Molecular pathogenesis of CNS tumors

Molecular pathogenesis of

CNS tumors

Imtiaz Ahmed

Present Scenario• Morphological diagnosis assisted by radiological

findings

• Tumor grade (WHO grade)

• Treatment according to tumor grade (Surgery, Chemotherapy, Radiotherapy)

• Targeted therapy

Today’s lecture• Epidemiology• Gliomas• Medulloblastoma• Ependymomas• Meningiomas• Primary CNS lymphomas• WHO 2007 classification• WHO 2016 classification

Hallmarks of cancer. (Adapted from Hanahan D, Weinberg RA.Hallmarks of cancer: the next generation. Cell 2011; 144:646.)

Epidemiology of CNS tumors

• Primary cerebral malignancy* -• 4 to 10/lac general population• 1.6% of all primary tumors• 2.3% of all cancer related deaths

• 2nd most common cancer in children• 20% of all cancers in children <15 yrs• Therapeutic X-irradiation has been unequivocally linked with

brain tumors (7–9 years)

*Francis Ali-Osman, Brain tumors, 2005

Distribution of Primary Brain and CNS Tumors by Behavior (N = 356,858), CBTRUS Statistical Report: NPCR and SEER, 2008-2012

Distributiona in Children (Age 0-14 years) of Primary Brain and CNS Tumors by CBTRUS Histology Groupings and Histology (N = 16,366), CBTRUS Statistical Report: NPCR and SEER, 2008-2012

Cell of Origin• Glial cells

• Neural stem cells (NSC): proposed that carcinogenesis isdependent 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

Gliomas

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%)

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

Gliomas• 1985 : Epidermal growth factor receptor (EGFR) gene

amplification in glioblastoma

• Subsequent discoveries : • Phosphatase and tensin homolog (PTEN) gene • Mutations in the TP53 gene• BRAF fusion • MGMT gene • IDH mutations

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

BRAF, v-raf murine sarcoma viral oncogene homolog B1 gene; CDKN2A/B, cyclin-dependent kinase inhibitors 2A and 2B genes; EGFR, epidermal growth factor receptor gene; GBM, glioblastoma multiforme; IDH, isocitrate dehydrogenase gene; mut., mutation; PTEN, phosphatase and tensin homolog gene; TP53, tumor protein p53 gene

Molecular Diagnostics of Gliomas—Nikiforova & Hamilton; Arch Pathol Lab Med—Vol 135, May 2011

Gliomas1. 1p/19q CODELETION:

• Loss of the short arm of chromosome 1 (1p), along with the long arm of chromosome 19 (19q); "genetic signature" of oligodendrogliomas

• Early genetic event in oligodendroglial tumorigenesis

• 80% to 90% in oligodendrogliomas (WHO grade II) • 60% in anaplastic oligodendrogliomas (WHO grade III) • 30% to 50% in oligoastrocytomas

• 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

Gliomas

• 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


• The CIC gene is a tumor suppressor gene present in the Chr 19

• Encodes for protein capicua homolog

• Member of the high mobility group (HMG)-box superfamily of transcriptional repressors

• Loss of CIC gene results in loss of transcription repressor function


• 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

• Cairncross et al.: (1998) : better response to procarbazine-lomustine-vincristine chemotherapy and a longer survival in patients with anaplastic oligodendroglioma

• Co-deletions (ie, 9p or 10q loss) may lead to poor outcome independent of the 1p/19q status

Gliomas2. IDH1 AND IDH2 MUTATIONS

• Mutations in the IDH1 gene were discovered in 2008 during a genome-wide analysis of 22 glioblastomas as a part of the Cancer Genome Atlas Project

• 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

Gliomas

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

Gliomas2. IDH1 AND IDH2 MUTATIONS

• 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

Gliomas

Primary glioblastoma (A-C); Secondary glioblastoma (D-F)

EGFR (A, D); p53 (B, E); IDH-1 (C, F)

Gliomas3. MGMT METHYLATION :

• The MGMT gene is located at chromosome 10q26 and encodes for a DNA repair protein

• 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• 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

MGMT: Methylguanine-DNA-methyltransferase

Gliomas3. MGMT METHYLATION :

• 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 has failed tocorrelate with disease outcome

CG : Cytosine/Guanine

Gliomas4. BRAF/KIAA1549 FUSION :

• 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• 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

Gliomas5. EGFR AND PTEN ALTERATIONS:

• 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• 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

Gliomas5. EGFR AND PTEN ALTERATIONS:

• 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

• Phosphatase and tensin homolog (PTEN) : 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

*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

Medulloblastoma

Medulloblastomas• Second most frequent BT in children after pilocytic astrocytoma

• First decade of life, second peak in the early 20s

• Genetic tumor syndrome: Turcot syndrome, Gorlin syndrome

• Embryonal tumor of the brain, analogous to Wilms tumor of the kidney

• Origin: stem cells located in the subependymal matrix and the external granular layer (EGL) of the cerebellum

• Medulloblastomas are tumors of the cerebellum, arising more frequently in the midline, especially in the posterior vermis, adjacent to the roof of the fourth ventricle

Medulloblastoma: molecular pathways and histopathological classifcation ; Anna Borowska, Jarosław Jóźwiak ; Arch Med Sci 2016; 12, 3: 659–666

Medulloblastomas• Molecular pathogenesis:

• Previously, thought to represent a subset of primitive neuroectodermal tumor (PNET) of the posterior fossa

• Gene expression profiling: distinct molecular profile and are distinct from other PNET tumors

• Five histological subtypes: 1. Classical type (CMB)2. Desmoplastic/nodular type (DN), 3. Medulloblastoma with extensive nodularity (MBEN), 4. Anaplastic type5. Large cell Medulloblastoma (LC)

• Four molecular subgroups:1. Wnt subgroup 2. Sonic hedgehog subgroup 3. Group 3 4. Group 4

Medulloblastomas1. WNT subgroup:

• The Wnt/β-catenin pathway participates in the control of vertebrate development

• Rarest subgroup of medulloblastoma, accounting for 11%

• Patients with Turcot syndrome: predisposition to Wnt MB

• Germline mutation of Apc gene

• Thought to arise from ‘mossy-fiber neuron precursors’, involved in the formation of synapses in the developing cerebellum


*DSV: DisheveledLRP: Low density lipoprotein receptor-related protein 1


• Includes mainly classic MB

• Large cell/anaplastic MB (good prognosis)

• Monosomy of chromosome 6 is present in about 100% of Wnt tumors

• Overall excellent long term prognosis (90% 5 year survival rate)


• IHC: monoclonal antibodies against the C-terminal domain of β-catenin

• CTNNB1 (β-catenin encoding gene) mutation analysis by direct gene sequencing

• Cantharidin and norcantharidin: drugs on trial against Wnt associated medulloblastoma

(A) Classic MB with nuclear β-catenin immunostaining

(B) Nodular MB with cytoplasmic β-catenin immunostaining

(C) Anaplastic MB with cytoplasmic β-catenin immunostaining

Medulloblastomas2. Shh Medulloblastomas

• Account for 28% of all medulloblastomas • Intermediate prognosis• Dichotomous age distribution: common in both children

(<4 years) and adults (>16 years) • Gorlin syndrome : germline mutations in PTCH gene*• Sonic hedgehog (Shh) pathway: plays a key role in normal

cerebellar development, induces proliferation of neuronal precursor cells in the developing cerebellum and other tissues

• Normal conditions: The Shh ligand is secreted by Purkinje neurons and promotes formation of the external germinal layer in the cerebellum

PTCH: patched 1


GLI: glioblastoma family protein


• Molecular analysis of sporadic medulloblastomas commonly shows Patched-1 (PTCH1) mutations

• Desmoplastic/nodular and MBEN are almost exclusively associated with Shh pathway activation

• IHC: GLI1, and GAB1 have been proposed

• Hh pathway inhibitor: Cyclopamine, Vismodegib, Saridegib • SMO inhibitors: SANT1–SANT4 • Arsenic compounds: targets GLI1

SFRP1: secreted frizzled related protein 1; GLI1: glioblastoma family protein; GAB1: GRB2-associated-binding protein 1

Medulloblastomas3. Group 3 Medulloblastomas

• 28% of all medulloblastomas • Associated with the worst prognosis of all the subgroups and are

frequently metastatic • Predominantly found in infants/children• Relatively little is known about the molecular pathogenesis • Associated with MYC amplification • Further categorized in to 3α and 3β, depending on MYC expression• 3α – tumors: increased MYC expression and worse prognosis• 3β – tumors: normal MYC expression and better prognosis• Mostly associated with classic or large cell/anaplastic morphology • Detected by transcriptional profiling, although IHC for NPR3* has

been proposed

*NPR3: Natriuretic peptide receptor


• Most common “typical” subgroup of medulloblastoma, accounting for around 34%

• Rarely affect infants (0–3 years) and mainly affect children, with a peak age of 10 years

• Intermediate prognosis• Classic histology • Associated with isochromosome 17q (2/3rd cases)• Associated with CDK6 and MYCN amplification but minimal

MYC over-expression • Chromosome X loss is seen in 80% of females


• Currently detected by gene expression profiling

• Immunohistochemistry for KCNA* has been proposed

*Potassium Voltage-Gated Channel Subfamily A

Ctnnb: catenin b1; APC: adenomatous polyposis coli; LC: Large cell; A: anaplastic; DN: diffuse nodular

Ependymoma

Ependymal Tumors Third most common pediatric brain tumors 50% of cases arising in children under 5 years of age Ependymal neuroepithelium of the ventricles and spinal

canal Occur in three distinct locations:

Supratentorial brain comprising the cerebral hemispheres Brain stem and cerebellum Spinal cord

Pediatric: Intracranial, cerebellum and brain stem Adult: Spinal cord

Ependymal Tumors1. Myxopapillary ependymoma Grade I

2. Subependymoma Grade I

3. Ependymoma Grade II

1. Cellular

2. Papillary

3. Clear cell

4. Tanycytic

4. Anaplastic ependymoma Grade III

Ependymal Tumors

1. Cytogenetic abnormalities

2. Molecular genetic abberations

3. Epigenetic modifications

4. Gene expression profiling studies

*Taylor MD, Poppleton H, Fuller C, et al. Radial glia cells are candidate stem cells of ependymoma [J]. Cancer Cell, 2005,8 (4):323-335

CDKN2A: cyclin-dependent kinase Inhibitor 2A

Ependymal Tumors2. Molecular Genetic Aberrations

Taylor et al*, aCGH profiles of 103 ependymomas, three molecularly distinct subtypes of ependymomas depending on tumor location:

1. Supratentorial ependymomas : CDKN2A deletion in >90% cases, poor prognosis

2. Spinal tumors: Deletion of chromosome 22q12,

3. Posterior fossa ependymomas : chromosome 1q gain, good prognosis

*Taylor MD, Poppleton H, Fuller C, et al. Radial glia cells are candidate stem cells of ependymoma [J]. Cancer Cell, 2005,8 (4):323-335


RELA fusion positive ependymomas: Subset of supratentorial ependymomas Fusion between C11orf95, a gene with unknown function, and

RELA gene on Chr 11q13 RELA encodes RelA (p65), protein which interacts with IκB and

p50 in the central signaling complex in the NF-κB pathway Amenable to targeted therapy

Supratentorial ependymomas of childhood carry C11orf95–RELA fusions leading to pathological activation of the NF κB signaling pathway: Acta ‑Neuropathol (2014) 127:609–611 RELA: v-rel avian reticuloendotheliosis viral oncogene homolog A


RELA fusion positive ependymomas:

*NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) ; IKK: IĸB kinase

RELA Fusion gene protein

Ependymal Tumors3. Epigenetics:

Methylation status of the hypermethylated in cancer 1 (HIC-1) putative tumor suppressor gene: down regulation in 81% of cases, correlated with non-spinal localization and pediatric age

The RAS association domain family 1 isoform A (RASSF1A) gene: silenced by methylation in 86% of ependymoma, results in loss of cell cycle control, enhanced genetic instability and cell motility, and resistance to K- Ras and TNF-α induced apoptosis

Ependymal Tumors4. Gene expression profiling:

112 abnormally expressed genes* in ependymoma WNT5A, TP53 homologue, TP63, ZIC1, VEGF and

Fibronectin1 Cell cycle, proliferation, adhesion, and extracellular matrix

regualtion

*Suarez Merino B, Hubank M, Revesz T, et al. Microarray analysis of pediatric ependymoma identifies a cluster of 112 candidate genes including four transcripts at 22q12.1 -q13.3 [J]. Neuro Oncol, 2005,7(1):20-31 **Taylor MD, Poppleton H, Fuller C, et al. Radial glia cells are candidate stem cells of ependymoma [J]. Cancer Cell, 2005,8 (4):323-335

Meningioma

Meningioma• Meningiomas are mostly benign, slow-growing tumors of the

CNS• Most common CNS tumor in adults• Originate from Arachnoidal cap cells • Annual incidence of meningiomas is 2.3 per 100,000 • Peaks in the 7th decade of life

• Associated risk factors: • Deletions of the neurofbromatosis Type 2 (NF2) gene• Ionizing radiation

Meningioma• Benign meningiomas are slow growing and have a 5-year

recurrence rate of 5% following gross-total resection

• Atypical meningiomas have 5-year recurrence rate of 40%

• Anaplastic meningiomas have recurrence rates of up to 80%

• Surgical resection and radiotherapy, mainstay treatment

Meningioma• Cytogenetic abnormality:

• Monosomy 22 is the most frequent genetic abnormality

• Association between the long arm of chromosome 22 (22q) and meningiomas was first studied in patients with NF2

• Bilateral vestibular schwannomas, multiple meningiomas, and other CNS tumors

• Allelic losses in 22q12.2: Nearly all NF2-associated meningiomas, and 70% of sporadic meningiomas

• NF2 gene encodes the tumor suppressor merlin, critical role in controlling cell growth and motility

NF2: Neurofibromatosis 2

Schematic diagram of merlin's role in tumourigenesis.

C. O. Hanemann Brain 2008;131:606-615


NDGR2: N-myc downregulated gene family


• Chr 1p deletions comprise the second most common chromosomal abnormality

• Found in:• 13%–26% of Grade I• 40%–76% of Grade II• 70%–100% of Grade III

• Loss of 1p is also associated with a 30% recurrence rate

Meningioma• Sex steroids:

• The incidence of meningiomas is more than 2-fold greater in women than in men

• Increased growth during pregnancy and the luteal phase of the menstrual cycle

• Expression of the progesterone receptor is most frequently observed

• Progesterone receptor is expressed in 81% of women and 40% of men with meningiomas

• Expression is highest in benign meningiomas (50%–80%) • PR status can help to describe the biological behavior of

meningiomas

CNS Lymphomas

Primary CNS lymphoma• Accounts for less than 5% of all primary brain tumors • Lymphoma occurring in the brain, leptomeninges, spinal cord, or

eyes without evidence of lymphoma outside the CNS • Majority are high- grade B -cell lymphomas• 95%-98% diagnosed as high -grade DLBCL• 05% of cases include Burkitt, Burkitt- like, and lymphoblastic B -cell

lymphomas as well as T -cell lymphomas

• Patients with AIDS develop PCNSL at a rate 3600 -foldhigher than the general population and have a lifetime risk of CNS lymphoma that approaches 20%

Primary CNS lymphoma• Age group: 60 yrs

• Intracranial mass lesion, 70% cases are supratentorial

• Basal ganglia, the corpus callosum, and/or the periventricular subependymal tissues

• Periventricular location, facilitating leptomeningeal seeding

• Extend across the corpus callosum and involve both cerebral hemispheres

Primary CNS lymphoma• Pathogenesis :

• Gain on chromosome 12 • EBV• Chemokines• Protooncogene mutation• Ectopic expression of Interleukin-4• Promoter hypermethylation of the CDKN2A gene• STAT6 overexpression• Unfolded protein response pathway• Somatic mutations in Ig variable region genes• Allelic deletions of the long arm of chromosome 6

Primary CNS lymphoma• Gain on chromosome 12 :

• Comparative genomic hybridization • Most frequent alteration • Gain in a region of 12q • MDM2, CDK4, and GLI1 overexpression

Primary CNS lymphoma• EBV:

• Immunocompromised individuals

• Proliferation of EBV infected B -cells is usually suppressed by normal T -cell immunity

• EBV infected clone may progress to malignant lymphoma

• EBV extracted from the CSF via PCR

Primary CNS lymphoma• Chemokines:

• Class of molecules that regulate the trafficking of leukocytes as well as their proliferation and adhesion

• BCA 1 (CXCL 13) : expressed at significant levels in PCNSL tumors

• Promotes B -cell homing to secondary lymphoid organs

• Helicobacter pylori–induced MALTomas as well as in gastric lymphoma

BCA 1: B-cell attracting chemokine1

Primary CNS lymphoma• Protooncogene mutation:

• Somatic mutation of PIM1 and MYC oncogene

• High level of expression of PIM1 and MYC proteins

• Ectopic expression of Interleukin-4:• Interleukin 4 is not expressed in the vasculature of normal brain

• Expression by tumor associated endothelia in PCNSL

• May contribute to the angiotropic growth pattern of lymphoma cells within the CNS

Primary CNS lymphoma• Hypermethylation of the CDKN2A gene:

• Established molecular event• Produces p14ARF

CDKN2A: cyclin-dependent kinase Inhibitor 2AHDM2: human double minute 2 (HDM2)

WHO 2007

WHO 2007

WHO 2016

WHO 2016

Summary• First impression is not the last impression

• Not only a morphological diagnosis, but also molecular data

• Prognosis and response to treatment

• 1p/19q assessment, IHC for IDH1 and β-catenin*

The Molecular Pathology of Primary Brain Tumors; David S. Hersh et al, Pathol Case Rev 2013;18: 210-220

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Molecular pathogenesis of CNS tumors