors, they do occur in smaller subsets of adult GBMs.28,95 BRAF V600E mutations are present in <5% of all GBMs. However, they are overrepresented in epithelioid GBMs, which have a much higher frequency (up to 50%) and can be detected by sequencing or BRAF V600E mutation-specific (VE1) IHC.95,104 Epithelioid GBMs (Fig. 9) are often peripheral masses, sometimes associated with the dura, and are characterized by malignant cells with abundant eosinophilic cytoplasm and well-defined cytoplasmic borders (epithelioid morphology) along with an eccentrically located nucleus with prominent nucleoli. These have been referred to as “rhabdoid” in the past and often elicit the consideration of melanoma.2,105 Less commonly, epithelioid GBMs harbor genetic alterations of conventional IDH-wt GBMs and its exact relationship to anaplastic PXA, a known mimicker with remarkably similar clinical, histologic, and molecular-genetic features, remains to be further elucidated.2,105,106

FIGURE 9

FIGURE 9

Approximately 5% to 9% of adult primary infiltrating astrocytomas that are IDH-wt are H3F3A-mutant.87,95 Among infiltrating gliomas arising in midline structures, including both children and adults, 1 study found that up to 64% of tumors harbored H3 K27M mutations.107 At present, H3 K27M-mutant diffuse gliomas arising in midline sites are graded as WHO grade IV tumors, as they are associated with poor outcomes despite morphologic heterogeneity.2 In the pediatric population, ∼80% of pontine tumors and 50% to 80% of thalamic and spinal cord tumors are H3 K27M-mutant; in adults up to 60% of diffuse midline gliomas are H3 K27M-mutant.69 The mutation-specific antibody that recognizes the mutant H3 K27M protein has become a critical component of the diagnostic work-up of diffuse gliomas.

Less than 5% of clinically diagnosed primary GBMs are IDH-mutant and most of these likely progressed from nonsymptomatic lower grade precursors.2,27,108 Nevertheless, the determination of the IDH mutation status is more relevant to the biological behavior of a GBM rather than the primary or secondary clinical designation.109 As expected from their evolution from grade II and III infiltrating astrocytomas, IDH-mutant GBMs frequently harbor TP53 and ATRX alterations; ∼85% of clinically designated secondary GBMs are IDH-mutant, with TP53 and ATRX mutations seen in 81% and 71%, respectively (Fig. 10).2,42,108 As these mutations occur early in gliomagenesis, additional alterations identified within IDH-mutant, secondary GBMs are likely acquired during biological progression and can serve as prognostic markers.33 Some characteristic CNAs have been observed in the progression of IDH-mutant GBMs, such as loss of chromosomes 9p (CDKN2A) and 10q (PTEN) (Fig. 10).33 Other potential mechanisms of progression of IDH-mutant GBMs include activation of the MYC and RTK/RAS/PI3K pathways as well as epigenetic alterations.33 Secondary IDH-mutant GBMs contain the highest number of alternating, intrachromosomal breakpoints (chromothripsis) and increasing CNAs and genomic instability have been associated with rapid progression of IDH-mutant astrocytomas, as have the G-CIMP-low subset.68,86,91

FIGURE 10

FIGURE 10

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Key Points: Glioblastoma

  • IDH-mutant GBMs arise in younger patients, have improved survival and have distinct genetic alterations compared to their IDH-wt counterparts
  • Characteristic genetic alterations of IDH-wt GBM include TERT-p mutations, +7/-10, homozygous CDKN2A deletions, amplification events including EGFR, PDGFRA, CDK4, and MDM2, and mutations of PTEN, NF1, and RB1
  • Cogains of chromosomes 19 and 20 have been associated with longer survival in IDH-wt GBM
  • Variants of IDH-wt GBM include gliosarcoma, giant cell GBM, and epithelioid GBM, each with distinct histologic findings or molecular-genetic signatures
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PEDIATRIC GLIOMAS ARE CLINICALLY AND GENETICALLY DISTINCT

In the pediatric population, low-grade neuroepithelial tumors usually exhibit clinical and radiologic findings distinct from those of adults, yet they may share morphologic similarities. Pilocytic astrocytomas (PA; WHO grade I) are the most common brain tumor in children and are commonly found in the cerebellum, followed by the cerebral hemispheres, deep midline structures, optic pathway, brainstem, and spinal cord.110 When they involve the temporal lobe, they are often associated with seizures, as are other low-grade tumors of childhood, including gangliogliomas (WHO grade I), dysembryoplastic neuroepithelial tumor (DNET, WHO grade I) and PXA (WHO grade II or III).2 The presence of Rosenthal fibers or eosinophilic granular bodies in the context of a low-grade glioma with a biphasic appearance is characteristic of PA, although numerous other morphologies are observed, including those resembling oligodendroglioma and those that are well differentiated and highly fibrillar throughout. In the cerebellum and to a lesser degree in other locations, the KIAA1549:BRAF fusion resulting from a tandem duplication in the chromosome 7q34 region is a defining genetic event and present in >70% of PAs (Fig. 11).111 PXA is a well-circumscribed or poorly infiltrating temporal lobe-predominant glioma that shows a combination of spindle-shaped cells, xanthomatous cells and pleomorphic, multinucleated astrocytes along with eosinophilic granular bodies. The presence of a BRAF V600E mutation is supportive, as they are present in 60% to 70% of the cases (Fig. 12).112 BRAF V600E mutations also frequently occur in other pediatric tumors including PAs (5% to 10%) gangliogliomas (20% to 40%), diffuse astrocytomas (23% to 43%), and DNET (30%).38,113,114

FIGURE 11

FIGURE 11

FIGURE 12

FIGURE 12

Most low-grade neuroepithelial tumors of childhood are characterized by a singular dominant somatic genetic event that affects protein coding, including genes such as NF1, RAF, or RAS, fibroblast growth factor receptor 1 (FGFR1), and V-Myb avian myeloblastosis viral oncogene homolog (MYB) or in its homolog, MYBL1. Typically such solitary alterations are mutually exclusive.38 Other alterations involve the genes encoding the neurotrophic tyrosine receptor kinase 2 (NTRK2), KRAS, the receptor tyrosine kinase adapter tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), and BRAF.113 Activation of the MAPK/ERK signaling pathway, a primary driver of pediatric low-grade gliomas, is mediated by alterations in BRAF, FGFR1, and NTRK gene family.38,115,116 In a study of 249 pediatric low-grade gliomas that included multiple histologic entities, 90% showed recurrent somatic alterations and 83% showed rearrangements or structural alterations.117

Several studies have pointed to important roles for MYB/MYBL1, FGFR1, and BRAF V600E alterations in pediatric low-grade gliomas and suggest a relationship between them and tumor histology.38,39,93,114,118,119 Qaddoumi et al118 reported a high frequency of FGFR1 alterations in tumors dominated by round, regular banal tumor cells (“oligodendroglial-like” morphology), including 82% of DNETs and 40% of diffuse oligodendroglial tumors. Tumors with astrocytic differentiation and “diffuse” growth patterns were characterized by MYB alterations—41% of pediatric diffuse astrocytomas showed MYB structural rearrangements and 87% of angiocentric gliomas showing the specific MYB-QKI fusion, a strong example of phenotype-genotype linkage. The MYB-QKI fusion appears to be a specific and solitary driver event promoting tumorigenesis in angiocentric gliomas through 3 mechanisms: generation of the oncogenic fusion protein; enhancer translocation driving MYB-QKI expression; and hemizygous loss of the tumor suppressor QKI.117 These findings clearly delineate the dissimilarities among pediatric and adult low-grade infiltrating gliomas, as the hallmark IDH mutations of adult lower grade diffuse gliomas are rare in the pediatric population and the mutations in the pediatric diseases are not typically seen in adults.39,114 TP53 mutations are similarly uncommon in pediatric diffuse gliomas (<5%).114 Most importantly, the IDH-wt designation in pediatric infiltrating low-grade gliomas does not necessarily imply a more biologically aggressive behavior as their rate of progression is significantly lower than histologically similar tumors arising in adults.39,118,120

Pediatric-type oligodendrogliomas represent a diagnostic challenge, as they can be histologically indistinguishable from their adult counterparts, yet do not harbor the defining IDH mutations and 1p/19q codeletion. Hence, pediatric oligodendrogliomas are not enriched with a unifying molecular signature. In a study of 50 cases of pediatric oligodendroglioma, only 18% had an IDHR132H mutation and 25% had 1p/19q codeletion with a tendency for those harboring these alterations (“adult-type” oligodendroglioma) to occur in older children and adolescents.120 In addition, nearly half of low-grade pediatric oligodendrogliomas lacked gross somatic cytogenetic alterations.121 As described above, FGFR1 alterations are more frequent in pediatric oligodendrogliomas, but occur in less than half.38,120 BRAF alterations are uncommon, which contrasts to diffuse leptomeningeal glioneuronal tumor (known also as disseminated oligodendroglial-like leptomeningeal tumor), a recently codified entity in the revised fourth edition of the WHO Classification that harbors concurrent KIAA1549:BRAF gene fusions and chromosome 1p deletions.2,8,121,122

The high-grade gliomas arising in childhood comprise a heterogenous group that are molecularly distinct from their adult counterparts, and often harbor specific biological drivers and potentially actionable alterations.116,123–126 In distinction from adults, pediatric high-grade gliomas nearly always arise de novo and only rarely are the result of progression of a lower grade lesion.114 In a study of 127 pediatric high-grade gliomas that included diffuse intrinsic pontine gliomas (DIPG) and non–brainstem gliomas, mutations targeting the RTK/RAS/PI3K pathway, histone modification, or chromatin remodeling and cell cycle regulation were, respectively, found in 68%, 73%, and 59% of cases.123 In this same study 47% had gene fusions, with recurrent NTRK fusions present in 40% of non–brainstem high-grade gliomas in children below 3 years of age.123 Compared with adult GBM’s, TERT-p mutations are much less frequent in childhood (∼3%).95,123

DIPG represents a specific form of pediatric high-grade glioma, typically occurring between 6 to 7 years of age and with a dismal median survival of 10 months.127 H3 K27M mutations are present in over 70% to 80% and PDGFRA amplifications are noted in 28% to 36%.127 Recurrent gain-of-function missense mutations in ACVR1 (also known as ALK2) are present in up to 32% of pediatric DIPGs and can co-occur with H3 K27M mutations.123,128 IDH mutations are not present in DIPGs.127,129 In contrast to the pediatric counterparts, adult brainstem infiltrating gliomas occur less frequently and have a better outcome.129

Korshunov and colleagues recently performed an integrated, large scale genomic and epigenetic analysis of 202 pediatric GBMs which unexpectedly showed that 20% displayed methylation profiles similar to either low-grade gliomas or PXAs, had a better OS and were enriched for PXA-associated molecular alterations including BRAF V600E mutations and homozygous CDKN2A deletions. The remaining 162 pediatric GBMs stratified into the following 4 subgroups: IDH1-mutant (6%), H3.3 G34-mutant (15%), H3.3/H3.1 K27-mutant (43%), and those GBMs that were wt for H3 and IDH (36%).124 Further studies of the heterogenous group of H3-/IDH-wt pediatric GBMs revealed 3 biologically and clinically distinct molecular subtypes with different genomic and epigenetic signatures and these were enriched for MYCN, PDGFRA, or EGFR amplifications.130

Mutations within the N-terminal tail of the histone variants H3.3 (encoded by the H3F3A and H3F3B genes), or H3.1 (encoded by the HIST1H3B and HIST1H3C genes) were first identified within midline pediatric diffuse astrocytomas but more recently have also been recognized in midline tumors of adolescents and adults.69,124,131 Two specific histone mutations in H3.3 are mutually exclusive with IDH mutations; 1 is present at amino acid 27 resulting in a substitution of lysine for methionine (K27M) and the second at position 34 resulting in a substitution of glycine for either arginine or valine (G34R/V).69,132,133 H3 K27M mutations lead to a global reduction of H3K27me3 which in combination with DNA hypomethylation are the major driving forces behind the gene expression pattern conferred by the mutation.134 Reduced H3K27me3 levels can be noted by IHC in those gliomas with H3 K27M mutations.134

H3 K27M mutations are strongly aligned with diffuse gliomas of the midline of younger children, with the classic presentation in the pons (DIPG) or thalamus. In this setting, H3 K27M mutations correlate with malignant behavior and are codified as diffuse midline gliomas, H3 K27M-mutant, WHO grade IV.2,135 The H3F3A G34R/V variant occurs in up to one third of cerebral hemispheric pediatric high-grade glioma and is typically noted in adolescents and young adults. Although almost always ultimately fatal, the prognosis associated with the G34R/V variant is not quite as dire as that of the K27M variant.39,69,116,127,132,133,136 TP53 and ATRX mutations occasionally co-occur with H3.3 mutations and have the highest correlation in G34R/V-mutants.95,133 H3 K27M mutations have more recently been described in high-grade astrocytomas arising in locations beyond the thalamus and brainstem, including those of third ventricle, cerebellum, pineal region, and the spinal cord in the pediatric and young adult population, further supporting the associations with younger age, aggressiveness, and midline preference.69,107,137 Another alteration affecting chromatin organization includes SETD2 mutations, a gene encoding a methyltransferase specific to lysine 36 (H3K36) of the histone H3 tail. Approximately 15% of pediatric and 8% of adult high-grade infiltrating astrocytomas arising in the cerebral hemispheres harbor SETD2 loss-of-function mutations that frequently co-occur with ATRX mutations.68,69,138

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Key Points: Pediatric Gliomas

  • Pediatric gliomas have distinct clinical behaviors and genetic profiles, often harboring a single driver event. IDH mutations are rare
  • Well-circumscribed or poorly infiltrative glial or glioneuronal neoplasms that arise in the pediatric population often carry BRAF V600E or BRAF fusions
  • H3 K27M mutations define the entity of “Diffuse midline glioma, H3 K27M-mutant, WHO grade IV” and occur in DIPG and other diffuse gliomas arising in the midline
  • High-grade gliomas with H3F3A G34R/V mutation occur in the cerebral hemispheres of adolescents
  • Other molecular-genetic alterations that commonly occur in pediatric gliomas include FGFR1, MYB/MYBL1, NTRK, and ACVR1 genes
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INTEGRATIVE DIAGNOSES AS THE NEW STANDARD OF PRACTICE IN SURGICAL NEUROPATHOLOGY

The elucidation of the genomic underpinnings of CNS neoplasia has led to the development of an array of biomarkers for robust and reproducible glioma classification. In addition to its diagnostic value, molecular profiling can also identify mutations that involve the germline or are therapeutically actionable.139 The classification of infiltrating gliomas has evolved in parallel with technologically advanced testing and bioinformatics platforms. Many biomarkers have been developed for routine use in diagnostic neuropathology and include IHC, cytogenomic testing platforms, and glioma-tailored NGS, among other molecular-genetic tests.140 Incorporating these test results into integrative diagnoses has markedly improved intraobserver and interobserver variability.9

Depending on the gene and its specific type of alteration, testing can be performed by IHC, FISH, or cytogenomic microarray, focused or high-throughput sequencing technologies, or multiplexed platforms. Gene sequencing is becoming more widely available and can be accomplished with a focused, single gene approach, a targeted gene panel, or whole exome or whole genome approach. Several authors have reported excellent results with customized glioma-tailored NGS panels capable of detecting single nucleotide variations, fusions, and CNAs with substantial concordance when compared with more traditional single biomarker methods.139,141,142 From a diagnostic perspective recurrently altered genes of interest include, but are certainly not limited to, IDH1, IDH2, TP53, ATRX, CIC, FUBP1, PIK3CA, PIK3R1, TERT-p, NOTCH1, DAXX, CDKN2A, EGFR, PTEN, NF1, RB1, BRAF, MET, MYB, MYBL1, MYC, CDK4, CDK6, MDM2, MDM4, KRAS, NRAS, FAT, PTPN11, FGFR1, FGFR3, NTRK, ACVR1, H3F3A, HIST1H3B, PDGFRA, and SETD2. It is fully expected that in the era of precision and personalized medicine NGS will become increasingly available for routine diagnostic neuropathology.113

As the quality and quantity of CNAs across glioma subtypes tend to correlate with classification, grade, progression, and prognosis, MIP array is diagnostically valuable (Fig. 5).4,59,143,144 Of note, FISH is also a widely available and commonly used method that assesses CNAs at a single locus and its clinical utility in diagnostic neuropathology has included detection of EGFR amplifications and deletions of PTEN in high-grade astrocytomas, for example, and 1p/19q codeletions in oligodendrogliomas.13 However, FISH is able to document only focal deletions targeted by specific probes and as whole-arm losses of 1p and 19q have become definitional for oligodendrogliomas it is expected that platforms assessing CNAs across the whole genome will increasingly become more available. As described above, a “false positive” detection rate of ∼6% is expected when using FISH as a marker for 1p and 19q codeletions in the setting of genomic instability in high-grade astrocytomas and this pitfall has been highlighted by several authors.52,145

One of the typical signatures of IDH-wt GBMs is the gain of chromosome 7, loss of chromosome 10, and loss of chromosome 9p, and this subset has been shown to have a poor prognosis (Fig. 7).143 This signature is especially informative in the setting of infiltrating IDH-wt astrocytomas WHO grades II and III, in which whole genome assessment of CNAs allows the identification of hallmark genetic alterations of IDH-wt primary GBM, correlating with a more aggressive behavior.87,146 In addition to the +7/-10 signature, EGFR and MYB amplifications and TERT-p, BRAF, and H3F3A mutations may allow for prognostic stratification of IDH-wt diffuse lower grade gliomas.87,146,147 Recurrent deletions occurring in IDH-wt GBMs are commonly seen at the following regions of interest (ROI): 17q11.2, 10q23.31, 9p21.3 and 13q14.2, corresponding to NF1, PTEN, CDKN2A/CDKN2B, and RB1 genes, respectively.2,95,97,99–101 More recently, Cimino et al71 also demonstrated that IDH-wt astrocytomas can be separated into prognostic groups on the basis of chromosome 1 gain, chromosome 19 gain, and CDK4/MDM2 coamplification. The recent description of whole chromosomal 19/20 cogain in long-term survivors with IDH-wt GBM opens the possibility that such a signature of better outcome can be integrated into stratification algorithms.102 Interestingly, chromosome 19/20 cogain is almost mutually exclusive with other CNAs, including deletions of chromosomes 13, 14, and 15.102

Among IDH-mutant gliomas, chromosome 7q gains are an early event, and appear to be specific to astrocytomas, as they are mutually exclusive with 1p/19q codeletion.143 IDH-mutant gliomas with TP53 mutations typically have at least one of the following CNAs: +7q, +8q, -9p, -11p, or +12p and these CNAs are associated with a poor prognosis and/or progression as they relate to gains or losses of MET, MYC, CDKN2A, CDKN1C, and KRAS, respectively.148 However, IDH-mutant astrocytomas are highly heterogenous regarding CNAs and no recurrent alteration at a frequency beyond 50% of all cases has been identified.71 As molecular alterations for subgroup risk-stratification continues to emerge, Cimino et al71 found that, independent of WHO grading, IDH-mutant astrocytomas could be separated into prognostically significant subclasses on the basis of CNAs that included CDK4 amplification, CDKN2A deletion, and chromosome 14 loss. IDH-mutant GBMs have an increased incidence of chromothripsis when compared with their precursor lesions and to IDH-wt tumors of all grades.91

Amplification events are often prognostically significant and are viewed as potential therapeutic targets in both pediatric and adult glioma.71,124 In IDH-wt GBMs, frequent amplifications include the following ROI: chromosome 7p11.2, 7q21.2, 7q31.2 (EGFR/CDK6/MET genes, respectively); chromosome 12q14.1 and 12q15 (CDK4/MDM2 genes, respectively); and chromosome 4q12 (PDGFRA).99 PDGFRA amplifications are also noted with increased frequency in higher grade IDH-mutant gliomas and appear to represent an independent prognostic factor in de novo IDH-mutant GBMs.149 Another important ROI 7q34 which includes the BRAF gene.

Many of the genetic events that drive gliomagenesis and represent diagnostic biomarkers can be assessed by IHC, a widely available and cost-efficient method with fast turn-around time. IHC for IDH1R132H, p53, ATRX, K27M, and BRAF (VE1) are commonly used for glioma classification; other immunostains including CIC, FUBP1, EGFR, EGFRvIII, and O6-methylguanine-DNA methyltransferase (MGMT) are available but are either used to lesser extents, are not recommended for clinical use or are not available for clinical use.28,87,131 A recent review by Tanboon et al131 addresses the utility of IHC for the diagnosis of gliomas.

A routine panel for the initial diagnostic work-up of diffuse gliomas involves IHC for IDH1R132H, p53, and ATRX. IDH mutations are not only critical for distinguishing between clinically distinct subtypes of gliomas but also to distinguish between glioma and florid reactive gliosis.35 Immunoreactivity for IDH1R132H IHC is strong evidence in favor of a diagnosis of IDH-mutant infiltrating glioma. The R132H variant of IDH1 mutation accounts for >90% of all IDH mutations and a highly sensitive and specific monoclonal antibody that recognizes the mutant protein in the cytoplasm is widely available.32,97 Gene sequencing analysis of IDH1 codon 132 and IDH2 codon 172 is recommended in the event of a negative or indeterminate result with IDH1R132H immunostain given the possibility of a non-R132H IDH mutation (<10% of cases) and the clinical significance of a IDH-wt designation in the context of an infiltrating glioma.13,131 However, it has recently been suggested that IDH sequencing analysis need not follow a negative IDH1R132H immunostain in GBMs arising in patients older than 55 years due to the rarity of variant mutations in older patients.2,3,150,151

In the TCGA analysis of diffuse gliomas, nearly two thirds of IDH-mutant WHO grade II and III infiltrating gliomas had intact 1p/19q and would qualify as IDH-mutant astrocytomas; of these 94% had mutations in TP53 and 86% had inactivation of ATRX.10 In the setting of an IDH mutation, the detection of strong p53 by IHC can be used as a surrogate for TP53 mutations and in support of an astrocytic lineage (Fig. 1), recognizing, however, that not all TP53 mutations manifest as strong nuclear immunoreactivity (Fig. 2). TP53 mutation leads to reduced degradation of the protein and nuclear accumulation of both mutant and wild-type gene products.13 Strong nuclear p53 IHC positivity in >10% tumor nuclei is a predictor of TP53 mutations, but is best evaluated in the context of morphology and results of other IHC in the diagnostic panel such as ATRX.131 Inactivating ATRX alterations commonly co-occur with TP53 mutations in the setting of IDH-mutant astrocytomas and these have become part of the molecular diagnostic algorithm for the refinement of diffuse glioma lineage.10 ATRX mutation results in a truncated protein and in abrogated protein expression, leading to loss of nuclear immunoreactivity of ATRX (Figs. 1, 2).13,28 When assessing ATRX status by IHC it is important to evaluate the immunoreactivity of non-neoplastic nuclei, such as those of endothelial cells and neurons, as an internal positive control.131

The entity of “diffuse midline glioma, H3 K27M-mutant” is now codified in the revised fourth edition of the WHO Classification, a name that highlights the tight coupling of this mutation to a specific form of high-grade glioma.2 H3 K27M mutations involving either the H3.3 or H3.1 histones can be detected by strong nuclear staining using K27M IHC with a sensitivity and specificity of approaching 100% (Fig. 6).107 The recent description in 2 cases of subclonal H3F3A K27M mutations accompanied by a mosaic expression pattern will require further studies to elucidate its biological and prognostic implications.152 Global reductions or loss of H3K27me3 nuclear staining are seen in H3F3A K27M-mutant tumors; however, its assessment is considered subjective and K27M IHC is a superior biomarker to detect such mutations.153 The mutation-specific BRAF V600E (VE1) IHC represents another clinically useful biomarker with a sensitivity of 100% and a specificity of 98% for the mutation when strong cytoplasmic immunoreactivity is considered as positive (Fig. 9).131 The respective frequencies of BRAF V600E mutations in PXAs, gangliogliomas, DNETs, and epithelioid GBM underscores its diagnostic utility.

In addition to the many genomic alterations that are diagnostically and prognostically relevant, methylation of the promoter for MGMT is one of the most clinically relevant prognostic and predictive biomarkers in adult GBM. MGMT is a DNA repair enzyme capable of restoring guanine from O6-methylguanine induced by alkylating agents commonly used to treat GBM such as temozolomide, thereby hampering its chemotherapeutic effects.95,97 Occurring in ∼40% of GBMs, MGMT promoter methylation correlates with an improved response to therapy and a survival benefit.97 MGMT promoter methylation is commonly assessed by methylation-specific polymerase chain reaction, the only prospectively validated method.13,97,154,155 Others have the utility of pyrosequencing assays.156 MGMT IHC is currently not recommended for clinical practice as MGMT expression has only modest correlation with MGMT promoter methylation status.97,131,157

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EPENDYMOMAS: MOLECULAR PROPERTIES DISTINGUISH SUBTYPES BASED ON LOCATION AND AGE

Ependymomas (EPN) are noninfiltrative gliomas that can arise in patients of any age. Their most common locations are in the posterior fossa (PF), spinal cord, and supratentorial (ST) compartment; they rarely arise outside the CNS. Perivascular pseudorosettes, true rosettes, and ependymal canals represent classic histologic features and well-defined histologic variants include papillary, clear cell, and tanycytic subtypes.2 WHO grade I myxopapillary EPN and subependymomas (SE) are members of the EPN family with specific clinicopathologic presentations and are codified separately.2 Biological behaviors of EPN vary tremendously depending on patient age and location, and the current grading schemes that are intended to stratify risk, especially those for grades II and III, are not optimal.2,158 It is now clear that EPNs are not all created equally, as marked genomic heterogeneity has been recognized across anatomic sites and patient ages, in keeping with known differences in behavior, despite similar histologies.158 The evolving molecular signatures of EPN represent a dramatic improvement in understanding and classification.

Several cytogenetic alterations have been described in EPN: gains of chromosome 1q have been consistently associated with worse outcomes in childhood PF EPN and loss of chromosome 9p (CDKN2A) was equally associated with aggressive behavior in ST tumors, leading to cytogenetic risk-stratification algorithms.159–162 Loss of chromosome 22q (NF2) characterizes the majority of spinal cord EPN, which is not surprising as NF2 mutations are also known to occur in this subset.162 Chromosome 22 loss without NF2 mutations have also been noted in group B PF EPN.163 PF EPN exhibit low mutation rates and gene amplification events are almost totally absent.162,164 Overall, genomic alterations including single nucleotide variations, insertions/deletions and focal CNAs are rare in EPN; however, structural variations are enriched in ST EPN.165

An important group of ST EPN is characterized by a C11orf95-RELA-fusion, an oncogenic driver event mediated through chromothripsis of chromosome 11q13.1 and resulting in NF-kB signaling activation (Fig. 13).165 This specific fusion was the most common recurrent genetic alteration in EPN, present in 70% of ST tumors. Neither chromothripsis of chromosome 11 nor the C11orf95-RELA-fusion has been documented in PF EPN.165 Gene and protein expression profiling demonstrated elevated levels of CCND1 and L1CAM in C11orf95-RELA-fused ST EPN, both in direct relationship with NF-kB activation, and L1CAM positivity by IHC correlates well with the fusion event.165 More recently, a subset of ST clear cell EPN with distinct clinicopathologic features was shown to be enriched for C11orf95-RELA-fusions.166

FIGURE 13

FIGURE 13

The molecular subgrouping of EPN across anatomic compartments has been shown to be superior to histologic grading in terms of risk-stratification. Nine molecular subgroups of EPN have been recognized by genome-wide methylation and gene expression profiling, each with varying clinical and histopathologic features.162 Three discrete groups were identified in each of the anatomic compartments as follows: (I) the ST compartment includes EPN with either RELA or YAP1 fusions, as well as a third group of SE that involve the lateral ventricles (in short, ST-EPN-RELA, ST-EPN-YAP1, ST-SE, respectively); (II) the PF included group A and B EPNs as well as SE that involves the fourth ventricle (in short, PF-EPN-A, PF-EPN-B, and PF-SE, respectively); (III) the spinal (SP) compartment included classic EPN in addition to myxopapillary ependymoma (MPE) arising in the region of the cauda equina and SE (in short, SP-EPN, SP-MPE, and SP-SE, respectively).162 Numerous important clinicopathologic associations were readily apparent as nearly all cases of PF-EPN-A occurred in children aged below 8-years old (median age, 3 y), whereas the vast majority of PF-EPN-B cases arose in older children and young adults.162,163 Approximately 75% of the RELA-fused EPN occurred in children with a median age of 8 years and the remaining quarter arose in adults. MPE and SE across all compartments occurred only in adults.162

Loss of chromosome 6 is frequently observed in SE (SP-SE and PF-SE) and PF-EPN-B tumors.162 Tumors in the PF-EPN-B class exhibit a high degree of genomic instability with frequent whole chromosomal or whole chromosomal arm gains and losses, whereas PF-EPN-A exhibit largely balanced genomes albeit with a propensity for chromosome 1q gain, a known independent factor of poor prognosis.162,163 The oncogenic driver of the ST-EPN-RELA tumors is the C11orf95-RELA-fusion and these are also enriched for homozygous CDKN2A deletions. YAP1-MAMLD1 and YAP1-FAM118B fusions were identified in the ST-EPN-YAP1 class of EPN and they also had focal CNAs of chromosome 11 around the YAP1 locus rather than the chromothripsis event characteristic of ST-EPN-RELA tumors.162 Poor clinical outcomes have been observed for ST-EPN-RELA and PF-EPN-A classes, which together represent up to two thirds of all EPN, with 10-year PFS and OS ∼50% and 20%, respectively.162 Witt et al163 similarly reported contrasting outcomes for PF EPN subgroups, with 5-year of PFS and OS of 47% and 69%, for group A tumors and 79% and 95% for group B tumors, respectively.

As distinct methylomes are observed in PF-EPN-A and PF-EPN-B tumors, others have attempted to correlate this finding with specific methylation markers. Using mass spectrometry-based technology, 1 group identified 3 genes with CpG hypermethylation in PF-EPN-A but not in PF-EPN-B, demonstrated distinct H3K27me3 signatures that could robustly stratify them and concluded that PF-EPN-A have a CIMP phenotype.164 Additional studies indicated that H3K27me3 immunostaining was significantly reduced in PF-EPN-A compared with PF-EPN-B tumors and suggested its use as an independent biomarker of poor prognosis.167,168 DNA methylation analysis also demonstrated that PF-EPN-A exhibited similarities to H3 K27M-mutant gliomas.167 Interestingly, 2 childhood PF-EPN-A harboring H3 K27M mutations (confirmed by NGS) that showed strong nuclear H3 K27M IHC and reduced nuclear labeling with H3K27me3 IHC where recently reported.169 Other markers are being pursued for more reliable risk-stratification.170–172

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Key Points: Ependymoma

  • Nine molecular subgroups of EPN with specific anatomic and clinical correlates have been described
  • RELA-fusion positive EPN occurs in the ST compartment and are clinically aggressive. L1CAM IHC is a useful marker
  • In the PF, PF-EPN-A have a poor prognosis compared with PF-EPN-B, with the former frequently showing chromosome 1q gain and reduced H3K27me3 immunoreactivity
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MEDULLOBLASTOMAS ARE BOTH HISTOLOGICALLY AND MOLECULARLY DEFINED

By definition, medulloblastoma is a WHO grade IV embryonal neoplasm arising within the cerebellum or dorsal brainstem, often growing into the fourth ventricle.2 The vast majority of our understanding of medulloblastoma has derived from those arising in infancy, childhood, and adolescent populations, yet adult medulloblastomas also occur and may behave differently.173–177 Four histologic subtypes of medulloblastomas have been recognized and their histopathologic classification is retained in the WHO Classification because of its time honored utility in the clinical setting.2 These include the (I) classic, (II) desmoplastic/nodular, (III) extensive nodularity, and (IV) large cell/anaplastic medulloblastoma. Within the past decade integrative, multiplatform analyses of transcriptomic, genomic, and epigenetic profiles have paved the way for a new molecular subclassification schema that currently includes 4 genetically defined consensus categories, each with distinct biological behaviors and independent prognostic value: (I) wingless (WNT)-activated, (II) Sonic Hedgehog (SHH)-activated, (III) group 3, and (IV) group 4 medulloblastomas.177–179 Group 3 and group 4 medulloblastomas are often considered together clinically as non-WNT/non-SHH medulloblastomas, since they are not easily segregated using standard biomarker approaches. Molecular subgroups appear to remain stable during disease progression and metastatic spread; however, subgroup-specific patterns of metastatic dissemination have been recently recognized.180,181 Although phenotype-genotype correlations continue to expand, it is widely accepted that nearly all WNT-activated medulloblastomas exhibit the classic morphology and that the desmoplastic/nodular medulloblastomas and medulloblastomas with extensively nodularity are almost always of the SHH-activated subgroup. However, approximately half of SHH-activated medulloblastomas will not exhibit desmoplastic/nodular histology. The large cell/anaplastic variants typically align with either the SHH-activated or group 3 molecular subgroups and the majority of non-WNT/non-SHH medulloblastomas exhibit classic morphology.178,179

WNT-activated medulloblastomas (Fig. 14) account for ∼10% of all cases, and up to 90% have CTNNB1 somatic mutations.182,183 Medulloblastomas that arise in the context of germline APC mutations (Turcot syndrome) also belong in this category, although these are uncommon.178,182 Monosomy of chromosome 6 is the hallmark CNA of WNT-activated medulloblastomas and is seen in about 85% of cases, whereas it is a rare occurrence other subgroups.178,179,182,184–186

FIGURE 14

FIGURE 14

SHH-activated medulloblastomas (Fig. 15) account for ∼30% of all cases and can be further stratified on the basis of TP53 mutations (TP53-mutant vs. TP53-wt). Multivariate analysis showed that TP53 status was the most important risk factor for this molecular subgroup and, when present, the possibility of germline mutation (Li-Fraumeni syndrome) should be considered do to its implications both for the patient and family members.187,188 TP53 mutations also occur in WNT-activated medulloblastomas, but only within SHH-activated medulloblastomas do they confer a worse prognosis. The TP53-mutant subset correlates with histologic anaplasia, a high rate of chromothripsis and tetraploidy, chromosome 17p loss, and amplification of MYCN.186,187,189 Other amplification events in SHH-activated medulloblastomas include GLI2, CCND2, MYCL1, PPM1D, YAP1, and MDM4.183

FIGURE 15

FIGURE 15

Mutations in PTCH1, SUFU, and SMO, as well as other genes associated with the SHH signaling pathway, characterize SHH-activated medulloblastomas.177,182 Losses of PTCH1 (chromosome 9q.22) are present in up to two thirds, but can also be noted in group 3.179,184 Germline mutations of the PTCH gene (Gorlin syndrome) are a predisposition for SHH-activated medulloblastomas in young children and genetic counseling may be warranted.178,182,188 Heterozygous TERT-p mutations are similarly overrepresented in SHH-activated medulloblastomas arising in adults, though they occur to a lesser degree in other subtypes.189,190

Loss of chromosome 17p is present in 25% of SHH-activated medulloblastomas and is associated with unfavorable outcomes. Gain of chromosome 3q, loss of 10q, and GLI2 amplifications have also been associated with poor outcomes in this subset, albeit with some contradicting results.184,185 Focal deletions of PTEN occur exclusively in pediatric SHH-activated medulloblastomas, contributing to PI3K pathway deregulation.182,183 PI3K/AKT pathway activation in adult SHH-activated medulloblastoma has also been associated with poor outcomes.189

The non-WNT/non-SHH medulloblastomas comprise 60% of all medulloblastomas. Group 3 tumors (∼20% of all cases) are characterized by MYC amplifications, whereas chromosome 17 alterations (predominantly an isochromosome 17) are found in ∼80% of group 4 medulloblastomas (∼40% of all cases).179 Within group 3 medulloblastomas PVT1-MYC fusions have been described in at least 60% of MYC-amplified cases and are thought to arise from chromothripsis of chromosome 8q.182,183 Among group 3 medulloblastomas, chromosome 1q gain, 17p loss, 17q gain, isochromosome 17q, and MYC amplification are associated with worse outcomes.185 Among group 4 medulloblastomas, most females exhibit loss of 1 copy of the X chromosome.182 MYCN and CDK6 amplifications also occur in group 4 medulloblastomas.184 Gains of chromosome 17 and loss of chromosome 11 have been associated with better outcomes in group 4 medulloblastomas.185

The current integrative diagnosis of medulloblastoma provides clinicians with a high degree of information that predicts outcome relatively well. Risk-stratification algorithms had historically included clinical variables such as patient demographics, extent of resection, presence of metastasis, histology, but now add molecular subtyping as well.188 Although the “gold standard” molecular subclassification can be achieved by technologically advanced platforms including sequencing, gene expression, transcriptomic and epigenetic analysis, IHC is a widely available tool that allows for a more practical approach in the routine clinical practice. Immunohistochemical work-up is often used to exclude other possible diagnosis including atypical teratoid/rhabdoid tumors (AT/RT) and embryonal tumor with multilayered rosettes (ETMR), but also to assign medulloblastomas within molecular categories. Nuclear immunoreactivity of beta-catenin IHC is a surrogate of WNT pathway activation; some authors have stressed a patchy pattern suggesting a cutoff of nuclear immunopositivity in 5% of tumor cells, whereas others have emphasized a “blanket appearance” of cytoplasmic and nuclear positivity in the majority of tumor cells in WNT-activated medulloblastomas (Fig. 14).179,191 GAB1 and YAP1 IHC are also very useful for medulloblastoma subclassification as immunoreactivity for these markers is not seen in non-WNT/non-SHH medulloblastomas.179,191 Although GAB1 immunoreactivity is only observed in SHH-activated medulloblastomas, YAP1 immunoreactivity can be observed in both WNT-activated and SHH-activated medulloblastomas (Figs. 14, 15). Hence, GAB1, YAP1, and p53 IHC are of diagnostic and prognostic utility for SHH-activated medulloblastomas.187 IHC for OTX2 is diagnostically useful as OTX2 amplification and overexpression occurs in non-WNT/non-SHH medulloblastomas; hence, OTX2 IHC is negative in WNT-activated and SHH-activated medulloblastomas.178,192 In 1 algorithm of biomarker testing for medulloblastoma, an initial panel of IHC includes beta-catenin, GAB1, YAP1, and INI1. If the results confirm SHH-activated medulloblastoma, p53 IHC is performed. If p53 IHC is equivoval, TP53 analysis by NGS may be needed, particularly if there are worrisome features such as cytologic anaplasia. Cytogenomic microarrays also have diagnostic utility, as numerous CNAs relevant to medulloblastoma can be assessed concurrently. A more targeted approach utilizing FISH testing for key CNAs such as monosomy 6, cMYC and MYCN amplifications may be suitable.

Molecular classes of medulloblastoma have distinct clinical settings and biomarker-driven subclassification allows for optimal risk-stratification. WNT-activated medulloblastomas have the best prognosis and this patient population may benefit from less intensive treatment; they are associated with a 5- and 10-year OS of 95% in children.178,182,184 Group 3 carries the worst OS across all age groups.184 The current consensus regarding risk-stratification of childhood medulloblastoma also recognized the WNT subgroup and nonmetastatic group 4 medulloblastomas with whole chromosome 11 loss or whole chromosome 17 gain as low-risk tumors that may benefit from reduced therapy.188 A recent study of children with irradiated medulloblastoma found significantly improved PFS in group 4 patients compared with initial reports and further prospective and validations studies will be required to determine if deescalation of aggressive adjuvant treatment regimens might be considered.193 On the opposite end of the spectrum, the current consensus of high-risk patients includes those with metastatic group 3 medulloblastoma and TP53-mutant SHH-activated tumors.188 It is expected that heterogeneity within each subgroup will continue to be elucidated and that new prognostic and predictive markers will emerge.

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EMBRYONAL TUMOR WITH MULTILAYERED ROSETTES, C19MC-ALTERED: CHROMOSOME 19Q13.42 ALTERATIONS AS A UNIFYING AND DEFINING EVENT

As the historic term “CNS primitive neuroectodermal tumor (PNET)” has become discouraged, these undifferentiated and “primitive-appearing” neoplasms are now referred to as embryonal tumors by the WHO and are considered grade IV.2 Following extensive molecular analysis that included genome-wide DNA methylation and copy number profiling, a single diagnostic category emerged that included the following 3 previously codified malignancies: embryonal tumor with abundant neuropil and true rosettes (ETANTR), ependymoblastoma, and medulloepithelioma.194 These tumors are now collectively referred to as ETMRs, which are unified by the morphologic finding of embryonal tumor cells forming multilayered rosettes and by the highly specific focal amplification and fusion of chromosome 19q13.42 (Fig. 16). This region encodes the largest human microRNA cluster, C19MC, and the alteration is not present in other pediatric tumors. With this new definition, ETMR is regarded as a universally aggressive embryonal tumor with median OS around 12 months.194–199 These tumors can arise anywhere within the ST or, to a lesser extent, the infratentorial compartments and preferentially affect a very young patient population with the vast majority of cases occurring before 4 years of age.194,199 As the name implies, well-defined multilayered and/or pseudostratified rosettes (ependymoblastic and medulloepithelial rosettes) are key histologic features of the entity along with variable proportions of small undifferentiated blue cells and neuropil that contribute to its heterogenous histomorphologic spectrum.2,194

FIGURE 16

FIGURE 16

Upregulation and overexpression of LIN28A, an RNA-binding protein, is highly correlated with the C19MC alteration. Although not entirely specific, strong LIN28A cytoplasmic immunoreactivity is a fairly sensitive marker of ETMRs, with the important caveat that a smaller proportion of high-grade gliomas and AT/RTs can be positive, albeit not as strongly and diffusely in the majority of cases.194,199–201 Thus, LIN28A immunoreactivity alone may be insufficient to establish a diagnosis of ETMR and other embryonal neoplasms such as AT/RT need to be ruled out. The hallmark focal high-level amplification at 19q13.42, identified by FISH or cytogenomic microarray, is the most robust diagnostic test for this entity (Fig. 16).194 Complex rearrangements within the same locus as well as fusion of C19MC to the promoter of the brain-specific TTYH1 gene (TTHYH1:C19MC) can also contribute to tumorigenesis by mediating high expression of the oncogenic microRNA cluster.194,197

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ATYPICAL TERATOID/RHABDOID TUMOR: A HIGHLY AGGRESSIVE PEDIATRIC TUMOR DEFINED BY SMARCB1 LOSS-OF-FUNCTION ALTERATIONS

The hallmark alteration of AT/RTs, a WHO grade IV embryonal tumor, is deletions or loss-of-function mutations of SMARCB1 (BAF47/INI1/hSNF5) at chromosome 22q11.2 or to a much lesser extent SMARCA4 (BRG1) at chromosome 19p13.2.202–205 AT/RTs account for 1% to 2% of all pediatric CNS tumors and typically arises in the ST or infratentorial compartments of young children (< 3 years) with a third of the cases arising within the first year of life.206 The worst outcomes are observed in patients below 6 months of age. However, a recent Canadian AT/RT registry study of patients in their first year of life suggested that high-dose chemotherapy was associated with better OS when compared with conventional chemotherapy approaches (5-year OS of 52.0%±8.3% vs. 10.7±4.5%, respectively).206 A study of AT/RT patients that included a wider age range (0 to 18 y) reported a median OS of 14.3 months and a 5-year OS rate of 29.9%.207 As a standard therapy is yet to be defined and the risk-benefit of radiotherapy is further refined, variation in treatment modalities for AT/RT patients likely account for the wide range of clinical outcomes that are reported, although it should be noted that outcomes have improved in recent years.208,209 Screening for germline mutations in AT/RT should be considered when making the diagnosis, particularly in the very young, as roughly one third of cases arise in the setting of Rhabdoid Tumor Predisposition Syndrome.206,209

AT/RTs are known to be mimickers of other neoplastic disease and they exhibit marked histomorphologic heterogeneity due to their differentiation along distinct phenotypic lines (teratoid), a finding highlighted by an indiscriminate immunoprofile.203,204 A small blue cell population can be prominent within AT/RTs, requiring caution to avoid an erroneous diagnosis of medulloblastoma when the tumor arises in the PF; the above discussed biomarkers in conjunction with INI1 IHC are diagnostically helpful.203 Differentiation of AT/RT from choroid plexus carcinoma poses an equally difficult diagnostic dilemma on histomorphologic assessment do to their overlapping features; however, INI1 and Kir7.1 IHC are of great utility in this differential diagnosis.210–212 Tumor cells with eccentric nuclei, vesicular chromatin, and prominent nucleoli in conjunction with eosinophilic cytoplasm and globular inclusions (rhabdoid cells) are, however, typical of AT/RT and an important diagnostic clue (Fig. 17).

FIGURE 17

FIGURE 17

The revised fourth edition of the WHO Classification requires loss of SMARCB1 or, to a much lesser extent, SMARCA4, to render a diagnosis of AT/RT.2,213 Although AT/RT is a heterogenous disease, they are unified under this genetic signature. SMARCB1 is a tumor suppressor and a component of the SWI/SNF chromatin remodeling complex and when altered, genome-wide deregulation ensues, affecting cell signaling, growth, proliferation, and differentiation pathways.205,214 Other than loss of SMARCB1, the genome of AT/RTs is not complex and exhibits a low mutational load as compared with other aggressive malignancies.215 A small subset of AT/RTs with intact SMARCB1 have loss-of-function alterations in SMARCA4, a separate component of the SWI/SNF chromatin remodeling complex.213 These alterations can be reliably assessed with INI1 and BRG1 IHC, respectively. INI1 is ubiquitously expressed in normal tissues and the vast majority of AT/RTs will show complete nuclear loss.210,216 In addition, alterations at chromosome 22q11.2 harboring SMARCB1 can be detected by FISH or cytogenomic microarray. It is important to recognize that the spectrum SMARCB1-defficient tumors has expanded to encompass other non-RTs, including carcinomas and sarcomas outside the CNS, such as epithelioid sarcomas, epithelioid malignant peripheral nerve sheath tumors, renal medullary carcinomas, and cribriform neuroepithelial tumors, among others.214,217–219 In addition, 3 patterns of expression (complete loss vs. mosaic expression vs. reduced expression) have been recognized, adding an additional layer of complexity to these diagnostic categories.214 The current WHO Classification recommends classifying a tumor as “CNS embryonal neoplasm with rhabdoid features” if it is morphologically compatible with an AT/RT, yet shows intact INI1 and BRG1 protein expression.

Recent transcription, methylation, and copy number profiling have identified molecular subgroups of AT/RTs characterized by activation of distinct signaling pathways, a finding that may explain the clinical heterogeneity of these aggressive tumors.215,220,221 One subset showed upregulation of Notch signaling pathway genes, with consistently increased expression of ASCL1, a Notch regulator. This group tended to arise supratentorially and had improved 5-year OS when compared with ASCL1-negative tumors.220 The prognostic significance of ASCL1 (assessed with IHC) was independent of treatment protocols.220 In contrast, upregulation of genes involved in the bone morphogenetic protein signaling pathway have been associated with infratentorial tumors and worse outcomes.220 More recently, 3 epigenetically defined subgroups of AT/RTs with differences in patient ages, tumor location, and type and extent of SMARCB1 alterations were described: (I) ATRT-TYR (overexpression of melanosomal markers including the enzyme tyrosinase), (II) ATRT-SHH (overexpression of genes in the SHH pathway, such as MYCN and GLI2, as well as genes in the Notch pathway, including ASCL1), and (III) ATRT-MYC (overexpression of the MYC oncogene and HOX cluster genes).215 These 3 epigenetically defined subsets of AT/RT, each with distinct clinical characteristics, were subsequently corroborated and subgroup-specific therapeutic sensitivities were demonstrated.221 The emerging subclassificaton of AT/RTs, together with clinical risk factors, may allow for targeted therapies as well as therapeutic stratification.209

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OTHER UNCOMMON EMBRYONAL TUMORS CAN ARISE WITHIN THE CENTRAL NERVOUS SYSTEM

Embryonal tumors other than those described above can also rarely present as primary CNS neoplams, most often in childhood. They too exhibit small poorly differentiated cells (small blue cell tumors) and share similar biological behaviors including an increased propensity for metastatic leptomeningeal dissemination. The revised fourth edition of the WHO recognizes that tumors corresponding to this category include medulloepitheliomas lacking C19MC amplification, CNS neuroblastomas, and CNS ganglioneuroblastomas.2 After careful consideration of alternate entities, a diagnosis of “CNS embryonal tumor, NOS” may be warranted in the appropriate clinicopathologic context. Before rendering any of these diagnoses, molecular-genetic and biomarker testing should be performed to rule out other tumors, such as medulloblastoma, C19MC-altered ETMR, AT/RT, and high-grade glioma. In this setting IHC for GAB1, YAP1, LIN28A, L1CAM, INI1, IDHR132H, H3K27M, and BRAF V600E can offer a practical approach and be of diagnostic utility.

Recently, Sturm et al222 described 4 new CNS tumor entities derived from DNA methylation, transcriptomic, and copy number profiling of CNS-PNETs, which were each defined by recurrent genetic alterations and distinct clinical and histomorphologic features. They designated these subclasses as (I) CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2), (II) CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC), (III) CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1), and (IV) CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR) (Fig. 18).222 Although there is histologic variability within these genetically defined entities, astroblastoma-like features and EPN-like features have been appreciated for the CNS HGNET-MN1 and CNS HGNET-BCOR subclasses, respectively.222–224 As more specific biological entities emerge it will allow for more cohesive clinical trials and open the door to possible new targeted therapies.

FIGURE 18

FIGURE 18

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Key Points: Embryonal Tumors

  • Four molecular classes of medulloblastoma are recognized: (I) WNT-activated, (II) SHH-activated, (III) group 3, and (IV) group 4
  • Monosomy 6 and CTNNB1 somatic mutations are the hallmark alterations of the WNT-activated class. Beta-catenin IHC may aid in diagnosis
  • SHH-activated medulloblastomas often show desmoplastic/nodular morphology and these can be further stratified on the basis of TP53 mutations. IHC for GAB1, YAP1, OTX2, and p53 are useful diagnostically
  • The group 3 and group 4 medulloblastomas are collectively known as non-WNT/non-SHH medulloblastomas. They often harbor MYC amplifications and chromosome 17 alterations
  • The highly specific focal amplification and fusion of chromosome 19q13.42 define “ETMR, C19MC-altered” LIN28A IHC is contributory
  • Deletions or loss-of-function mutations of SMARCB1 (INI1) or SMARCA4 (BRG1) are required for the diagnosis of AT/RTs
  • Four discrete new entities are defined by recurrent genetic alterations and include CNS NB-FOXR2, CNS EFT-CIC, HGNET-MN1, and CNS HGNET-BCOR
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CHANGES IN UNCOMMON TUMORS OF THE CENTRAL NERVOUS SYSTEM

Advances in molecular profiling of primary CNS tumors have not been confined to the entities described above and significant findings have been reported for meningiomas, primary CNS melanocytic neoplasms, peripheral nerve sheath tumors, hematopoietic neoplasms, tumors of the sellar region, and other mesenchymal neoplasms (recently reviewed by Sahm et al225). Many of these remarkable changes are already reflected in the most recent edition of the WHO classification and are routinely used for diagnostic purposes. For example, the presence of NAB2-STAT6 fusions in most solitary fibrous tumors/hemangiopericytomas allows for the use of STAT6 IHC for definitive diagnosis. Similarly, GNAQ and GNA11 mutations confirm a diagnosis of primary CNS melanocytic neoplasms and exclude other diagnostic possibilities such as melanotic schwannoma (malignant melanotic schwannian tumor) or metastatic melanoma. Other uncommon neoplasms that arise within the CNS harbor mutations that may be targetable. For example, many cases of Erdheim-Chester disease will have a BRAF V600E mutations and inflammatory myofibroblastic tumor may have ALK and RET rearrangements, or much less commonly a TFG-ROS1 fusion as we recently showed (published in abstract form).226 In such cases, neuropathologists play a key role in uncovering genetic signatures of prognostic and predictive value.

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CONCLUSIONS

Neuropathologists, molecular pathologists, and clinicians now work closely together to provide optimal clinical care by utilizing molecular biomarkers for diagnostic and treatment purposes.227–230 Without doubt, the expanded molecular profiling of CNS tumors has improved our diagnostic algorithms, risk-stratification schemas and has opened the door to the development of targeted therapies.

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ACKNOWLEDGMENTS

The authors are appreciative of Jennifer E. Hauenstein, MS, from the Emory Oncology Cytogenetics laboratory who kindly provided all the MIP array illustrations. The authors also acknowledge Brent A. Orr, MD, PhD (St. Jude Children’s Research Hospital) for his collaboration and the courtesy of providing the FISH images shown in Figures 13 and 16. The authors thank Arie Perry, MD, and David Solomon, MD, PhD (University of California, San Francisco) for performing and analyzing the NGS molecular studies that led to the integrative diagnosis of the CNS HGNET-BCOR case illustrated in Figure 18. The authors equally thank Matthew J. Schniederjan, MD, for providing exemplary cases of childhood CNS neoplasia and Stewart G. Neill, MD, Debra F. Saxe, PhD, and Michael R. Rossi, PhD, for their expertise in the interpretation of MIP arrays that were submitted by the Emory Neuropathology Division.

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Keywords:

infiltrating glioma; neuroepithelial tumor; glioblastoma; ependymoma; medulloblastoma; atypical teratoid/rhabdoid tumor; embryonal tumor with multilayered rosettes; embryonal neoplasm; molecular-genetic; biomarker; IDH; 1p/19q codeletion; RELA-fusion; C19MC; INI1; K27M; BRAF

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