Role of Glioma markers for neurosurgeon

1. Dr. Shahnawaz Alam
Guided by: Dr. Vikas Chandra Jha
HOD, Dept. of Neurosurgery
Moderated by: Dr. Saraj kumar Singh
Faculty, Dept. of Neurosurgery
Glioma Molecular Markers
The 2016 WHO Update

2. Present Scenario
• Epidemiology of Gliomas
• Morphological diagnosis assisted by radiological findings
• Treatment according to tumor grade (Surgery, Chemotherapy, Radiotherapy)
• Targeted therapy
• WHO 2016 classification updates

3. Introduction
• For the first time, the WHO classification of CNS tumors 2016 uses molecular
parameters in addition to histology to define many tumor entities, thus
formulating a concept for how CNS tumor diagnoses should be structured in
the molecular era.
• Accurate prognostication is crucial for managing patients as treatments may
be associated with high morbidity and the benefits of high risk interventions
must be judged by the treating clinicians.
• Prognostic biomarker is a tumour-specific trait that predicts clinical outcome
regardless of treatment given.
• Predictive biomarker predicts clinical response to a specific treatment or drug
class.

4. Gliomas
• Gliomas (a primary tumor of glial cell origin) are the most common
intracranial neoplasm.
• Astrocytomas, glioblastomas, and oligodendrogliomas accounting for more
than 80%.
• Grade I to Grade IV tumors.
• GBMs: most aggressive and deadly of these tumors, are the most common
of the gliomas (55%).

5. Cell of Origin
• Glial cells.
• Neural stem cells (NSC): proposed that carcinogenesis is dependent on a small
population of cells termed ‘‘cancer stem cells*’’(CSCs).
• Genes that are expressed in NSC are Nestin, EGFR, PTEN,
Hedgehog etc : Neurogenesis and Gliogenesis.
• Aberrant activation of developmental genetic programs in NSCs gives rise to
CNS tumors.
*Ignatova et al. 2002; Shen et al. 2004

6. Distribution of Primary Brain and CNS Gliomasb by Histology Subtypes (N = 97,910), CBTRUS Statistical
Report: NPCR and SEER, 2008-2012

7. Pathways Of Gliomagenesis

9. HIF1: hypoxia inducible factor 1; NADP: nicotinamide adenine dinucleotide phosphate;
NADPH: reduced nicotinamide adenine dinucleotide phosphate; wt, wild type

10. IDH mutants create Oncometabolite

12. Paediatric Gliomas
• TP53 and PIK3CA mutations occur in all high grade gliomas,<10% of
childhood GBM harbor EGFR amplifications or PTEN mutations.
• IDH mutations are found in <5% of pediatric GBM although become more
common in adolescents.
• K27M mutations have been shown to occur in over 70% of pediatric
diffuse intrinsic pontine gliomas (DIPG) and confer a worse OS when
compared to patients with wild type H3.3.
• A tandem duplication at 7q34 is seen in a high proportion of pilocytic
astrocytomas. This rearrangement creates a fusion gene (KIAA1549:BRAF)
with constitutive BRAF kinase activity and putative abnormal activation of
MAPK/ERK pathway.

13. Diffuse midline glioma, H3 K27M-mutant:
• WHO Grade IV
• Formerly termed diffuse intrinsic pontine
glioma
• KIAA1549: BRAF fusion was an independent
prognostic marker for significantly improved 5 year
PFS for pilocytic astrocytomas as well as Grade II
diffuse and pilomyxoid astrocytomas.

14. Molecular Markers: Glioma
• 1p/19q co-deletion in oligodendroglial tumors.
• Mutations in the IDH1/2 genes in diffuse gliomas.
• Hypermethylation of the MGMT-gene promoter in glioblastomas.
• Alterations in the EGFR and PTEN genes, and 10q deletions in GBMs.
• BRAF alterations in pilocytic astrocytomas
MGMT: O6-methylguanine DNA methyltransferase ;, BRAF :v-raf murine sarcoma viral oncogene
homologe B1, IDH: isocitrate dehydrogenase; PTEN: Phosphatase and tensin homolog

15. IDH-1 & 2 Mutations
• IDH-1 in cytosol, IDH-2 in mitochondria to generate NADPH from
NADP, by oxidative decarboxylation of isocitrate to alpha-
ketoglutarate; maintain cellular redox state.
• Mutant IDH will cause conversion of alpha KG to 2-hydroxyglutarate;
important in hypoxic pathway.
• Presence of the mutation is associated with young age, a secondary-type GBM,
and increased overall survival; 60% to 90% of secondary glioblastomas that
developed from lower-grade tumors.
• IDH1 mutations are rare in primary GBMs and are completely absent in
pilocytic astrocytomas.
• Mutations in IDH2 gene were detected in a smaller proportion of gliomas
(5%), mostly in oligodendroglial tumors.

16. IDH Status and Survival in gliomas

17. • Sanger sequencing analysis: most commonly used method for detection of
IDH1 and IDH2 mutations. It allows for detection of all mutational variants.
• Pyrosequencing : better sensitivity than Sanger sequencing.
• Real-time PCR amplification: fast, less laborious, and more sensitive;
allows detection of as little as 10% mutant alleles or 20% of cells with
mutations in a background of normal DNA.
• Immunohistochemistry: monoclonal antibodies for detection of IDH1
R132H mutation. Convenient detection of mutations in tissue sections. IHC
will miss approximately 10% of gliomas carrying less-common mutations of
IDH1 and all of the IDH2 mutations.

18. MGMT-Promoter Methylation
• The MGMT gene is located at chromosome 10q26 and codes for a ubiquitously
expressed suicide DNA repair enzyme that removes alkyl adducts from the O6-
position of guanine.
• O6-alkylated guanine leads to double-strand breaks and base mispairing,
thereby inducing apoptosis and cell death, MGMT protects normal cells from
carcinogens.
• Epigenetic silencing of this gene by promoter hypermethylation leads to reduced
expression of the MGMT protein.
• MGMT gene silencing improves survival in patients with glioblastoma who are
treated concurrently with alkylating drug temozolomide and radiation therapy.
• Prognostic and predictive marker.
MGMT: Methylguanine-DNA methyltransferase

19. • It also protects tumor cells from normally lethal effects of chemotherapy
with alkylating agents such as temozolomide.
• Methylation of the MGMT promoter is found in 35%-45% of malignant
gliomas (WHO grades III and IV) and in about 80% of WHO grade II
gliomas.
• Hegi and colleagues (2005): reported that 49% of patients with
glioblastoma and methylated MGMT were alive at 2 years after treatment
with temozolomide and radiotherapy, as compared with 15% of patients
with unmethylated MGMT.

20. • Most of the methods for MGMT analysis are based on evaluation of the
methylation status of the ‘CG island’of the MGMT gene.
• Methylation-specific PCR (MSP) : methylation status at 6 to 9 CpGs.
• Real-time PCR.
• Methylation-specific Pyrosequencing.
• IHC: assessment of MGMT methylation by IHC hasfailed to correlate
with disease outcome.
*CG : Cytosine/Guanine

21. 1p19q-Codeletion
• Complete deletion of both the short arm of chromosome 1 (1p) and the long arm
of chromosome 19 (19q) (1p/19q co-deletion) is the molecular genetic signature
of oligodendroglioma; Early genetic event in oligodendroglial tumorigenesis.
• The presence of 1p/19q co-deletion is a strong independent prognostic biomarker
associated with improved survival in both diffuse low-grade and anaplastic
tumours, among all diffuse gliomas, patients with 1p/19q- co deletion have the
most favourable prognosis.
• 80% to 90% in oligodendrogliomas (WHO grade II); 60% in anaplastic
oligodendrogliomas (WHO grade III); 30% to 50% in oligoastrocytomas.

22. • 1p/19q-codeletion has predictive value for response to chemotherapy in
anaplastic oligodendrogliomas.
• Partial loss of chromosome 1p in oligodendrogliomas has an opposite
prognostic significance when compared with tumors that have a
complete 1p/19q loss.
• Almost all oligodendrogliomas with a 1p/19q codeletion are also positive
for IDH1 or IDH2 mutations.
• Cairncross et al.: (1998) : better response to procarbazine-lomustine-
vincristine chemotherapy and a longer survival in patients with anaplastic
oligodendroglioma.
• Co-deletions (I:e, 9p or 10q loss) may lead to poor outcome independent
of the 1p/19q status.

23. • The first allele is lost (1st Hit) due to an imbalanced reciprocal translocation
between chromosomes 1 and 19.
• The second allele is disrupted (2nd Hit) by a somatic mutation capable of
inhibiting protein Function.

24.  The status of the 1p/19q loci detected by:
• FISH
• PCR
• Loss of heterozygosity (LOH) analysis or virtual karyotyping
• Comparative genomic hybridization array
• Single nucleotide polymorphism array

25. EGFR-Mutation

26. • EGFR is a receptor tyrosine kinase. Ligand binding by EGF promotes receptor
dimerization and autophosphorylation of the cytoplasmic domain.
• Such EGFR activation is thought to promote cellular proliferation via activation of
the MAPK and PI3K-Akt pathways.
• It is well known up to 65% of so-called primary glioblastomas showepidermal
growth factor receptor (7p12) amplification,over- expression, and/or mutations of this
pathway.
• Such glioblas-tomas are part of the “classic” expression subtype occurring mutually
exclusively with IDH-mutated secondary GBM.
• The majority of GBMs with EGFR amplification also contain the mutant EGFR
gene, EGFRvIII , which is typically expressed in about 30% of newly diagnosed
GBM patients.
• The EGFRvIII is characterized by the deletion of exons 2 – 7, resulting in a sense
mutation that has a truncated extracellular domain with ligand-independent
constitutive activity.

27. • Cell surface receptors for Endothelial growth factors.
• EGFR affects cell proliferation and growth through the activation of
downstream effector molecules in the MAPK and PI3K-AKT pathways.
• EGFR gene : located on chromosome 7p12.
• Activation of EGFR signaling through gene amplification or
mutations is found in about 30% to 40% of primary glioblastomas.
• Mutant EGFR: characterized by a deletion of 267 amino acids in the
extracellular domain of the EGFR protein.

28. • Truncated protein: EGFRvIII receptor : lacks an extracellular domain
but remains constitutively activated.
• Detection of either EGFR amplification or EGFRvIII is indicative of high-
grade glioma and can be used diagnostically.
• Attractive target for new therapies in gliomas/ anti-EGFR tyrosine kinase
inhibitors
• anti-EGFRvIII vaccine: addition of vaccine to radiation and
chemotherapy resulted in increased overall survival*
• EGFR amplification: FISH/ EGFRvIII analysis: performed by RT-PCR
amplification
*Heimberger AB, Sampson JH. The PEPvIII-KLH (CDX-110) vaccine in glioblastoma multiforme patients. Expert Opin Biol Ther. 2009;9(8):1087–1098. 84.
Yoshimoto K, Dang J, Zhu S, et al. Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples. Clin Cancer Res. 2008;
14(2):488–493

29. PTEN-Mutation
• Phosphatase and tensin homolog (PTEN), located on chromosome 10q23.3, is
one of the most commonly lost or downregulated genes implicated in brain,
breast cancer, and prostate tumors.
• PTEN is a tumor suppressor gene, playing important roles in the regulation
of cell proliferation, adhesion and invasion, apoptosis, and DNA damage
repair.
• PTEN has independently been shown to play roles in both neurogenesis and
gliogenesis.
• PTEN loss results in disrupted regulation of cell size or cell number in the
brain.
• The loss of PTEN expression has been indicated to be an early event in
glioma, with mutations occurring in between 5% and 40% of glioma cases.

30. • Studies have suggested that PTEN gene mutations in glioma are
associated with poor survival.
• Tumor suppressor gene located on the long arm of chromosome 10
• Counteracts one of the most critical cancer-promoting pathways, the
PI3K-AKT signaling pathway
• Genetic alterations: LOH at 10q frequently found in high-grade gliomas
(15-40%)
• Poor prognostic marker for anaplastic astrocytomas and glioblastoma
• Detected in FFPE tissue by LOH analysis or FISH

31. PDGFRA
• Discovered as a serum growth factor for fibroblasts, smooth muscle cells,
and glial cells, the PDGF family has become one of the most extensively
studied growth factor families.
• Ligands binds to the receptors, homo- and heterodimerization of the receptors
occur. This in turn leads to transphosphorylation of the intracellular domains
and receptor activation.
• Once activated, intracellular mediators dock to phosphotyrosine residues in the
receptor, which leads to downstream activation of intracellular signaling
pathways.

32. • In GBM, platelet-derived growth factor receptor alpha (PDGFRA) is the
most commonly altered receptor tyrosine kinase after EGFR.
• GBM with amplified PDGFRA have been shown to be associated with
either amplified EGFR or amplified MET (the hepatocyte growth factor
receptor).
• PDGFRA amplification has recently been shown to be associated with
a poor prognosis in IDH1 mutant GBM and have a negative
prognostic value in Grade III gliomas.

33. • Part of the mitogen-activated protein kinase (MAPK) pathway.
• Serine/threonine kinase, modulates cell proliferation and survival.
• First BRAF mutation reported in papillary thyroid carcinomas.
• In gliomas: BRAF activation is by gene duplication or point mutation.
• Fusion between the KIAA1549 and BRAF genes.
BRAF

34. • Identified in 60% to 80% of pilocytic astrocytomas.
• Rare in diffuse astrocytic gliomas.
• Prognostic significance is still under investigation.
• RAF inhibitors (vemurafenib and dabrafenib).
• Interphase FISH: currently the best method for testing for this fusion.
• IHC : anti-BRAF V600E (VE1) antibody.

36. The WHO 2016 Update
• Integrated diagnoses using, for the first time, molecular parameters in addition
to histology to define tumor entities.
• Improves classification, objectivity and treatment.
• Breaks with over 100 years of tradition of defining disease based on light
microscopy.
• Genotype trumps histologic phenotype (still need a pathologist for glioma
diagnosis and grade).
• Oligodendroglioma, IDH-mutant and 1p/19q- codeleted (even if it looks
astrocytic).

37. • Diffuse astrocytoma, IDH-mutant (even if it looks like an oligodendroglioma).
• Oligoastrocytomas should become vanishingly rare.
• Ependymoma, RELA fusion-positive; Upregulates the NF-κB pathway.
• PNET has been deleted, this tumor is now typically embryonal tumor with
multi-layered rosettes, C19MC-altered(ETMR).
• Medulloblastoma, WNT-activated/ SHH-activated and TP53-mutant.

38. SUMMARY
• Only IDH mutation status (prognostic) and MGMT methylation status and 1p/19q co-
deletion (predictive) are currently routinely used for evaluation of glioma patients by
clinicians.
• Gliomas with mutated IDH1 and IDH2 have improved prognosis compared to
gliomas with wild-type IDH.
• IDH1 mutations often occur with a TP53 mutation in astrocytic tumors, and these
tumors rarely demonstrate loss of chromosomes 1p and 19q.
• IDH mutation is seen in virtually all oligodendrogliomas with 1p/19q co-deletion, and
these tumors rarely demonstrate p53 mutation.
• MGMT-promoter methylation not only predict an improved response to temozolomide,
but may represent a surrogate marker of a more treatment-responsive tumour in
general.
• Among all diffuse gliomas, patients with 1p/19q- co-deletion have the most
favourable prognosis.

39. • PTEN gene mutations in glioma are associated with poor survival.
• The ongoing development of targeted therapies as mono and
combination treatments necessitates the discovery of optimal
molecular predictive biomarkers.
• Not only a morphological diagnosis, but also molecular data
• Prognosis and response to treatment
• 1p/19q assessment, IHC for IDH1 and β-catenin
• Molecular data is guiding pathologic diagnoses, leading to better, more
targeted treatment strategies.
• “Setting the stage for progress.” / Future work: Subdivide the “NOS”
categories.

40. Thank You