Translating Molecular Biomarkers of Gliomas to Clinical Practice

Acknowledgments

The authors are grateful to their NYU Langone Medical Center colleagues Matija Snuderl, MD, for contributing illustrated cases of cytogenetics analysis and methylation array; Cyrus Hedvat, MD, PhD, for providing an illustrated case of loss of heterozygosity analysis; and Elad Mashiach for assisting with the preparation of images and diagrams, and the editing of the chapter.

Glioma classification and grading have traditionally been based on the histomorphology of the tumors. Recent advances have identified new molecular markers with diagnostic, prognostic, and/or predictive (ie, therapeutic) significance ( Box 4.1 , Table 4.1 ). Since the publication of the World Health Organization (WHO) guidelines in 2007, there has been a rapid expansion of molecular data on central nervous system (CNS) tumors that has improved clinicians’ diagnostic, prognostic, and therapeutic abilities. Although most of this information has not yet been translated into tangible clinical advances, many changes have been implemented in the revised WHO guidelines for the classification of tumors of the CNS (2016) for gliomas. This chapter reviews recently identified genetic markers that have had a significant impact on the molecular classification of gliomas. Many have been shown to be essential in better diagnosing CNS tumors, reliably determining the prognosis, and allowing better clinical management. Based on the revised WHO classification, each tumor type is discussed separately, accompanied by the relevant molecular profiles.

Box 4.1

The National Institutes of Health Biomarkers Definitions Working Group defined a biomarker as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” Biomarkers in gliomas have been investigated particularly for their use in identifying patients with a disease or a disease subtype (diagnostic biomarkers), stratifying the patients’ prognoses and natural history of the disease (prognostic biomarkers), and identifying patients who may achieve a particular outcome based on a particular treatment and attempting to personalize clinical treatment (predictive biomarkers). Several of the biomarkers discussed in this chapter have distinct roles as diagnostic, prognostic and/or predictive biomarkers, and these are discussed in the text and summarized in Table 4.1 and Figs. 4.1 and 4.5 .

Definition and types of biomarkers

Table 4.1

Overview of common chromosomal, genetic, epigenetic and phenotypic alterations in gliomas and their use as biomarkers

Gene/Phenotype ^a	Gene Family/Alternative Name	Chromosomal Location	Driver Gene ^b	Typical Mutation ^c	Copy Number Alteration	Translocation Partner	Detection Method	Adult Glioma Tumor Type	Pediatric Glioma Tumor Type	Biomarker Clinical Utility
Chromosomal
—	—	1p/19q	—	—	Codeletion	—	FISH, LOH, MLPA, 450K-MA	OD, AOD	—	Diag, Prog, Pred (chemo + radiotherapy)
CIC	Transcription repressor	19q13.2	TSG	R215Q/W	Del	—	IHC (loss of staining), others	OD, AOD	—	Diag, Prog
FUBP1	DNA-binding protein	1p31.1	TSG	Many	Del	—	IHC (loss of staining), others	OD, AOD	—	Diag, Prog
—	—	7 or 7q	—	—	Single copy gain	—	FISH, others	DA, AA, GBM	—	—
—	—	10 or 10q	—	—	Single copy loss	—	FISH, others	GBM	—	—
Genetic
ACVR1	RSTK	2q23-q24	TSG	Few	—	—	qRT-PCR, Seq	—	Midline HGG, DIPG	—
BRAF	RAF kinase	7q34	ONC	V600E	—	—	MS-IHC, qRT-PCR, Seq	PXA, EGBM	PA, PXA, cortical HGG	Diag
BRAF	—	—	—	—	Amp	KIAA1549, others	FISH, others; qRT-PCR, Seq	PA	PA, PMA	Diag
CDKN2A	Kinase inhibitor/p14, p16	9p21	TSG	—	Del	—	FISH, MLPA, 450K-MA	OD, AOD, DA, AA, GBM	PXA	—
CDKN2B	Kinase inhibitor	9p21	—	—	Del	—	FISH, MLPA, 450K-MA	OD, AOD, DA, AA, GBM	PXA	—
EGFR	RTK	7p12	ONC	—	Amp	SEPT14	FISH, others; qRT-PCR, Seq	Classic GBM	Cortical HGG	Diag
EGFR	—	—	—	EGFRvIII, A289D/T/V	—	—	MS-IHC, qRT-PCR, Seq	Classic GBM	—	Diag
FGFR1	RTK/CD331	8p11.23-p11.22	—	K656E	—	TACC1	qRT-PCR, Seq; FISH, others	—	PA, midline HGG/DIPG	—
FGFR3	RTK/CD333	4p16.3	ONC	—	—	TACC3	FISH, qRT-PCR, Seq	GBM	—	—
IDH1	Dehydrogenase	2q34	ONC	R132H, others	—	—	MS-IHC, qRT-PCR, Seq	OD, AOD, DA, AA, GBM	Cortical HGG	Diag, Prog
IDH2	Dehydrogenase	15q26.1	ONC	R172K, others	—	—	qRT-PCR, Seq	OD, AOD, DA, AA, GBM	—	Diag, Prog
MDM2	Ubiquitin protein ligase	12q13-q14	ONC	Few	Amp	—	FISH, MLPA, 450K-MA	GBM	—	—
MDM4	p53 regulator	1q32	ONC	Few	Amp	—	FISH, MLPA, 450K-MA	GBM	—	—
MET	RTK	7q31	ONC	—	Amp	—	FISH, MLPA, 450K-MA	GBM	—	—
MYC	Transcription factor	8q24	ONC	—	Amp	—	FISH, MLPA, 450K-MA	Astrocytoma, GBM	—	—
NF1	RAS negative regulator	17q11.2	TSG	Many	Del	—	qRT-PCR, Seq; FISH, others	Mesenchymal GBM	PA, midline HGG	—
NOTCH1	receptor	9q34.3	TSG	F357del	Amp	—	qRT-PCR, Seq; FISH, others	OD	—	—
NTRK2	RTK	9q22.1	—	—	Amp	QKI	FISH, others; qRT-PCR, Seq	—	PA, non-brainstem HGG	—
PDGFRA	RTK/CD140a	4q12	ONC	Many	Amp	KDR	qRT-PCR, Seq; FISH, others	Proneural GBM	Midline HGG, DIPG	Prog
PIK3CA	PI3 kinase	3q26.3	ONC	H1047L/R/Y	Amp	—	qRT-PCR, Seq; FISH, others	OD, AOD, GBM	Midline HGG, DIPG	—
PIK3R1	Regulatory subunit of PI3 kinase	5q13.1	TSG	G376R	Del	—	qRT-PCR, Seq; FISH, others	OD, AOD, GBM	Midline HGG, DIPG	—
PTEN	Phosphatase	10q23	TSG	R130 ^d/Q	Del	—	qRT-PCR, Seq; FISH, others	Astrocytoma, classical GBM	—	Prog
PTPN11	Phosphatase	12q24.1	ONC	Many	—	—	qRT-PCR, Seq	—	PA	—
RB1	Ligand	13q14.2	TSG	R445 ^d, X445_splice	Del	—	qRT-PCR, Seq; FISH, others	Mesenchymal GBM	—	—
TERT	Telomerase	5p15.33	—	Promoter	—	—	qRT-PCR, Seq	OD, AOD, astrocytoma, GBM	—	Diag, Prog
TP53	Transcription factor	17p13.1	TSG	R273C/H/L, R248Q/W	—	—	(IHC), qRT-PCR, Seq	Astrocytoma, GBM	Midline/cortical HGG	—
Epigenetic
ATRX	Chromatin remodeler	Xq21.1	TSG	F2113fs	—	—	IHC (loss of staining), others	DA, AA, GBM	Cortical HGG	Diag, Prog
DAXX	Chromatin remodeler	6p21.3	TSG	—	Amp	—	FISH, MLPA, 450K-MA	—	Cortical HGG	—
HIST1H3B	Histone	6p22.2	ONC	H3.1 K27M	—	—	qRT-PCR, Seq	—	Midline HGG, DIPG	Diag, Prog
H3F3A	Histone	1q42.12	ONC	H3.3 K27M	—	—	MS-IHC, qRT-PCR, Seq	Midline HGG, DIPG	Midline HGG, DIPG	Diag, Prog
H3F3A	Histone	1q42.12	ONC	H3.3 G34R/V	—	—	MS-IHC, qRT-PCR, Seq	—	Cortical HGG	Diag, Prog
MGMT	DNA cysteine MT	10q26		Promoter methylation	—	—	MS-PCR, 450K-MA	GBM	—	Prog, Pred (temozolomide)
SETD2	Histone lysine MT	3p21.31	TSG	Many	Del	—	qRT-PCR, Seq; FISH, others	—	Cortical HGG	—
TET2	Demethylase	4q24	TSG	Few	—	—	qRT-PCR, Seq	GBM	—	—
Phenotypic
2-HG	2-hydroxyglutarate	—	—	—	—	—	MRS, mass spectrometry	OD, AOD, DA, AA, GBM	—	—
G-CIMP	Glioma–CpG island methylator phenotype	—	—	—	—	—	450K-MA	OD, AOD, DA, AA, GBM	—	—

Abbreviations: AA, anaplastic astrocytoma; AOD, anaplastic oligodendroglioma; CD, cluster of differentiation; DA, diffuse astrocytoma; Diag, diagnostic biomarker; DIPG, diffuse intrinsic pontine glioma; EGBM, epithelioid glioblastoma; EGFR; epidermal growth factor receptor; EGFRvIII, deleted exons 2 to 7 EGFR; FISH, fluorescence in situ hybridization; fs, frame shift; GBM, glioblastoma; HGG, high-grade (III–IV) glioma; IHC, immunohistochemistry; LGG, low-grade (I-II) glioma; LOH, loss of heterozygosity; MLPA, multiplex ligation-dependent probe amplification; MRS, magnetic resonance spectroscopy; MS-IHC, mutation-specific immunohistochemistry; MS-PCR, methylation-specific polymerase chain reaction; MT, methyltransferase; OD, oligodendroglioma; ONC, oncogene; PA, pilocytic astrocytoma; PMA, pilomyxoid astrocytoma; Pred, predictive biomarker; Prog, prognostic biomarker; PXA, pleomorphic xanthoastrocytoma; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; RSTK, receptor serine/threonine kinase; RTK, receptor tyrosine kinase; Seq, targeted nucleotide sequencing; TSG, tumor suppressor gene; 450K-MA, 450K CpG methylation array.

a Gene symbols, gene families, and chromosomal locations according to the Human Genome Organisation Gene Nomenclature Committee ( www.genenames.org ) and Catalogue of Somatic Mutations in Cancer ( cancer.sanger.ac.uk ).

b Driver genes that contain driver gene mutations as defined by Vogelstein and colleagues.

c Gene mutations and copy number alterations based on the Merged Cohort of LGG and GBM (The Cancer Genome Atlas [TCGA], 2016) database (1102 samples) generated by TCGA Research Network ( http://www.cbioportal.org/index.do ). In addition, the translocation partners for BRAF, EGFR, FGFR1, NTRK2, and PDGFRA are listed.

d Change to a termination codon (nonsense mutation).

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.