Translating Molecular Biomarkers of Gliomas to Clinical Practice

Adult diffuse gliomas

Adult diffuse gliomas are infiltrating glial neoplasms that include astrocytomas and oligodendrogliomas. In the 2007 edition of the WHO Classification of Tumours of the Central Nervous System , these entities were diagnosed and classified as grade II (diffuse) or grade III (anaplastic) based on histologic features. In cases in which a morphologic distinction between these two entities was not clear, a diagnosis of oligoastrocytoma was appropriate. Following major advances in our understanding of molecular gliomagenesis, the revised WHO Classification of Tumours of the Central Nervous System (2016) has refined the diagnostic criteria for astrocytomas and oligodendrogliomas by incorporating clinically relevant molecular information about the mutation status of isocitrate dehydrogenase 1/2 ( IDH1/2 ), and alpha thalassemia/mental retardation syndrome X-linked ( ATRX ) genes and codeletion of chromosome arms 1p and 19q. After an initial IDH mutation, oligodendrogliomas are thought to develop via subsequent telomerase reverse transcriptase ( TERT ) promoter mutations and codeletion of 1p/19q, whereas IDH -mutant astrocytomas develop with subsequent alterations of TP53 and/or ATRX. The diagnosis of oligoastrocytoma is now strongly discouraged ( Fig. 4.1 ).

Point mutations in cytosolic IDH1 and mitochondrial IDH2 most commonly by substitution of arginine to histidine (R132H) or to lysine (R172K), respectively, alter their catalytic activity such that they produce high levels of the oncometabolite 2-hydroxyglutarate (2-HG), instead of α-ketoglutarate. The presence of 2-HG results in disruption of tet methylcytosine dioxygenase 2 (TET2) activity, leading to aberrant histone regulation and development of the glioma–CpG island methylator phenotype (G-CIMP).

G-CIMP is an epigenetic molecular profile that was noted and named after the observation of a subset of gliomas within The Cancer Genome Atlas (TCGA) database that showed concerted hypermethylation at a large number of loci. In general, CIMP gliomas are lower-grade, often IDH -mutated, tumors. Mutation of IDH is the molecular basis for the G-CIMP phenotype. Overall, IDH -mutant, G-CIMP high infiltrating gliomas are associated with a favorable prognosis compared with IDH wild-type tumors. IDH status is an even stronger predictor of patient outcome than histologic grade in infiltrating gliomas.

IDH mutations can be detected by immunohistochemical analysis of formalin-fixed, paraffin-embedded (FFPE) tissue using the IDH1 R132H mutant-specific antibody ( Fig. 4.2 A, B ). Direct Sanger sequencing, although requiring more tissue specimens, has the advantage over immunohistochemistry (IHC) of not only detecting IDH1 R132H but also detecting other noncanonical IDH mutations. This technique is highly sensitive, but is limited because the specimens must contain at least 50% neoplastic cells to ensure reliability. Another method, pyrosequencing, has a higher sensitivity than Sanger sequencing because it can detect as little as 10% mutant alleles. Moreover, clinical efforts have been undertaken to determine whether IDH mutations can be detected indirectly, and magnetic resonance spectroscopy has been proposed as a reliable technique to achieve this goal by detecting the levels of 2-HG.

Oligodendrogliomas

Changes to the diagnostic criteria of oligodendroglioma and anaplastic oligodendroglioma include the requirement of both an IDH mutation and a 1p/19q codeletion (see Fig. 4.1 ). There are 4 distinct types of oligodendroglioma tumors: (1) IDH mutant and 1p/19q codeleted, (2) NOS, (3) anaplastic IDH mutant and 1p/19q codeleted, and (4) anaplastic NOS (when only classic histology is available). The diagnosis of oligodendroglioma NOS should only be given after further analysis excludes potential diagnoses such as dysembryoplastic neuroepithelial tumor, clear cell ependymoma, neurocytoma, pilocytic astrocytoma, and several other tumor types that are histologically similar to oligodendroglioma.

Loss of 1p/19q has more than just a diagnostic significance. It has been associated with a favorable prognosis and an increased sensitivity to chemotherapy. Moreover, 1p/19q-codeleted anaplastic oligodendrogliomas that show polysomy for chromosomes 1p and 19q have intermediate survival between 1p/19q-retained tumors and 1p/19q-codeleted oligodendrogliomas in a euploid background. Codeletion of 1p/19q can be detected by fluorescence in situ hybridization (FISH), loss of heterozygosity (LOH) capillary gel electrophoresis, or as a change in copy number in the 450K CpG methylation array (450K-MA) results ( Figs. 4.3 and 4.4 A ). However, only FISH allows the enumeration of absolute numbers of chromosomes and determination of ploidy status.

Several other recurrent molecular alterations can be found in oligodendrogliomas. CIC (Capicua transcriptional repressor), a gene that resides in chromosome 19q, plays a pivotal role in regulating RAS (rat sarcoma oncogene)/MAPK (mitogen-activated protein kinase) signaling. Somatic mutations in CIC are present in as many as 69% of oligodendrogliomas. FUBP1 (far-upstream element binding protein) is a gene located on chromosome 1p. It has been suggested that inactivating somatic mutations in this gene (along with CIC ) are also associated with the development of oligodendroglioma. Tumors with such alterations seem to cluster with 1p/19q-codeleted oligodendrogliomas. Recent work has identified that TERT is overexpressed in oligodendrogliomas (see Fig. 4.1 ). Oligodendrogliomas are therefore now recognized to contain at least 4 different recurrent molecular genetic correlates: IDH mutation, 1p/19q loss, CIC mutation, and TERT promoter mutation. Mutations in the NOTCH1 gene (discussed later) were also observed in 31% of oligodendrogliomas.

Adult glioblastomas

Glioblastomas are high-grade, infiltrative astrocytomas with atypical nuclei, mitotic activity, extensive vascular proliferation, and/or necrosis. According to the revised WHO classification of CNS tumors, glioblastomas can be molecularly classified as IDH wild-type, IDH -mutant glioblastomas or NOS (see Fig. 4.1 ). Histologic IDH wild-type variants include giant cell ( Fig. 4.2 E, F), gliosarcoma, and the newly described epithelioid glioblastoma. About 90% of glioblastomas (usually primary) present as IDH wild-type; glioblastomas are found to be IDH mutated in about 10% of cases, and these are most often secondary glioblastomas that have progressed from lower-grade IDH -mutant gliomas.

Recent efforts to molecularly subclassify glioblastomas based on genetic and epigenetic expression profiling have consistently identified the classical, mesenchymal and proneural subtypes characterized by epidermal growth factor receptor ( EGFR ) amplification, neurofibromin 1 ( NF1 ) loss, and platelet-derived growth factor receptor A ( PDGFRA ) amplification, respectively. Notably, the proneural subtype was also associated with IDH1/2 mutations, G-CIMP phenotype as well as TP53 mutations. Additional mutations in histone H3F3A-K27 and H3F3A-G34 have defined epigenetic subgroups of glioblastoma in young adult (and pediatric) patients, which are addressed later.

IDH -mutated glioblastomas typically arise in younger patients following secondary transformation of a lower-grade astrocytoma. As such, the molecular features in IDH -mutant glioblastomas are often similar to those seen in IDH -mutant diffuse or anaplastic astrocytomas. IDH -mutant glioblastomas often harbor concurrent ATRX and TP53 mutations. Despite their high-grade histology, IDH -mutant glioblastomas show clinical behavior similar to that seen in lower-grade IDH -mutant astrocytomas. In contrast, IDH wild-type glioblastomas most commonly present as de novo tumors arising in patients older than 55 years. These tumors not only show vast histomorphologic heterogeneity but also harbor a wide variety of genetic alterations (see Fig. 4.1 ; Fig. 4.4 C). Data from TCGA show frequent alterations in 3 core signaling pathways: the CDK4/6-p16 ^INK4a -RB1-E2F, p14 ^ARF -MDM2-MDM4-p53, and RTK-RAS-PI3K pathways. These pathways and their common associated alterations are discussed below.