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Home , Fibrillary astrocytoma, Oligoastrocytoma

This article reviews the molecular

classification of diffuse

in the adult population.

Flight Deck Circa 1969. Photograph courtesy of Craig Damlo. www.soapboxrocket.com.

Impending Impact of Molecular Pathology on Classifying Adult Diffuse Gliomas Robert J. Macaulay, MD

Background: Progress in molecular oncology during the last decade has enabled investigators to more precisely define and group gliomas. The impacts of isocitrate dehydrogenase (IDH) mutation (mut) status and other molecular markers on the classification, prognostication, and management of diffuse gliomas are likely to be far-reaching. Methods: Clinical experience and the medical literature were used to assess the current status of categorization and the likely impact of the pending revision of the classification scheme of the World Health Organization (WHO). Results: IDH-mut is a defining event in most adult fibrillary (FAs) and nearly all oligodendro- gliomas (ODs). The IDH-mut status of most gliomas can be established by immunohistochemistry for the most common mutant of IDH1 (R132H). IDH wild-type (wt) diffuse gliomas include several familiar entities — in particular, (GBM) and most pediatric gliomas — as well as an assortment of less well-defined entities. The codeletion of 1p/19q distinguishes OD from FA, which, by contrast, shows frequent loss of the α thalassemia/mental retardation syndrome X-linked protein. Mixed are typically classifiable as either OD or FA using molecular testing. Conclusions: The current practice of designating IDH-mut WHO grade 4 as secondary GBM will likely be discouraged, and primary or de novo GBM, which is always IDH-wt, may lose this qualification. Histologically, low- or intermediate-grade IDH-wt gliomas with molecular changes characteristic of GBM might justify the designation of GBM WHO grade 3. Mixed is losing popularity as a diagnostic term because most cases will fall into either the FA or OD category. Distinguishing IDH-mut from IDH-wt tumors in clinical trials is likely to clarify sensitivity rates or tumor resistance among subgroups, thus suggesting opportunities for targeted therapy.

Introduction eries in molecular biology during the last decade. Insight into the pathobiology of primary brain tumors Important genetic, epigenetic, and proteomic alter- has accompanied technological and analytic discov- ations have been documented in a variety of low- and high-grade gliomas with clinical implications. Gliomas, which constitute the majority of tumors derived from From the Department of Anatomic Pathology, H. Lee Moffitt Cancer 1 Center & Research Institute, Tampa, Florida. intra-axial elements, may arise from undifferentiated Submitted February 2, 2015; accepted March 11, 2015. or partially differentiated , , 2 Address correspondence to Robert J. Macaulay, MD, Department of or ependymal cells. Although histology remains a Anatomic Pathology, Moffitt Cancer Center, 12902 Magnolia Drive, mainstay of diagnosis, overlapping morphological Tampa, FL 33612. E-mail: [email protected] features can often be resolved without subjectivity; No significant relationship exists between the author and the com- panies/organizations whose products or services may be referenced for example, mixed oligoastrocytoma (MOA) vs ei- in this article. ther (FA) or

200 Cancer Control April 2015, Vol. 22, No. 2 (OD), small cell glioblastoma (GBM) vs anaplastic OD, linked to tumorigenesis.21 Therefore, 2HG can be des- and (EP) vs FA. ignated as an oncometabolite. Because IDH alterations Many gliomas exhibit characteristic genetic, chro- are fundamental to pathobiology, disease progres- mosomal, and/or biochemical signatures indicative of sion, diagnosis, response to adjuvant treatment, and distinct pathophysiology, presaging a reproducible clinical outcome, the classification of gliomas should and reliable molecular overlay to histopathological first acknowledge IDH-mut vs IDH-wt status. This diagnosis.3 Pertinent ancillary studies beyond histo- approach would solidify the analysis of subtypes of pathology may include immunohistochemical testing, IDH-mut tumors,22 allow more objective classification of chromosomal assessment, traditional and array-based IDH-wt entities, and highlight the molecular distinc- genetics,4 epigenetic analysis, and ribonucleic acid tions between pediatric and adult tumors exhibiting (RNA) profiling (both messenger RNA [mRNA]5 and similar morphological features.23,24 noncoding micro-RNA [miRNA]6-8). The World Health Organization (WHO) classification of central nervous Common Morphological Entities system tumors will likely incorporate these findings The 3 morphological entities that exhibit IDH-mut are in its soon-to-be released update.9 Such an improved FA (grades 2–4), OD (grades 2 and 3), and MOA (grade classification system may contribute to the improved 2 or 3).13 All other brain tumors are IDH-wt, includ- clinical investigation of new and targeted therapies ing most pediatric cases, of any to combat the continued guarded outlook for most age, pleomorphic xanthoastrocytoma, subependymal primary brain .1 Of particular interest are giant cell astrocytoma, EP, , and me- the implications of isocitrate dehydrogenase (IDH) ningioma.25 IDH-wt diffuse astrocytomas, including mutation (mut) status. de novo GBM, ,26 thalamic,27 and brainstem astrocytoma, harbor a variety of genetic Two Groups of Gliomas changes, and they should be regarded as distinct from The traditional categorization of gliomas into their their IDH-mut counterparts despite being morpholog- morphological subgroups (astrocytic, oligodendrog- ically indistinguishable. Conversely, cases of OD are lial, and ependymal) typically precedes textbook dis- almost always IDH-mut.22 Rare IDH-wt exceptions,28 cussions of particular entities. This is likely to change. including most pediatric cases,23 should be segregated In a seminal, genome-wide study, Parsons et al10 docu- within this diagnostic category until their pathogene- mented IDH1 mutations in subsets of glial neoplasms. sis is resolved. Morphologically MOAs are frequently It was soon recognized that mutations of IDH1 are IDH-mut and subsequently classifiable as either FA frequent in FAs, ODs, and MOAs,11,12 particularly at or OD following further molecular testing.29 The ap- amino acid residue 132 (arginine), which is replaced propriate pathological diagnosis for the rare IDH-wt by histidine in nearly 90% of tumors bearing an IDH1 MOA remains unsettled; however, such tumors should mutation.13 This residue may also be replaced by cys- still be segregated from the IDH-mut categories. teine,14 serine, or other amino acids,15 and mutations may rarely occur at other loci. IDH2 may also be Immunohistochemistry affected, albeit at a different locus (residue 172). Mu- Once assigned to the diffuse glioma category by rou- tations of other IDH isoforms have not been reported tine pathological assessment, testing for the IDH1 to accompany gliomagenesis. Thus, the finding of an R132H mutation may be easily conducted using a IDH1 or IDH2 mutation can be termed IDH-mut, and commercially available antibody test that recognizes the absence of a mutation in either gene is designat- the altered epitope.30 A positive result has high sen- ed as IDH wild type (wt). Acute myeloid leukemia, sitivity and specificity rates for IDH-mut R132H,22,31 chondrosarcoma,16 and giant cell tumor of the bone17 although reports exist of cross-reactivity with R132L may also exhibit IDH mutations. and R132M mutations.15,31 A total of 10% to 15% of IDH-mut cases are immunonegative, and such cases Accumulation of 2-Hydroxyglutarate can be sent for direct sequencing or hotspot analysis Early events in developing neoplasms involve the for both IDH1 and IDH2; IDH1 R132C is the most acquisition of aberrations in proliferation-related in- common immunonegative mutation.19 tracellular signaling pathways.18 IDH normally par- ticipates in the oxidative decarboxylation of isoci- IDH1/2 Mutations Account for Most Fibrillary trate to α ketoglutarate. The mutant form lacks this Astrocytomas important enzymatic property and instead generates A patient with low-grade astrocytoma typically pres- an abundance of 2-hydroxyglutarate (2HG).19 Excess ents with new-onset , , focal neu- 2HG interferes with chromatin-modifying activity, thus rological deficits, or all of these symptoms. Neuro- leading to global DNA hypermethylation and the gli- imaging typically shows a hemispheric hypointense, oma-CpG island methylation phenotype,20 which is nonenhancing mass that — if followed without sur-

April 2015, Vol. 22, No. 2 Cancer Control 201 gical intervention — may show minimal growth in the early stages. If magnetic resonance spectroscopy reveals a peak attributable to the accumulation of 2HG, then that result strongly predicts IDH-mut and is correlated with improved outcomes.32 His- topathology shows an astrocytic proliferation ex- A B hibiting coarse fibrillary processes and sufficient nuclear irregularity and hyperchromatism to justify designation as a (Fig A–C). Approximately 70% of these tumors will exhibit IDH-mut (Fig D) as well as other changes.11 The gradual progression to high-grade astrocytoma is typical, although the time to progression is unpredicatable.12 When WHO grade 4 features are documented, the designation of C D secondary glioblastoma has found favor. (Whether this term is appropriate, even at first diagnosis,33 is discussed below.) Cases with features reminiscent of oligodendroglioma (small round monomorphic cells with perinuclear haloes) may prompt a preliminary diagnosis of MOA or the more generic diffuse gli- oma (not otherwise specified) until further testing E F 29 resolves the diagnosis. Fig A–F. — Representative images from a left parasagittal-enhancing mass in a man 37 years of age. The tumor was moderately cellular with (A) perinuclear TP53 Mutations Common in IDH-mut halos (× 20), (B) mitoses (× 40), vascular proliferation, and necrosis (not shown). (C) Neoplastic cells showed perinuclear and process positivity for Astrocytomas GFAP (× 20). The Ki67 index was 2% (not shown). (D) IDH1-mutant R132H Mutations in TP53 have been recognized in astrocyto- immunohistochemistry was strongly positive, and the mutation was con- ma, ranging from 30% to 50% of studied cases,34 and firmed with molecular testing (× 20). (E) Immunostaining for p53 protein showed overexpression in a significant minority of neoplastic cells (× 20). alterations in related genes are frequently detected (F) There was the conspicuous absence of ATRX expression in the neoplastic in all grades of diffuse astrocytomas.35 Interactions cells. Immunopositive, small background cells are likely normal oligodendro- between a p53 alteration and IDH-mut in the earliest cytes or lymphocytes (× 20). Codeletion of 1p/19q was not detected; MGMT promoter methylation was demonstrated. The diagnosis was astrocytoma stages of tumor initiation have been suggested by World Health Organization grade 4, isocitrate dehydrogenase mutation (also the predilection for the uncommon R132C mutation known as secondary glioblastoma). to occur in Li–Fraumeni syndrome (germline muta- tion of p53 with tumor predisposition).14 Nuclear p53 WHO Grade 4 IDH-mut Fibrillary overexpression is common in astrocytoma, is readily Astrocytoma detected with immunohistochemistry (Fig E), and of- WHO grade 4 FA with an IDH-mut accounts for fewer ten correlates with TP53 mutation; however, as a di- than 10% of WHO grade 4 gliomas,33 typically prompt- agnostic36 or prognostic37 marker, its utility is limited, ing the designation of secondary GBM11,22,33,42,43; and it has been supplanted by IDH, α thalassemia/ however, the weight of evidence, both molecular mental retardation syndrome X-linked (ATRX), and and clinical, suggests that this term is outdated. It 1p/19q testing. is likely that IDH-mut WHO grade 4 astrocytoma arises from a different precursor pool than IDH-wt ATRX Loss Characterizes IDH-mut GBM.44 IDH-mut gliomas follow a distinct molecular Astrocytomas trajectory from IDH-wt tumors.33 The ambiguity of Mutations in the telomere maintenance protein the term “primary GBM” has been emphasized by ATRX appear to distinguish IDH-mut FA from tu- the occasional de novo presentation of an IDH-mut mors that lack this change — in particular, OD.38 WHO grade 4 astrocytoma or conversely by IDH-wt Absence of ATRX protein (Fig F) by immunohis- low-grade glioma progressing quickly to GBM.45 In tochemistry is evident in 25% to 30% of adult and addition, compared with those with IDH-wt GBM, pediatric high- and low-grade astrocytomas.39 Loss patients with IDH-mut WHO grade 4 FA are younger of ATRX expression and IDH-mut typifies more and have longer survival times.33,45 Therefore, it is than 95% of adult diffuse astrocytomas, and it is clear that IDH1-mut FAs — whatever the grade — almost mutually exclusive with 1p/19q codeletion.40 and IDH1-wt gliomas are different diseases.18 Be- Classification based on IDH-mut status and ATRX cause GBM is a familiar term for pathologists and expression will likely be a key recommendation in clinicians, it will likely be retained, but this term may the next WHO classification update.40,41 be restricted to morphologically appropriate gliomas

202 Cancer Control April 2015, Vol. 22, No. 2 that lack IDH-mut. It will be interesting to determine therapy are improved. If IDH status is unknown, then whether the classic histopathological WHO grading array-based genetic testing for IDH1 and IDH2 can be system developed for diffuse astrocytoma when IDH considered in patients for whom an increased likeli- status was not being tested46 will require modifica- hood of a positive result exists,4 particularly for those tion for IDH-mut astrocytomas. tumors that exhibit features such as oligodendroglial morphology or loss of ATRX expression. Characterizing Oligodendroglioma The classic OD appearance of small round cells Complex Molecular Signature of IDH-wt GBM arranged in honeycomb-like nests with “chicken Among the myriad molecular alterations in GBM, wire”-like coarse, branching vasculature, microcalci- EGFR mutations, deletions, and protein overexpres- fications, and perinuclear haloes has withstood the sion are common. Clinical trials targeting the epi- scrutiny of advancing technologies. 1p/19q codele- dermal growth factor receptor vIII mutant form,55 in tion47 is prognostic,12 predictive,48,49 and diagnostic.50 which exons 2 to 7 are lost, have shown promise, but In addition, more than 90% of 1p-/19q- ODs are also detecting the mutation, either by immunohistochem- IDH-mut.11 Among IDH-mut gliomas, mutations in the istry, reverse transcription–polymerase chain reaction, promoter of TERT are confined to OD24; conversely, or multiplex ligation-dependent probe amplification, OD lacks ATRX mutations.38 For practical purposes, is of dubious significance in multivariate models.56 grading of OD is restricted to grades 2 and 3 because WT1 is variably expressed in gliomas, and although it patient outcomes are considerably better for OD than correlates with worse outcome, its effect is overshad- for IDH-wt gliomas.51 Surgical cases with ambiguous owed by age and IDH-mut status.57 The significance features can be initially designated as MOA or simply of the loss of PTEN is unsettled in GBM,58 but it may as diffuse glioma. Support is growing for the notion be a poor prognostic indicator in diffuse gliomas, par- that most MOAs can be placed into either the FA or ticularly in tumors lacking 1p/19q codeletion.59 TERT OD category using IDH status, ATRX expression, and promoter mutations are detectable in about one-half 1p/19q codeletion testing, and that a genuine mixture of IDH-wt GBMs and only those lacking ATRX mu- of the 2 is a rare occurrence.52 tation; TERT promoter mutations in IDH-mut tumors are restricted to OD.24 Other changes, such as p16, Diffuse IDH-wt Gliomas in Adults NF, Rb, or CDKN2A, have yet to achieve diagnostic About 30% of WHO low-grade adult diffuse astro- or therapeutic significance. Therefore, the molecu- cytomas and fewer than 10% of morphologically di- lar pathogenesis of sporadic GBM in adults remains agnosed OD are IDH-wt11; by contrast, the majority uncertain. Sophisticated expression profiling, which of newly encountered GBM is IDH-wt.10 Among the allows grouping into proneural, neural, mesenchymal, IDH-wt low-grade astrocytoma group, some of these and classical phenotypes, has yet to reach the point cases harbor chromosomal and molecular alterations, of diagnostic utility, possibly due to considerable in- thus aligning them with de novo GBM.3 The remaining tratumoral heterogeneity.60 cases possess a variety of genetic and biochemical alterations, some of which are more akin to pediatric Intraoperative Diagnosis: High-Grade Glioma gliomas, particularly BRAF alterations and histone A pathologist handling an aggressive neoplasm with modifications,27,53 facilitating further subclassification. ring enhancement and central necrosis may provide an intraoperative diagnosis of GBM if morpholog- IDH-wt Glioblastoma ically appropriate. However, IDH-mut FA may un- The most common primary , GBM, is also dergo anaplastic transformation following disease the most lethal.1 Histologically, GBM is populated by progression, or it may have histological features sim- anaplastic astrocytes with mitotic activity, glomeru- ilar to IDH-wt GBM at presentation; alternatively, OD loid vascular proliferation, and geographic necrosis may acquire mitotic activity, vascular proliferation, with (or without) peripheral pseudopalisading. The and necrosis and yet still follow a clinical course molecular landscape of GBM is complex and likely more consistent with WHO grade 3. IDH-mut tu- accommodates several distinct entities,54 as well as mors maintain their molecular signatures over time considerable intratumoral heterogeneity5 and overlap. and subsequent resections, and they have improved Most GBMs arise rapidly and are clinically designated outcomes relative to IDH-wt GBM with similar grad- as being primary or de novo,42 whereas individuals ing features. Because these cases are morpholog- with a lower-grade IDH-mut astrocytoma may prog- ically indistinguishable on intraoperative smears ress to grade 4 (secondary GBM).33,51 Future classifica- and frozen sections, an intraoperative diagnosis of tion may eliminate the “secondary” GBM designation high-grade glioma for practical purposes would en- because progression-free and overall survival rates as sure the appropriate handling of diagnostic material. well as responses to adjuvant or radio- The final diagnosis could range from FA grades 3 or

April 2015, Vol. 22, No. 2 Cancer Control 203 4, OD grade 3, EP grade 3, GBM, or primitive neuro- dichotomy from the outset of study design risks a ectodermal tumor/medulloblastoma (depending on misinterpretation of results; for example, a compar- location). It remains to be determined whether the ison of micro-ribonucleic acid (RNA) in low- com- preoperative demonstration of a 2-hydroxyglutarate pared with high-grade gliomas may be better inter- peak on magnetic resonance spectroscopy is suffi- preted as comparing IDH-mut vs IDH-wt tumors.3,8 cient evidence of IDH-mut or conversely whether its By contrast, a trial of the intratumoral heterogene- absence is sufficient to imply IDH-wt.32 ity of messenger RNA expression identified IDH-wt status as a fundamental study inclusion criterion.5 Glioblastoma WHO Grade 3 Glioblastoma remains an essential diagnostic cat- Considerable interest exists in the biology and clinical egory and will likely be subdivided on a molecular behavior of low- or intermediate-grade diffuse astro- basis, but, ideally, it would exclude IDH-mut tumors. cytic IDH-wt neoplasms in adults. Some examples that The diagnostic term secondary glioblastoma might be share molecular features with GBM can be considered replaced with astrocytoma World Health Organization as being “missed” GBM due to undersampling. Other (WHO) grade 4 and restricted to IDH-mut tumors. patients may present during the early phase of tumor The diagnostic term de novo glioblastoma could then progression and before florid mitotic activity, vascular be shortened to glioblastoma WHO grade 4 and re- proliferation, and necrosis have developed; biopsy stricted to IDH-wt tumors. Histologically lower-grade findings may indicate FA WHO grade 2 or 3.40 The neoplasms of evidently similar biology may qualify tumors with molecular findings of GBM will more for the designation of glioblastoma WHO grade 3 if rapidly advance than their IDH-mut counterparts, al- they possess the molecular signature of glioblastoma though their overall outcome is still better than GBM.51 but lack vascular proliferation and necrosis. 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