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Review Articles

Advances in Diagnostic Immunohistochemistry for Primary Tumors of the Central Nervous System

Meredith, David M. MD, PhD

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Advances In Anatomic Pathology: May 2020 - Volume 27 - Issue 3 - p 206-219
doi: 10.1097/PAP.0000000000000225
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Abstract

Recent advances in molecular testing have uncovered a wide genetic diversity among neoplasms of central nervous system (CNS). These findings have allowed for refinement of diagnostic criteria, paving the way for the 2016 WHO CNS tumor classification update. In many instances, H&E appearances are no longer sufficient for proper classification and prognosis determination. With advanced molecular testing playing such an integral role in the diagnosis of CNS neoplasms, there is risk of incurring significant delay and cost to obtain the requisite data for proper classification. As a result, many novel immunohistochemical markers have been identified to detect either directly or indirectly many diagnostically relevant genomic alterations.

The development of several mutation-specific antibodies in particular has greatly facilitated the classification of diffuse gliomas, which may show overlapping histologic features but radically different clinical behaviors. Subclassifying embryonal tumors has also benefitted from markers that detect either the diagnostic cytogenetic aberration or resultant upregulated pathway components. Advances in developmental biology have also identified novel lineage markers for tumors of ependymal and pineal origin. Finally, loss of expression of certain markers in rare meningioma subtypes are not only diagnostic but also associated with germline alterations.

This review will focus on the above immunohistochemical markers and their integration with clinical, morphologic, and genomic findings in the diagnostic workup of several newly defined CNS tumors.

DIFFUSE GLIOMAS

One of the most significant discoveries in glioma biology was the identification of gain-of-function mutations in isocitrate dehydrogenase (IDH) enzymes that drive oncogenesis in a subset of gliomas. Mutations in codon 132 of IDH1 and 172 of IDH2 result in the production of the oncometabolite 2-hydroxyglutarate, which in turn induces genome-wide promoter hypermethylation and silencing of genes critical for terminal cell differentiation.1–3 Paradoxically, IDH1/2 mutations also slow the clinical progression of gliomas, resulting in dramatically improved survival outcomes compared with histologically equivalent IDH-wildtype gliomas.4 As such, it has become essential to determine IDH1/2 status during initial workup for proper treatment planning and clinical trial enrollment.

Isocitrate Dehydrogenase–mutant Gliomas

Oligodendroglioma

As of the 2016 WHO update, oligodendrogliomas are strictly defined genetically as possessing whole arm codeletion of both chromosomes 1p and 19q and mutation in either IDH1 or IDH2.5 They are diffusely infiltrating and slow growing and represent ∼6% of all glial neoplasms.6 Tumors tend to arise in younger adults with peak incidence in the fourth and fifth decades of life; they are rarely encountered in children.6 Males are affected somewhat more frequently than females (M:F 1.3:1).6 They mostly involve the white matter of the cerebral hemispheres, with rare occurrence in other CNS locations.5

Tumors contain a monomorphic population of cells with round nuclei and characteristic perinuclear haloes (Fig. 1A). A delicate branching network of capillaries, microcalcifications, and microcystic change are also typically encountered. Grading criteria for oligodendrogliomas remain somewhat ill-defined. Low-grade tumors usually exhibit minimal mitotic activity and pleomorphism, whereas anaplastic oligodendrogliomas generally have abundant mitoses, as well as vascular proliferation and necrosis.7

FIGURE 1
FIGURE 1:
Immunoprofiles of IDH-mutant and ATRX-mutant gliomas. A, Oligodendroglioma with characteristic monotonous population of rounded tumor cells with perinuclear haloes and delicate branching vasculature. Immunohistochemistry with a mutant-specific antibody to IDH1 R132H (B) is positive in the majority of cases. ATRX expression is retained (C), and p53 staining shows scattered weak positive cells (D), both of which are consistent with lack of mutations in their respective genes. IDH-mutant astrocytomas exhibit a greater degree of pleomorphism and nuclear hyperchromasia (E) and show a triple mutant pattern for IDH1 R132H (F), ATRX (G), and p53 (H). Occasional IDH-wildtype gliomas, such as giant cell glioblastomas (I), may show loss of ATRX (K) and upregulation of p53 (L) despite having no IDH1 R132H mutation (J). Diffuse midline gliomas (M) may demonstrate variable morphologic appearances, but usually show an astrocytic morphology with high-grade features. They are negative for IDH1 R132H (N) and may show loss of ATRX (O); however, nuclear positivity for the mutation-specific H3F3A K27M antibody can be diagnostic (P).

Mutation in IDH1 or IDH2 is universal, with roughly 90% of tumors containing the IDH1 R132H variant, detectable by a mutation-specific IDH1 R132H antibody.8,9 The remaining 10% contain IDH2 R172K or alternate missense mutations at codon 132 in IDH1.9 Of note, 2 pan-IDH1/2 mutation-specific antibodies have been developed that are able to detect some, but not all pathogenic variants in both proteins.10 Mutations in CIC and TERT promoter are common events in oligodendrogliomas, whereas FUBP1 variants are encountered less frequently.11–13 High-grade tumors may also exhibit deletion of CDKN2A and mutations in NOTCH1 and components of the PI3K pathway.12,14 Mutations in ATRX and TP53 are extremely rare and should be considered for practical purposes as mutually exclusive with 1p/19q codeletion.12,14 Currently, FISH or chromosomal microarray testing are required for detection of 1p/19q codeletion; however, the combination of immunohistochemistry for IDH1 R132H, ATRX, and p53 can greatly assist in the diagnosis (Figs. 1B–D).

Reported clinical outcomes for oligodendrogliomas are variable, but more recent studies that incorporate current genetic definitions and adjuvant radiochemotherapy regimens have achieved mean overall survival in excess of 15 years for low-grade tumors, and over 10 years for anaplastic oligodendrogliomas.15

Astrocytoma

IDH-mutant gliomas without 1p/19q codeletion are by definition astrocytomas, irrespective of histologic appearance, and account for ∼15% to 20% of all gliomas.5,6 Most IDH-mutant astrocytomas present as low-grade lesions in the fourth and fifth decades and predictably progress to higher grade lesions over time. Similar to oligodendrogliomas, IDH-mutant astrocytomas occur preferentially in males (M:F 1.3:1) and localize to the white matter of the cerebral hemispheres.6

Tumor cells possess atypical hyperchromatic nuclei and fibrillar cytoplasm that usually lacks perinuclear haloes (Fig. 1E). Gemistocytic forms with abundant eosinophilic cytoplasm and eccentric nuclei that resemble normal reactive astrocytes may be variably abundant. The background matrix is typically loosely structured and may show extensive microcyst formation. Low-grade tumors (diffuse astrocytoma) exhibit mild pleomorphism and cellularity with extremely rare, if any, mitotic figures. Anaplastic astrocytomas generally show greater nuclear atypia and cellularity with overtly elevated mitotic activity. The presence of microvascular proliferation and/or necrosis in addition to these features warrants classification as glioblastoma.5 IDH-mutant glioblastomas most often arise through malignant progression of a lower grade astrocytoma and account for <10% of all glioblastomas.16–20

Like oligodendrogliomas, the vast majority of tumors are IDH1 R132H mutant and label with a mutation-specific antibody (Fig. 1F).8 IDH-mutant astrocytomas are also defined by concomitant loss-of-function mutations in ATRX and TP53, which are present in over 90% of cases.21–28ATRX mutations are usually nonsense or truncating frameshift mutations that result in loss of staining by immunohistochemistry (Fig. 1G); however, retained expression may be encountered in some ATRX mutants with especially distal truncations.29 Mutations in TERT promoter are mutually exclusive with ATRX mutation and indicate wildtype IDH1/2 status in the absence of 1p/19q codeletion.14,30,31 Nonetheless, a small subset of IDH-wildtype astrocytomas (eg, giant cell glioblastomas and histone-mutant gliomas) show inactivation of both ATRX and TP53 (Figs. 1I–L),32,33 which should prompt sequencing studies to exclude the presence of an alternate IDH1/2 mutation. Missense inactivating mutations in TP53 are most common, either in combination with 17p deletion or as copy-neutral loss of heterozygosity, and can be detected by strong, diffuse nuclear staining for p53 (Fig. 1H).14 By contrast, homozygous deletions of TP53 will show complete loss of staining in tumor cells. Genetic alterations typical of IDH-wildtype gliomas, including receptor tyrosine kinase (RTK) amplification and losses of PTEN and CDKN2A, are uncommon, but are usually seen in IDH-mutant glioblastomas when present.34–37

Clinical outcomes for IDH-mutant astrocytomas are superior to histologically matched IDH-wildtype tumors, with estimated median survivals for grade II and III tumors being 10.9 and 9.3 years, respectively.38 The prognosis for IDH-mutant glioblastoma is significantly worse, with overall survival reported at 27 to 31 months following standard therapy.16,17

Emerging Subtypes of Isocitrate Dehydrogenase-wildtype Gliomas

IDH1/2 wildtype gliomas comprise over 50% of primary malignant brain tumors, with glioblastoma being by far the most common.6 Numerous genomic studies have revealed a surprising diversity in these tumors, especially in gliomas that tend to arise in a younger patient population, leading to the integration of several novel entities into the 2016 WHO classification of CNS tumors.

Diffuse Midline Glioma, H3 K27M Mutant

This genetically defined subset of high-grade IDH-wildtype astrocytomas occurs predominantly in children and young adults and includes the former entity diffuse intrinsic pontine glioma.5 Tumors arise in midline locations, namely the brainstem, spinal cord, and thalamus, and possess the defining K27M (alternatively known as K28M) missense mutation within either H3F3A or HIST1H3B/C.33,39–43

Histologically, these tumors exhibit an infiltrative growth pattern and astrocytic morphology (Fig. 1M). High-grade features are usually present; however, they are not necessary for the diagnosis, and some examples may lack overt mitotic activity. Staining for OLIG2 is a consistent feature, though GFAP positivity is variable and may be limited. A mutation-specific antibody against H3F3A K27M can aid in the diagnosis, which demonstrates strong and diffuse nuclear staining (Fig. 1P).44,45 This antibody can also demonstrate nonspecific staining in macrophages and other inflammatory cells, necessitating careful interpretation and on occasion additional confirmatory studies. Because mutation at the lysine 27 position may also affect methylation at this residue, loss of immunostaining by tri-methyl-histone H3 K27 (H3K27me3) can be used in combination with H3F3A K27M positivity, although loss of H3K27me3 alone is not specific for this tumor type.44–46TP53 mutations are common in these tumors (∼50%), co-occurring with loss-of-function mutations ATRX in about 15% of cases (Fig. 1O).33,39,42 Mutations in IDH1/2 and positivity for IDH1 R132H are not encountered, however (Fig. 1N). Amplifications of RTKs are also common, most often of PDGFRA, CDK4/6, and CCND1-3.33,39 The prognosis for this tumor type is especially grim, with a 2-year survival rate of <10%.47,48

Despite the requirement of H3 K27M mutation in this entity, it must be noted that this mutation is observed in a subset of noninfiltrative gliomas and glioneuronal tumors. Examples of tumors with classic histopathology of pilocytic astrocytoma, ganglioglioma, and even ependymoma have been encountered and do not exhibit the same aggressive clinical behavior as diffuse midline gliomas.49–57 Many such tumors contain additional driver mutations/fusions in BRAF or FGFR1 and appear to lack RTK amplification and loss of tumor suppressors.51,52,58 As such, H3 K27M mutant tumors with a noninfiltrative growth pattern should not be classified as diffuse midline glioma, WHO grade IV.59

Epithelioid Glioblastoma

A rare variant of IDH-wildtype glioblastoma with a broad age incidence, these high-grade astrocytic tumors exhibit a predominant component of cells with epithelioid, rhabdoid, or gemistocytic morphology (Fig. 2A).60 Typical features of glioblastoma, including elevated mitotic activity, microvascular proliferation, and necrosis, are also required for the diagnosis. A subset of tumors may exhibit histologic overlap with anaplastic pleomorphic xanthoastrocytomas (ie, showing xanthomatous change and marked nuclear pleomorphism); however, features of low-grade pleomorphic xanthoastrocytoma or other glioneuronal tumors, including Rosenthal fibers and eosinophilic granular bodies, are not present.61

FIGURE 2
FIGURE 2:
Diagnostic immunohistochemistry in novel glial entities. A, Epithelioid glioblastoma contains a preponderance of epithelioid, rhabdoid, or gemistocytic astrocytes along with typical high-grade features. Features characteristic of low-grade gliomas or glioneuronal tumors, such as Rosenthal fibers and eosinophilic granular bodies are absent. B, Immunostaining for BRAF V600E is positive in over half of cases and portends a slightly more indolent course. C, Angiocentric gliomas contain elongate cells with fibrillar, bipolar processes that occasionally form perivascular pseudorosettes. Nuclear staining for MYB (D) functions as a surrogate marker for the defining MYB-QKI fusion gene. E, Ependymomas with RELA rearrangements contain characteristic round to ovoid cells arranged in perivascular pseudorosettes with diffuse membranous positivity for L1CAM (F) and nuclear RELA/p65 (G). H, Nuclear FOXJ1 staining confirms the ependymal origin of this tumor.

Tumors predominantly arise in the cerebral hemispheres, though examples occurring in the lateral ventricles, deep nuclei, and posterior fossa have been described.62–66 On imaging, tumors may appear well circumscribed and contain both solid and cystic areas.61,62,64,67

Tumor cells are usually positive for S100 and GFAP, though the latter may be present only focally.61,62,68,69 Staining for cytokeratins and epithelial membrane antigen (EMA) may also be seen, producing a differential diagnosis that includes metastatic carcinoma, melanoma, and even ependymoma. SMARCB1 (INI1) staining is universally intact, and myogenic and melanocytic markers are routinely negative.61,62,68–70

Approximately half of epithelioid glioblastomas possess BRAF V600E mutations, which may be detected by the VE1 mutation-specific BRAF V600E antibody (Fig. 2B).61,71 This subset of epithelioid glioblastomas cluster with pleomorphic xanthoastrocytomas by methylation analysis, preferentially occur in children and young adults, and are slightly less aggressive (overall survival ∼34 mo).61,72

Tumors lacking BRAF mutation overlap clinically and genetically with conventional IDH-wildtype glioblastomas.72 Tumors in older adults frequently show gain of 7q, deletion of 10q, homozygous deletion of CDKN2A, and TERT promoter mutation and exhibit an overall survival of 11 months.72BRAF wildtype tumors that occur predominantly in children and young adults frequently show amplifications of PDGFRA and MYCN, along with deletion of 10q and regions of chromothripsis.72BRAF V600E and TERT promoter mutations were not observed in this subgroup.72 Outcomes in this subgroup are also poor, with a median overall survival of 18 months.72

Angiocentric Glioma

Angiocentric gliomas typically occur within the cerebral cortex of children and young adults as well-demarcated T2 hyperintense mass lesions and are strongly associated with intractable epilepsy.73 Tumors are composed of an infiltrative monomorphic population of elongate bipolar cells that occasionally form characteristic perivascular pseudorosettes (Fig. 2C). Mitoses are usually rare or altogether absent, and microvascular proliferation and necrosis are not observed.

Genetically, these tumors are defined by rearrangements of MYB, with MYB-QKI fusions being the most common and resulting from an interstitial deletion on chromosome 6q.49,74–76 Immunohistochemistry for MYB is a sensitive marker for these tumors, showing strong and diffuse expression (Fig. 2D)49; however, focal MYB expression can be detected in several other low-grade gliomas and must be interpreted with caution.77 Tumor cells also show consistent positivity for GFAP and dot-like EMA, suggesting a possible ependymal origin.73

Angiocentric gliomas are designated as WHO grade I, and surgical resection is usually curative.73

Ependymomas Defined by Fusion Events

Until recently, the diagnosis of ependymoma relied heavily on the observation of characteristic tumor morphology, namely glial cells with round to oval nuclei arranged in perivascular pseudorosettes. Although ependymoma cells stereotypically stain positively for GFAP and a dot-like pattern of EMA, this profile is not completely sensitive or specific. Recent studies, however, have revealed overexpression of the master regulator of ciliogenesis, FOXJ1, in normal tissues and neoplasms of ependymal and choroid plexus origin.78–83 Consequently, immunohistochemistry for FOXJ1 is highly specific for marking ependymomas and choroid plexus tumors of all subtypes, showing somewhat reduced expression in higher grade lesions (Fig. 2H).82,83

Subclassification of ependymal neoplasms is largely dependent upon combining classic histologic features with broad copy number alterations.84–87 Cortical ependymomas, however, uniquely exhibit recurrent fusion events involving either RELA or YAP1.84,88–90 Ependymomas with RELA rearrangements account for ∼70% of cases arising in the supratentorial compartment and are most common in children, though many occur in adulthood.84,88

C11orf95-RELA transcripts are by far the most common, producing in-frame fusions of exons 1-2 of C11orf95 with the entire coding region of RELA.88 Either C11orf95 or RELA may exhibit different fusion partners, and chromothripsis involving 11q13 is a common finding.88 The resulting oncoprotein drives nuclear translocation of RELA/p65 and constitutive activation of the NF-κB pathway, with upregulation of downstream target genes such as L1CAM and CCND1.88 Immunohistochemistry for L1CAM demonstrates diffuse membranous positivity, and nuclear staining for RELA/p65 and CCND1 is routinely positive in this tumor (Figs. 2F, G).88,90–92

The importance of identifying ependymomas with RELA rearrangements has been highlighted in initial reports showing more aggressive clinical behavior compared with other supratentorial ependymomas, with 5-year overall survival of 75%.84,85

A minority of supratentorial ependymomas have been shown to possess YAP1 fusion events, the most common being YAP1-MAMLD1.84,88,90 This subset occurs predominantly in young children and has thus far been associated with excellent clinical outcome.84,85

EMBRYONAL TUMORS

Tremendous strides have been made recently uncovering a wealth of genomic insight into the diverse spectrum of embryonal tumors, identifying many novel entities with unique clinicopathologic features that were previously classified under the now obsolete designation “primitive neuroectodermal tumor.” As with many tumors that arise preferentially in childhood, most of these entities exhibit recurrent point mutations or structural variants, several of which may not be present on most currently available sequencing platforms. Fortunately, surrogate immunohistochemical markers for diagnostic mutations, fusion proteins, and activated downstream pathway components are available to assist in the diagnosis of this challenging group of tumors (summarized in Table 1).

TABLE 1
TABLE 1:
Summary of Immunohistochemical Profiles of Embryonal Tumors

Genetic Subgroups of Medulloblastoma

Medulloblastoma is by far the commonest of the CNS embryonal tumors, comprising 25% of all pediatric intracranial neoplasms,93 and extensive study has identified 4 unique molecular subgroups with distinct clinical characteristics. Definitive subgroup assignment requires multimodal advanced molecular testing, including examination of copy number, mutational, transcriptional, and methylation profiling; however, select immunohistochemical markers can be applied to identify subsets of tumors with activation of the WNT or SHH pathways.

WNT-activated Medulloblastoma

These tumors account for ∼10% of all medulloblastomas, occurring predominantly in older children and young adults.94–96 Unique among medulloblastomas, tumors of this subgroup frequently arise from the dorsal brainstem and extend into the fourth ventricle.97–99 Their prognosis is excellent, with complete remission being routinely achieved with standard therapy.100

They almost always exhibit classic medulloblastoma histology, in which undifferentiated embryonal cells are arranged in dense syncytial sheets with occasional Homer Wright rosettes and regions of neurocytic differentiation and reduced cellularity (Fig. 3A). Perinodular collagen deposition and desmoplasia are never encountered. Rare tumors may show large cell or anaplastic features; however, the biological potential of these tumors is uncertain.101,102

FIGURE 3
FIGURE 3:
Diagnostic immunohistochemistry in embryonal tumors. A, WNT-activated medulloblastomas show classic morphology with sheets of poorly differentiated cells and occasional Homer Wright rosettes. B, Positive nuclear staining for β-catenin is usually only present in focal clusters but is diagnostic of this medulloblastoma subtype. C, Pineoblastoma also contains sheets of embryonal cells with minimal architecture; however, strong nuclear staining for the pineal lineage CRX is routinely observed (D). E, Atypical teratoid/rhabdoid tumor has variable morphologic appearances, with a predominantly embryonal component. Scattered rhabdoid cells (inset) are not always present, but loss of SMARCB1 (F) is diagnostic.

Genetically, these tumors are characterized by activating mutations within exon 3 of CTNNB1 along with monosomy of chromosome 6.103–107 Immunohistochemistry for nuclear β-catenin is a reliable marker for this subgroup, showing at least focal positivity in 85% of tumors (Fig. 3B).101,108,109 While recurrent mutations in TP53 have been described in WNT-activated tumors, they appear to carry no distinct prognostic significance.110

SHH-activated Medulloblastoma

This subgroup constitutes ∼30% of all medulloblastomas and shows a bimodal age distribution with peaks in early childhood and young adults.104–106 Tumors tend to arise in the cerebellar hemispheres, and unlike WNT-activated medulloblastomas, TP53 mutational status has a dramatic impact on their clinicopathologic features.110

SHH-activated and TP53 Wildtype

Medulloblastomas within this category characteristically show a biphasic desmoplastic/nodular histology, in which reticulin-free nodules of lower density tumor showing neurocytic differentiation and decreased proliferation are interspersed among regions of densely packed undifferentiated cells with high mitotic rate and a reticulin-rich matrix.5 Occasional tumors may exhibit classic or large cell/anaplastic features.

Activation of the SHH pathway usually occurs via loss-of-function mutations in the negative regulators PTCH1 or SUFU, with activating mutations of SMO or amplification of GLI2 occurring much less often.105–107,111 Upregulation of filamin A, GRB2-associated binding protein 1 (GAB1), and the transcriptional coactivator yes-associated protein 1 (YAP1) are observed in SHH-activated tumors, all of which can be detected by immunohistochemistry.101 Whereas cytoplasmic GAB1 staining is a specific marker for SHH-activated tumors, positivity for filamin A (cytoplasmic) and YAP1 (nuclear and cytoplasmic) is also present in WNT-activated medulloblastomas.101 Neuronal markers, such as synaptophysin and NeuN, are commonly positive in nodular regions, and diffuse p53 staining is not seen. The prognosis for these tumors is intermediate, with a 5-year overall survival of 76% in 1 study.110

SHH-activated and TP53 Mutant

In contrast to TP53 wildtype SHH-activated medulloblastomas, these tumors preferentially occur in children, occasionally in the setting of Li-Fraumeni syndrome.110,112 Microscopically, they show aggressive large cell/anaplastic histology, with marked pleomorphism, cell wrapping, prominent nucleoli, and abundant mitotic and apoptotic figures. Strong, diffuse p53 staining is usually observed, indicative of the underlying TP53 mutation. SHH activation is achieved through amplifications of GLI2, MYCN, or SHH instead of mutations in PTCH1, SUFU, and SMO.105,107,111 Prognosis is poor, with a 5-year overall survival estimated at 41%.110

Non-WNT/Non-SHH Medulloblastoma

Tumors that do not show genetic evidence of WNT or SHH pathway activation may be further subdivided into groups 3 and 4 based largely on their transcriptome and methylation profiles. Together they comprise the largest fraction of medulloblastomas, with group 3 accounting for roughly 20% of all medulloblastomas, and 40% belong to group 4.104–106

Recurrent genetic alterations have been described for each group; however, definitive subclassification can be challenging in the absence of comprehensive molecular analysis. The combination of chromosome 17p loss and 17q gain, or the presence of isodicentric 17q is present in the majority (∼80%) of non-WNT/non-SHH medulloblastomas.101,104,105 Amplification of MYC is unique to group 3; however, this is only observed in the minority of cases.104,105,107

Most non-WNT/non-SHH medulloblastomas show classic histology, though a subset of group 3 tumors may exhibit large cell/anaplastic features. Neither group possesses a characteristic immunophenotype. Synaptophysin is variably positive, but GAB1, YAP1, filamin A, and nuclear β-catenin are universally negative.101

Clinical outcomes for group 3 tumors are usually poor, whereas group 4 tumors have an intermediate prognosis depending on the presence of metastatic disease at presentation.100,113

Pineoblastoma

Pineoblastoma is a rare embryonal malignancy that may occur at any age but are most common during the first 2 decades.5 They arise within the pineal region and may show marked extension into adjacent structures that can obscure their site of origin.

Histologically, these tumors exhibit a classic small round blue cell tumor appearance with diffuse sheets of primitive cells (Fig. 3C). Occasional Homer Wright and Flexner-Wintersteiner rosettes may also be seen. Variable immunostaining for neuronal markers may be observed, and glial markers are generally negative, marking entrapped glial cells when present.114 SMARCB1 expression is universally retained. Positivity for the master regulator of retinal photoreceptor differentiation, CRX, is an extremely sensitive and specific marker for pineal lineage differentiation, marking 100% of pineal parenchymal tumors, including pineoblastoma (Fig. 3D).83,115–117 CRX is also a specific marker for retinoblastoma, and rare instances of medulloblastomas may show positivity as well.115–117

Prognosis for pineoblastoma is generally better than other supratentorial embryonal tumors, with some modern trials reporting 5-year overall survival rates in excess of 80%.118–122

Atypical Teratoid/Rhabdoid Tumor

Atypical teratoid/rhabdoid tumor (AT/RT) is another rare embryonal malignancy that predominantly occurs in infancy, comprising approximately 1% to 2% of all brain tumors in the pediatric population and >10% in infants.123–125 Tumors may arise anywhere in the neuroaxis, with a slight predilection for the supratentorial compartment.126,127

Microscopically, AT/RTs exhibit a wide spectrum of growth patterns and primitive differentiation; however, an embryonal appearing small cell component is usually present (Fig. 3E).128,129 The classic histologic finding in these tumors is the presence of cells with rhabdoid morphology, showing eccentrically placed nuclei with vesicular chromatin and prominent nucleoli, abundant eosinophilic cytoplasm with a dense core, and sharply demarcated cell borders. These cells may be present in small foci or altogether absent in many cases, however. AT/RTs may show immunoreactivity for a variety for antibodies, including EMA, SMA, GFAP, cytokeratins, and synaptophysin, though germ cell markers are typically negative.

These tumors are defined by genetic inactivation (either by mutation or deletion) of SMARCB1, which can be detected by loss of nuclear staining for SMARCB1 (Fig. 3F).130–132 Case reports have also described instances of AT/RTs with retained SMARCB1 expression, that instead show loss of SMARCA4.133,134 Immunostaining for SMARCA4 (BRG1) may thus be helpful in such cases that show typical AT/RT morphology and intact SMARCB1 expression.

AT/RT is extremely aggressive and usually rapidly fatal, with most studies reporting overall survival rates under 12 months; however, a subset of patients may experience multiyear survival with aggressive multimodality therapy.126,135–140

Embryonal Tumor With Multilayered Rosettes

This distinct subset of aggressive embryonal tumors occurs in young children under 4 years of age, affecting males and females equally.141–143 Tumors show a predilection for the supratentorial compartment, but involvement of the cerebellum and brainstem has been observed in ∼30% of cases.141,142

They may show several distinct growth patterns, each being formerly identified as distinct entities: embryonal tumor with abundant neuropil and true rosettes (ETANTR), ependymoblastoma, and medulloepithelioma. The ETANTR pattern shows a biphasic architecture with clusters of small, primitive cells arranged in multilayered rosettes interspersed with large paucicellular regions of neuropil that may contain a differentiated neuronal or ganglion cell component (Fig. 4A). The ependymoblastoma pattern lacks regions of neuropil and neuronal differentiation, instead exhibiting sheets of small-sized to medium-sized primitive cells among multilayered rosette structures. The medulloepithelioma pattern contains embryonal cells forming papillary and tubular structures, reminiscent of early neural tube architecture.

FIGURE 4
FIGURE 4:
Rare embryonal tumor entities. A, An example of ETMR with ETANTR growth pattern containing clusters of embryonal cells embedded in large regions of neuropil. B, Cytoplasmic staining for LIN28 is present in the poorly differentiated component. C, CNS HGNET-BCOR contains cells with glial morphology and frequent perivascular pseudorosettes. This example exhibits vague Homer Wright rosette formation. D, Diffuse nuclear positivity for BCOR is indicative of BCOR rearrangements.

Immunohistochemical staining for neuronal markers, such as synaptophysin, NFP, and NeuN and GFAP may be seen within regions of neuropil and differentiated elements. Poorly differentiated areas and rosettes are generally negative for these markers, instead showing focal positivity for epithelial markers, including cytokeratins and EMA. SMARCB1 is universally retained. Regardless of histologic pattern, these tumors stain diffusely for LIN28 (Fig. 4B), though nonspecific staining may be encountered in AT/RT, germ cell tumors, and some gliomas.142–147

The defining genetic lesion is a focal amplification on chromosome 19 of the microRNA cluster at 19q13.42 (C19MC), which may occur alongside loss of 6q and gains of chromosomes 2, 7q, and 11q.142,143,148,149 Regardless of therapeutic approach, overall survival is dismal with an average of 12 months.141–143

High-grade Neuroepithelial Tumors With Recurrent Gene Fusions

Comprehensive genetic analysis of a large cohort of tumors previously classified as CNS primitive neuroectodermal tumors revealed several distinct molecular subgroups showing recurrent structural variants.150 Each of the entities described below exhibit relatively similar clinical characteristics, with tumors most often presenting in the first 2 decades (median age, under 5 y) in a nearly equal male:female ratio.150 Most tumors also showed no obvious anatomic predilection.150

Fusions involving FOXR2 were reproducibly detected in tumors that showed varying degrees of neuronal differentiation (CNS NB-FOXR2), including tumors alternately classified as CNS neuroblastomas or ganglioneuroblastomas.150 These tumors showed multiple structural alterations that resulted in marked upregulation of FOXR2, including recurrent JMJD1C-FOXR2 fusions and various tandem duplications that juxtapose the promoter region of neighboring genes to activate FOXR2 expression.150 These tumors interestingly show robust expression of both OLIG2 and synaptophysin, a unique feature among this group of tumors.150 This tumor subgroup has an intermediate prognosis, with a 5-year overall survival of ∼65%.150

Another subgroup showed recurrent rearrangements in MN1 (CNS HGNET-MN1), most often with the partner BEND2 via a t(22;X)(q12;p22) translocation.150 These tumors occur far more often in females (>5:1 female:male ratio) and present later in young adulthood.150 While most tumors occur in the cerebral hemispheres, examples arising in the cerebellum and spinal cord may be encountered.150 This group shows a diverse spectrum of growth patterns ranging from solid to pseudopapillary architecture with frequent dense stromal hyalinization and included many tumors classified as astroblastomas by histology.150 Lesions are usually well circumscribed and contain ovoid tumor cells that are usually negative for glial and neurocytic markers.150 Overall survival in this cohort is excellent, with 100% of patients alive at 96 months.150

The “CNS EFT-CIC” subgroup shows significant overlap both genetically and histologically with CIC-rearranged round cell sarcomas that arise in the periphery. Both tumor types show recurrent CIC fusion events that produce dramatic upregulation of the ETS family of transcription factors.150–152 Presence of CIC-NUTM1 fusion is characteristic of this group and results in upregulation of NUTM1 expression.150 As a result, NUTM1 immunohistochemistry is highly sensitive and specific for detection of these tumors, while staining for glial and neuronal markers is rarely observed.150 These tumors have a similar prognosis as CNS NB-FOXR2, with a 5-year overall survival of ∼60%.150

The most aggressive HGNET subgroup (CNS HGNET-BCOR) is characterized by distal internal tandem duplications within exon 15 of BCOR.150,153 Tumors present in the first years of life and may occur anywhere within the CNS. Histologically, tumor cells usually show glial morphology with oval to tapered nuclei and fibrillar processes (Fig. 4C). Perivascular pseudorosettes are also often encountered. Despite these features, staining for OLIG2 and synaptophysin is routinely negative.150,153 Nonetheless, this entity shows robust upregulation of BCOR and subsequent activation of the WNT pathway, detected by nuclear staining for BCOR and β-catenin (Fig. 4D).150,153 This entity carries a dismal prognosis, showing rapid fatality within a few months of diagnosis.150,153

Meningioma Subtypes Associated With Germline Mutations

The combination of cytogenetic and mutational profiling of meningiomas has revealed a strong association of genomic signatures with several unique histologic growth patterns. The presence of chromosomal polysomies in angiomatous meningiomas, NF2 deficiency in fibrous meningiomas, KLF4/TRAF7 mutations in secretory meningiomas, and multiple broad losses in higher grade meningiomas all require molecular testing to definitively assess.154–158 The discovery of somatostatin receptor 2A (SSTR2A) expression in meningiomas has proved a useful adjunct to distinguish meningiomas from histologic mimics159,160; however, until only recently have subtype specific markers been identified for a subset of higher grade meningiomas.

Clear Cell Meningioma

Clear cell meningiomas represent an extremely rare variant of meningiomas with an increased rate of occurrence commensurate with WHO grade II designation.161 Descriptions of this tumor type are largely confined to small case series; however, these tumors occur most often in children and young adults with a predilection for the cerebellopontine angle and spinal cord.161–166

Histologically, the tumors show epithelioid cell morphology with prominent cytoplasmic clearing and distinctive “blocky” interstitial and perivascular deposits of collagen (Fig. 5A). Other characteristic features of meningioma, including whorl formation and psammoma bodies, are usually absent. As with other meningioma variants, tumor cells routinely stain with both EMA and SSTR2A.164,167

FIGURE 5
FIGURE 5:
Meningioma subtypes associated with germline syndromes. A, Clear cell meningiomas contain epithelioid cells with clear or palely eosinophilic cytoplasm and prominent interstitial blocky collagen. Immunohistochemistry for SMARCE1 is consistently negative in both sporadic and familial examples (B). C, Rhabdoid meningiomas possess a dominant component of rhabdoid cells with abundant, dense eosinophilic cytoplasm and eccentric nuclei with prominent nucleoli. D, BAP1 staining is lost in a subset of tumors and may indicate the presence of BAP1 cancer predisposition syndrome.

Studies investigating familial forms of disease revealed an association with SMARCE1 loss function.168–171 SMARCE1 is a member of the SWI/SNF chromatin remodeling complex whose loss is implicated in a subset of cases of Coffin-Siris syndrome.172,173 Subsequent studies have demonstrated strict correlation between SMARCE1 loss and clear cell meningioma histology (Fig. 5B).167 Importantly, tumors showing focal clear cell change do not exhibit loss of SMARCE1 by immunohistochemistry.167

Rhabdoid Meningioma

These tumors constitute a subset of anaplastic meningiomas (WHO grade III), often showing overt features of malignancy and an aggressive clinical course.174–176 They are characterized by a preponderance of cells with classic rhabdoid morphology (round cells possessing an eccentric nucleus with open chromatin and a single prominent nucleolus and a dense eosinophilic cytoplasmic inclusion) and high mitotic rate (Fig. 5C). Immunohistochemistry for EMA and SSTR2A is usually positive, and SMARCB1 expression is retained.159,174

Comprehensive genomic analysis of a large cohort of rhabdoid meningiomas and meningiomas with focal rhabdoid features uncovered a novel association between BAP1 (BRCA-1 associated protein 1) deficiency and aggressive clinical course.177 Tumors with homozygous inactivating mutations or deletions show loss of staining for BAP1 (Fig. 5D).177 Furthermore, a subset of patients showed germline BAP1 mutations, placing rhabdoid meningiomas within the BAP1 cancer predisposition syndrome.177 Patients with tumors showing BAP1 loss of expression, therefore, should be referred for germline testing.

CONCLUSIONS

Definitive classification of CNS tumors increasingly requires an integrated approach that combines conventional light microscopy with advanced molecular diagnostics. Extensive genomic characterization of primary CNS neoplasms has uncovered numerous class-defining alterations that strongly correlate with clinical behavior and response to therapy.

The development of mutation-specific antibodies to IDH1 R132H, BRAF V600E, and H3F3A K27M have greatly assisted classification of diffuse gliomas. In addition, the presence of certain structural rearrangements may be inferred from strong immunopositivity to the implicated protein (eg, MYB in angiocentric gliomas, RELA/p65 in RELA-rearranged ependymomas, and BCOR in CNS HGNET-BCOR) or complete absence of normal protein expression (eg, ATRX in gliomas and SMARCB1 in AT/RT). Supporting evidence for other alterations for which direct immunohistochemistry is not possible may be provided by demonstrating upregulation of downstream pathways (eg, nuclear β-catenin in WNT-activated medulloblastoma and LIN28 positivity in ETMR). Lastly, advances in developmental biology have also facilitated the identification of the tissue-specific master regulators CRX and FOXJ1 in pineal and ependymal tumors, respectively, which can be especially helpful in the diagnosis of those entities.

Although molecular testing remains the gold standard for classification of several CNS tumors, advances in the development of immunohistochemical markers can facilitate a more accurate and efficient diagnosis in many instances.

ACKNOWLEDGMENTS

The author wishes to thank Dr Sanda Alexandrescu, Department of Pathology, Boston Children’s Hospital, Harvard Medical School and Dr Sandro Santagata, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School for contributing several of the cases illustrated.

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Keywords:

immunohistochemistry; glioma; embryonal; meningioma; ependymoma; CNS

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